(19) TZZ ¥_T

(11) EP 2 394 657 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 14.12.2011 Bulletin 2011/50 A61K 39/00 (2006.01)

(21) Application number: 11075205.2

(22) Date of filing: 12.10.2008

(84) Designated Contracting States: • Moseman, Elliott Ashley AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Jamaica Plain HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT Massachusetts 02130 (US) RO SE SI SK TR • Zhang, Liangfang Cambridge (30) Priority: 12.10.2007 US 979596 P Massachusetts 02130 (US) • Basto, Pamela (62) Document number(s) of the earlier application(s) in Somerville accordance with Art. 76 EPC: Massachusetts 02144 (US) 08839738.5 / 2 217 269 • Iannacone, Matteo Boston (71) Applicant: Massachusetts Institute Of Technology Massachusetts 02108 (US) Cambridge, MA 02139-4307 (US) • Alexis, Frank Greenville, SC 29607-7405 (US) (72) Inventors: • Von Andrian, Ulrich H. (74) Representative: Bassett, Richard Simon et al Chestnut Hill Potter Clarkson LLP Massachusetts 02467 (US) Park View House • Farokhzad, Omid 58 The Ropewalk Chestnut Hill Nottingham Massachusetts 02467 (US) NG1 5DD (GB) • Langer, Robert Newton Remarks: Massachusetts 02459 (US) This application was filed on 05-09-2011 as a • Junt, Tobias divisional application to the application mentioned 73614 Schorndorf (DE) under INID code 62.

(54) Vaccine nanotechnology

(57) The present invention provides compositions and systems for delivery of nanocarriers to cells of the immune system. The invention provides vaccine nano- carriers capable of stimulating an immune response in T cells and/or in B cells, in some embodiments, comprising at least one immunomodulatory agent, and optionally comprising at least one targeting moiety and optionally at least one immunostimulatory agent. The invention pro- vides pharmaceutical compositions comprising vaccine nanocarriers. The present invention provides methods of designing, manufacturing, and using vaccine nanocar- riers and pharmaceutical compositions thereof. The in- vention provides methods of prophylaxis and/or treat- ment of diseases, disorders, and conditions comprising administering at least one vaccine nanocarrier to a sub- ject in need thereof. EP 2 394 657 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 394 657 A1 2

Description responses. For the treatment and prevention of infectious diseases, there is a need for improved vaccine compo- Related Applications sitions that are highly immunogenic but do not cause dis- ease. [0001] This application claims the benefit under 35 5 U.S.C. §119 of U.S. provisional application serial number Summary of the Invention 60/979,596, filed October 12, 2007. The entire contents of which is herein incorporated by reference. [0006] The present invention provides synthetic nano- carriers for modulating the immune system. The synthet- Government Support 10 ic nanocarriers comprise one or more of an immunomod- ulatory agent, an immunostimulatory agent, and a tar- [0002] The United States Government has provided geting agent (also referred to herein as "targeting moie- grant support utilized in the development of the present ty"). The immunomudulatory agent induces an immune invention. In particular, National Institutes of Health (con- response in B and/or T cells. The immunostimulatory tract numbers AI069259, AI072252, CA 119349, and 15 agent helps stimulate the immune system (in a manner HL56949) and National Institutes of Health/National In- that can ultimately enhance, suppress, direct, or redirect stitute of Biomedical Imaging and BioEngineering (con- an immune response). Immunostimulatory agents, tract number EB 003647) have supported development therefore, include immune suppressants and agents that of this invention. The United States Government has cer- induce regulatory T cells. Such agents can, in some em- tain rights in the invention. 20 bodiments, promote the acquisition of tolerance. The tar- geting agent recognizes one or more targets associated Background of the Invention with a particular organ, tissue, cell, and/or subcellular locale. The nanocarriers are useful in pharmaceutical [0003] Many current vaccines against microbial path- preparations and kits for the prophylaxis and/or treatment ogens comprise live attenuated or non-virulent strains of 25 of diseases, disorders, or conditions susceptible to treat- the causative microorganisms. Many vaccines comprise ment by immune system modulation. Such conditions killed or otherwise inactivated microorganisms. Other include those diseases, disorders, or conditions modified vaccines utilize purified components of pathogen lysates, by enhancing the immune response specifically or non- such as surface carbohydrates or recombinant patho- specifically, suppressing the immune response specifi- gen-derived proteins. Vaccines that utilize live attenuat- 30 cally or nonspecifically, or directing/redirecting the im- ed or inactivated pathogens typically yield a vigorous im- mune response specifically or nonspecifically. mune response, but their use has limitations. For exam- [0007] As will be recognized by those skilled in the art, ple, live vaccine strains can sometimes cause infectious immune system modulation is useful, among other pathologies, especially when administered to immune- things, in connection with medical treatments, such as, compromised recipients. Moreover, many pathogens, 35 for example, for prophylaxis and/or treatment of infec- particularly viruses, undergo continuous rapid mutations tious disease, cancer, allergy, asthma (including allergic in their genome, which allow them to escape immune asthma), autoimmune disease (including rheumatoid ar- responses to antigenically distinct vaccine strains. thritis), immune suppression in connection with trans- [0004] Given the difficulty of vaccine development, plants to ameliorate transplant rejection, immunization many vaccines are in extremely short supply. For exam- 40 against addictive substances, and immunization against ple, as of October 2007, there are influenza, varicella, biohazards and other toxic substances. Immune system and hepatitis A vaccine shortages in the United States. modulation also is useful as a tool in industrial and aca- In some instances, vaccine shortages occur because not demic research settings, such as, for example, to immu- enough manufacturers devote their facilities to vaccine nize an animal to produce antibodies. The nanocarriers production to keep up with demand. In some cases, vac- 45 of the invention can be used for all these purposes. cine shortages are attributed to low potency of the vac- [0008] One aspect of the invention is the provision of cine, which means a large amount of vaccine product vaccines. A vaccine according to the invention typically must be administered to each individual in order to contains an antigen. In one embodiment, the antigen is achieve a prophylactic effect. For example, some vac- physically ’bound’ to the nanocarrier by covalent or non- cines cannot be administered as an intact organism 50 covalent means. Noncovalently bound includes, for ex- (even if attenuated or killed) because they cause infec- ample, ionic bonding, hydrophobic bonding, physical en- tious pathologies. Instead, such vaccines usually com- trapment, and the like, all described in greater detail be- prise purified pathogen components, which typically low. Such nanocarriers which themselves carry an anti- leads to a much less potent immune response. gen are included in the category referred to below as [0005] Thus, there is a need in the art for systems and 55 vaccine nanocarriers. In another embodiment, the nano- methods for producing highly immunogenic, effective carrier has bound to it an immunostimulatory agent for vaccines. There is also a need for improved vaccine com- enhancing, suppressing, directing, or redirecting an im- positions that can potently induce long-lasting immune mune response, preferably to an antigen. In this case,

2 3 EP 2 394 657 A1 4 the antigen may be mixed with the preparation of agent enhanced if covalently bound to nanocarriers. It also has bound nanocarrier to which the immunostimulatory agent been discovered unexpectedly that nanocarriers can is bound form the vaccine. The antigen, of course may help target an immunomodulatory agent or immunostim- also be bound to a nanocarrier, including as discussed ulatory agent to appropriate immune cells even without below, the same nanocarrier to which the immunostim- 5 a targeting agent. ulatory agent is bound. [0012] The systems described herein permit the ma- [0009] It is contemplated that the antigen, in some em- nipulation of the parameters affecting the immune sys- bodiments, is delivered passively (e.g., where a subject tem in a manner which results in improved immune mod- is exposed environmentally to an allergen). In this in- ulation. One important aspect of the invention is that the stance, a nanocarrier with bound immunostimulatory 10 nanocarriers can be controlled in terms of size, density agent could be administered without the antigen, the an- of agent, degree and location of targeting, degradation, tigen being delivered environmentally. For example, the release of agent, etc. A variety of aspects of the invention immunostimulatory agent could be an agent that redi- achieve one or more of these benefits, described in more rects the immune system from a Th2 response to a Th detail below. In particular, below are described immune 1 response. In some embodiments, therefore, the com- 15 modulating preparations, synthetic nanocarrier compo- bination of the administered agent/nanocarrier and the nents of such preparations, specific and preferred nano- environmentally delivered antigen act to redirect the im- carriers, specific and preferred immunomodulatory, im- mune response away from Th2 response which is, gen- munstimulatory, and targeting agents, component parts erally, associated with IgE production and driven by the and building blocks of nanocarriers of the invention, as cytokine IL-4 toward a Th 1 predominant response (which 20 well as methods for manufacturing such nanocarriers, is associated with IgG production and is driven by IL-12 including a preferred method involving self-assembled and interferon-gamma). In some embodiments, the ad- nanocarriers. In addition, preparations and systems for ministered agent/nanocarrier and the environmentally generating robust immune modulation in connection with delivered antigen, therefore, reduce the presence of IgE weak antigens and antigens not recognized by T cells antibodies and thereby treat allergy. It also is possible to 25 (e.g., carbohydrate and small molecule antigens) are de- administer an immunostimulatory agent bound to nano- scribed. In some aspects, a composition comprising a carriers as monotherapy even in the absence of expected nanocarrier (e.g., one that targets a specific organ, tis- environmental exposure to antigen, to redirect the im- sue, cell, or subcellular locale) is provided. In some em- mune response toward Th 1 or to affect aspects of the bodiments, the nanocarrier targets one or more second- immune system which can be manipulated independent 30 ary lymphoid tissues or organs. In some embodiments, of antigen administration, such as eosinophil infiltration. the secondary lympoid tissue or organ is the lymph [0010] The preparations of the invention in many in- nodes, spleen, Peyer’s patches, appendix, or tonsils. stances will include one or more nanocarriers. In some [0013] The scaffold of the nanocarrier (and which the embodiments, the preparation includes a nanocarrier agents provided herein may be associated with or en- bound to one or more, but not all, of an immunomodula- 35 capulated by) can be composed of polymer and/or non- tory agent, an immunostimulatory agent, and a targeting polymer molecules. Accordingly, the nanocarrier scaffold agent. In some embodiments, the preparation is a mix- can be protein-based, nucleic acid based, or carbohy- ture of nanocarriers with subpopulations carrying one or drate-based. The scaffold, in some embodiments, is more, but not all, of an immunomodulatory agent, an im- macromolecular. In some embodiments, the scaffold is munostimulatory agent, and a targeting agent. In some 40 composed of amino acids or nucleic acids. In some em- embodiments, the preparation is a mixture of different bodiments, the scaffold is composed of crosslinking nanocarriers, each nanocarrier carrying one or more, but chains of molecules, such as nucleic acids. In some em- not all, of an immunomodulatory agent, an immunostim- bodiments, the scaffold is composed of RNAi crosslink- ulatory agent, and a targeting agent. The preparations ing chains. In some embodiments, the scaffold is likewise may be one of nanocarriers, wherein each na- 45 polyamino-based. A nanocarrier can be, but is not limited nocarrier has bound to it all of an immunomodulatory to, one or a plurality of lipid-based nanoparticles, poly- agent, an immunostimulatory agent, and a targeting meric nanoparticles, metallic nanoparticles, surfactant- agent. In this instance, the nanocarriers themselves, based emulsions, dendrimers, and/or nanoparticles that apart from the agents they deliver, may be the same or are developed using a combination of nanomaterials different. 50 such as lipid-polymer nanoparticles. [0011] Important is the discovery that the nanocarriers [0014] In some embodiments, the nanocarrier is com- of the invention are powerful at stimulating the immune posed of one or more polymers. In some embodiments, system. Important is the discovery that the nanocarriers the one or more polymers is a water soluble, non-adhe- can be fashioned to mimic, and from an immunological sive polymer. In some embodiments, polymer is polyeth- standpoint, improve on, what the immune system ’sees’ 55 ylene glycol (PEG) or polyethylene oxide (PEO). In some when exposed to antigens in nature or in prior vaccine embodiments, the polymer is polyalkylene glycol or poly- technology. In this respect, it has been discovered un- alkylene oxide. In some embodiments, the one or more expectedly that the activity of adjuvants can be markedly polymers is a biodegradable polymer. In some embodi-

3 5 EP 2 394 657 A1 6 ments, the one or more polymers is a biocompatible pol- 80%, 90%, or more of the nanocarriers of a population ymer that is a conjugate of a water soluble, non-adhesive of nanocarriers have a diameter that is less than 500 nM. polymer and a biodegradable polymer. In some embod- In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, iments, the biodegradable polymer is polylactic acid 70%, 80%, 90%, or more of the nanocarriers of a popu- (PLA), poly(glycolic acid) (PGA), or poly(lactic acid/gly- 5 lation of nanocarriers have a diameter that is greater than colic acid) (PLGA). In some embodiments, the nanocar- 50 nm but less than 500 nm. In some embodiments, 10%, rier is composed of PEG-PLGA polymers. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more [0015] In some embodiments, the nanocarrier is of the nanocarriers of a population of nanocarriers have formed by self-assembly. Self-assembly refers to the a diameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 process of the formation of a nanocarrier using compo- 10 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, nents that will orient themselves in a predictable manner 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or forming nanocarriers predictably and reproducably. In 475 nm. In some embodiments, 10%, 20%, 30%, 40%, some embodiments, the nanocarriers are formed using 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers amphiphillic biomaterials which orient themselves with of a population of nanocarriers have a diameter that is respect to one another to form nanocarriers of predictable 15 between 100-400 nm, 100-300 nm, 100-250 nm, or dimension, constituents, and placement of constituents. 100-200 nm. In some embodiments, 10%, 20%, 30%, According to the invention, the amphiphillic biomaterials 40%, 50%, 60%, 70%, 80%, 90%, or more of the nano- may have attached to them immunomodulatory agents, carriers of a population of nanocarriers have a diameter immunostimulatory agents and/or targeting agents such that is between 60-400 nm, 60-350 nm, 60-300 nm, that when the nanocarriers self assemble, there is a re- 20 60-250 nm, or 60-200 nm. In some of the foregoing em- producible pattern of localization and density of the bodiments, the nanocarriers are nanoparticles. agents on/in the nanocarrier. [0019] The nanocarrier provided herein can be used [0016] In some embodiments, the nanocarrier is a mi- to modulate an immune response (e.g., enhance, sup- croparticle, nanoparticle, or picoparticle. In some embod- presse, direct, or redirect) and comprises at least one of iments, the microparticle, nanoparticle, or picoparticle is 25 an immunomodulatory agent, an immunostimulatory self-assembled. agent, and a targeting agent. In some embodiments, the [0017] In some embodiments, the nanocarrier has a nanocarrier comprises at least one of a B cell antigen, a positive zeta potential. In some embodiments, the nano- T cell antigen, an immunostimulatory agent, and a tar- carrier has a net positive charge at neutral pH. In some geting agent. In some embodiments, the nanocarrier embodiments, the nanocarrier comprises one or more 30 comprises at least two of a B cell antigen, a T cell antigen, amine moieties at its surface. In some embodiments, the an immunostimulatory agent, and a targeting agent. In amine moiety is a primary, secondary, tertiary, or qua- some embodiments, the nanocarrier comprises at least ternary amine. In some embodiments, the amine moiety three of a B cell antigen, a T cell antigen, an immunos- is an aliphatic amine. In some embodiments, the nano- timulatory agent, and a targeting agent. In some embod- carrier comprises an amine-containing polymer. In some 35 iments, the nanocarrier comprises all of a B cell antigen, embodiments, the nanocarrier comprises an amine-con- a T cell antigen, an immunostimulatory agent, and a tar- taining lipid. In some embodiments, the nanocarrier com- geting agent. prises a protein or a peptide that is positively charged at [0020] In some embodiments, the nanocarrier com- neutral pH. In some embodiments, the nanocarrier is a prises a B cell antigen. The B cell antigen may be on the latex particle. In some embodiments, the nanocarrier with 40 surface of the nanocarrier, encapsulated within the na- the one or more amine moieties on its surface has a net nocarrier, or both. In some embodiments, the B cell an- positive charge at neutral pH. tigen is on the surface of the nanocarrier at a density [0018] The nanocarriers of the compositions provided which activates B cell receptors. In some embodiments, herein, in some embodiments, have a mean geometric the B cell antigen is associated with the nanocarrier. In diameter that is less than 500 nm. In some embodiments, 45 some embodiments, the B cell antigen is covalently as- the nanocarriers have mean geometric diameter that is sociated with the nanocarrier. In some embodiments, the greater than 50 nm but less than 500 nm. In some em- B cell antigen is non-covalently associated with the na- bodiments, the mean geometric diameter of a population nocarrier. In some embodiments, the nanocarrier further of nanocarriers is about 60 nm, 75 nm, 100 nm, 125 nm, comprises a targeting moiety. In some embodiments, the 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 50 B cell antigen is a poorly immunogenic antigen. In some nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, embodiments, the B cell antigen is a small molecule. In or 475 nm. In some embodiments, the mean geometric some embodiments, the B cell antigen is an addictive diameter is between 100-400 nm, 100-300 nm, 100-250 substance. Is some embodiments, the B cell antigen is nm, or 100-200 nm. In some embodiments, the mean a toxin. In some embodiments, the toxin for inclusion in geometric diameter is between 60-400 nm, 60-350 nm, 55 a nanocarrier is the complete molecule or a portion there- 60-300 nm, 60-250 nm, or 60-200 nm. In some embod- of. In some embodiments the B cell antigen is not a T iments, the mean geometric diameter is between 75-250 cell antigen. In some embodiments, the B cell antigen is nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, a carbohydrate. In some embodiments, the B cell antigen

4 7 EP 2 394 657 A1 8 is a degenerative disease antigen, an infectious disease is non-covalently associated with the nanocarrier. antigen, a cancer antigen, an atopic disease antigen, an [0025] In some embodiments, where a nanocarrier autoimmune disease antigen, an alloantigen, a xenoan- comprises both a B cell antigen and a T cell antigen, the tigen, an allergen, an addictive substance, or a metabolic nanocarrier further comprises targeting agent. In some disease enzyme or enzymatic product. 5 embodiments, the targeting agent is on the surface of [0021] An allergen refers to a substance (antigen) that the nanocarrier. In some embodiments, the targeting can induce an allergic response in a susceptible subject. agent is associated with the nanocarrier. In some em- The list of allergens is enormous and includes pollens, bodiments, the targeting agent is covalently associated insect venoms, animal dander dust, fungal spores and with the nanocarrier. In some embodiments, the targeting drugs (e.g. penicillin). Allergens also include food aller- 10 agent is non-covalently associated with the nanocarrier. gens. [0026] In some embodiments, where a nanocarrier [0022] In some embodiments, the nanocarrier com- comprises both a B cell antigen and a T cell antigen, the prises a T cell antigen. In some embodiments, the T cell nanocarrier further comprises an immunostimulatory antigen is on the surface of the nanocarrier, encapsulated agent and a targeting agent. In some embodiments, the within the nanocarrier, or both. In some embodiments, 15 immunostimulatory agent is on the surface of the nano- the T cell antigen is associated with the nanocarrier. In carrier (e.g., covalently or non-covalently associated) some embodiments, the T cell antigen is covalently as- and/or is encapsulated within the nanocarrier (e.g., cov- sociated with the nanocarrier. In some embodiments, the alently or non-covalently associated), while the targeting T cell antigen is non-covalently associated with the na- agent is on the surface of the nanocarrier (e.g., covalently nocarrier. In some embodiments, the antigen is a degen- 20 or non-covalently associated). erative disease antigen, an infectious disease antigen, [0027] In some embodiments, the nanocarrier com- a cancer antigen, an atopic disease antigen, an autoim- prises an immunostimulatory agent. In some embodi- mune disease antigen, an alloantigen, a xenoantigen, an ments, the immunostimulatory agent is on the surface of allergen, an addictive substance, or a metabolic disease the nanocarrier. In some embodiments, the immunostim- enzyme or enzymatic product. In some embodiments the 25 ulatory agent is encapsulated within the nanocarrier. In T cell antigen is a ’universal’ T cell antigen (i.e., one which some embodiments, the immunostimulatory agent is can be used with an unrelated B cell antigen, including both on the surface of the nanocarrier and encapsulated a carbohydrate, to stimulate T cell help). In some em- within the nanocarrier. In some embodiments, the immu- bodiments, the nanocarrier further comprises a targeting nostimulatory agent on the surface of the nanocarrier is moiety. 30 different from the immunostimulatory agent encapsulat- [0023] In some embodiments, the nanocarrier com- ed within the nanocarrier. In some embodiments, the im- prises both a B cell antigen and a T cell antigen. In some munostimulatory agent on the surface of and encapsu- embodiments, the B cell antigen and the T cell antigen lated within the nanocarrier is the same. are different antigens. In some embodiments, the B cell [0028] In some embodiments, the nanocarrier com- antigen and the T cell antigen are the same antigen. In 35 prises more than one species of immunostimulatory some embodiments, the B cell antigen is on the surface agents, in which case the immunostimulatory agents are of the nanocarrier (e.g., covalently or non-covalently as- different. sociated) or is both on the surface of the nanocarrier [0029] In some embodiments, the nanocarrier com- (e.g., covalently or non-covalently associated) and en- prises an immunostimulatory agent and an antigen. In capsulated within the nanocarrier (e.g., covalently or 40 some embodiments, the antigen is a B cell antigen or a non-covalently associated), while the T cell antigen is on T cell antigen. In some embodiments, the immunostim- the surface of the nanocarrier (e.g., covalently or non- ulatory agent is an immunosuppressant (suppresses an covalently associated), is encapsulated within the nano- immune response). In some embodiments, the immuno- carrier (e.g., covalently or non-covalently associated), or suppressant is cyclosporin, a steroid, methotrexate or is both on the surface of the nanocarrier (e.g., covalently 45 any agent that interferes with T cell activation. In some or non-covalently associated) and encapsulated within embodiments, the immunostimulatory agent induces the nanocarrier (e.g., covalently or non-covalently asso- regulatory T cells (e.g., TGF-β, rapamycin or retinoic ac- ciated). id). In some embodiments, the immunosuppressant or [0024] In some embodiments, where a nanocarrier agent that induces regulatory T cells promotes the ac- comprises both a B cell antigen and a T cell antigen, the 50 quisition of tolerance to an antigen. The nanocarrier, in nanocarrier further comprises an immunostimulatory some embodiments, further comprises a targeting agent. agent. In some embodiments, the immunostimulatory In some embodiments, the nanocarrier can be used to agent is on the surface of the nanocarrier and/or is en- suppress the immune system and/or promote tolerance capsulated within the nanocarrier. In some embodi- in a subject. ments, the immunostimulatory agent is associated with 55 [0030] In some embodiments where the nanocarrier the nanocarrier. In some embodiments, the immunostim- comprises an immunostimulatory agent, the nanocarrier ulatory agent is covalently associated with the nanocar- further comprises a B cell antigen and/or a T cell antigen. rier. In some embodiments, the immunostimulatory agent In some embodiments, the B cell antigen is a poorly im-

5 9 EP 2 394 657 A1 10 munogenic antigen. In some embodiments, the B cell conjugate/conjugates or non-covalent conjugate/cconju- antigen is a small molecule. In some embodiments, the gates or any combination thereof. In some embodiments, B cell antigen is a carbohydrate. In some embodiments, the antigen is a B cell antigen. In some embodiments, the B cell antigen is an addictive substance. Is some the nanocarrier further comprises a conjugate of a poly- embodiments, the B cell antigen is a toxin. In some em- 5 mer and a T cell antigen. In some embodiments, such a bodiments, the T cell antigen is a degenerative disease conjugate is a covalent or non-covalent conjugate. In antigen, an infectious disease antigen, a cancer antigen, some embodiments, the antigen is a T cell antigen. In an atopic disease antigen, an autoimmune disease an- some embodiments, the nanocarrier further comprises tigen, an alloantigen, a xenoantigen, an allergen, an ad- a conjugate of a polymer and a B cell antigen. In some dictive substance, or a metabolic disease enzyme or en- 10 embodiments, such a conjugate is a covalent or non- zymatic product. In some embodiments, the T cell anti- covalent conjugate. gen is an universal T cell antigen. In some embodiments, [0034] In some aspects, a composition comprising a the nanocarrier further comprises a targeting agent. nanocarrier comprising a molecule or molecules of the [0031] The nanocarrier, in some embodiments, can be following formula X-L1-Y-L2-Z, wherein X is a biodegrad- used to induce or enhance an immune response to a 15 able polymer, Y is a water soluble, non-adhesive poly- poorly immunogenic antigen (e.g., a small molecule or mer, Z is a targeting moiety, an immunomodulatory carbohydrate) in a subject. In some embodiments, the agent, an immunostimulatory agent, or a pharmaceutical nanocarrier can be be used to induce or enhance an agent, and L1 and L2 are bonds or linking molecules, immune response to an addictive substance in a subject. wherein either Y or Z, but not both Y and Z, can be absent, In some embodiments, the nanocarrier can be used to 20 is provided. In some embodiments, the nanocarrier com- induce or enhance an immune response to a toxin in a prises an antigen, an immunostimulatory agent, or both. subject. The nanocarrier, in some embodiments, can be In some embodiments, the pharmaceutical agent is an used to treat a subject that has or is susceptible to an antigen. In some embodiments, the antigen is a degen- addiction. The nanocarrier, in some embodiments, can erative disease antigen, an infectious disease antigen, be used to treat a subject that has been or will be exposed 25 a cancer antigen, an atopic disease antigen, an autoim- to a toxin. In some embodiments, the nanocarrier can be mune disease antigen, an alloantigen, a xenoantigen, an used to treat and/or prevent infectious disease, cancer, allergen, an addictive substance, or a metabolic disease allergy, asthma (including allergic asthma), or autoim- enzyme or enzymatic product. Z may be any antigen de- mune disease (including rheumatoid arthritis). In other scribed herein. In some embodiments, Z is a targeting embodiments, the nanocarriers can be used for immune 30 moiety. In some embodiments, Z is a targeting moiety suppression in connection with transplants to ameliorate that binds a receptor expressed on the surface of a cell. transplant rejection. In some embodiments, Z is a targeting moiety that binds [0032] In some embodiments, the nanocarrier com- a soluble receptor. In some embodiments, the soluble prises a targeting moiety. In some embodiments, the tar- receptor is a complement protein or a pre-existing anti- geting moiety is on the surface of the nanocarrier. In some 35 body. In some embodiments, the targeting moiety is for embodiments, the targeting moiety is associated with the delivery of the nanocarrier to antigen presenting cells, T nanocarrier. In some embodiments, the targeting moiety cells or B cells. In some embodiments, the antigen pre- is covalently associated with the nanocarrier. In some senting cells are dendritic cells (DCs), follicular dendritic embodiments, the targeting moiety is non-covalently as- cells (FDCs), or macrophages. In some embodiments, sociated with the nanocarrier. 40 the macrophages are subcapsular sinus macrophages [0033] In some aspects a composition comprising a (SCS-Mphs). In some embodiments, the Y is PEG or nanocarrier comprising (a) a conjugate of a polymer and PEO. In some embodiments, Y is polyalkylene glycol or an antigen, (b) a conjugate of a polymer and an immu- polyalkylene oxide. In some embodiments, X is PLGA, nostimulatory agent, and/or (c) a conjugate of a polymer PLA or PGA. In some embodiments, Z is absent. and a targeting moiety is provided. In some embodi- 45 [0035] In some aspects, a composition comprising a ments, the nanocarrier comprises a conjugate of a pol- nanocarrier comprising an immunostimulatory agent is ymer and an antigen and a conjugate of a polymer and provided. In some embodiments, the composition further an immunostimulatory agent. In some embodiments, the comprises an antigen and/or a targeting moiety. In some nanocarrier comprises a conjugate of a polymer and an embodiments, at least one of the antigen, targeting moi- antigen and a conjugate of a polymer and a targeting 50 ety, and immunostimulatory agent is conjugated to a wa- moiety. In some embodiments, the nanocarrier compris- ter soluble, non-adhesive polymer. In some embodi- es a conjugate of a polymer and an immunostimulatory ments, at least one of the antigen, targeting moiety, and agent and a conjugate of a polymer and a targeting moi- immunostimulatory agent is conjugated to a biodegrad- ety. In some embodiments, the nanocarrier comprises a able polymer. In some embodiments, at least one of the conjugate of a polymer and an antigen, a conjugate of a 55 antigen, targeting moiety, and immunostimulatory agent polymer and an immunostimulatory agent and a conju- is conjugated to a biocompatible polymer. In some em- gate of a polymer and a targeting moiety. In some em- bodiments, the biocompatible polymer is a conjugate of bodiments, the conjugate or conjugates is/are covalent a water soluble, non-adhesive polymer conjugated to a

6 11 EP 2 394 657 A1 12 biodegradable polymer. In some embodiments, the an- biocompatible polymer. In some embodiments, the nic- tigen is a B cell antigen. In some embodiments, the B otine is conjugated to a biocompatible polymer. In some cell antigen is not a T cell antigen. In some embodiments, embodiments, the immunostimulatory agent is on the the nanocarrier further comprises a T cell antigen. In surface of the nanocarrier, is encapsulated within the na- some embodiments, the antigen is a T cell antigen. 5 nocarrier, or is both on the surface of the nanocarrier and [0036] In some aspects, a composition comprising a encapsulated within the nanocarrier. In some embodi- nanocarrier comprising a small molecule, an immunos- ments, the immunostimulatory agent is conjugated to a timulatory agent, and a T cell antigen is provided. In some polymer. In some embodiments, the polymer is a water embodiments, the small molecule is on the surface of the soluble, non-adhesive polymer, a biodegradable poly- nanocarrier or is both on the surface of the nanocarrier 10 mer, or a biocompatible polymer biodegradable polymer. and encapsulated within the nanocarrier. In some em- In some embodiments, immunostimulatory agent is con- bodiments, the small molecule is an addictive substance. jugated to a biodegradable polymer. In some embodi- In some embodiments, the addictive substance is nico- ments, the targeting moiety is conjugated to a polymer. tine. In some embodiments, the small molecule is a toxin. In some embodiments, the polymer is a water soluble, In some embodiments, the toxin is from a chemical weap- 15 non-adhesive polymer, a biodegradable polymer, or a on, an agent of biowarfare, or a hazardous environmental biocompatible polymer biodegradable polymer. In some agent. In some embodiments, the small molecule is con- embodiments, targeting moiety is conjugated to a bio- jugated to a polymer. In some embodiments, the polymer compatible polymer. In some embodiments, the water is a water soluble, non-adhesive polymer, a biodegrad- soluble, non-adhesive polymer is PEG or PEO. In some able polymer, or a biocompatible polymer. In some em- 20 embodiments the water soluble, non-adhesive polymer bodiments, the polymer is a biocompatible polymer. In is polyalkylene glycol or polyalkylene oxide. In some em- some embodiments, the immunostimulatory agent is on bodiments, the biodegradable polymer is PLGA, PLA, or the surface of the nanocarrier or is both on the surface PGA. In some embodiments, the biocompatible polymer of the nanocarrier and encapsulated within the nanocar- is a conjugate of a water soluble, non-adhesive polymer rier. In some embodiments, the immunostimulatory agent 25 and a biodegradable polymer. is conjugated to a polymer. In some embodiments, the [0038] In some embodiments of any of the nanocarri- polymer is a water soluble, non-adhesive polymer, a bi- ers provided herein, an immunostimulatory agent is en- odegradable polymer, or a biocompatible polymer bio- capsulated within the nanocarrier. In some of these em- degradable polymer. In some embodiments, the polymer bodiments, the immunostimulatory agent is R848, a is water soluble, non-adhesive polymer or a biodegrad- 30 TLR9 agonist (e.g., a CpG/CpG-containing nucleic acid). able polymer. In some embodiments, the nanocarrier fur- Such nanocarriers, in some embodiments, may be used ther comprises a targeting moiety. In some embodi- to activate CD4 T cells and/or CD8 T cells. In some em- ments, the targeting moiety is conjugated to a polymer. bodiments, the immunostimulatory agent, e.g., R848 or In some embodiments, the polymer is a water soluble, TLR9 agonist, is not conjugated. In some embodiments, non-adhesive polymer, a biodegradable polymer, or a 35 the immunostimulatory agent, e.g., R848 or TLR9 ago- biocompatible polymer biodegradable polymer. In some nist, is conjugated to a polymer. In some embodiments, embodiments, the polymer is a biocompatible polymer. the conjugation is covalent. In some embodiments, the In some embodiments, the water soluble, non-adhesive conjugation is non-covalent. In some embodiments, the polymer is PEG or PEO. In some embodiments the water polymer is a water soluble, non-adhesive polymer. In soluble, non-adhesive polymer is polyalkylene glycol or 40 some embodiments, the polymer is a biodegradable pol- polyalkylene oxide. In some embodiments, the biode- ymer. In some embodiments, the polymer is a biocom- gradable polymer is PLGA, PLA, or PGA. In some em- patible polymer. In some embodiments, the polymer is bodiments, the biocompatible polymer is a conjugate of PEG-PLA or PLA. In any of these embodiments, the na- a water soluble, non-adhesive polymer and a biodegrad- nocarrier can further comprise a T cell antigen. able polymer. 45 [0039] In some aspects, a composition comprising a [0037] In some embodiments, a composition compris- nanocarrier comprising a poorly immunogenic antigen, ing a nanocarrier comprising nicotine, an immunostimu- an immunostimulatory agent, and a T cell antigen is pro- latory agent, a T cell antigen, and a targeting moiety is vided. In some embodiments, the poorly immunogenic provided. In some embodiments, the immunostimulatory antigen is on the surface of the nanocarrier or is both on agent is a TLR 7/8 agonist. In some embodiments, the 50 the surface of the nanocarrier and encapsulated within immunostimulatory agent is R848 (also referred to as the nanocarrier. In some embodiments, the poorly im- CL097) or imiquimod. In some embodiments, the nicotine munogenic antigen is a small molecule or a carbohy- is on the surface of the nanocarrier or is both on the drate. In some embodiments, the poorly immunogenic surface of the nanocarrier and encapsulated within the antigen is an addictive substance. In some embodi- nanocarrier. In some embodiments, the nicotine is con- 55 ments, the poorly immunogenic antigen is a toxin. In jugated to a polymer, preferably covalently conjugated. some embodiments, the poorly immunogenic antigen is In some embodiments, the polymer is a water soluble, covalently conjugated to a polymer. In some embodi- non-adhesive polymer, a biodegradable polymer, or a ments, the polymer is a water soluble, non-adhesive pol-

7 13 EP 2 394 657 A1 14 ymer, a biodegradable polymer, or a biocompatible pol- is a B cell antigen or a T cell antigen. ymer biodegradable polymer. In some embodiments, the [0045] In some embodiments, Z is a degenerative dis- immunostimulatory agent is on the surface of the nano- ease antigen, an infectious disease antigen, a cancer carrier or is both on the surface of the nanocarrier and antigen, an atopic disease antigen, an autoimmune dis- encapsulated within the nanocarrier. In some embodi- 5 ease antigen, an alloantigen, a xenoantigen, an allergen, ments, the immunostimulatory agent is covalently con- a hapten, an addictive substance, or a metabolic disease jugated to a polymer. In some embodiments, the polymer enzyme or enzymatic product. In some embodiments, Z is a water soluble, non-adhesive polymer, a biodegrad- is any of the B cell antigens described herein. In some able polymer, or a biocompatible polymer biodegradable embodiments, Z is any of the T cell antigens provided polymer. In some embodiments, the nanocarrier further 10 herein. comprises a targeting moiety. In some embodiments, the [0046] In some embodiments, Z is a targeting moiety targeting moiety is covalently conjugated to a polymer. that binds a receptor expressed on the surface of a cell. In some embodiments, the polymer is a water soluble, In some embodiments, Z is a targeting moiety that binds non-adhesive polymer, a biodegradable polymer, or a a soluble receptor. In some embodiments, the soluble biocompatible polymer biodegradable polymer. 15 receptor is complement or a pre-existing antibody. In [0040] In some aspects, a composition comprising a some embodiments, the targeting moiety is for targeting nanocarrier that targets a specific cell, tissue or organ antigen presenting cells, T cells or B cells. and modulates an immune response comprising a B cell [0047] In some embodiments, Y is PEG or PEO. In antigen on its surface at a density that activates B cells some embodiments, Y is polyalkylene glycol or poly- and a immunostimulatory agent is provided. In some em- 20 alkylene oxide. bodiments, the nanocarrier further comprises a targeting [0048] In some embodiments, X is PLGA, PGA, or moiety. In some embodiments, the composition is a phar- PLA. maceutical composition and further comprises a phar- [0049] In some embodiments, Z is absent. In some maceutically acceptable carrier. In some embodiments, embodiments, Y is absent. the pharmaceutical composition is a vaccine composi- 25 [0050] In some aspects, a pharmaceutical composi- tion. tion comprising a conjugate of a immunostimulatory [0041] In some aspects, a composition, such as a phar- agent and a polymer is provided. In some embodiments, maceutical composition, comprising an antigen present- the conjugate is a covalent conjugate. In some embodi- ing cell-targeting moiety and a nanocarrier is provided. ments, the conjugate is a non-covalent conjugate. In In some embodiments, the antigen presenting cell-tar- 30 some embodiments, the polymer is a water soluble, non- geting moiety and nanocarrier are conjugated. In some adhesive polymer, a biodegradable polymer, or a bio- embodiments, the conjugate is a covalent conjugate. In compatible polymer. In some embodiments, the polymer some embodiments, the conjugate is a non-covalent con- is a biocompatible polymer. In some embodiments, the jugate. biocompatible polymer is a biodegradable polymer or a [0042] In some aspects, a composition, such as a phar- 35 water soluble, non-adhesive polymer. In some embodi- maceutical composition, comprising an immunostimula- ments, the biocompatible polymer is a conjugate of a tory agent and a nanocarrier is provided. In some em- water soluble, non-adhesive polymer and a biodegrada- bodiments, the immunostimulatory agent and nanocar- ble polymer. In some embodiments, the polymer is syn- rier are conjugated. In some embodiments, the conjugate thetic. In some embodiments, the pharmaceutical com- is a covalent conjugate. In some embodiments, the con- 40 position comprises one or more nanocarriers wherein the jugate is a non-covalent conjugate. conjugate is a component of the one or more nanocarri- [0043] In some aspects, a composition comprising a ers. In some embodiments, the composition further com- molecule with the formula X-L1-Y-L2-Z, wherein X is a prises an antigen. In some embodiments, the pharma- biodegradable polymer, Y is a water soluble, non-adhe- ceutical composition does not comprise an antigen. In sive polymer, Z is a targeting moiety, an immunostimu- 45 some embodiments, the composition further comprises latory agent, or a pharmaceutical agent, and L1 and L2 a targeting agent. are bonds or linking molecules, wherein either Y or Z, [0051] In some aspects, a vaccine composition com- but not both Y and Z, can be absent is provided. prising a conjugate of an immunostimulatory agent and [0044] In some aspects, a composition comprising a a polymer is provided. In some embodiments, the con- molecule with the formula T-L1-X-L2-Y-L3-Z, where T is 50 jugate is a covalent conjugate. In some embodiments, a T cell antigen, X is a biodegradable polymer, Y is a the conjugate is a non-covalent conjugate. In some em- water soluble, non-adhesive polymer, Z is an Z is a tar- bodiments, the polymer is a water soluble, non-adhesive geting moiety, an immunostimulatory agent, or a phar- polymer, a biodegradable polymer, or a biocompatible maceutical agent, wherein L1, L2, and L3 are bonds or polymer. In some embodiments, the water soluble, non- linking molecules, and wherein any one or two of T, Y, 55 adhesive polymer is polyethylene glycol. In some em- and Z, but not all three of T, Y, and Z, can be absent is bodiments, the polymer is a biocompatible polymer. In provided. In some embodiments, the pharmaceutical some embodiments, the biocompatible polymer is a bi- agent is an antigen. In some embodiments, the antigen odegradable polymer or a water soluble, non-adhesive

8 15 EP 2 394 657 A1 16 polymer. In some embodiments, the biocompatible pol- chloroethyl)sulfide, Bis(2-chloroethylthio)methane, Ses- ymer is a conjugate of a water soluble, non-adhesive quimustard: 1,2-Bis(2-chloroethylthio)ethane, 1,3-Bis(2- polymer and a biodegradable polymer. In some embod- chloroethylthio)-n-propane, 1,4-Bis(2-chloroethylth- iments, the polymer is synthetic. In some embodiments, io)-n-butane, 1,5-Bis(2-chloroethylthio)-n-pentane, Bis the pharmaceutical composition comprises one or more 5 (2-chloroethylthiomethyl)ether, O-Mustard: Bis(2-chlo- nanocarriers wherein the conjugate is a component of roethylthioethyl)ether, Lewisites: Lewisite 1: 2-Chlorovi- the one or more nanocarriers. In some embodiments, the nyldichloroarsine, Lewisite 2: Bis(2-chlorovinyl)chloro- composition further comprises an antigen. In some em- arsine, Lewisite 3: Tris(2-chlorovinyl)arsine, Nitrogen bodiments, the pharmaceutical composition does not mustards: HN1: Bis(2-chloroethyl)ethylamine, HN2: Bis comprise an antigen. In some embodiments, the compo- 10 (2-chloroethyl)methylamine, HN3: Tris(2-chloroethyl) sition further comprises a targeting agent. amine,Saxitoxin,Ricin,Amiton:O,O-DiethylS-(2-(diethyl- [0052] In some embodiments, the B cell antigen is a amino)ethyl)phosphorothiolate and corresponding protein or peptide. In some embodiments, the B cell an- alkylated or protonated salts, PFIB: 1,1,3,3,3-Pen- tigen is a non-protein antigen (i.e., not a protein or pep- tafluoro-2-(trifluoromethyl)-1-propene, 3-Quinuclidinyl tide). In some embodiments, the protein or peptide is 15 benzilate (BZ), Phosgene: Carbonyl dichloride, Cyano- from an infectious agent. In some embodiments, the in- gen chloride, Hydrogen cyanide and Chloropicrin: fectious agent is a bacterium, fungus, virus, protozoan, Trichloronitromethane. In some embodiments, the toxin or parasite. In some embodiments, the virus is a pox for inclusion in a nanocarrier is a complete molecule of virus, smallpox virus, ebola virus, marburg virus, dengue any of the foregoing or a portion thereof. fever virus, influenza virus, parainfluenza virus, respira- 20 [0056] In some embodiments, the B cell antigen is a tory syncytial virus, rubeola virus, human immunodefi- biohazard or hazardous environmental agent. In some ciency virus, human papillomavirus, varicella-zoster vi- embodiments, the hazardous environmental agent is ar- rus, herpes simplex virus, cytomegalovirus, Epstein-Barr senic, lead, mercury, vinyl chloride, polychlorinated bi- virus, JC virus, rhabdovirus, rotavirus, rhinovirus, aden- phenyls, benzene, polycyclic aromatic hydrocarbons, ovirus, papillomavirus, parvovirus, picornavirus, poliovi- 25 cadmium, benzo(a)pyrene, benzo(b)fluoranthene, chlo- rus, virus that causes mumps, virus that causes rabies, roform, dichlor-diphenyl-trichlorethylene (DDT), P,P’-, reovirus, rubella virus, togavirus, orthomyxovirus, retro- aroclor 1254, aroclor 1260, dibenzo(a,h)anthracene, virus, hepadnavirus, coxsackievirus, equine encephalitis trichloroethylene, dieldrin, chromium hexavalent, or p,p’- virus, Japanese encephalitis virus, yellow fever virus, Rift dichlorodiphenyldichloroethene (DDE, P,P’). Valley fever virus, hepatitis A virus, hepatitis B virus, hep- 30 [0057] In some embodiments, the B cell antigen is a atitis C virus, hepatitis D virus, or hepatitis E virus. carbohydrate. In some embodiments, the carbohydrate [0053] In some embodiments, the B cell antigen is a is from an infectious agent. In some embodiments, the small molecule. In some embodiments, the small mole- infectious agent is a bacterium, fungus, virus, protozoan, cule is an abused substance, an addictive substance, or or parasite. In some embodiments, the bacterium is a a toxin. 35 Pseudomonas, Pneumococcus, E. coli, Staphylococ- [0054] In some embodiments, the B cell antigen is an cus, Streptococcus, Treponema, Borrelia, Chlamydia, addictive substance. In some embodiments, the addic- Haemophilus, Clostridium, Salmonella, Legionella, Vi- tive substance is nicotine, a narcotic, a hallucinogen, a brio or Enterococci bacterium or a Mycobacterium. In stimulant, a cough suppressant, a tranquilizer, or a sed- some embodiments, the virus is a pox virus, smallpox ative. In some embodiments, the B cell antigen is an opi- 40 virus, ebola virus, marburg virus, dengue fever virus, in- od or benzodiazepine. fluenza virus, parainfluenza virus, respiratory syncytial [0055] In some embodiments, the B cell antigen is a virus, rubeola virus, human immunodeficiency virus, hu- toxin. In some embodiments, the toxin is from a chemical man papillomavirus, varicella-zoster virus, herpes sim- weapon. In some embodiments, the toxin from a chem- plex virus, cytomegalovirus, Epstein-Barr virus, JC virus, ical weapon is botulinum toxin or phosphene. Toxins from 45 rhabdovirus, rotavirus, rhinovirus, adenovirus, papillo- a chemical weapon also include, but are not limited to, mavirus, parvovirus, picornavirus, poliovirus, virus that O-Alkyl (

9 17 EP 2 394 657 A1 18 the cytokine is TNF, IL-1, or IL-6. In some embodiments, coxsackievirus, equine encephalitis virus, Japanese en- the self antigen is cholesteryl ester transfer protein (CE- cephalitis virus, yellow fever virus, Rift Valley fever virus, TP), the Aβ protein associated with Alzheimer’s, a pro- hepatitis A virus, hepatitis B virus, hepatitis C virus, hep- teolytic enzyme that processes the pathological form of atitis D virus, or hepatitis E virus. the Aβ protein, LDL associated with atherosclerosis, or 5 [0062] In some embodiments, the B cell antigen is a a coreceptor for HIV-1. In some embodiments, the pro- poorly immunogenic antigen. In some embodiments, the teolytic enzyme that processes the pathological form of poorly immunogenic antigen is a non-protein antigen. In the Aβ protein is beta-secretase. In some embodiments, some embodiments, the poorly immunogenic antigen is the LDL associated with atherosclerosis is oxidized or a carbohydrate or small molecule. In some embodi- minimally modified. In some embodiments, the corecep- 10 ments, the poorly immunogenic antigen is an abused tor for HIV-1 is CCR5. In some embodiments, the self substance, addictive substance, or toxin. In some em- antigen is an autoimmune disease antigen. bodiments, the toxin is from a chemical weapon. In some [0059] In some embodiments, the B cell antigen is a embodiments, the poorly immunogenic antigen is a haz- degenerative disease antigen, an infectious disease an- ardous environmental agent. In some embodiments, the tigen, a cancer antigen, an atopic disease antigen, an 15 poorly immunogenic antigen is a self antigen. autoimmune disease antigen, or a metabolic disease en- [0063] In general, the T cell antigen is a protein or pep- zyme or enzymatic product thereof. tide. In some embodiments, the T cell antigen is a de- [0060] In some embodiments, the antigen is a cancer generative disease antigen, an infectious disease anti- antigen. Is some embodiments, the cancer antigen is Me- gen, a cancer antigen, an atopic disease antigen, an au- lan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adeno- 20 toimmune disease antigen, an alloantigen, a xenoanti- sine deaminase-binding protein (ADAbp), cyclophilin b, gen, an allergen, a contact sensitizer, a hapten, or a met- Colorectal associated antigen (CRC)-C017-1A/GA733, abolic disease enzyme or enzymatic product. Carcinoembryonic Antigen (CEA) and its immunogenic [0064] In some embodiments, the T cell antigen is from epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Spe- an infectious agent. In some embodiments, the infectious cific Antigen (PSA) and its immunogenic epitopes PSA- 25 agent is a bacterium, fungus, virus, protozoan, or para- 1, PSA-2, and PSA-3, prostate-specific membrane anti- site. In some embodiments, the infectious disease anti- gen (PSMA), T-cell receptor/CD3-zeta chain, MAGE- gen is a viral antigen. In some embodiments, the viral family of tumor antigens (e.g., MAGE-A1, MAGE-A2, antigen is an antigen from a pox virus, smallpox virus, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE- ebola virus, marburg virus, dengue fever virus, influenza A7, MAGE-A8, MAGE-A9, MAGE-A 10, MAGE-A11, 30 virus, parainfluenza virus, respiratory syncytial virus, ru- MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 beola virus, human immunodeficiency virus, human pap- (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, illomavirus, varicella-zoster virus, herpes simplex virus, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE- cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovi- family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE- rus, rotavirus, rhinovirus, adenovirus, papillomavirus, 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, 35 parvovirus, picornavirus, poliovirus, virus that causes GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM- mumps, virus that causes rabies, reovirus, rubella virus, 1, CDK4, tyrosinase, p53, MUC family, HER2/neu, togavirus, orthomyxovirus, retrovirus, hepadnavirus, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β- coxsackievirus, equine encephalitis virus, Japanese en- catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, cephalitis virus, yellow fever virus, Rift Valley fever virus, NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX- 40 hepatitis A virus, hepatitis B virus, hepatitis C virus, hep- 2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, atitis D virus, or hepatitis E virus. cdc27, adenomatous polyposis coli protein (APC), fodrin, [0065] In some embodiments, T cell antigen is a uni- P1A, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 versal T cell antigen. In some embodiments, the universal gangliosides, viral products such as human papilloma T cell antigen is one or more peptides derived from tet- virus proteins, Smad family of tumor antigens, Imp-1, 45 anus toxoid, Epstein-Barr virus, or influenza virus. EBV-encoded nuclear antigen (EBNA)-1, or c-erbB-2. [0066] In some embodiments, immunostimulatory [0061] In some embodiments, the infectious disease agents are interleukins, interferon, cytokines, etc. In antigen is a viral antigen. In some embodiments, the viral some embodiments, the immunostimulatory agent is a antigen is an antigen from a pox virus, smallpox virus, toll-like receptor (TLR) agonist, cytokine receptor ago- ebola virus, marburg virus, dengue fever virus, influenza 50 nist, CD40 agonist, Fc receptor agonist, CpG-containing virus, parainfluenza virus, respiratory syncytial virus, ru- immunostimulatory nucleic acid, complement receptor beola virus, human immunodeficiency virus, human pap- agonist, or an adjuvant. In some embodiments, the TLR illomavirus, varicella-zoster virus, herpes simplex virus, agonist is a TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR- cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovi- 6, TLR-7, TLR-8, TLR-9, or TLR-10 agonist. In some em- rus, rotavirus, rhinovirus, adenovirus, papillomavirus, 55 bodiments, the Fc receptor agonist is a Fc-gamma re- parvovirus, picornavirus, poliovirus, virus that causes ceptor agonist. In some embodiments, the complement mumps, virus that causes rabies, reovirus, rubella virus, receptor agonist binds to CD21 or CD35. In some em- togavirus, orthomyxovirus, retrovirus, hepadnavirus, bodiments, the complement receptor agonist induces en-

10 19 EP 2 394 657 A1 20 dogenous complement opsonization of the nanocarrier. ments, the targeting moiety is the soluble receptor, CRFc. In some embodiments, the cytokine receptor agonist is In some embodiments, CRFc can be used to target mac- a cytokine. In some embodiments, the cytokine receptor rophages in the subcapsular sinus but not macrophages agonist is a small molecule, antibody, fusion protein, or of the medulla. In some embodiments, the targeting moi- aptamer. In some embodiments, the immunostimulatory 5 ety is one or more amine moieties. agent is an adjuvant. In some embodiments, the adjuvant [0070] In some aspects, the compositions provided induces cytokine biosynthesis. In some embodiments, herein are immunogenic. the adjuvant is alum, MF59, R848, cholera toxin, [0071] In some aspects, a method comprising admin- squalene, phosphate adjuvants, or tetrachlorodecaox- istering any of the compositions provided herein to a sub- ide. In some embodiments, the adjuvant is monophos- 10 ject in an amount effective to modulate an immune re- phoryl lipid A (MPL, SmithKline Beecham); saponins in- sponse is provided. In some embodiments, the compo- cluding QS21 (SmithKline Beecham); immunostimulato- sition is in an amount effective to induce or enhance an ry oligonucleotides (e.g., CpG immunostimulatory oligo- immune response. In some embodiments, the composi- nucleotides first described by Kreig et al., Nature 374: tion is in an amount effective to suppress an immune 546-9, 1995); incomplete Freund’s adjuvant; complete 15 response. In some embodiments, the composition is in Freund’s adjuvant; montanide; vitamin E and various wa- an amount effective to direct or redirect an immune re- ter-in-oil emulsions prepared from biodegradable oils sponse. In some embodiments, the method is for proph- such as squalene and/or tocopherol, Quil A, Ribi Detox, ylaxis and/or treatment of the conditions identified herein. CRL-1005, or L-121. [0072] In some embodiments, where the method is to [0067] In specific embodiments, an immunostimulato- 20 induce or enhance an immune response, the subject has ry agent may be a natural or synthetic agonist for a Toll- or is susceptible to having cancer, an infectious disease, like receptor (TLR). In specific embodiments, an immu- a non-autoimmune metabolic or degenerative disease, nostimulatory agent may be a ligand for toll-like receptor an atopic disease, an allergic disease, or an addiction. (TLR)-7, such as CpGs, which induce type I interferon In some embodiments, the subject has been exposed to production; an agonist for the DC surface molecule 25 or may be exposed to a toxin. In some embodiments, the CD40; an agent that promotes DC maturation; a TLR-4 subject has been exposed to or may be exposed to a agonist; a cytokine; proinflammatory stimuli released toxin from a chemical weapon. In some embodiments, from necrotic cells (e.g. urate crystals); activated com- the subject has been exposed to or may be exposed to ponents of the complement cascade (e.g. CD21, CD35, a toxin from a hazardous environmental substance. In etc.); and so forth. 30 some embodiments, the nanocarrier comprises a B-cell [0068] In some embodiments, the targeting moiety antigen, an immunostimulatory agent, and a T cell anti- binds a receptor expressed on the surface of a cell. In gen, such as an universal T cell antigen. In some em- some embodiments, the targeting moiety binds a soluble bodiments, the nanocarrier further comprises a targeting receptor. In some embodiments, the soluble receptor is moiety. a complement protein or a pre-existing antibody. In some 35 [0073] In some embodiments, where the method is for embodiments, the targeting moiety is for delivery of the treating or preventing an addiction (or for treating a sub- nanocarrier to antigen presenting cells, T cells, or B cells. ject exposed to or who may be exposed to a toxin), the In some embodiments, the antigen presenting cells are nanocarrier comprises the addictive substance or toxin, macrophages. In some embodiments, the macrophages an adjuvant, and a T cell. In some embodiments, the are subcapsular sinus macrophages. In some embodi- 40 method raises high titer antibodies that bind and neutral- ments, the antigen presenting cells are dendritic cells. In ize the offending agent before it reaches its effector site some embodiments, the antigen presenting cells are fol- (e.g., the brain). In some embodiments, the addictive licular dendritic cells. substance or toxin is at a high density on the surface of [0069] In some embodiments, the targeting moiety is the nanocarrier. a molecule that binds to CD11 b, CD169, mannose re- 45 [0074] In some embodiments, the infectious disease, ceptor, DEC-205, CD11c, CD21/CD35, CX3CR1, or a is a chronic viral infection. In some embodiments, the Fc receptor. In some embodiments, the targeting moiety chronic viral infection is HIV, HPV, HBV, or HCV infection. is a molecule that binds to CD169, CX3CR1, or a Fc In some embodiments, the infectious disease is or is receptor. In some embodiments, the molecule that binds caused by a bacterial infection. In some embodiments, to CD169 is an anti-CD169 antibody. In some embodi- 50 the subject has or is susceptible to having a Pseu- ments, the molecule that binds CX3CR1 is CX3CL1 (frac- domonas infection, a Pneumococcus infection, tubercu- talkine). In some embodiments, the targeting moiety losis, malaria, leishmaniasis, H. pylori, a Staphylococcus comprises the Fc portion of an immunoglobulin. In some infection, or a Salmonella infection. In some embodi- embodiments, the targeting moiety comprises the Fc por- ments, the infectious disease is or is caused by a fungal tion of an IgG. In some embodiments, the Fc portion of 55 infection. In some embodiments, the infectious disease an immunoglobulin is a human Fc portion of an immu- is or is caused by a parasitic infection. In some embod- noglobulin. In some embodiments, the Fc portion of an iments, the infectious disease is or is caused by a pro- IgG is a human Fc portion of an IgG. In some embodi- tozoan infection. In some embodiments, the subject has

11 21 EP 2 394 657 A1 22 or is susceptible to having influenza. embodiments, an immunomodulatory agent is an anti- [0075] In some embodiments, where the method is to gen. In some embodiments, an immunomodulatory suppress or redirect an immune response, the subject agent is used for vaccines. has or is susceptible to having an allergic disease or an [0080] In some embodiments, an immunomodulatory autoimmune disease. In some embodiments, the subject 5 agent is any protein and/or other antigen derived from a has a food allergy. In some embodiments, the subject is pathogen. The pathogen may be a virus, bacterium, fun- allergic to milk or other milk components (e.g., lactose), gus, protozoan, parasite, etc. In some embodiments, an eggs, peanuts, tree nuts (walnuts, cashews, etc.), fish, immunomodulatory agent may be in the form of whole shellfish, soy, or wheat. In some embodiments, the meth- killed organisms, peptides, proteins, glycoproteins, glyc- od is to tolerize a subject to an antigen, such as an aller- 10 opeptides, proteoglycans, carbohydrates, or combina- gen. In some embodiments, the autoimmune disease is tions thereof. disease is lupus, multiple sclerosis, rheumatoid arthritis, [0081] In some embodiments, all of the immunomod- diabetes mellitus type I, inflammatory bowel disease, thy- ulatory agents of a vaccine nanocarrier are identical to roiditis, or celiac disease. In some embodiments, the sub- one another. In some embodiments, all of the immu- ject has had or will have a transplant, and the method 15 nomodulatory agents of a vaccine nanocarrier are differ- can be to prevent or ameliorate transplant rejection. In ent. In some embodiments, a vaccine nanocarrier com- some embodiments, the nanocarrier comprises an anti- prises exactly one distinct type (i.e., species) of immu- gen and an immune suppressant or an agent that induces nomodulatory agent. For example, when the immu- regulatory T cells. In some embodiments, the nanocarrier nomodulatory agent is an antigen, all of the antigens that further comprises a targeting moiety. Generally, where 20 are in the vaccine nanocarrier are the same. In some the method is one to suppress an immune response, the embodiments, a vaccine nanocarrier comprises exactly antigen is provided in the absence of an adjuvant. two distinct types of immunomodulatory agents. In some [0076] In some embodiments for treating an allergy, embodiments, a vaccine nanocarrier comprises greater the nanocarrier includes an allergen and an immunos- than two distinct types of immunomodulatory agents. timulatory agent (such as an adjuvant, e.g., a TLR ago- 25 [0082] In some embodiments, a vaccine nanocarrier nist). comprises a single type of immunomodulatory agent that [0077] In some embodiments, the composition is ad- stimulates an immune response in B cells. In some em- ministered in an amount effective to modify an immune bodiments, a vaccine nanocarrier comprises a single response (e.g., from a Th2 to a Th1 immune response). type of immunomodulatory agent that stimulates an im- In some embodiments, the subject has or is susceptible 30 mune response in T cells. In some embodiments, a vac- to having an allergic disease. In some embodiments, the cine nanocarrier comprises two types of immunomodu- nanocarrier comprises an antigen, such as an allergen, latory agents, wherein the first immunomodulatory agent and an immunostimulatory agent. In some embodiments, stimulates B cells, and the second immunomodulatory the nanocarrier further comprises a targeting moiety. agent stimulates T cells. In certain embodiments, any of [0078] In some aspects, vaccine nanocarriers for de- 35 the aforementioned agents could stimulate both B cells livery of immunomodulatory agents to the cells of the and T cells, but this is not necessarily so. In certain em- immune system are provided. In some embodiments, bodiments, the aforementioned immunomodulatory vaccine nanocarriers comprise at least one immunomod- agents stimulates only B cells or T cells, respectively. In ulatory agent that is capable of inducing an immune re- some embodiments, a vaccine nanocarrier comprises sponse in B cells and/or in T cells. In certain embodi- 40 greater than two types of immunomodulatory agents, ments, immunomodulatory agents presented on nano- wherein one or more types of immunomodulatory agents carrier surfaces stimulate B cells, and immunomodula- stimulate B cells, and one or more types of immunomod- tory agents encapsulated within the nanocarriers are ulatory agents stimulate T cells. processed and presented to T cells. In some embodi- [0083] In some embodiments, a vaccine nanocarrier ments, vaccine nanocarriers comprise at least one tar- 45 includes a lipid membrane (e.g. lipid bilayer, lipid mon- geting moiety that is useful for selective delivery of the olayer, etc.). At least one immunomodulatory agent may vaccine nanocarrier to specific antigen-presenting cells be associated with the lipid membrane. In some embod- (APCs). iments, at least one immunomodulatory agent is embed- [0079] In some embodiments, an immunomodulatory ded within the lipid membrane, embedded within the lu- agent may comprise isolated and/or recombinant pro- 50 men of a lipid bilayer, associated with the interior surface teins or peptides, carbohydrates, glycoproteins, glyco- of the lipid membrane, and/or encapsulated with the lipid peptides, proteoglycans, inactivated organisms and vi- membrane of a vaccine nanocarrier. ruses, dead organisms and virus, genetically altered or- [0084] In some embodiments, a vaccine nanocarrier ganisms or viruses, and cell extracts. In some embodi- includes a polymer (e.g. a polymeric core). The immu- ments, an immunomodulatory agent may comprise nu- 55 nomodulatory agent may be associated with the polymer, cleic acids, carbohydrates, lipids, and/or small mole- and in some embodiments, at least one type of immu- cules. In some embodiments, an immunomodulatory nomodulatory agent is associated with the polymer. In agent is one that elicits an immune response. In some some embodiments, the immunomodulatory agent is em-

12 23 EP 2 394 657 A1 24 bedded within the polymer, associated with the interior CD 11b (i.e. CD 11b/CD18, Mac-1, CR3 or αMβ2 in- surface of the polymer, and/or encapsulated within the tegrin), Fc receptor, and/or the mannose receptor (i. e. polymer of a vaccine nanocarrier, and, in some embod- a multi-valent lectin), proteins which are all prominently iments, at least one type of immunomodulatory agent is expressed on SCS-Mph. Examples of such markers are embedded within the polymer, associated with the inte- 5 provided elsewhere herein. rior surface of the polymer, and/or encapsulated within [0090] In certain embodiments, a target is a B cell the polymer of a vaccine nanocarrier. marker. In some embodiments, B cell markers may in- [0085] In some embodiments, inventive vaccine nano- clude complement receptors, CR1 (i.e. CD35) or CR2 carriers comprise less than less than 90% by weight, less (i.e. CD21), proteins which are expressed on B cells. In than 75% by weight, less than 50% by weight, less than 10 some embodiments, B cell targeting can be accom- 40% by weight, less than 30% by weight, less than 20% plished by B cell markers such as CD 19, CD20, and/or by weight, less than 15% by weight, less than 10% by CD22. In some embodiments, B cell targeting can be weight, less than 5% by weight, less than 1% by weight, accomplished by B cell markers such as CD40, CD52, or less than 0.5% by weight of the immunomodulatory CD80, CXCR5, VLA-4, class II MHC, surface IgM or IgD, agent. 15 APRL, and/or BAFF-R. Examples of such markers are [0086] In some embodiments, vaccine nanocarriers provided elsewhere herein. are associated with at least one targeting moiety. In some [0091] In certain embodiments, a target is a FDC mark- embodiments, a targeting moiety may be a nucleic acid, er. In some embodiments, FDC markers include comple- polypeptide, peptide, glycoprotein, glycopeptide, prote- ment receptors, CR1 (i.e. CD35) or CR2 (i.e. CD21), pro- oglycan, carbohydrate, lipid, small molecule, etc. For ex- 20 teins which are expressed on FDCs. Examples of such ample, a targeting moiety can be a nucleic acid targeting markers are provided elsewhere herein. moiety (e.g. an aptamer, Spiegelmer®, etc.) that binds [0092] In some embodiments, a vaccine nanocarrier to a cell type specific marker. In some embodiments, a comprises a single type of targeting moiety that directs targeting moiety may be a naturally occurring or synthetic delivery of the vaccine nanocarrier to a single cell type ligand for a cell surface protein, e.g., DEC-205, CD169, 25 (e.g. delivery to SCS-Mph only). In some embodiments, CD11b, etc. Examples of targeting moieties also include a vaccine nanocarrier comprises a single type of target- those provided elsewhere herein, such as those de- ing moiety that directs delivery of the vaccine nanocarrier scribed above. to multiple cell types (e.g. delivery to both SCS-Mphs [0087] In accordance with the present invention, a tar- and FDCs, or to both SCS-Mphs and DCs). In some em- geting moiety recognizes one or more "targets" or "mark- 30 bodiments, a vaccine nanocarrier comprises two types ers" associated with a particular organ, tissue, cell, of targeting moieties, wherein the first type of targeting and/or subcellular locale. In some embodiments, a target moiety directs delivery of the vaccine nanocarrier to one may be a marker that is exclusively or primarily associ- cell type, and the second type of targeting moiety directs ated with one or a few cell types, with one or a few dis- delivery of the vaccine nanocarrier to a second cell type. eases, and/or with one or a few developmental stages. 35 For example, in some embodiments, the first type of tar- Examples of cells that are targeted include antigen pre- geting moiety directs delivery to SCS-Mphs, and the sec- senting cells (APCs), such as dendritic cells, follicular ond type of targeting moiety directs delivery to DCs. As dendritic cells, and macrophages. One example of a another example, the first type of targeting moiety directs macrophage is a subcapsular sinuc macrophage. Other delivery to SCS-Mphs, and the second type of targeting cells that are targeted include T cells and B cells. In some 40 moiety directs delivery to FDCs. embodiments, a target can comprise a protein, a carbo- [0093] In some embodiments, inventive vaccine nano- hydrate, a lipid, and/or a nucleic acid. In some embodi- carriers comprise less than 50% by weight, less than 40% ments, a target is a tumor marker. In some embodiments, by weight, less than 30% by weight, less than 20% by a target is an APC marker. In certain embodiments, a weight, less than 15% by weight, less than 10% by target is a T cell marker. In some embodiments, the tar- 45 weight, less than 5% by weight, less than 1% by weight, geting moieties target secondary lymphoid tissues or or- or less than 0.5% by weight of the targeting moiety. gans. Secondary lympoid tissues or organs include [0094] In some embodiments, vaccine nanocarriers lymph nodes, the spleen, Peyer’s patches, the appendix, may transport one or more types of immunostimulatory or tonsils. agents which can help stimulate immune responses. In [0088] In certain embodiments, a target is a dendritic 50 some embodiments, immunostimulatory agents boost cell marker. In some embodiments, DC markers include immune responses by activating APCs to enhance their DC-205, CD11c, class II MHC, CD80, CD86, DC-SIGN, immunostimulatory capacity. In some embodiments, im- CD11b, BDCA-1, BDCA-2, BDCA-4, Siglec-H, CX3CR1, munostimulatory agents boost immune responses by and/or Langerin. Examples of such markers are provided amplifying lymphocyte responses to specific antigens. In elsewhere herein. 55 some embodiments, immunostimulatory agents boost [0089] In certain embodiments, a target is a subcap- immune responses by inducing the local release of me- sular sinus macrophage marker. In some embodiments, diators, such as cytokines from a variety of cell types. SCS-Mph markers include CD 169 (i.e. sialoadhesin), [0095] In some embodiments, a vaccine nanocarrier

13 25 EP 2 394 657 A1 26 comprises a single type of immunostimulatory agent that some embodiments, inventive nanocarriers have a great- stimulates both B cells and T cells. In some embodi- est dimension (e.g., diameter) of 100 nm or less. In some ments, a vaccine nanocarrier comprises two types of im- embodiments, inventive nanocarriers have a greatest di- munostimulatory agents, wherein the first type of immu- mension ranging between 25 nm and 200 nm. In some nostimulatory agent stimulates B cells, and the second 5 embodiments, inventive nanocarriers have a greatest di- type of immunostimulatory agent stimulates T cells. In mension ranging between 20 nm and 100 nm. some embodiments, a vaccine nanocarrier comprises [0101] A variety of different nanocarriers can be used greater than two types of immunostimulatory agents, in accordance with the present invention. In some em- wherein one or more types of immunostimulatory agents bodiments, nanocarriers are spheres or spheroids. In stimulate B cells, and one or more types of immunostim- 10 some embodiments, nanocarriers are flat or plate- ulatory agents stimulate T cells. shaped. In some embodiments, nanocarriers are cubes [0096] In some embodiments, various assays can be or cuboids. In some embodiments, nanocarriers are utilized in order to determine whether an immune re- ovals or ellipses. In some embodiments, nanocarriers sponse has been modulated in a B cell or group of B cells are cylinders, cones, or pyramids. Nanocarriers may be or in a T cell or group of T cells. In some embodiments, 15 solid or hollow and may comprise one or more layers. In the assay assesses whether or not the cell or group of some embodiments, each layer has a unique composi- cells has/have become "activated". tion and unique properties relative to the other layer(s). [0097] In some embodiments, various assays can be To give but one example, nanocarriers may have a core/ utilized in order to determine whether an immune re- shell structure, wherein the core is one layer (e.g. a pol- sponse has been stimulated in a T cell or group of T cells. 20 ymeric core) and the shell is a second layer (e.g. a lipid In some embodiments, stimulation of an immune re- bilayer or monolayer). Nanocarriers may comprise a plu- sponse in T cells can be determined by measuring anti- rality of different layers. In some embodiments, one layer gen-induced production of cytokines by T cells. In some may be substantially cross-linked, a second layer is not embodiments, stimulation of an immune response in T substantially cross-linked, and so forth. In some embod- cells can be determined by measuring antigen-induced 25 iments, one, a few, or all of the different layers may com- proliferation of T cells. In some embodiments, an immune prise one or more immunomodulatory agents, targeting response in T cells is determined to be stimulated if cel- moieties, immunostimulatory agents, and/or combina- lular markers of T cell activation are expressed at differ- tions thereof. In some embodiments, one layer compris- ent levels (e.g. higher or lower levels) relative to unstim- es an immunomodulatory agent, targeting moiety, and/or ulated cells. 30 immunostimulatory agent, a second layer does not com- [0098] In some embodiments, various assays can be prise an immunomodulatory agent, targeting moiety, utilized in order to determine whether an immune re- and/or immunostimulatory agent, and so forth. In some sponse has been stimulated in a B cell or group of B cells. embodiments, each individual layer comprises a different In some embodiments, stimulation of an immune re- immunomodulatory agent, targeting moiety, immunos- sponse in B cells can be determined by measuring anti- 35 timulatory agent, and/or combination thereof. body titers, antibody affinities, antibody performance in [0102] In some embodiments, nanocarriers may op- neutralization assays, class-switch recombination, affin- tionally comprise one or more lipids. In some embodi- ity maturation of antigen-specific antibodies, develop- ments, a nanocarrier is a liposome. In some embodi- ment of memory B cells, development of long-lived plas- ments, a nanocarrier comprises a lipid bilayer. In some ma cells that can produce large amounts of high-affinity 40 embodiments, a nanocarrier comprises a lipid monolay- antibodies for extended periods of time, germinal center er. In some embodiments, a nanocarrier is a micelle. In reactions, and/or antibody performance in neutralization some embodiments, a nanocarrier comprises a core of assays. a polymeric matrix surrounded by a lipid layer (e.g. lipid [0099] A vaccine nanocarrier is an entity that compris- bilayer, lipid monolayer, etc.). In some embodiments, a es, for example, at least one immunomodulatory agent 45 nanocarrier comprises a non-polymeric core (e.g. metal which is capable of stimulating an immune response in particle, quantum dot, ceramic particle, bone particle, vi- B cells and/or T cells. Any vaccine nanocarrier can be ral particle, etc.) surrounded by a lipid layer (e.g. lipid used in accordance with the present invention. bilayer, lipid monolayer, etc.). [0100] In some embodiments, a nanocarrier has a [0103] In some embodiments, a nanocarrier compris- greatest dimension (e.g., diameter) of less than 100 mi- 50 es one or more polymers. In some embodiments, a pol- crons (mm). In some embodiments, inventive nanocarri- ymeric matrix can be surrounded by a coating layer (e.g. ers have a greatest dimension (e.g., diameter) of 300 nm liposome, lipid monolayer, micelle, etc.). In some em- or less. In some embodiments, inventive nanocarriers bodiments, an immunomodulatory agent, targeting moi- have a greatest dimension (e.g., diameter) of 250 nm or ety, and/or immunostimulatory agent can be associated less. In some embodiments, inventive nanocarriers have 55 with the polymeric matrix. In such embodiments, the im- a greatest dimension (e.g., diameter) of 200 nm or less. munomodulatory agent, targeting moiety, and/or immu- In some embodiments, inventive nanocarriers have a nostimulatory agent is effectively encapsulated within the greatest dimension (e.g., diameter) of 150 nm or less. In nanocarrier.

14 27 EP 2 394 657 A1 28

[0104] In some embodiments, an immunomodulatory moiety, and/or immunostimulatory agent is associated agent, targeting moiety, and/or immunostimulatory agent with the surface of, encapsulated within, surrounded by, can be covalently associated with a nanocarrier. In some and/or dispersed throughout an aggregate of non-poly- embodiments, covalent association is mediated by a link- meric components. er. In some embodiments, an immunomodulatory agent, 5 [0109] In some embodiments, nanocarriers may op- targeting moiety, and/or immunostimulatory agent is non- tionally comprise one or more amphiphilic entities (i.e., covalently associated with a nanocarrier. For example, entities that possess both hydrophilic and hydrophobic in some embodiments, an immunomodulatory agent, tar- properties). In some embodiments, an amphiphilic entity geting moiety, and/or immunostimulatory agent is encap- can promote the production of nanocarriers with in- sulated within, surrounded by, and/or dispersed through- 10 creased stability, improved uniformity, or increased vis- out a polymeric matrix, a lipid membrane, etc. Alterna- cosity. tively or additionally, an immunomodulatory agent, tar- [0110] In some embodiments, a nanocarrier compris- geting moiety, and/or immunostimulatory agent may be es one or more nanoparticles associated with the exterior associated with a polymeric matrix, a lipid membrane, surface of and/or encapsulated within the nanocarrier. etc. by hydrophobic interactions, charge interactions, 15 [0111] Nanocarriers may be prepared using any meth- van der Waals forces, etc. od known in the art. For example, particulate nanocarrier [0105] A wide variety of polymers and methods for formulations can be formed by methods such as nano- forming polymeric matrices therefrom are known in the precipitation, flow focusing using fluidic channels, spray art of drug delivery. In general, a polymeric matrix com- drying, single and double emulsion solvent evaporation, prises one or more polymers. Any polymer may be used 20 solvent extraction, phase separation, milling, microemul- in accordance with the present invention. Polymers may sion procedures, microfabrication, nanofabrication, sac- be natural or unnatural (synthetic) polymers. Polymers rificial layers, simple and complex coacervation, as well may be homopolymers or copolymers comprising two or as other methods well known to those of ordinary skill in more monomers. In terms of sequence, copolymers may the art. Alternatively or additionally, aqueous and organic be random, block, or comprise a combination of random 25 solvent syntheses for monodisperse semiconductor, and block sequences. Polymers in accordance with the conductive, magnetic, organic, and other nanoparticles present invention may be organic polymers. In some em- may be utilized. bodiments, the polymers are dendritic polymers or [0112] In some embodiments, immunomodulatory blends of polymers. agents, targeting moieties, and/or immunostimulatory [0106] Examples of polymers include polyethylenes, 30 agents, are not covalently associated with a nanocarrier. polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhy- For example, nanocarriers may comprise a polymeric drides (e.g. poly(sebacic anhydride)), polyhydroxyacids matrix, and immunomodulatory agents, targeting moie- (e.g. poly(β-hydroxyalkanoate)), polypropylfumerates, ties, and/or immunostimulatory agents, etc. are associ- polycaprolactones, polyamides (e.g. polycaprolactam), ated with the surface of, encapsulated within, and/or dis- polyacetals, polyethers, polyesters (e.g. polylactide, pol- 35 tributed throughout the polymeric matrix of an inventive yglycolide), poly(orthoesters), polycyanoacrylates, poly- nanocarrier. Immunomodulatory agents may be re- vinyl alcohols, polyurethanes, polyphosphazenes, poly- leased by diffusion, degradation of the nanocarrier, acrylates, polymethacrylates, polyureas, polystyrenes, and/or a combination thereof. In some embodiments, pol- and polyamines. ymer(s) of the nanocarrier degrade by bulk erosion. In [0107] In some embodiments, nanocarriers comprise 40 some embodiments, polymer(s) of the nanocarrier de- immunomodulatory agents embedded within reverse mi- grade by surface erosion. celles. To give but one example, a liposome nanocarrier [0113] In some embodiments, immunomodulatory may comprise hydrophobic immunomodulatory agents agents, targeting moieties, and/or immunostimulatory embedded within the liposome membrane, and hy- agents are covalently associated with a particle. In some drophilic immunomodulatory agents embedded with re- 45 embodiments, covalent association is mediated by one verse micelles found in the interior of the liposomal na- or more linkers. Any suitable linker can be used in ac- nocarrier. cordance with the present invention. In some embodi- [0108] In some embodiments, a nanocarrier does not ments, the linker is a cleavable linker (e.g., an ester link- include a polymeric component. In some embodiments, age, an amide linkage, a disulfide linkage, etc.). nanocarriers comprise metal particles, quantum dots, ce- 50 [0114] In some embodiments, nanocarriers are made ramic particles, bone particles, viral particles, etc. In by self-assembly. As an example, lipids are mixed with some embodiments, an immunomodulatory agent, tar- a lipophilic immunomodulatory agent, and then formed geting moiety, and/or immunostimulatory agent is asso- into thin films on a solid surface. A hydrophilic immu- ciated with the surface of such a non-polymeric nanocar- nomodulatory agent is dissolved in an aqueous solution, rier. In some embodiments, a non-polymeric nanocarrier 55 which is added to the lipid films to hydrolyze lipids under is an aggregate of non-polymeric components, such as vortex. Liposomes with lipophilic immunomodulatory an aggregate of metal atoms (e.g. gold atoms). In some agents incorporated into the bilayer wall and hydrophilic embodiments, an immunomodulatory agent, targeting immunomodulatory agents inside the liposome lumen

15 29 EP 2 394 657 A1 30 are spontaneously assembled. In certain embodiments, [0118] In some embodiments, inventive prophylactic pre-formulated polymeric nanoparticles are mixed with and/or therapeutic protocols involve administering a ther- small liposomes under gentle vortex to induce liposome apeutically effective amount of one or more inventive vac- fusion onto polymeric nanoparticle surface. cine nanocarriers to a subject such that an immune re- [0115] As another example, a hydrophilic immu- 5 sponse is modulated (e.g., stimulated in both T cells nomodulatory agent to be encapsulated is first incorpo- and/or B cells). rated into reverse micelles by mixing with naturally de- [0119] The present invention provides novel composi- rived and non-toxic amphiphilic entities in a volatile, wa- tions comprising a therapeutically effective amount of ter-miscible organic solvent. In some embodiments, a one or more vaccine nanocarriers and one or more phar- biodegradable polymer is added after reverse micelle for- 10 maceutically acceptable excipients. In some embodi- mation is complete. The resulting biodegradable poly- ments, the present invention provides for pharmaceutical mer-reverse micelle mixture is combined with a polymer- compositions comprising inventive vaccine nanocarriers insoluble hydrophilic non-solvent to form nanoparticles as described herein. The composition may include more by the rapid diffusion of the solvent into the non-solvent than one type of nanocarrier, each type having different and evaporation of the organic solvent. 15 constituents (e.g., immunomodulatory agents, targeting [0116] In some embodiments, lipid monolayer stabi- agents, immunostimulatory agents, excipients, etc.). In lized polymeric nanocarriers are used to deliver one or accordance with some embodiments, a method of ad- a plurality of immunomodulatory agents. In certain em- ministering a pharmaceutical composition comprising in- bodiments, a hydrophilic immunomodulatory molecule is ventive compositions to a subject (e.g. human) in need first chemically conjugated to the polar headgroup of a 20 thereof is provided. lipid. The conjugate is mixed with a certain ratio of un- [0120] In some embodiments, a therapeutically effec- conjugated lipid molecules in an aqueous solution con- tive amount of an inventive vaccine nanocarrier compo- taining one or more water-miscible solvents. A biode- sition is delivered to a patient and/or animal prior to, si- gradable polymeric material is mixed with the hydropho- multaneously with, and/or after diagnosis with a disease, bic immunomodulatory agents to be encapsulated in a 25 disorder, and/or condition. In some embodiments, a ther- water miscible or partially water miscible organic solvent. apeutic amount of an inventive vaccine nanocarrier com- The resulting polymer solution is added to the aqueous position is delivered to a patient and/or animal prior to, solution of conjugated and unconjugated lipid to yield simultaneously with, and/or after onset of symptoms of nanoparticles by the rapid diffusion of the organic solvent a disease, disorder, and/or condition. In certain embod- into the water and evaporation of the organic solvent. 30 iments, a therapeutic amount of an inventive vaccine na- [0117] The compositions and methods described nocarrier composition is administered to a patient and/or herein can be used for the prophylaxis and/or treatment animal prior to exposure to an infectious agent. In certain of any infectious disease, disorder, and/or condition. Ex- embodiments, a therapeutic amount of an inventive vac- amples of other diseases, disorders, and/or conditions cine nanocarrier composition is administered to a patient are provided elsewhere herein. In some embodiments, 35 and/or animal after exposure to an infectious agent. In vaccine nanocarriers in accordance with the present in- certain embodiments, a therapeutic amount of an inven- vention may be used to treat, alleviate, ameliorate, re- tive vaccine nanocarrier composition is administered to lieve, delay onset of, inhibit progression of, reduce se- a patient and/or animal prior to exposure to an addictive verity of, and/or reduce incidence of one or more symp- substance or a toxin. In certain embodiments, a thera- toms or features of a disease, disorder, and/or condition. 40 peutic amount of an inventive vaccine nanocarrier com- In some embodiments, inventive vaccine nanocarriers position is administered to a patient and/or animal after may be used to treat, alleviate, ameliorate, relieve, delay exposure to an addictive substance or a toxin. onset of, inhibit progression of, reduce severity of, and/or [0121] In some embodiments, the pharmaceutical reduce incidence of one or more symptoms or features compositions of the present invention are administered of microbial infection (e.g. bacterial infection, fungal in- 45 by a variety of routes, including oral, intravenous, intra- fection, viral infection, parasitic infection, etc.). In some muscular, intraarterial, intramedullary, intrathecal, sub- embodiments, the prophylaxis and/or treatment of micro- cutaneous, intraventricular, transdermal, interdermal, bial infection comprises administering a therapeutically rectal, intravaginal, intraperitoneal, topical (as by pow- effective amount of inventive vaccine nanocarriers to a ders, ointments, creams, and/or drops), transdermal, subject in need thereof, in such amounts and for such 50 mucosal, nasal, buccal, enteral, sublingual; by intratra- time as is necessary to achieve the desired result. In cheal instillation, bronchial instillation, and/or inhalation; certain embodiments of the present invention, a "thera- and/or as an oral spray, nasal spray, and/or aerosol. In peutically effective amount" of an inventive vaccine na- certain embodiments, the composition is administered nocarrier is that amount effective for treating, alleviating, orally. In certain embodiments, the composition is ad- ameliorating, relieving, delaying onset of, inhibiting pro- 55 ministered parenterally. In certain embodiments, the gression of, reducing severity of, and/or reducing inci- composition is administered via intramuscular injection. dence of one or more symptoms or features of disease, [0122] In certain embodiments, vaccine nanocarriers disorder, and/or condition provided herein. which delay the onset and/or progression of a disease,

16 31 EP 2 394 657 A1 32 disorder, and/or condition (e.g., a particular microbial in- composite vaccine carries internal T cell antigens, adju- fection) may be administered in combination with one or vants (not shown) and targeting moieties for DCs, FDC more additional therapeutic agents which treat the symp- and SCS-Mph together with surface antigen for B cell toms of the disease, disorder, and/or condition. For ex- recognition. Upon s.c. or i.m. injection, the material ample, the vaccine nanocarriers may be combined with 5 reaches lymph nodes via draining lymph vessels and ac- the use of an anti-cancer agent, anti-inflammatory agent, cumulates on each APC (for clarity, only APC-specific antibiotic, or anti-viral agent. targeting moieties are shown, but each APC acquires the [0123] The invention provides a variety of kits compris- entire complex). DCs internalize and digest the complex ing one or more of the nanocarriers of the invention. For and present antigenic peptides in MHC class I and class example, the invention provides a kit comprising an in- 10 II to CD8 and CD4 T cells, respectively. The activated T ventive nanocarrier and instructions for use. A kit may cells differentiate into effector/memory (TEff/Mem) cells comprise multiple different nanocarriers. A kit may com- that mediate cellular immune responses. TFH cells pro- prise any of a number of additional components or rea- vide help to B cells that were initially stimulated by antigen gents in any combination. According to certain embodi- on SCS-Mph and in the process have acquired and proc- 15 ments of the invention, a kit may include, for example, essed T cell antigens for restimulation of TFH. The help (i) a nanocarrier comprising at least one immunomodu- provided by TFH cells allows the development of a GC latory agent, wherein the at least one immunomodulatory reaction during which B cells proliferate and generate agent is capable of stimulating both a T cell and/or B cell high-affinity antibodies. response, at least one targeting moiety, and/or at least [0128] Figure 2: SCS-Mph bind lymph-borne viral par- one immunostimulatory agent; (ii) instructions for admin- 20 ticles and present them to follicular B cells. (A) Immuno- istering the nanocarrier to a subject in need thereof. In histochemical staining of the cortex of a mouse popliteal certain embodiments, a kit may include, for example, (i) lymph node stained with anti-CD 169 and counter- at least one immunomodulatory agent, wherein the at stained with wheat germ agglutinin. The lymph node was least one immunomodulatory agent is capable of stimu- harvested 30 minutes after footpad injection of red fluo- lating both a T cell and B cell response; (ii) at least one 25 rescent vesicular stomatitis virus (VSV). In the subcap- targeting moiety; (iii) at least one immunostimulatory sular sinus of the draining lymph node, the red virus colo- agent; (iv) a polymeric matrix precursor; (v) lipids and calized exclusively with CD169+ macrophages. (B) Elec- amphiphilic entities; (vi) instructions for preparing inven- tron micrograph of a lymph node macrophage (Mph) and tive vaccine nanocarriers from individual components a follicular B cell (B1) below the floor of the subcapsular (i)-(v). 30 sinus (SCSf) 30 minutes after VSV injection shows VSV [0124] In some embodiments, the kit comprises an in- at the surface and within a phagolysosome of the Mph ventive nanocarrier and instructions for mixing. Such kits, and at the interface between Mph and B cells (arrow- in some embodiments, also include an immunostimula- heads). (C) Injection of VSV into the footpad of untreated tory agent and/or an antigen. The nanocarrier of such mice (B6) results in rapid downregulation of surface-ex- kits may comprise an immunomodulatory agent (e.g., a 35 pressed IgM on virus-specific B cells, a sign of B cell T cell antigen, such as a universal T cell antigen) and/or activation. Depletion of SCS-Mph after footpad injection a targeting moiety. The T cell antigen and/or the targeting of clodronate liposomes (CLL) abolished B cell activa- moiety may be on the surface of the nanocarrier. In some tion, indicating that SCS-Mph are essential to present embodiments, the immunomodulatory agent and the an- particulate antigen to B cells. tigen are the same. In some embodiments, they are dif- 40 [0129] Figure 3: An exemplary liposome nanocarrier ferent. with a lipophilic immunomodulatory agent incorporated [0125] In any of the foregoing embodiments described in the membrane, and a hydrophilic immunomodulatory above, the word conjugated means covalently or non- agent encapsulated within the liposome. covalently conjugated, unless the context clearly indi- [0130] Figure 4: An exemplary nanoparticle-stabilized cates otherwise. In any of the foregoing embodiments 45 liposome nanocarrier with a lipophilic immunomodulato- described above, the word encapsulated means physi- ry agent incorporated into the membrane, and a hy- cally trapped within, whether by admixture, by a shell drophilic immunomodulating agent encapsulated within surrounding a core, by covalent bonding internal of the the liposome. surface of the nanocarrier, and the like. [0131] Figure 5: An exemplary liposome-polymer na- [0126] This application refers to various issued pat- 50 nocarrier with a lipophilic immunomodulatory agent in- ents, published patent applications, journal articles, and corporated into the membrane, and a hydrophobic im- other publications; all of which are incorporated herein munomodulating agent encapsulated within the polymer- by reference. ic nanoparticle. [0132] Figure 6: An exemplary nanoparticle-stabilized Brief Description of the Drawing 55 liposome-polymer nanocarrier with a lipophilic immu- nomodulatory agent incorporated into the membrane, [0127] Figure 1: Combined vaccine targeting strategy and a hydrophobic immunomodulating agent encapsu- for optimal humoral and cellular immune response. The lated within the polymeric nanoparticle.

17 33 EP 2 394 657 A1 34

[0133] Figure 7: An exemplary liposome-polymer na- LNs from nai¨ve C57BL/6 mice showing co-expression of nocarrier containing reverse micelles with a lipophilic im- selected markers on CD169+ cells (arrowheads). Scale munomodulatory agent incorporated into the membrane, bars: 125 mm in the left column and 20 mm in all other and a hydrophilic immunomodulatory agent encapsulat- columns. ed within the reverse micelles. 5 [0139] Figure 13: Morphological changes in popliteal [0134] Figure 8: An exemplary nanoparticle-stabilized LNs following CLL treatment. (A) Confocal micrographs liposome-polymer nanocarrier containing reverse mi- of popliteal LNs (top three rows) and spleens (bottom celles with a lipophilic immunomodulatory agent incor- row) of untreated control mice (-CLL, left column) and porated into the membrane, and a hydrophilic immu- animals that had received CLL footpad injections 610 nomodulatory agent encapsulated inside the liposome. 10 days earlier. CLL treatment depleted CD169+ macro- [0135] Figure 9: An exemplary lipid-stabilized poly- phages in the LN (top row), but not in spleens; Lyve-1+ meric nanocarrier with a hydrophilic immunomodulatory medullary lymphatic endothelial cells (second row) and agent conjugated to the lipid monolayer, and a hydro- cortical CD 1 1chiph dendritic cells (third row) were not phobic immunomodulatory agent encapsulated inside affected. (B) Cellular subset frequency in popliteal LNs the polymer core. 15 with and without CLL treatment, data are from n = 3 mice [0136] Figure 10: An exemplary lipid-stabilized poly- and shown as mean 6 SEM; *: p < 0.05, **: p < 0.01; meric nanocarrier containing reverse micelles with a hy- unpaired student’s t-test. (C) Frequency of different I- drophilic immunomodulatory agent conjugated to the li- Ab+CD11b+ leukocyte subsets in popliteal LNs at 610 pid monolayer, and a hydrophilic immunomodulatory days after footpad injection of 50 ml CLL. Each symbol agent encapsulated inside the polymer core. 20 represents pooled popliteal LNs from one mouse. Subset [0137] Figure 11: Capture of lymph-borne VSV by SCS frequencies among total mononuclear cells in popliteal macrophages. (A) MP-IVM micrographs of VSV in a pop- LNs were assessed by flow cytometry after gating on I- liteal LN (numbers: minutes after footpad injection; scale Ab+CD11b+ cells as shown in Figure 12A. (D) Immuno- bar: 100 mm). (B) VSV accumulation in a C57BL/6→Act histochemical analysis of popliteal LNs without treatment (EGFP) recipient 3 hours after injection (scale bar: 50 25 (-CLL) or 7 days after footpad injection of CLL (+CLL). mm). (C) Electron micrographs of VSV in LN 5 minutes Scale bars: 300 mm. (E) Ultrastructure of the SCS in a after injection. Center micrograph is shown schematically representative popliteal LN 7 days after CLL treatment (left) and at higher magnification (right). Arrowheads and 5 minutes after footpad injection of 20 mg VSV-IND. identify VSV particles (scale bars: 2 mm). (D) Confocal Note the complete absence of SCS macrophages and micrographs of VSV-draining LN (30 minutes). Scale 30 viral particles. Scale bar: 2 mm. bars: 100 mm (left), 15 mm (right). (E) VSV titers in pop- [0140] Figure 14: Retention of fluorescent viruses and liteal LNs 2 hours after injection into wildtype, C3-defi- latex nanoparticles in popliteal LNs. (A) Confocal micro- cient or CLL-depleted mice. ***: p < 0.001 (two-way ANO- graphs of popliteal LNs 30 minutes after footpad injection VA, Bonferroni’s post-test). (F) VSV capture in DH- of Alexa-568-labeled adenovirus (AdV). Frozen sections LMP2a mice. *: p < 0.05 (unpaired t-test). (G) VSV titers 35 were stained with FITC-α-CD169 and Alexa-647-α-B220 after footpad injection in untreated and CLL-treated mice to identify B cells. Scale bars: 100 mm (left panel) and (one of two similar experiments; n = 3). ProxLN: inguinal, 15 mm (right panel). (B) Transmission electron micro- paraaortic LNs; BrachLN: brachial LN. (H) Viral titers in graphs of AdV particles captured by a SCS macrophage. lymph, spleen and blood after TD cannulation; *: p < 0.05 The top panel shows an annotated schematic drawing (unpaired t-test). Horizontal bars in (E-H) indicate means. 40 of the low magnification overview (middle panel). The [0138] Figure 12: Characterization of CD169+ macro- boxed area in the middle panel is enlarged in the lower phages in peripheral LNs. (A-C) Lineage marker expres- panel, arrowheads denote electron-dense, spherical sion analysis of pooled mononuclear cells from LNs of AdV particles. Scale bars: 2 mm (top and middle panel) naïve C57BL/6 mice. (A) After gating on the CD 169+ and 1 mm (lower panel). (C-D) Confocal micrographs of population (middle panel), cells were analyzed for ex- 45 popliteal LNs from C57BL/6 mice 30 minutes after foot- pression of the two macrophage-associated surface pad injection of 20 mg Alexa-568 labeled UV-inactivated markers, I-Ab (MHC class II) and CD11b (bottom panel). AdV (C) or VV (D). Fluorescent viruses accumulated in Staining with an isotype control for anti-CD169 is shown the cortical SCS above B follicles identified by FITC-α- in the top panel. (B) CD 169+I-Ab+CD11 b+ cells were B220 staining and also in the medulla where viruses were further analyzed for expression of CD68, F4/80, CD11c, 50 not only bound by CD169+ macrophages, but also by and Gr-1. Gates were drawn to identify marker+ cells, LYVE-1+ lymphatic endothelial cells. Scale bars indicate except for CD11c staining where the marker was posi- 125 mm (left panel) and 25 mm (right panel). (E) Confocal tioned to identify conventional CD 11chigh dendritic cells micrograph of a popliteal LN 30 minutes after hind foot- (overlay). Numbers indicate percentage of CD169+I- pad injection of Alexa-568 labeled VSV and approximate- Ab+CD11b+ cells under the histogram gate. Data are rep- 55 ly 1011 Crimson Fluospheres (200 nm diameter). Frozen resentative of 35 experiments with similar results. (C) LN sections were counter-stained with FITC-α-CD169. Quantitative analyses of data in panel (B), error bars rep- Note that the Latex beads, unlike VSV, were poorly re- resent SEM. (D-G) Confocal micrographs of popliteal tained in draining LNs. Scale bar: 125 mm.

18 35 EP 2 394 657 A1 36

[0141] Figure 15: Effect of CLL footpad injection on days after immunization by footpad injection of 10 mg VSV distribution in draining LNs. Confocal micrographs UV-VSV or UV-VSV-AlexaFluor-488-IND. (E) Calcium show the localization of fluorescent VSV particles in pop- flux in VI10YEN B cells exposed to supernatant from VSV liteal LNs without (A) or 7 days after (B) CLL treatment. stocks. Supernatant was generated by ultracentrifuga- B follicles were identified by FITC-α-B220 staining. In the 5 tion through a sucrose cushion resulting in approximately medulla (boxed area), VSV was bound by LYVE-1+ cells 10,000-fold reduction in viral titers and was used on B that were not affected by CLL treatment. Scale bars: 125 cells either undiluted (top right) or at 1:100 dilution (bot- mm (left column) and 25 mm (right column). tom right). As a control, VSV stock solution was diluted [0142] Figure 16: SCS macrophages present lymph- to equivalent viral titers (MOI; left panels). The results derived AdV to follicular B lymphocytes. (A) Confocal mi- 10 demonstrate the presence of antigenic VSV-G that is not crograph of CD169+ macrophages in the SCS above a associated with virus particles in our virus preparation. B follicle in a popliteal LN. Frozen sections were coun- [0145] Figure 19: VSV-induced adhesion of VI10YEN terstained with wheatgerm agglutinin (WGA) to identify B cells to ICAM-1 and VCAM-1. (A,B) Adhesion of puri- extracellular matrix and with α-B220 to detect B cells. fied naïve and VSV-IND activated (30 minute exposure) Note that some B cells reside in the SCS, and one B cell 15 VI10YEN B cells to plastic plates coated with the indicat- appears to migrate between the follicle and the SCS (ar- ed concentrations of recombinant ICAM-1-Fc (A) or rowhead). Scale bar: 25 mm. (B) Electron micrograph VCAM-1-Fc (B). Pooled data of two triplicate experi- and (C) schematic drawing of a SCS macrophage and ments are shown. Horizontal bars represent means. (C, surrounding cells in a popliteal LN 30 minutes after foot- D) Confocal micrographs of ICAM-1 and VCAM-1 ex- pad injection of AdV. Scale bar: 2 mm. The boxes drawn 20 pression in popliteal LNs of C57BL/6 mice. Scale bars: in (C) indicate areas of higher magnification shown in 50 mm. (E) Adhesion of purified naïve wildtype and panels (D) and (E). These panels show two examples of VI10YEN B cells to plastic dishes coated with the indi- AdV particles at the interface between the SCS macro- cated pfu-equivalent concentrations of UV-inactivated phage and B cells (arrowheads). Asterisks denote other VSV-IND. Data represent means 6 SEM of triplicates. macrophage-associated AdV particles. Scale bars: 500 25 [0146] Figure 20: SCS macrophages are required for nm. early activation of VSV-specific B cells in LNs. (A) Con- [0143] Figure 17: Macrophage-mediated transfer of focal micrograph shows MHC-II colocalization with VSV- lymph-borne VSV across the SCS floor alters virus-spe- IND (30 minutes after injection) in VI10YENxMHCII cific B cell behavior. (A) Electron micrographs and sche- (EGFP) B cells at the SCS (arrowhead), not the deep matic drawing (middle) showing a macrophage penetrat- 30 follicle (asterisk). Scale bar: 25 mm. (B) Distance of VSV- ing the SCS floor of a popliteal LN 30 minutes after VSV associated and VSV-free VI10YENxMHCII(EGFP) B injection. Scale bars: 10 mm (left) and 2 mm (right). Arrow: cells to the SCS. Horizontal lines: medians. (C) BCR ex- vacuole with digested VSV. Arrowheads: virions in con- pression kinetics on VI10YEN and (D) polyclonal B cells tact zone between macrophage and B cells. (B) MP-IVM after VSV-IND footpad injection. (E) BCR expression on of polyclonal and VI10YEN B cells in popliteal LNs. Scale 35 VI10YEN cells in CLL-treated or untreated popliteal LNs bars: 50 mm. (C) Regional ratios of VI10YEN B cells/ after VSV-IND injection (20 mg). Mean fluorescence in- control B cells following VSV injection. Results are from tensities were normalized to virus-free values (dashed 3 movies/group. (D,E) Localization of VI10YEN B cells line). Means 6 SEM (35 mice). (F) Confocal micro- in popliteal LNs relative to the SCS. **: p < 0.01 (one- graph of VI10YEN B cells in control and (G) CLL-treated way ANOVA with Bonferroni’s post-test). 40 popliteal LNs 6 hours after VSV-IND injection (0.4 mg). [0144] Figure 18: Characteristics of VSV serotypes Scale bar: 125 mm. (H) VI10YEN B cell frequency at T/B and VSV-IND-specific VI I OYEN B cells. (A) SDS-PAGE borders and in follicles 6 hours after VSV-IND injection gels (12%) of purified VSV lysates. Top: VSV-IND and at indicated doses. Means 6 SEM; n = 3-4 follicles/2 VSV-NJ. The N and P proteins co-migrate in VSV-NJ, mice; *: p < 0.05; **: p < 0.01; ***: p < 0.001 (t-test). approximate molecular weights are shown in parenthe- 45 [0147] Figure 21: VI10YEN B cell motility in draining ses. (B) Binding of Alexa-488 labeled VSV-IND (middle LNs following virus injection. Median 3D instantaneous row) or VSV-NJ (bottom row) to B cells from C57BL/6 velocities of wildtype (triangles) and VI10YEN B cells (cir- mice (left column) or VI10YEN mice (right column). The cles) in deep follicles and the SCS/superficial follicle upper row shows control staining with the anti-idiotypic about 535 min after VSV footpad injection. Horizontal antibody 35.61 to the VI10YEN BCR (Dang and Rock, 50 bars represent means; *: p < 0.05; **: p < 0.01 (one-way 1991, J. Immunol., 146:3273). (C) Intracellular calcium ANOVA with Bonferroni’s post test). Note that specific B flux in CD43ncg purified, Fluo-LOJO loaded B cells from cells slow down throughout the entire follicle, likely as a VI I OYEN mice (upper row) or C57BL/6 mice (lower row). consequence of free VSV-G in our preparation (see Fig- Events were collected continuously over time, asterisks ure 18). Control experiments showed similar B cell mo- indicate the timepoint when antibodies or virus were add- 55 tility parameters in CLL-treated and nontreated popLNs. ed. Virus particles were used at 1000/B cell, anti- [0148] Figure 22: Timecourse of activation marker in- 6 IgM-(Fab)2 at 10 mg/10 B cells. (D) Neutralization assay duction on VI10YEN B cells in virus-draining and non- for total Ig and IgG in serum of C57BL/6 mice 4 and 10 draining LNs following injection of VSV-IND. VI10YEN B

19 37 EP 2 394 657 A1 38 cells were fluorescently tagged with CMTMR and trans- CD169. Images are oriented so that the medulla (weak, ferred to naive mice that were injected 18 hours later with diffuse staining with anti-CD169) faces to the right and 20 mg UV-inactivated VSV-IND (time 0 hours). The drain- the subcapsular sinus (SCS) region (bright anti-CD 169) ing popliteal LN (popLN) and a distal brachial LN faces to the left. Note that the red amine modified parti- (brachLN) were harvested after the indicated time inter- 5 cles prominently localize to the SCS, while blue carboxy vals to generate single-cell suspensions. CD69 and modified beads are primarily retained in the medulla. CD86 expression on B cells was assessed by flow cy- [0154] Figure 28: (A) Antigen-bearing targeted nano- tometry after gating on (A) B220+CMTMR+VI10YEN particles are highly immunogenic and induce high anti- cells or (B) B220+CMTMR-endogenous control B cells. body titers. (B) The induced immune response elicited [0149] Figure 23: Confocal (left and middle columns) 10 by nanoparticle vaccines confers potent protection from and MP-IVM micrographs (right column) of popliteal LNs a lethal dose of VSV. of mice that had received adoptive transfers of a mixture [0155] Figure 29: In vivo T cell activation by immu- of CMTMR-labeled VI10YEN B cells and CMAC-labeled nomodulatory nanoparticles. (A) Effect of NPs on CD4 T polyclonal B cells (in the right column). On the following cell activation. (B) Effect of NPs on CD8 T cell response day, 20 mg UV-inactivated VSV-IND was injected in a 15 mixed with CpG adjuvant (TLR9 agonist). (C) Effect of footpad and the draining popliteal LNs were either sur- co-encapsulated adjuvant on CD8 T cell activation. gically prepared for MP-IVM or harvested for confocal [0156] Figure 30: Shows an exemplary nicotine con- analysis of frozen sections at the indicated time points. jugation strategy. MP-IVM images show that VSV-specific, but not polyclo- [0157] Figure 31: Shows an exemplary R848 conjuga- nal B cells made contact with VSV in the SCS as early 20 tion strategy. as 30 minutes after virus injection. VI10YEN B cells re- located to the T/B border at 6 hours following injection. Definitions Scale bars: 150 mm in the left column and 25 mm in the other columns. [0158] Abused substance: As used herein, the term [0150] Figure 24: In vivo targeting of SCS-Mph using 25 "abused substance" is any substance taken by a subject Fc fragments from human IgG. (A) The FACS histograms (e.g., a human) for purposes other than those for which on the left document the binding of fluorescent PEG- it is indicated or in a manner or in quantities other than PLGA nanoparticles (~100 nm diameter) to lymph node directed by a physician. In some embodiments, the macrophages. (B) Fc-nanoparticle (NP) targets SCS- abused substance is a drug, such as an illegal drug. In Mph and follicular dendritic cells. 30 certain embodiments, the abused substance is an over- [0151] Figure 25: Identification of the chemokine re- the-counter drug. In some embodiments, the abused ceptor CX3CR1 (fractalkine receptor) on macrophages substance is a prescription drug. The abused substance, in lymph node subcapsular sinus (SCS), but not in mac- in some embodiments, is an addictive substance. In rophages in the medulla. The micrograph on the right is some embodiments, the abused substance has mood- a 3D projection of a lymph node from a double-knockin 35 altering effects, and, therefore, includes inhalants and mouse where green fluorescent protein (GFP) is ex- solvents. In other embodiments, the abused substance pressed in the CX3CR 1 locus, while red fluorescent pro- is one that has no mood-altering effects or intoxication tein (RFP) reports the expression of another chemokine properties, and, therefore, includes anabolic steroids. receptor, CCR2. SCS-Mph are readily identifiable by Abused substances include, but are not limited to, can- their prominent green fluorescence, while medullary 40 nabinoids (e.g., hashish, marijuana), depressants (e.g., macrophages express primarily RFP. barbituates, benodiazepines, flunitrazepam (Rohypnol), [0152] Figure 26: SCS-Mph express the chemokine GHB, methaqualone (quaaludes)), dissociative anes- receptor CX3CR1. The graph shows a FACS plot of a thetics (e.g., ketamine, PCP), hallucinogens (e.g, LSD, single cell suspension from a lymph node of a knockin mescaline, psilocybin), opioids and morphine derivatives mouse that was genetically engineered to express GFP 45 (e.g., codeine, fentanyl, heroin, morphine, opium), stim- from the CX3CR1 locus. SCS-Mphs are identified by ulants (amphetamine, cocaine, Ecstacy (MDMA), meth- staining with a soluble receptor, CRFc, which binds mac- amphetamine, methylphenidate (Ritalin), nicotine), ana- rophages in the SCS, but not the medulla. The CRFc bolic steriods, and inhalants. In some embodiments, the negative CX3CR1-expressing (i.e., GFP-high) cells are abused substance for inclusion in a nanocarrier is the conventional dendritic cells that express this chemokine 50 complete molecule or a portion thereof. receptor. [0159] Addictive substance: As used herein, the term [0153] Figure 27: Fluorescent micrographs of frozen "addictive substance" is a substance that causes obses- sections from mouse popliteal lymph nodes 24h after sion, compulsion, or physical dependence or psycholog- footpad injection of 0.2 mm diameter Latex beads surface ical dependence. In some embodiments, the addictive modified with either amine (left and middle panel) or car- 55 substance is an illegal drug. In other embodiment, the boxy moieties (right panel). Both sets of beads were pur- addictive substance is an over-the-counter drug. In still chased from Invitrogen (Cat. no. F8763 and F8805). Sec- other embodiments, the addictive substance is a pre- tions on left and right were counterstained with anti- scription drug. Addictive substances include, but are not

20 39 EP 2 394 657 A1 40 limited to, cocaine, heroin, marijuana, methampheta- classes: IgG, IgM, IgA, IgD, and IgE. As used herein, the mines, and nicotine. In some embodiments, the addictive terms "antibody fragment" or "characteristic portion of an substance for inclusion in a nanocarrier is the complete antibody" are used interchangeably and refer to any de- molecule or a portion thereof. rivative of an antibody which is less than full-length. An [0160] Amino acid: As used herein, term "amino acid," 5 antibody fragment can retain at least a significant portion in its broadest sense, refers to any compound and/or of the full-length antibody’s specific binding ability. Ex- substance that can be incorporated into a polypeptide amples of such antibody fragments include, but are not chain. In some embodiments, an amino acid has the gen- limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv diabody, eral structure H2NC(H)(R)COOH. In some embodi- and Fd fragments. Antibody fragment also include Fc ments, an amino acid is a naturally-occurring amino acid. 10 fragments. An antibody fragment may be produced by In some embodiments, an amino acid is a synthetic ami- any means. For example, an antibody fragment may be no acid; in some embodiments, an amino acid is a D- enzymatically or chemically produced by fragmentation amino acid; in some embodiments, an amino acid is an of an intact antibody and/or it may be recombinantly pro- L-amino acid. "Standard amino acid" or "natural amino duced from a gene encoding the partial antibody se- acid" refers to any of the twenty standard L-amino acids 15 quence. Alternatively or additionally, an antibody frag- commonly found in naturally occurring peptides. "Non- ment may be wholly or partially synthetically produced. standard amino acid" refers to any amino acid, other than An antibody fragment may optionally comprise a single the standard amino acids, regardless of whether it is pre- chain antibody fragment. Alternatively or additionally, an pared synthetically or obtained from a natural source. As antibody fragment may comprise multiple chains which used herein, "non-natural amino acid" encompasses 20 are linked together, for example, by disulfide linkages. chemically produced or modified amino acids, including An antibody fragment may optionally comprise a multi- but not limited to salts, amino acid derivatives (such as molecular complex. A functional antibody fragment will amides), and/or substitutions. Amino acids, including typically comprise at least about 50 amino acids and carboxy- and/or amino-terminal amino acids in peptides, more typically will comprise at least about 200 amino can be modified by methylation, amidation, acetylation, 25 acids. and/or substitution with other chemical groups that can [0163] Approximately: As used herein, the terms "ap- change the peptide’s circulating half-life without adverse- proximately" or "about" in reference to a number are gen- ly affecting their activity. Amino acids may participate in erally taken to include numbers that fall within a range of a disulfide bond. The term "amino acid" is used inter- 5%, 10%, 15%, or 20% in either direction (greater than changeably with "amino acid residue," and may refer to 30 or less than) of the number unless otherwise stated or a free amino acid and/or to an amino acid residue of a otherwise evident from the context (except where such peptide. It will be apparent from the context in which the number would be less than 0% or exceed 100% of a term is used whether it refers to a free amino acid or a possible value). residue of a peptide. [0164] Associated with: As used herein, the term "as- [0161] Animal: As used herein, the term "animal" refers 35 sociated with" refers to the state of two or more entities to any member of the animal kingdom. In some embod- which are linked by a direct or indirect covalent or non- iments, "animal" refers to humans, at any stage of de- covalent interaction. In some embodiments, an associa- velopment. In some embodiments, "animal" refers to tion is covalent. In some embodiments, a covalent asso- non-human animals, at any stage of development. In cer- ciation is mediated by a linker moiety. In some embodi- tain embodiments, the non-human animal is a mammal 40 ments, an association is non-covalent (e.g., charge in- (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, teractions, affinity interactions, metal coordination, phys- a cat, a sheep, cattle, a primate, and/or a pig). In some ical adsorption, host-guest interactions, hydrophobic in- embodiments, animals include, but are not limited to, teractions, TT stacking interactions, hydrogen bonding mammals, birds, reptiles, amphibians, fish, and/or interactions, van der Waals interactions, magnetic inter- worms. In some embodiments, an animal may be a trans- 45 actions, electrostatic interactions, dipole-dipole interac- genic animal, genetically-engineered animal, and/or a tions, etc.). For example, in some embodiments, an entity clone. (e.g., immunomodulatory agent, targeting moiety, immu- [0162] Antibody: As used herein, the term "antibody" nostimulatory agent, nanoparticle, etc.) may be covalent- refers to any immunoglobulin, whether natural or wholly ly associated with a vaccine nanocarrier. In some em- or partially synthetically produced. All derivatives thereof 50 bodiments, an entity (e.g., immunomodulatory agent, tar- which maintain specific binding ability are also included geting moiety, immunostimulatory agent, nanoparticle, in the term. The term also covers any protein having a etc.) may be non-covalently associated with a vaccine binding domain which is homologous or largely homolo- nanocarrier. For example, the entity may be associated gous to an immunoglobulin binding domain. Such pro- with the surface of, encapsulated within, surrounded by, teins may be derived from natural sources, or partly or 55 and/or distributed throughout a lipid bilayer, lipid monol- wholly synthetically produced. An antibody may be mon- ayer, polymeric matrix, etc. of an inventive vaccine na- oclonal or polyclonal. An antibody may be a member of nocarrier. any immunoglobulin class, including any of the human [0165] Biocompatible: As used herein, the term "bio-

21 41 EP 2 394 657 A1 42 compatible" refers to substances that are not toxic to they may not actually pose a risk. Hazardous environ- cells. In some embodiments, a substance is considered mental agents include, but are not limited to, arsenic, to be "biocompatible" if its addition to cells in vivo does lead, mercury, vinyl chloride, polychlorinated biphenyls, not induce inflammation and/or other adverse effects in benzene, polycyclic aromatic hydrocarbons, cadmium, vivo. In some embodiments, a substance is considered 5 benzo(a)pyrene, benzo(b)fluoranthene, chloroform, to be "biocompatible" if its addition to cells in vitro or in DDT, P,P’-, aroclor 1254, aroclor 1260, dibenzo(a,h)an- vivo results in less than or equal to about 50%, about thracene, trichloroethylene, dieldrin, chromium hexava- 45%, about 40%, about 35%, about 30%, about 25%, lent, and DDE, P,P’. In some embodiments, the hazard- about 20%, about 15%, about 10%, about 5%, or less ous environmental agent for inclusion in a nanocarrier is than about 5% cell death. 10 the complete molecule or a portion thereof. [0166] Biodegradable: As used herein, the term "bio- [0170] In vitro: As used herein, the term "in vitro" refers degradable" refers to substances that are degraded un- to events that occur in an artificial environment, e.g.,in der physiological conditions. In some embodiments, a a test tube or reaction vessel, in cell culture, etc., rather biodegradable substance is a substance that is broken than within an organism (e.g., animal, plant, and/or mi- down by cellular machinery. In some embodiments, a 15 crobe). biodegradable substance is a substance that is broken [0171] In vivo: As used herein, the term "in vivo" refers down by chemical processes. to events that occur within an organism (e.g., animal, [0167] B cell antigen: As used herein, the term "B cell plant, and/or microbe). antigen" refers to any antigen that is recognized by and [0172] Immunostimulatory agent: As used herein, the triggers an immune response in a B cell. In some em- 20 term "immunostimulatory agent" refers to an agent that bodiments, an antigen that is a B cell antigen is also a T modulates an immune response to an antigen but is not cell antigen. In certain embodiments, the B cell antigen the antigen or derived from the antigen. "Modulate", as is not also a T cell antigen. In certain embodiments, when used herein, refers to inducing, enhancing, suppressing, a nanocarrier, as provided herein, comprises both a B directing, or redirecting an immune response. Such cell antigen and a T cell antigen, the B cell antigen and 25 agents include immunostimulatory agents that stimulate T cell antigen are not the same antigen, although each (or boost) an immune response to an antigen but, as of the B cell and T cell antigens may be, in some embod- defined above, is not the antigen or derived from the an- iments, both a B cell antigen and a T cell antigen. In other tigen. Immunostimulatory agents, therefore, include ad- embodiments, the B cell antigen and T cell antigen of the juvants. In some embodiments, the immunostimulatory nanocarrier are the same. 30 agent is on the surface of the nanocarrier and/or is en- [0168] Cell type: As used herein, the term "cell type" capsulated within the nanocarrier. In some embodi- refers to a form of cell having a distinct set of morpho- ments, the immunostimulatory agent on the surface of logical, biochemical, and/or functional characteristics the nanocarrier is different from the immunostimulatory that define the cell type. One of skill in the art will recog- agent encapsulated within the nanocarrier. In some em- nize that a cell type can be defined with varying levels of 35 bodiments, the nanocarrier comprises more than one specificity. For example, T cells and B cells are distinct type of immunostimulatory agent. In some embodiments, cell types, which can be distinguished from one another the more than one type of immunostimulatory agent act but share certain features that are characteristic of the on different pathways. Examples of immunostimulatory broader "lymphocyte" cell type of which both are mem- agents include those provided elsewhere herein. bers. Typically, cells of different types may be distin- 40 [0173] Nucleic acid: As used herein, the term "nucleic guished from one another based on their differential ex- acid," in its broadest sense, refers to any compound pression of a variety of genes which are referred to in the and/or substance that is or can be incorporated into an art as "markers" of a particular cell type or types (e.g., oligonucleotide chain. In some embodiments, a nucleic cell types of a particular lineage). In some embodiments, acid is a compound and/or substance that is or can be cells of different types may be distinguished from one 45 incorporated into an oligonucleotide chain via a phos- another based on their differential functions. A "cell type- phodiester linkage. In some embodiments, "nucleic acid" specific marker" is a gene product or modified version refers to individual nucleic acid residues (e.g., nucle- thereof that is expressed at a significantly greater level otides and/or nucleosides). In some embodiments, "nu- by one or more cell types than by all or most other cell cleic acid" refers to an oligonucleotide chain comprising types and whose expression is characteristic of that cell 50 individual nucleic acid residues. As used herein, the type. Many cell type specific markers are recognized as terms "oligonucleotide" and "polynucleotide" can be used such in the art. interchangeably. In some embodiments, "nucleic acid" [0169] Hazardous environmental agent: As used encompasses RNA as well as single and/or double- herein, the term "hazardous environmental agent" refers stranded DNA and/or cDNA. Furthermore, the terms "nu- to any hazardous substance found in the environment. 55 cleic acid", "DNA", "RNA", and/or similar terms include Such substances are generally believed to pose a health nucleic acid analogs, i.e., analogs having other than a risk. Hazardous environmental agents include substanc- phosphodiester backbone. For example, the so-called es that are thought to pose a health risk even though "peptide nucleic acids," which are known in the art and

22 43 EP 2 394 657 A1 44 have peptide bonds instead of phosphodiester bonds in in, in some embodiments, have a mean geometric diam- the backbone, are considered within the scope of the eter that is less than 500 nm. In some embodiments, the present invention. The term "nucleotide sequence en- nanocarriers have mean geometric diameter that is coding an amino acid sequence" includes all nucleotide greater than 50 nm but less than 500 nm. In some em- sequences that are degenerate versions of each other 5 bodiments, the mean geometric diameter of a population and/or encode the same amino acid sequence. Nucle- of nanocarriers is about 60 nm, 75 nm, 100 nm, 125 nm, otide sequences that encode proteins and/or RNA may 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 include introns. Nucleic acids can be purified from natural nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, sources, produced using recombinant expression sys- or 475 nm. In some embodiments, the mean geometric tems and optionally purified, chemically synthesized, etc. 10 diameter is between 100-400 nm, 100-300 nm, 100-250 Where appropriate, e.g., in the case of chemically syn- nm, or 100-200 nm. In some embodiments, the mean thesized molecules, nucleic acids can comprise nucleo- geometric diameter is between 60-400 nm, 60-350 nm, side analogs such as analogs having chemically modified 60-300 nm, 60-250 nm, or 60-200 nm. In some embod- bases or sugars, backbone modifications, etc. A nucleic iments, the mean geometric diameter is between 75-250 acid sequence is presented in the 5’ to 3’ direction unless 15 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, otherwise indicated. The term "nucleic acid segment" is 80%, 90%, or more of the nanocarriers of a population used herein to refer to a nucleic acid sequence that is a of nanocarriers have a diameter that is less than 500 nM. portion of a longer nucleic acid sequence. In many em- In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, bodiments, a nucleic acid segment comprises at least 3, 70%, 80%, 90%, or more of the nanocarriers of a popu- 4, 5, 6, 7, 8, 9, 10, or more residues. In some embodi- 20 lation of nanocarriers have a diameter that is greater than ments, a nucleic acid is or comprises natural nucleosides 50 nm but less than 500 nm. In some embodiments, 10%, (e.g., adenosine, thymidine, guanosine, cytidine, uridine, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more deoxyadenosine, deoxythymidine, deoxyguanosine, of the nanocarriers of a population of nanocarriers have and deoxycytidine); nucleoside analogs (e.g., 2-ami- a diameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 noadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimi- 25 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, dine, 3-methyl adenosine, 5-methylcytidine, C-5 propy- 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or nyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, 475 nm. In some embodiments, 10%, 20%, 30%, 40%, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers propynyl-uridine, C5-propynylcytidine, C5-methylcyti- of a population of nanocarriers have a diameter that is dine, 2-aminoadenosine, 7-deazaadenosine, 7-deaza- 30 between 100-400 nm, 100-300 nm, 100-250 nm, or guanosine, 8-oxoadenosine, 8-oxoguanosine, O 100-200 nm. In some embodiments, 10%, 20%, 30%, (6)-methylguanine, and 2-thiocytidine); chemically mod- 40%, 50%, 60%, 70%, 80%, 90%, or more of the nano- ified bases; biologically modified bases (e.g., methylated carriers of a population of nanocarriers have a diameter bases); intercalated bases; modified sugars (e.g., 2’- that is between 60-400 nm, 60-350 nm, 60-300 nm, fluororibose, ribose, 2’-deoxyribose, arabinose, and hex- 35 60-250 nm, or 60-200 nm. ose); and/or modified phosphate groups (e.g., phospho- [0175] Poorly immunogenic antigen: As used herein, rothioates and 5’-N-phosphoramidite linkages). the term "poorly immunogenic antigen" refers to an an- [0174] Particle: As used herein, a "particle" refers to tigen that does not trigger any or a sufficient level of a any entity having a diameter of less than 10 microns (mm). desired immune response. "Sufficient", as used herein, Typically, particles have a longest dimension (e.g., di- 40 refers to the ability to elicit a detectable or protective im- ameter) of 1000 nm or less. In some embodiments, par- mune response when administered in a composition that ticles have a diameter of 300 nm or less. Particles include does not employ a nanocarrier described herein, e.g., as microparticles, nanoparticles, and picoparticles. In some free antigen mixed with adjuvant in the absence of a na- embodiments, nanoparticles have a diameter of 200 nm nocarrier. In some embodiments, the desired immune or less. In some embodiments, nanoparticles have a di- 45 response is to treat or prevent a disease or condition. In ameter of 100 nm or less. In some embodiments, nano- certain embodiments, the desired immune response is particles have a diameter of 50 nm or less. In some em- to alleviate one or more symptoms of a disease or con- bodiments, nanoparticles have a diameter of 30 nm or dition. Poorly immunogenic antigens include, but are not less. In some embodiments, nanoparticles have a diam- limited to, self antigens, small molecules, and carbohy- eter of 20 nm or less. In some embodiments, nanoparti- 50 drates. cles have a diameter of 10 nm or less. In some embod- [0176] Self antigen: As used herein, the term "self an- iments, particles can be a matrix of polymers. In some tigen" refers to a normal substance in the body of an embodiments, particles can be a non-polymeric particle animal that when an immune response against the anti- (e.g., a metal particle, quantum dot, ceramic, inorganic gen within the animal is triggered, autoimmunity (e.g., an material, bone, etc.). Particles may also be liposomes 55 autoimmune disease) can result. A self antigen can be and/or micelles. As used herein, the term "nanoparticle" a protein or peptide, lipoprotein, lipid, carbohydrate, or a refers to any particle having a diameter of less than 1000 nucleic acid. The nucleic acid can be a DNA or RNA. Self nm. The nanocarriers of the compositions provided here- antigens include, but are not limited to enzymes, struc-

23 45 EP 2 394 657 A1 46 tural proteins, secreted proteins, cell surface receptors, "patient" refers to any organism to which a composition and cytokines. In some embodiments, the self antigen is of this invention may be administered, e.g., for experi- a cytokine, and the cytokine is TNF, IL-1, or IL-6. In some mental, diagnostic, and/or therapeutic purposes. Typical embodiments, the self antigen is cholesteryl ester trans- subjects include animals (e.g., mammals such as mice, fer protein (CETP), a serum protein responsible for cho- 5 rats, rabbits, non-human primates, and humans) and/or lesterol transfer from high-density lipoprotein (HDL) to plants. low-density lipoprotein cholesterol (LDL), the Aβ protein [0180] Suffering from: An individual who is "suffering associated with Alzheimer’s, a proteolytic enzyme that from" a disease, disorder, and/or condition has been di- processes the pathological form of the Aβ protein, LDL agnosed with, can be diagnosed with, or displays one or associated with atherosclerosis, or a coreceptor for HIV- 10 more symptoms of the disease, disorder, and/or condi- 1. In some embodiments, the proteolytic enzyme that tion. processes the pathological form of the Aβ protein is beta- [0181] Susceptible to: An individual who is "suscepti- secretase. In some embodiments, the LDL associated ble to" a disease, disorder, and/or condition has not been with atherosclerosis is oxidized or minimally modified. In diagnosed with and/or may not exhibit symptoms of the some embodiments, the coreceptor for HIV-1 is CCR5. 15 disease, disorder, and/or condition. In some embodi- [0177] Small molecule: In general, a "small molecule" ments, a disease, disorder, and/or condition is associat- is understood in the art to be an organic molecule that is ed with a microbial infection (e.g., bacterial infection, viral less than about 2000 g/mol in size. In some embodi- infection, fungal infection, parasitic infection, etc.). In ments, the small molecule is less than about 1500 g/mol some embodiments, an individual who is susceptible to or less than about 1000 g/mol. In some embodiments, 20 a microbial infection may be exposed to a microbe (e.g., the small molecule is less than about 800 g/mol or less by ingestion, inhalation, physical contact, etc.). In some than about 500 g/mol. In some embodiments, small mol- embodiments, an individual who is susceptible to a mi- ecules are non-polymeric and/or non-oligomeric. In some crobial infection may be exposed to an individual who is embodiments, small molecules, are not proteins, pep- infected with the microbe. In some embodiments, an in- tides, or amino acids. In some embodiments, small mol- 25 dividual who is susceptible to a microbial infection is one ecules are not nucleic acids or nucleotides. In some em- who is in a location where the microbe is prevalent (e.g., bodiments, small molecules are not saccharides or one who is traveling to a location where the microbe is polysaccharides. prevalent). In some embodiments, an individual who is [0178] Specific binding: As used herein, the term "spe- susceptible to a disease, disorder, and/or condition will cific binding" refers to non-covalent physical association 30 develop the disease, disorder, and/or condition. In some of a first and a second moiety wherein the association embodiments, an individual who is susceptible to a dis- between the first and second moieties is at least 2 times ease, disorder, and/or condition will not develop the dis- as strong, at least 5 times as strong as, at least 10 times ease, disorder, and/or condition. In some embodiments, as strong as, at least 50 times as strong as, at least 100 the subject has or is susceptible to having cancer, an times as strong as, or stronger than the association of 35 infectious disease, a non-autoimmune metabolic or de- either moiety with most or all other moieties present in generative disease, or an addiction. In some embodi- the environment in which binding occurs. Binding of two ments, the subject has or is susceptible to having a bac- or more entities may be considered specific if the equi- terial, fungal, protozoan, parisitic, or viral infection. The -3 -4 librium dissociation constant, Kd,is10 M or less, 10 cause of such infection can be any of the organisms as M or less, 10-5 M or less, 10-6 M or less, 10-7 M or less, 40 provided herein. In some embodiments, the subject has 10-8 M or less, 10-9 M or less, 10-10 M or less, 10-11 M or is susceptible to tuberculosis, malaria, leishmaniasis, or less, or 10-12 M or less under the conditions employed, H. pylori, a Staphylococcus infection, or a Salmonella e.g., under physiological conditions such as those inside infection. In some embodiments, the subject has or is a cell or consistent with cell survival. In some embodi- susceptible to having influenza. In some embodiments, ments, specific binding can be accomplished by a plu- 45 the subject has or is susceptible to an autoimmune dis- rality of weaker interactions (e.g., a plurality of individual ease, an allergy, or asthma. interactions, wherein each individual interaction is char- [0182] T cell antigen: As used herein, the term "T cell -3 acterized by a Kd of greater than 10 M). In some em- antigen" refers to any antigen that is recognized by and bodiments, specific binding, which can be referred to as triggers an immune response in a T cell (e.g., an antigen "molecular recognition," is a saturable binding interaction 50 that is specifically recognized by a T cell receptor on a T between two entities that is dependent on complemen- cell via presentation of the antigen or portion thereof tary orientation of functional groups on each entity. Ex- bound to a major histocompatiability complex molecule amples of specific binding interactions include aptamer- (MHC). In some embodiments, an antigen that is a T cell aptamer target interactions, antibody-antigen interac- antigen is also a B cell antigen. In other embodiments, tions, avidin-biotin interactions, ligand-receptor interac- 55 the T cell antigen is not also a B cell antigen. T cells tions, metal-chelate interactions, hybridization between antigens generally are proteins or peptides. T cell anti- complementary nucleic acids, etc. gens may be an antigen that stimulates a CD8+ T cell [0179] Subject: As used herein, the term "subject" or response, a CD4+ T cell response, or both. The nano-

24 47 EP 2 394 657 A1 48 carriers, therefore, in some embodiments can effectively ment protein or a pre-existing antibody. In further em- stimulate both types of responses. bodiments, the targeting moiety is for delivery of the na- [0183] Target: As used herein, the term "target" or nocarrier to antigen presenting cells, T cells, or B cells. "marker" refers to any entity that is capable of specifically In some embodiments, the antigen presenting cells are binding to a particular targeting moiety. In some embod- 5 macrophages. In other embodiments, the macrophages iments, targets are specifically associated with one or are subcapsular sinus macrophages. In still other em- more particular tissue types. In some embodiments, tar- bodiments, the antigen presenting cells are dendritic gets are specifically associated with one or more partic- cells. In some embodiments, the antigen presenting cells ular cell types. For example, a cell type specific marker are follicular dendritic cells. Specific non-limiting exam- is typically expressed at levels at least 2 fold greater in 10 ples of targeting moieties include molecules that bind to that cell type than in a reference population of cells. In CD11b, CD169, mannose receptor, DEC-205, CD11c, some embodiments, the cell type specific marker is CD21/CD35, CX3CR1, or a Fc receptor. In some em- present at levels at least 3 fold, at least 4 fold, at least 5 bodiments, the molecule that binds any of the foregoing fold, at least 6 fold, at least 7 fold, at least 8 fold, at least is an antibody or antigen-binding fragment thereof (e.g., 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, 15 an anti-CD169 antibody). In some embodiments, the or at least 1000 fold greater than its average expression molecule that binds a Fc receptor is one that comprises in a reference population. Detection or measurement of the Fc portion of an immunoglobulin (e.g., IgG). In other a cell type specific marker may make it possible to dis- embodiments, the Fc portion of an immunoglobulin is is tinguish the cell type or types of interest from cells of a human Fc portion. In some embodiments, the molecule many, most, or all other types. In some embodiments, a 20 that binds CX3CR1 is CX3CL1 (fractalkine). Targeting target can comprise a protein, a carbohydrate, a lipid, moieties that bind CD169 include anti-CD169 antibodies and/or a nucleic acid, as described herein. and ligands of CD169, e.g., sialylated CD227, CD43, [0184] Targeted: A substance is considered to be "tar- CD206, or portions of these ligands that retain binding geted" for the purposes described herein if it specifically function, e.g., soluble portions. binds to a target. In some embodiments, a targeting moi- 25 [0186] Therapeutically effective amount: As used ety specifically binds to a target under stringent condi- herein, the term "therapeutically effective amount" tions. An inventive nanocarrier, such as a vaccine nano- means an amount of a therapeutic, prophylactic, and/or carrier, comprising a targeting moiety is considered to diagnostic agent (e.g., inventive vaccine nanocarrier) be "targeted" if the targeting moiety specifically binds to that is sufficient, when administered to a subject suffering a target, thereby delivering the entire nanocarrier to a 30 from or susceptible to a disease, disorder, and/or condi- specific organ, tissue, cell, and/or subcellular locale. tion, to treat, alleviate, ameliorate, relieve, alleviate [0185] Targeting moiety: As used herein, the term "tar- symptoms of, prevent, delay onset of, inhibit progression geting moiety" refers to any moiety that binds to a com- of, reduce severity of, and/or reduce incidence of the dis- ponent of a cell. In some embodiments, the targeting moi- ease, disorder, and/or condition. The term is also intend- ety specifically binds to a component of a cell. Such a 35 ed to refer to an amount of nanocarrier or composition component is referred to as a "target" or a "marker." A thereof provided herein that modulates an immune re- targeting moiety may be a polypeptide, glycoprotein, nu- sponse in a subject. cleic acid, small molecule, carbohydrate, lipid, etc. In [0187] Therapeutic agent: As used herein, the term some embodiments, a targeting moiety is an antibody or "therapeutic agent" refers to any agent that, when ad- characteristic portion thereof. In some embodiments, a 40 ministered to a subject, has a therapeutic, prophylactic, targeting moiety is a receptor or characteristic portion and/or diagnostic effect and/or elicits a desired biological thereof. In some embodiments, a targeting moiety is a and/or pharmacological effect. ligand or characteristic portion thereof. In some embod- [0188] Treating: As used herein, the term "treating" re- iments, a targeting moiety is a nucleic acid targeting moi- fers to partially or completely alleviating, ameliorating, ety (e.g., an aptamer) that binds to a cell type specific 45 relieving, delaying onset of, inhibiting progression of, re- marker. In some embodiments, a targeting moiety is a ducing severity of, and/or reducing incidence of one or small molecule. The targeting moiety in some embodi- more symptoms or features of a particular disease, dis- ments is on the surface of the nanocarrier. In other em- order, and/or condition. For example, "treating" a micro- bodiments, the targeting moiety is encapsulated within bial infection may refer to inhibiting survival, growth, the nanocarrier. In still other embodiments, the targeting 50 and/or spread of the microbe. Treatment may be admin- moiety is associated with the nanocarrier. In some em- istered to a subject who does not exhibit signs of a dis- bodiments, the targeting moiety is covalently associated ease, disorder, and/or condition and/or to a subject who with the nanocarrier. In other embodiments, the targeting exhibits only early signs of a disease, disorder, and/or moiety is non-covalently associated with the nanocarrier. condition for the purpose of decreasing the risk of devel- In yet other embodiments, the targeting moiety binds a 55 oping pathology associated with the disease, disorder, receptor expressed on the surface of a cell. The targeting and/or condition. In some embodiments, treatment com- moiety, in some embodiments, binds a soluble receptor. prises delivery of an inventive vaccine nanocarrier to a In some embodiments, the soluble receptor is a comple- subject.

25 49 EP 2 394 657 A1 50

[0189] Universal T cell antigen: As used herein, the particles. term "universal T cell antigen" refers to a T cell antigen [0191] Water soluble, non-adhesive polymer: As used that can promote T cell help and enhance an immune herein, the term "water soluble, non-adhesive polymer" response to a completely unrelated antigen. Universal T refers to a polymer that is soluble in water and that can cell antigens include tetanus toxoid, as well as one or 5 confer reduced biofouling properties. In some embodi- more peptides derived from tetanus toxoid, Epstein-Barr ments, the water soluble, non-adhesive polymer is pol- virus, or influenza virus. yethylene glycol, polyethylene oxide, polyalkylene gly- Universal T cell antigens also include a component of col, and polyalkylene oxide. influenza virus, such as hemagglutinin, neuraminidase, or nuclear protein, or one or more peptides derived there- 10 Detailed Description of Certain Preferred Embodi- from. ments of the Invention [0190] Vaccine Nanocarrier: As used herein, the term "vaccine nanocarrier" refers to an entity comprising at Vaccines least one immunomodulatory agent or immunostimula- tory agent. In certain embodiments, a vaccine nanocar- 15 [0192] Vaccinations are typically either passive or ac- rier includes at least two types of immunomodulatory tive in nature. In general, active vaccinations involve the agents. In some embodiments, the immunomodulatory exposure of a subject’s immune system to one or more agents are antigens, and the vaccine nanocarrier com- agents that are recognized as unwanted, undesired, prises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens. The and/or foreign and elicit an endogenous immune re- different antigens can be or be derived from completely 20 sponse resulting in the activation of antigen-specific na- different antigenic molecules, or the different antigens ive lymphocytes that then give rise to antibody-secreting can be different epitopes from the same antigenic mole- B cells or antigen-specific effector and memory T cells cule. In other embodiments, the vaccine nanocarrier or both. This approach can result in long-lived protective comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different immunity that may be boosted from time to time by re- epitopes from the same ’ antigenic molecule. A vaccine 25 newed exposure to the same antigenic material. The nanocarrier may be any form of particle. A vaccine na- prospect of longevity of a successful immune response nocarrier, in some embodiments, is capable of stimulat- to active vaccination makes this strategy more desirable ing an immune response in T cells and/or B cells. In other in most clinical settings than passive vaccination where- embodiments, the vaccine nanocarrier is capable of en- by a recipient is injected with preformed antibodies or hancing, suppressing, directing, or redirecting an im- 30 with antigen-specific effector lymphocytes, which may mune response. In some embodiments, any assay avail- confer rapid ad hoc protection, but typically do not es- able in the art may be used to determine whether T cells tablish persistent immunity. and/or B cells have been stimulated. In some embodi- [0193] A large variety of vaccine formulations are be- ments, T cell stimulation may be assayed by monitoring ing or have been employed in humans. The most com- antigen-induced production of cytokines, antigen-in- 35 mon route of administration in humans is by intramuscu- duced proliferation of T cells, and/or antigen-induced lar (i.m.) injection, but vaccines may also be applied oral- changes in protein expression. In some embodiments, ly, intranasally, subcutaneously, inhalationly, or intrave- B cell stimulation may be assayed by monitoring antibody nously. In most cases, vaccine-derived antigens are in- titers, antibody affinities, antibody performance in neu- itially presented to naive lymphocytes in regional lymph tralization assays, class-switch recombination, affinity 40 nodes. maturation of antigen-specific antibodies, development [0194] Some current vaccines against, e.g., microbial of memory B cells, development of long-lived plasma pathogens, consist of live attenuated or non-virulent var- cells that can produce large amounts of high-affinity an- iant strains of microorganisms, or killed or otherwise in- tibodies for extended periods of time, germinal center activated organisms. Other vaccines utilize more or less reactions, and/or antibody performance in neutralization 45 purified components of pathogen lysates, such as sur- assays. In some embodiments, a vaccine nanocarrier face carbohydrates or recombinant pathogen-derived further comprises at least one targeting moiety that can proteins that are sometimes fused to other molecules, help deliver the vaccine nanocarrier to a particular target particularly proteins that can confer adjuvant activity. (e.g., organ, tissue, cell, and/or subcellular locale) within [0195] Vaccines used for intramuscular injections are a subject. In some embodiments, a vaccine nanocarrier 50 typically administered with an adjuvant carrier, most fre- further comprises at least one immunostimulatory agent quently alum (i.e., aluminum potassium sulphate), that that can help stimulate an immune response in T cells is thought to establish a depot for prolonged release of and/or B cells. In some embodiments, a vaccine nano- antigenic material, but also exerts immunomodulatory carrier further comprises at least one nanoparticle that activities, such as skewing toward Th2 responses by allows for tunable membrane rigidity and controllable li- 55 mechanisms that are incompletely understood (Lindblad, posome stability. In some embodiments, vaccine nano- 2004, Immunol. Cell. Biol., 82:497; and Jordan et al., carriers comprise lipids, amphiphilic compounds, poly- 2004, Science, 304:1808; both of which are incorporated mers, sugars, polymeric matrices, and/or non-polymeric herein by reference).

26 51 EP 2 394 657 A1 52

[0196] Vaccines that utilize live attenuated or inacti- that measure tens to hundreds of nanometers in diameter vated pathogens typically yield a vigorous immune re- and induce potent cellular and antibody responses are sponse, but their use has limitations. For example, live captured and retained on the surface of macrophages in vaccine strains can sometimes cause infectious pathol- the subcapsular sinus of draining lymph nodes (i.e., sub- ogies, especially when administered to immune-compro- 5 capsular sinus macrophages, abbreviated SCS-Mph). mised recipients. Moreover, many pathogens, particular- These macrophages are involved in the efficient early ly viruses, undergo continuous rapid mutations in their presentation of intact viral particles to follicular B cells. genome, which allow them to escape immune responses In some embodiments, inventive nanocarriers mimic viral to antigenically distinct vaccine strains. However, most particles and target SCS-Mph. As shown in Example 1, or all pathogens are thought to possess certain antigenic 10 upon subcutaneous injection of Cy5 encapsulated poly determinants that are not easily mutated because they (lactic-coglycolic acid) (PLGA) nanoparticles (50 nm - are associated with essential functions. Antibodies di- 150 nm) that are surface stabilized with a monolayer of rected against these conserved epitopes, rather than lipid and polyethylene glycol, the injected nanoparticles more variable, non-essential epitopes can protect readily enter lymphatics and are bound in the subcapsu- against highly mutable viruses (Baba et al., 2000, Nat. 15 lar sinus of draining lymph nodes similar to lymph-borne Med., 6:200; incorporated herein by reference). Vaccines viruses. Similar nanocarriers carrying immunomodulato- based on live or killed intact pathogens do not necessarily ry agent(s) that stimulate B cells and/or T cells are par- promote the recognition of these critical epitopes, but ticularly useful in vaccinating a subject. may essentially "distract" the immune system to focus [0199] Thus, the present invention encompasses the its assault on highly variable determinants. Thus, the 20 recognition that nanocarriers, such as lymph-borne virus- present invention encompasses the recognition that an sized nanocarriers carrying an immunomodulatory agent engineered vaccine nanocarrier that mimics the highly can be recognized in lymph nodes as if they were viruses immunogenic particulate nature of viral particles, but and may elicit a potent immune response, for example, presents selectively essential, immutable epitopes, when the particles include immunomodulatory agent(s) could yield much more potent and "escape-proof" neu- 25 that are recognized by B cells and/or T cells. tralizing antibody and effector T cell responses than intact [0200] By carrying immunomodulatory agents on the microorganisms. surface and/or loading similar or distinct immunomodu- [0197] The precise mechanisms by which vaccines latory agents inside, nanocarriers can simultaneously de- stimulate antibody responses in draining lymph nodes liver these immunomodulatory agents to distinct cells of (or fail to do so) are still incompletely understood. B and 30 the immune system and stimulate them. In certain em- T cells are initially sequestered in distinct anatomic re- bodiments, immunomodulatory agents presented on na- gions, the superficially located B follicles and the sur- nocarrier surfaces stimulate B cells, and immunomodu- rounding paracortex and deep cortex, respectively. Upon latory agents encapsulated within the nanocarriers are antigen challenge, antigen-specific B cells in follicles as processed by antigen-presenting cells (APCs), such as well as CD4 T cells in the T cell area become activated 35 dendritic cells (DCs), in lymphoid tissues (and by B cells and then migrate toward the border zone between the after activation) and presented to T cells. In some em- two compartments. B cells that have phagocytosed bodiments, by modifying the surface of nanocarriers with lymph-borne antigens process the acquired material and a targeting moiety (e.g., antibody or fragment thereof, begin to present antigenic peptides in MHC class-II sur- peptide or polypeptide, Affibody®, Nanobody™ AdNec- face molecules that are then recognized by the activated 40 tin™, Avimer™, aptamer, Spiegelmer®, small molecule, CD4 T cells (the TFH cells). Antigen-recognition allows lipid, carbohydrate, etc.), nanocarriers can selectively the TFH cells to provide help to B cells, which constitutes deliver immunomodulatory agents to specific antigen a potent survival signal and triggers the formation of ger- presenting cells, such as DCs, SCS-Mph, FDCs, T Cells, minal centers (GCs) within B follicles. The GC reaction B cells, and/or combinations thereof. A nanocarrier can promotes class-switch recombination, affinity maturation 45 be, but is not limited to, one or a plurality of lipid nano- of antigen-specific antibodies, and the formation of mem- particles, polymeric nanoparticles, metallic nanoparti- ory B cells and long-lived plasma cells that can produce cles, surfactant-based emulsions, dendrimers, and/or large amounts of high-affinity antibodies for extended pe- nanoparticles that are developed using a combination of riods of time. Thus, the present invention encompasses nanomaterials such as lipid-polymer nanoparticles. Vac- the recognition that a vaccine nanocarrier may have com- 50 cine nanocarriers are described in further detail in the ponents that allow antigenic material to be efficiently rec- section entitled "Vaccine Nanocarriers." ognized by both B and T cells and to induce vigorous GC reactions (Figure 1). T Cells [0198] The present invention describes systems for developing vaccine nanocarriers for vaccine delivery that 55 [0201] The present invention provides vaccine nano- can overcome these aforementioned limitations of cur- carriers for delivery of, for example, immunomodulatory rent vaccine technology. The present invention encom- agents to the cells of the immune system. In some em- passes the recognition that lymph-borne viral particles bodiments, vaccine nanocarriers comprise at least one

27 53 EP 2 394 657 A1 54 immunomodulatory agent which can be delivered to Mycoplasma species, Mycoplasma pneumoniae, Neis- APCs, which then process and deliver the immunomod- seria species, Neisseria meningitidis, Neisseria gonor- ulatory agent(s) to T cells. rhoeae, Pneumococcus species, Pseudomonas spe- [0202] Professional APCs are very efficient at internal- cies, Pseudomonas aeruginosa, Salmonella species, izing antigen, either by phagocytosis or by endocytosis, 5 Salmonella typhi, Salmonella enterica, Rickettsia spe- and then display a fragment of the antigen, bound to ei- cies, Rickettsia ricketsii, Rickettsia typhi, Shigella spe- ther a class II major histocompatibility complex (class II cies, Staphylococcus species, Staphylococcus aureus, MHC) molecule or a class I MHC molecule on the APC Streptococcus species, Streptococccus pnuemoniae, membrane. CD4 T cells recognize and interact with the Streptococcus pyrogenes, Streptococcus mutans, antigen-class II MHC molecule complex on the APC 10 Treponema species, Treponema pallidum, a Vibrio spe- membrane, whereas CD8 T cells recognize and interact cies, Vibrio cholerae, Yersinia pestis, and the like. with the antigen-class I MHC molecule complex. An ad- [0205] In some embodiments, immunomodulatory ditional co-stimulatory signal as well as modulating cy- agents may include antigens of viral organisms such as tokines are then produced by the APC, leading to T cell pox viruses, smallpox (variola), ebola virus, hepadnavi- activation. 15 rus, marburg virus, dengue fever virus, influenza A and B, parainfluenza, respiratory syncytial virus, measles (ru- Immunomodulatory Agents beola virus), human immunodeficiency virus (HIV), hu- man papillomavirus (HPV), varicella-zoster, herpes sim- [0203] The present invention provides vaccine nano- plex 1 and 2, cytomegalovirus, Epstein-Barr virus, JC carriers comprising one or more immunomodulatory 20 virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, or- agents. In some embodiments, inventive nanocarriers thomyxovirus, papillomavirus, parvovirus, picornavirus, comprising one or more immunomodulatory agents are poliovirus, mumps, rabies, reovirus, rubella, togavirus, used as vaccines. In some embodiments, an immu- retrovirus, coxsackieviruses, equine encephalitis, Japa- nomodulatory agent may comprise isolated and/or re- nese encephalitis, yellow fever, Rift Valley fever, hepa- combinant proteins or peptides, carbohydrates, glyco- 25 titis A, B, C, D, and E virus, and the like. Viral organisms proteins, glycopeptides, proteoglycans, inactivated or- include those that are dsDNA viruses, ssDNA viruses, ganisms and viruses, dead organisms and virus, genet- dsRNA viruses, (+) ssRNA viruses (-) sRNA viruses, ss- ically altered organisms or viruses, and cell extracts. In RNA-RT viruses, and dsDNA-RT viruses. some embodiments, an immunomodulatory agent may [0206] In some embodiments, immunomodulatory comprise nucleic acids, carbohydrates, lipids, and/or 30 agents may include antigens of fungal, protozoan, and/or small molecules. In some embodiments, an immu- parasitic organisms such as Aspergillus species, Cand- nomodulatory agent is one that elicits an immune re- ida species, Candida albicans, Candida tropicalis, Cryp- sponse. In other embodiments, an immunomodulatory tococcus species, Cryptococcus neoformans, Entamoe- agent is a polynucleotide that encodes a protein or pep- ba histolylica, Histoplasma capsulatum, Leishmania tide that when the protein or peptide is expressed an 35 speceis, Nocardia asteroides, Plasmodium falciparum, immune response is elicited. In some embodiments, an Toxoplasma gondii, Trichomonas vaginalis, Toxoplas- immunomodulatory agent is an antigen. In some embod- ma species, Trypanosoma brucei, Schistosoma manso- iments, an immunomodulatory agent is a protein or pep- ni, and the like. tide. In some embodiments, an immunomodulatory agent [0207] In some embodiments, immunomodulatory is used for vaccines. 40 agents may include E1 and/or E2 proteins of HCV. In [0204] In some embodiments, an immunomodulatory some embodiments, immunomodulatory agents may in- agent is any protein and/or other antigen derived from a clude gp120 of HIV. In some embodiments, immunomod- pathogen. The pathogen may be a virus, bacterium, fun- ulatory agents may include hemagglutinin and/or neu- gus, protozoan, parasite, etc. In some embodiments, im- raminidase of influenza virus. In some embodiments, im- munomodulatory agents may include antigens of bacte- 45 munomodulatory agents may include pneumococcal rial organisms such as Borrelia species, Bacillus anthra- polysaccharide or family 1 and/or family 2 PspA of Strep- cis, Borrelia burgdorferi, Bordetella pertussis, Camphy- tococcus pneumoniae or capsular polysaccharides lobacter jejuni, Chlamydia species, Chlamydial psittaci, types 5 and 8 or microbial surface components recog- Chlamydial trachomatis, Clostridium species, Clostrid- nizing adhesive matrix molecule of Stapylococcus au- ium tetani, Clostridium botulinum, Clostridium perfrin- 50 reus. In some embodiments, immunomodulatory agents gens, Corynebacterium diphtheriae, Coxiella species, may include mannan of Candida albicans or cryptococcal an Enterococcus species, Erlichia species, Escherichia capsular polysaccharide of Cryptococcus neoformans. coli, Francisella tularensis, Haemophilus species, Hae- In some embodiments, immunomodulatory agents may mophilus influenzae, Haemophilus parainfluenzae, include PfEMP1 of Plasmodium falciparum or other par- Lactobacillus species, a Legionella species, Legionella 55 asite-derived antigens expressed on plasmodium-infect- pneumophila, Leptospirosis interrogans, Listeria spe- ed red blood cells or GRA7 of Toxoplasma gondi. cies, Listeria monocytogenes, Mycobacterium species, [0208] Any of the antigens described herein may be in Mycobacterium tuberculosis, Mycobacterium leprae, the form of whole killed organisms, peptides, proteins,

28 55 EP 2 394 657 A1 56 glycoproteins, glycopeptides, proteoglycans, nucleic ac- nanocarrier comprises two or more types of immunomod- ids that encode a protein or peptide, carbohydrates, small ulatory agent which are all derived from a single genus, molecules, or combinations thereof. species, and strain of microorganism. [0209] In some embodiments, an immunomodulatory [0214] In some embodiments, a vaccine nanocarrier agent is derived from a microorganism for which at least 5 comprises two or more types of immunomodulatory one vaccine already exists. In some embodiments, an agents which are derived from different strains of a single immunomodulatory agent is derived from a microorgan- species of microorganism. In some embodiments, a vac- ism for which no vaccines have been developed. cine nanocarrier comprises two or more types of immu- [0210] In some embodiments, a vaccine nanocarrier nomodulatory agents which are derived from different comprises at least one type of immunomodulatory agent. 10 species of the same genus of microorganism. In other In some embodiments, all of the immunomodulatory embodiments, a vaccine nanocarrier comprises two or agents of a vaccine nanocarrier are identical to one an- more types of immunomodulatory agents each derived other. In some embodiments, a vaccine nanocarrier com- from different genera of microorganism. prises a number of different immunomodulatory agents. [0215] In some embodiments, a vaccine nanocarrier In some embodiments, a vaccine nanocarrier comprises 15 comprises a single type of immunomodulatory agent that multiple individual immunomodulatory agents, all of stimulates an immune response in both B cells and T which are the same. In some embodiments, a vaccine cells. In some embodiments, a vaccine nanocarrier com- nanocarrier comprises exactly one type of immunomod- prises two types of immunomodulatory agents, wherein ulatory agent. In some embodiments, a vaccine nano- the first immunomodulatory agent stimulates B cells, and carrier comprises exactly two distinct types of immu- 20 the second type of immunomodulatory agent stimulates nomodulatory agents. In some embodiments, a vaccine T cells. In certain embodiments, one or both agents may nanocarrier comprises greater than two distinct types of stimulate T cells and B cells. In some embodiments, a immunomodulatory agents. In some embodiments, a vaccine nanocarrier comprises greater than two types of vaccine nanocarrier comprises 3, 4, 5, 6, 7, 8, 9, 10, or immunomodulatory agents, wherein one or more types more distinct types of immunomodulatory agents. 25 of immunomodulatory agents stimulate B cells, and one [0211] In some embodiments, a vaccine nanocarrier or more types of immunomodulatory agents stimulate T comprises two types of immunomodulatory agents which cells. are both derived from a single genus of microorganism. [0216] In some embodiments, a vaccine nanocarrier In some embodiments, a vaccine nanocarrier comprises comprises at least one type of immunomodulatory agent two types of immunomodulatory agents which are both 30 that is associated with the exterior surface of the vaccine derived from a single genus and species of microorgan- nanocarrier. In some embodiments, the association is ism. In some embodiments, a vaccine nanocarrier com- covalent. In some embodiments, the covalent associa- prises two types of immunomodulatory agents which are tion is mediated by one or more linkers. In some embod- both derived from a single genus, species, and strain of iments, the association is non-covalent. In some embod- microorganism. In some embodiments, a vaccine nano- 35 iments, the non-covalent association is mediated by carrier comprises two types of immunomodulatory charge interactions, affinity interactions, metal coordina- agents which are both derived from a single clone of a tion, physical adsorption, host-guest interactions, hydro- microorganism. phobic interactions, TT stacking interactions, hydrogen [0212] In some embodiments, a vaccine nanocarrier bonding interactions, van der Waals interactions, mag- comprises more than two types of immunomodulatory 40 netic interactions, electrostatic interactions, dipole-di- agents which are all derived from a single genus of mi- pole interactions, and/or combinations thereof. For a croorganism. In some embodiments, a vaccine nanocar- more detailed description of how an immunomodulatory rier comprises more than two types of immunomodula- agent may be associated with a vaccine nanocarrier, tory agents which are all derived from a single genus and please see the section below entitled "Production of Vac- species of microorganism. In some embodiments, a vac- 45 cine Nanocarriers." cine nanocarrier comprises more than two types of im- [0217] In some embodiments, a vaccine nanocarrier munomodulatory agents which are all derived from a sin- includes a lipid membrane (e.g., lipid bilayer, lipid mon- gle genus, species, and strain of microorganism. In some olayer, etc.). At least one immunomodulatory agent may embodiments, a vaccine nanocarrier comprises more be associated with the lipid membrane. In some embod- than two types of immunomodulatory agents which are 50 iments, at least one immunomodulatory agent is embed- all derived from a single clone of a microorganism. ded within the lipid membrane. In some embodiments, [0213] In some embodiments, a vaccine nanocarrier at least one immunomodulatory agent is embedded with- comprises two or more types of immunomodulatory in the lumen of a lipid bilayer. In some embodiments, a agent which are all derived from a single genus of micro- vaccine nanocarrier comprises at least one immunomod- organism. In some embodiments, a vaccine nanocarrier 55 ulatory agent that is associated with the interior surface comprises two or more types of immunomodulatory of the lipid membrane. In some embodiments, at least agent which are all derived from a single genus and spe- one immunomodulatory agent is encapsulated within the cies of microorganism. In some embodiments, a vaccine lipid membrane of a vaccine nanocarrier. In some em-

29 57 EP 2 394 657 A1 58 bodiments, at least one type of immunomodulatory agent antigen may be taken from an unrelated source, such as may be located at multiple locations of a vaccine nano- an infectious agent to which wide-spread immunity al- carrier. For example, a first type of immunomodulatory ready exists (e.g., tetanus toxoid or a common compo- agent may be embedded within a lipid membrane, and nent of influenza virus, such as hemagglutinin, neurami- a second type of immunomodulatory agent may be en- 5 nidase, or nuclear protein). In the latter case, the vaccine capsulated within the lipid membrane of a vaccine nano- exploits the presence of memory T cells that have arisen carrier. To give another example, a first type of immu- in response to prior infections or vaccinations. Memory nomodulatory agent may be associated with the exterior cells in general react more rapidly and vigorously to an- surface of a lipid membrane, and a second type of im- tigen rechallenge and, therefore, may provide a superior munomodulatory agent may be associated with the inte- 10 source of help to B cells. rior surface of the lipid membrane of a vaccine nanocar- [0221] Other T cell antigens include, but are not limited rier. In some embodiments, a first type of immunomod- to, degenerative disease antigens, infectious disease an- ulatory agent may be embedded within the lumen of a tigens, cancer antigens, allergens, alloantigens, atopic lipid bilayer of a vaccine nanocarrier, and the lipid bilayer disease antigens, autoimmune disease antigens, con- may encapsulate a polymeric matrix throughout which a 15 tact sensitizers, haptens, xenoantigens, or metabolic dis- second type of immunomodulatory agent is distributed. ease enzymes or enzymatic products thereof. In some In some embodiments, a first type of immunomodulatory embodiments, the infectious disease antigen is a viral agent and a second type of immunomodulatory agent antigen, which includes any antigen derived from any of may be in the same locale of a vaccine nanocarrier (e.g., the viruses described herein. Examples of T cell antigens they may both be associated with the exterior surface of 20 include those provided elsewhere herein. a vaccine nanocarrier; they may both be encapsulated [0222] In some embodiments, T cell antigens are in- within the vaccine nanocarrier; etc.). corporated into nanocarriers as intact proteins. In some [0218] In some embodiments, a vaccine nanocarrier embodiments, T cell antigens are incorporated into na- includes a polymer (e.g., a polymeric core). At least one nocarriers as modified proteins. In some embodiments, type of immunomodulatory agent may be associated with 25 T cell antigens are incorporated into nanocarriers as mu- the polymer. In some embodiments, at least one type of tated proteins. In some embodiments, T cell antigens are immunomodulatory agent is embedded within the poly- provided as a collection of overlapping peptides, which mer. In some embodiments, a vaccine nanocarrier com- can boost antigen incorporation into MHC class II com- prises at least one type of immunomodulatory agent that plexes and, therefore, further promote a helper response. is associated with the interior surface of the polymer. In 30 In some embodiments, T cell antigens are provided as some embodiments, at least one type of immunomodu- a collection of non-overlapping peptides, which can boost latory agent is encapsulated with the polymer of a vaccine antigen incorporation into MHC class II complexes and, nanocarrier. In some embodiments, at least one type of therefore, further promote a helper response. In some immunomodulatory agent may be located at multiple lo- embodiments, T cell antigens are provided as nucleic cations of a vaccine nanocarrier. For example, a first type 35 acids that encode the antigens. of immunomodulatory agent may be embedded within a [0223] In some embodiments, inventive nanocarriers, polymer, and a second type of immunomodulatory agent such as vaccine nanocarriers, comprise less than less may be encapsulated within a lipid membrane surround- than 90% by weight, less than 75% by weight, less than ing the polymeric core of a vaccine nanocarrier. To give 50% by weight, less than 40% by weight, less than 30% another example, a first type of immunomodulatory agent 40 by weight, less than 20% by weight, less than 15% by may be associated with the exterior surface of a polymer, weight, less than 10% by weight, less than 5% by weight, and a second type of immunomodulatory agent may be less than 1% by weight, or less than 0.5% by weight of embedded within the polymer of a vaccine nanocarrier. the immunomodulatory agent. [0219] One of ordinary skill in the art will recognize that the preceding examples are only representative of the 45 Targeting Moieties many different ways in which multiple immunomodulato- ry agents may be associated with different locales of vac- [0224] In some embodiments, inventive nanocarriers cine nanocarriers. Multiple immunomodulatory agents comprise one or more targeting moieties. In certain em- may be located at any combination of locales of vaccine bodiments of the invention, nanocarriers are associated nanocarriers. Additionally, the aforementioned examples 50 with one or more targeting moieties. A targeting moiety can also apply to the other agents of a nanocarrier (e.g., is any moiety that binds to a component associated with a immunostimulatory agent). an organ, tissue, cell, extracellular matrix, and/or subcel- [0220] In some embodiments, the immunomodulatory lular locale. In some embodiments, such a component is agent is a T cell antigen, and the T cell antigen is derived referred to as a "target" or a "marker," and these are from the same pathogen against which vaccination is 55 discussed in further detail below. intended. In this case, an initially small number of naive [0225] A targeting moiety may be a nucleic acid, T cells are stimulated to generate pathogen-specific ef- polypeptide, glycoprotein, carbohydrate, lipid, small mol- fector and memory T cells. In some embodiments, the ecule, etc. For example, a targeting moiety can be a nu-

30 59 EP 2 394 657 A1 60 cleic acid targeting moiety (e.g. an aptamer, Spiegelm- nucleic acid; RNA nucleic acid; modified DNA nucleic er®, etc.) that binds to a cell type specific marker. In gen- acid; modified RNA nucleic acid; nucleic acid that in- eral, an aptamer is an oligonucleotide (e.g., DNA, RNA, cludes any combination of DNA, RNA, modified DNA, or an analog or derivative thereof) that binds to a partic- and modified RNA; etc. ular target, such as a polypeptide. In some embodiments, 5 [0228] In some embodiments, a targeting moiety could a targeting moiety may be a naturally occurring or syn- be the surface glycoprotein molecule from VSV. VSV thetic ligand for a cell surface receptor, e.g., a growth comprises a single surface molecule, VSV-G, which is a factor, hormone, LDL, transferrin, etc. A targeting moiety toll-like receptor agonist. VSV is efficiently targeted to can be an antibody, which term is intended to include cells of the immune system, so in some embodiments, antibody fragments, characteristic portions of antibodies, 10 vaccine nanocarriers could comprise the VSV surface single chain antibodies, etc. Synthetic binding proteins molecule in order to target vaccine nanocarriers to cells such as Affibodies®, Nanobodies™, AdNectins™, Avim- of the immune system. ers™, etc., can be used. Peptide targeting moieties can [0229] In some embodiments, a target is a tumor mark- be identified, e.g., using procedures such as phage dis- er. In some embodiments, a tumor marker is an antigen play. This widely used technique has been used to iden- 15 that is expressed in tumor cells but not in healthy and/or tify cell specific ligands for a variety of different cell types. normal cells. In some embodiments, a tumor marker is [0226] In accordance with the present invention, a tar- an antigen that is more prevalent in tumor cells than in geting moiety recognizes one or more "targets" or "mark- healthy and/or normal cells. Exemplary tumor markers ers" associated with a particular organ, tissue, cell, include, but are not limited to, gp100; Melan-A; tyrosi- and/or subcellular locale. In some embodiments, a target 20 nase; PSMA; HER-2/neu; MUC-1; topoisomerase IIα; si- may be a marker that is exclusively or primarily associ- alyl-Tn; carcinoembryonic antigen; ErbB-3-binding pro- ated with one or a few cell types, with one or a few dis- tein-1; alpha-fetoprotein; and the cancer-testis antigens eases, and/or with one or a few developmental stages. MAGE-A1, MAGE A4, and NY-ESO-1. A cell type specific marker is typically expressed at levels [0230] In some embodiments, a target is an APC mark- at least 2 fold greater in that cell type than in a reference 25 er. In some embodiments, an APC target is an antigen population of cells which may consist, for example, of a that is expressed in APCs but not in non-APCs. In some mixture containing an approximately equal amount of embodiments, an APC target is an antigen that is more cells (e.g., approximately equal numbers of cells, approx- prevalent in APCs than in non-APCs. Exemplary APC imately equal volume of cells, approximately equal mass markers include, but are not limited to, CD11c, CD11b, of cells, etc.). In some embodiments, the cell type specific 30 CD 14, CD40, CD45, CD 163, CD 169 (sialoadhesin), marker is present at levels at least 3 fold, at least 4 fold, DEC205 (CD205), MHC class II, DC-SIGN, CD21/CD35, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 and Fc γ RI, PD-L2. In some embodiments, APC markers fold, at least 9 fold, at least 10 fold, at least 50 fold, at include any of DC and/or macrophage markers, exam- least 100 fold, at least 500 fold, at least 1000 fold, at least ples of which are described herein. 5000 fold, or at least 10,000 fold greater than its average 35 [0231] In certain embodiments, a target is a DC mark- expression in a reference population. Detection or meas- er. In some embodiments, a DC target is an antigen that urement of a cell type specific marker may make it pos- is expressed in DCs but not in non-DCs. In some em- sible to distinguish the cell type or types of interest from bodiments, a DC target is an antigen that is more prev- cells of many, most, or all other types. alent in DCs than in non-DCs. Exemplary DC markers [0227] In some embodiments, a target can comprise 40 are listed below in the section entitled "Dendritic Cells" a protein, a carbohydrate, a lipid, and/or a nucleic acid. and include those provided elsewhere herein. In certain embodiments, a target can comprise a protein [0232] In certain embodiments, a target is a T cell and/or characteristic portion thereof, such as a tumor- marker. In some embodiments, a T cell target is an an- marker, integrin, cell surface receptor, transmembrane tigen that is expressed in T cells but not in non-T cells. protein, intercellular protein, ion channel, membrane 45 In some embodiments, a T cell target is an antigen that transporter protein, enzyme, antibody, chimeric protein, is more prevalent in T cells than in non-T cells. Exemplary glycoprotein, etc. In certain embodiments, a target can T cell markers are listed below in the section entitled "T comprise a carbohydrate and/or characteristic portion Cell Targeting Moieties" and and include those provided thereof, such as a glycoprotein, sugar (e.g., monosac- elsewhere herein. charide, disaccharide, polysaccharide), glycocalyx (i.e., 50 [0233] In some embodiments, a target is preferentially the carbohydrate-rich peripheral zone on the outside sur- expressed in particular cell types. For example, expres- face of most eukaryotic cells) etc. In certain embodi- sion of an APC, DC, and/or T cell target in APCs, DCs, ments, a target can comprise a lipid and/or characteristic and/or T cells is at least 2-fold, at least 3-fold, at least 4- portion thereof, such as an oil, fatty acid, glyceride, hor- fold, at least 5-fold, at least 10-fold, at least 20-fold, at mone, steroid (e.g., cholesterol, bile acid), vitamin (e.g. 55 least 50-fold, at least 100-fold, at least 500-fold, or at vitamin E), phospholipid, sphingolipid, lipoprotein, etc. In least 1000-fold overexpressed in APCs, DCs, and/or T certain embodiments, a target can comprise a nucleic cells relative to a reference population. In some embod- acid and/or characteristic portion thereof, such as a DNA iments, a reference population may comprise non-APCs,

31 61 EP 2 394 657 A1 62

FDCs, and/or T cells. DC maturation can be induced by many signals, such as [0234] In some embodiments, expression of an APC, certain inflammatory cytokines, ligation of DC-expressed DC, and/or T cell target in activated APCs, DCs, and/or CD40, agonists for TLRs, (e.g., bacterial endotoxin), im- T cells is at least 2-fold, at least 3-fold, at least 4-fold, at mune complexes, activated complement, necrotic cells, least 5-fold, at least 10-fold, at least 20-fold, at least 50- 5 apoptotic cells, free urate, urate crystals, and/or HMGB- fold, at least 100-fold, at least 500-fold, or at least 1000- 1. fold overexpressed in activated APCs, DCs, and/or T [0239] DEC-205 (i.e., CD205) is a surface-expressed cells relative to a reference population. In some embod- multi-functional lectin that is selectively expressed on iments, a reference population may comprise non-acti- DCs and thymic epithelial cells in lymphoid tissues. In vated APCs, DCs, and/or T cells. 10 vivo experiments with subcutaneously injected chimeric [0235] In some embodiments, inventive nanocarriers, α-DEC-205 monoclonal antibodies have shown that lig- such as vaccine nanocarriers, comprise less than 50% and binding to DEC-205 induces efficient internalization by weight, less than 40% by weight, less than 30% by and subsequent processing of the endocytosed material weight, less than 20% by weight, less than 15% by for presentation in MHC molecules in both mice and hu- weight, less than 10% by weight, less than 5% by weight, 15 mans (Hawiger et al., 2001, J. Exp. Med. 194:769; Bon- less than 1% by weight, or less than 0.5% by weight of ifaz et al., 2002, J. Exp. Med., 196:1627; and Bozzacco the targeting moiety. et al., 2007, Proc. Natl. Acad. Sci., USA, 104:1289; each [0236] In some embodiments, targeting moieties are of which is incorporated herein by reference). Upon intra- covalently associated with a nanocarrier. In some em- cutaneous or subcutaneous injection, the chimeric anti- bodiments, covalent association is mediated by a linker. 20 body is transported via lymph vessels to the draining In some embodiments, targeting moieties are not cova- lymph nodes where it binds specifically to resident DCs, lently associated with a nanocarrier. For example, tar- thus providing the means to target antigens to resting geting moieties may be associated with the surface of, DCs without causing their maturation. The targeted DCs encapsulated within, surrounded by, and/or distributed will then induce T cell tolerance to the presented antigen, throughout the polymeric matrix of an inventive particle. 25 rather than immunity. However, when DEC-205 is tar- For example, in some embodiments, a targeting moiety geted together with an immunostimulatory agent that in- can be encapsulated within, surrounded by, and/or dis- duces DC maturation (e.g., α-CD40 or one or more lig- persed throughout the liposomal membrane and/or pol- ands for DC-expressed TLRs; discussed in further detail ymeric matrix of a nanocarrier. Alternatively or addition- below in the section entitled "Immunostimulatory ally, a targeting moiety can be associated with a nano- 30 Agents"), then the vaccine acts as a potent immunostim- carrier by charge interactions, affinity interactions, metal ulant promoting preferentially cytotoxic and Th1-type ef- coordination, physical adsorption, host-guest interac- fector T cell responses. tions, hydrophobic interactions, TT stacking interactions, [0240] DC targeting can be accomplished by moieties hydrogen bonding interactions, van der Waals interac- that bind DC-205, CD 11 c, class II MHC, CD80, CD86, tions, magnetic interactions, electrostatic interactions, di- 35 DC-SIGN, CD11b, BDCA-1, BDCA-2, BDCA-4, Siglec- pole-dipole interactions, and/or combinations thereof. H, CX3CR1, and/or Langerin. Association of targeting moieties with vaccine nanocar- [0241] In some embodiments, DC targeting can be ac- riers is described in further detail below, in the section complished by any targeting moiety that specifically entitled "Production of Vaccine Nanocarriers." binds to any entity (e.g., protein, lipid, carbohydrate, [0237] Dendritic Cells 40 small molecule, etc.) that is prominently expressed [0238] Dendritic Cells (DCs) are a type of myeloid leu- and/or present on DCs (i.e., a DC marker). Exemplary kocytes; they are among the most potent antigen pre- DC markers include, but are not limited to, CD1a (R4, senting cells for T lymphocytes. Resting DCs reside in T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); many tissues, including lymph nodes, in an immature, CD1e (R2); CD11b (αM Integrin chain, CR3, Mo1, C3niR, tolerogenic state, i.e., they present intermediate to high 45 Mac-1); CD11c (αX Integrin, p150, 95, AXb2); CDw117 levels of peptide-MHC complexes, but with little or no (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); costimulatory molecules and without secreting cytokines CD 35 (CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, that T cells need to differentiate into effector cells. T cells PASIV); CD39 (ATPdehydrogenase, NTPdehydroge- that are presented with a specific antigen by immature nase-1); CD40 (Bp50); CD45 (LCA, T200, B220, Ly5); DCs begin to proliferate for a few days, but then they die 50 CD45RA; CD4SRB; CD45RC; CD45RO (UCHL-1); by apoptosis or become unresponsive to further activa- CD49d (VLA-4α, α4 Integrin); CD49e (VLA-5α, α5 In- tion. The ensuing depletion of antigen-specific T cell re- tegrin); CD58 (LFA-3); CD64 (FcγRI); CD72 (Ly-19.2, Ly- sponses renders the host selectively tolerant to this an- 32.2, Lyb-2); CD73 (Ecto-5’nucloticlase); CD74 (Ii, invar- tigen. By contrast, when DCs acquire antigens while they iant chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); are exposed to maturation stimuli, the cells rapidly up- 55 CD83 (HB15); CD85a (ILT5, LIR3, HL9); CD85d (ILT4, regulate MHC and costimulatory molecules and secrete LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, several cytokines. The now mature DCs are potent in- LIR5, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97 ducers of effector T cells and immunological memory. (BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-

32 63 EP 2 394 657 A1 64

CSFRα); CD120a (TMFRI, p55); CD 120b (TNFRII, p75, tegrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α, TNFR p80); CD123 (IL-3Rα); CD 139; CD 148 (HPTP- α6 Integrin gplc); CD50 (ICAM-3); CD52 (CAMPATH-1, η, p260, DEP-1); CD 150 (SLAM, IPO-3); CD156b (TA- HES); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CE, ADAM17, cSVP); CD157 (Mo5, BST-1); CD167a CD56 (Leu-19, NKH-1, NCAM); CD57 (HNK1, Leu-7); (DDR1, trkE, cak); CD168 (RHAMM, IHABP, HMMR); 5 CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); MIRL, P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD171 (L1CAM, NILE); CD172 (SIRP-1α, MyD-1); CD60c (7-O acetyl GD3); CD62L (L-selectin, LAM-1, CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD184 LECAM-1, MEL-14, Leu8, TQ1); CD73 (Ecto-5’-nucloti- (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); dase); CD75 (sialo-masked Lactosamine); CD75S (α2, CD197 (CCR7 (ws CDw197)); CDw197 (CCR7, EBI1, 10 6 sialylated Lactosamine); CD81 (TAPA-1); CD82 (4F9, BLR2); CD200 (OX2); CD205 (DEC-205); CD206 C33, IA-4, KAI1, R2); CD84 (P75, GR6); CD85a (ILT5, (MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209 LIR3, HL9); CD85j (ILT2, LIR1, MIR7); CD87 (uPAR); (DC-SIGN); CDw218a(IL18Rα); CDw218b (IL8Rβ); CDw92 (p70); CD94 (Kp43); CD95 (APO-1, FAS, CD227 (MUC 1, PUM, PEM, EMA); CD230 (Prion Protein TNFRSF6); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, (PrP)); CD252 (OX40L, TNF (ligand) superfamily, mem- 15 E2); CD99R (CD99 Mab restricted); CD100 (SEMA4D); ber 4); CD258 (LIGHT, TNF (ligand) superfamily, mem- CD102 (ICAM-2); CD108 (SEMA7A, JMH blood group ber 14); CD265 (TRANCE-R, TNF-R superfamily, mem- antigen); CDw119 (IFNγR, IFNγRa); CD120a (TNFRI, ber 11a); CD271 (NGFR, p75, TNFR superfamily, mem- p55); CD120b (TNFRII, p75, TNFR p80); CD121a (Type ber 16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); 1 IL-1R); CD121b (Type 2 IL-1R); CD 122 (IL2Rβ); CD275 (B7H2, ICOSL); CD276 (B7H3); CD277 (BT3.1, 20 CD124 (IL-4Rα); CD126 (IL-6Rα); CD127 (p90, IL-7R, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-like re- IL-7Rα); CD128a (IL-8Ra, CXCR1, (Tentatively re- ceptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 named as CD181)); CD128b (IL-8Rb, CXCR2, (Tenta- (LEPR); CD298 (ATP1B3, Na K ATPase β3 submit); tively renamed as CD182)); CD130 (gp130); CD132 CD300a (CMRF-35H); CD300c (CMRF-35A); CD301 (Common γ chain, IL-2Rγ); CD147 (Basigin, EMMPRIN, (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2); 25 M6, OX47); CD148 (HPTP-η, p260, DEP-1); CD150 CD304 (BDCA4); CD312 (EMR2); CD317 (BST2); (SLAM, IPO-3); CD153 (CD3OL, TNSF8); CD156b (TA- CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68 CE, ADAM17, cSVP); CD158a (KIR2DL1, p58.1); (gp110, Macrosialin); class II MHC; BDCA-1; Siglec-H; CD158b1 (KIR2DL2, p58.2); CD158b2 (KIR2DL3, wherein the names listed in parentheses represent alter- p58.3); CD158c(KIR2DS6, KIRX); CD158|e1/e2 native names. 30 (KIR3DLI/S1, p70); CD159F (KIR2DL5); CD158g [0242] T Cell Targeting Moieties (KIR2DS5); CD158h (KIR2DS1, p50.1); CD158i [0243] In some embodiments, T cell targeting can be (KIR2DS4, p50.3); CD158j (KIR2DS2, p50.2); CCD158k accomplished by any targeting moiety that specifically (KIR3DL2, p140); CD159a (NKG2A); CD160 (BY55, binds to any entity (e.g., protein, lipid, carbohydrate, NK1, NK28); CD161 (NKR, NKRP1A); CD162 (PSGL- small molecule, etc.) that is prominently expressed 35 1); CD164 (MGC-24, MUC-24); CD 171 (L1 CAM, NILE); and/or present on T cells (i.e., a T cell marker). Exemplary CD172g (SIRPg); CD181 (CXCR 1, (Formerly known as T cell markers include, but are not limited to, CD2 (E- CD128a)); CD182 (CXCR2, (Formerly known as rosette R, T11, LFA-2); CD3 (T3); CD3 α; CD3 β; CD3 CD128b)); CD183 (CXCR3, GPR9); CD184 (CXCR4, ε; CD4 (L3T4, W3/25, T4); CD5 (T1, Tp67, Leu-1, LY-1); NPY3R); CD185 (CXCR5); CD186 (CXCR6); CD191 CD6 (T12); CD7 (gp40, Leu 9); CD8a (Leu2, T8, Lyt2,3); 40 (CCR1); CD192 (CCR2); CD193 (CCR3); CD195 CD8b (CD8, Leu2, Lyt3); CD11a (LFA-1α, α Integrin (CCR5); CD196 (CCR6); CD197 (CCR7 (was CDw197)); chain); CD11b (αM Integrin chain, CR3, Mo1, C3niR, CDw197 (CCR7, EBI1, BLR2); CDw198 (CCR8); Mac-1); CD11c (αX Integrin, p150, 95, AXb2); CD15s CDw199 (CCR9); CD205 (DEC-205); CDw210 (CK); (Sialyl Lewis X); CD15u (3’ sulpho Lewis X); CD15su (6 CDw217 (CK); CDw218a (IL18Rα); CDw218b (IL18Rβ); sulpho-sialyl Lewis X); CD16b (FcgRIIIb); CDw17 (Lac- 45 CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, tosylceramide, LacCer); CD18 (Integrin β2 CD11a, b, c IGFII-R); CD223 (LAG-3); CD224 (GGT); CD225 β-subunit); CD26 (DPP IV ectoeneyme, ADA binding pro- (Leu13); CD226 (DNAM-1, PTA1); CD229 (Ly9); CD230 tein); CD27 (T14, S152); CD28 (Tp44, T44); CD29 (Prion Protein (PrP)); CD244 (2B4, P38, NAIL); CD245 (Platelet GP11a, β-1 integrin, GP); CD31 (PECAM-1, En- (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL- docam); CD35 (CR1, C3b/C4b receptor); CD37 50 R1, TNF-R superfamily, member 10a); CD262 (TRAIL- (gp52-40); CD38 (ADP-ribosyl/cyclase, T10); CD43 (Sia- R2, TNF-R superfamily, member 10b); CD263 (TRAIL- lophorin, Leukosialin); CD44 (ECMRII, H-CAM, Pgp-1); R3, TNF-R superfamily, member 10c); CD264 (TRAIL- CD45 (LCA, T200, B220, Ly5); CD45RA (p561ck, R4, TNF-R superfamily, member 10d); CD265 p59fyn, Src kinases); CD45RB (p561ck, p59fyn, Src ki- (TRANCE-R, TNF-R superfamily, member 11a); CD268 nases); CD45RC (p561ck, p59fyn, Src kinases); CD46 55 (BAFFR, TNF-R superfamily, member 13C); CD272 (BT- (MCP); CD47 (gp42, IAP, OA3, Neurophillin); CD47R LA); CD275 (B7H2, ICOSL); CD277 (BT3.1, B7 family: (MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); Butyrophilin 3); CD294 (CRTH2, PGRD2, G protein-cou- CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, α4 In- pled receptor 44); CD295 (LEPR); CD296 (ART1, ADP-

33 65 EP 2 394 657 A1 66 ribosyltransferase 1); CD298 (ATP I B3, Na K ATPase base pairing which is interrupted by structures (e.g., hair- β3 subunit); CD300a (CMRF-35H); CD300c (CMRF- pin loops) that disrupt base pairing. 35A); CD305 (LAIR1); CD314 (NKG2D); CD316 (EW12); [0247] In some embodiments, a nucleic acid targeting CD317 (BST2); CD319 (CRACC, SLAMF7); CD321 moiety is a Spiegelmer®. In general, Spiegelmers® are (JAM1); CD322 (JAM2); CDw328 (Siglec7); and CD68 5 high-affinity L-enantiomeric oligonucleotide ligands that (gp 110, Macrosialin); wherein the names listed in pa- display high resistance to enzymatic degradation com- rentheses represent alternative names. pared with D-oligonucleotides. In some embodiments, [0244] In some embodiments, T cell targeting can be Spiegelmers® can be designed and utilized just as an accomplished by any targeting moiety that binds, such aptamer would be designed and utilized. as specifically binds, to any entity (e.g., protein, lipid, car- 10 [0248] One of ordinary skill in the art will recognize that bohydrate, small molecule, etc.) that is prominently ex- any nucleic acid that is capable of specifically binding to pressed and/or present on T cells upon activation (i.e., a target, as described herein, can be used in accordance activated T cell targets). Exemplary activated T cell tar- with the present invention. geting moieties include, but are not limited to, CD1a (RA, [0249] Nucleic acids of the present invention (including T6, HTA-1); CD1b (R1); Cd1c (M241,R7); CD1d (R3); 15 nucleic acid targeting moieties and/or functional RNAs CD9 (p24, DRAP-1, MRP-1); CD25 (Tac antigen, IL-2Rα, to be delivered, e.g., RNAi agents, ribozymes, tRNAs, p55); CD30 (Ber-H2, Ki-1); CD39 (ATPdehydrogenase, etc., described in further detail below) may be prepared NTPdehydrogenase-1); CD45RO (UCHL-1); CD49a according to any available technique including, but not (VLA-1α, α1 Integrin); CD49b (VLA-2α, gpla, α2 In- limited to chemical synthesis, enzymatic synthesis, en- tegrin); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 20 zymatic or chemical cleavage of a longer precursor, etc. (Ki-24, CD27 ligand); CD74 (Ii, invariant chain); CD80 Methods of synthesizing RNAs are known in the art (see, (B7, B7-1, BB1); CD86 (B7-2/B70); CD96 (TACTILE); e.g., Gait, M.J. (ed.) Oligonucleotide synthesis: α prac- CD97 (BL-KDD/F12); CD101 (IGSF2, P126, V7); CD103 tical approach, Oxford [Oxfordshire], Washington, DC: (HML-1, Integrin αE, ITGAE); CD107a (LAMP-1); IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide CD107b (LAMP-2); CD109 (8A3, E123 7D1); CD 134 25 synthesis: methods and applications, Methods in molec- (OX40, TNFRSF4); CDw137 (4-1 BB, ILA); CD 146 (Muc ular biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana 18, S-endo, MCAM, Mel-CAM); CD152 (CTLA-4); Press, 2005; both of which are incorporated herein by CD154 (CD40L, gp39, TRAP-1, T-BAM); CD166 (AL- reference). CAM, KG-CAM, SC-1, BEN, DM-GRASP); CD178 (Fas [0250] The nucleic acid that forms the nucleic acid tar- Ligand); CD227 (MUC1, PUM, PEM, EMA); CD253 30 geting moiety may comprise naturally occurring nucleo- (TRAIL, TNF (ligand) superfamily, member 10); CD254 sides, modified nucleosides, naturally occurring nucleo- (TRANCE, RANKL, TNF (ligand) superfamily, member sides with hydrocarbon linkers (e.g., an alkylene) or a 11); CD258 (LIGHT, TMF (ligand) superfamily, member polyether linker (e.g., a PEG linker) inserted between 14); CD267 (TACI, TNF-R superfamily, member 13B); one or more nucleosides, modified nucleosides with hy- CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD278 35 drocarbon or PEG linkers inserted between one or more (ICOS); CD279 (PD1); and CD312 (EMR2); wherein the nucleosides, or a combination of thereof. In some em- names listed in parentheses represent alternative bodiments, nucleotides or modified nucleotides of the nu- names. cleic acid targeting moiety can be replaced with a hydro- [0245] Molecular Characteristics of Targeting Moie- carbon linker or a polyether linker provided that the bind- ties 40 ing affinity and selectivity of the nucleic acid targeting [0246] Nucleic Acid Targeting Moieties. As used here- moiety is not substantially reduced by the substitution in, a "nucleic acid targeting moiety" is a nucleic acid that (e.g., the dissociation constant of the nucleic acid target- binds selectively to a target. In some embodiments, a ing moiety for the target should not be greater than about nucleic acid targeting moiety is a nucleic acid aptamer. 1 3 10-3 M). An aptamer is typically a polynucleotide that binds to a 45 [0251] It will be appreciated by those of ordinary skill specific target structure that is associated with a partic- in the art that nucleic acids in accordance with the present ular organ, tissue, cell, extracellular matrix component, invention may comprise nucleotides entirely of the types and/or subcellular locale. In general, the targeting func- found in naturally occurring nucleic acids, or may instead tion of the aptamer is based on the three-dimensional include one or more nucleotide analogs or have a struc- structure of the aptamer. In some embodiments, binding 50 ture that otherwise differs from that of a naturally occur- of an aptamer to a target is typically mediated by the ring nucleic acid. U.S. Patents 6,403,779; 6,399,754; interaction between the two- and/or three-dimensional 6,225,460; 6,127,533; 6,031,086; 6,005,087; 5,977,089; structures of both the aptamer and the target. In some and references therein disclose a wide variety of specific embodiments, binding of an aptamer to a target is not nucleotide analogs and modifications that may be used. solely based on the primary sequence of the aptamer, 55 See Crooke, S. (ed.) Antisense Drug Technology: Prin- but depends on the three-dimensional structure(s) of the ciples, Strategies, and Applications (1st ed), Marcel aptamer and/or target. In some embodiments, aptamers Dekker; ISBN: 0824705661, 1 st edition (2001); incorpo- bind to their targets via complementary Watson-Crick rated herein by reference; and references therein. For

34 67 EP 2 394 657 A1 68 example, 2’-modifications include halo, alkoxy and ally- affected. The modified region may be at the 5’-end and/or loxy groups. In some embodiments, the 2’-OH group is the 3’-end of one or both strands. For example, modified replaced by a group selected from H, OR, R, halo, SH, aptamers in which approximately 1 to approximately 5 SR1,NH2,NHR,NR2 or CN, wherein R is C1-C6 alkyl, residues at the 5’ and/or 3’ end of either of both strands alkenyl, or alkynyl, and halo is F, Cl, Br or I. Examples 5 are nucleotide analogs and/or have a backbone modifi- of modified linkages include phosphorothioate and 5’-N- cation can be employed. The modification may be a 5’ phosphoramidite linkages. or 3’ terminal modification. A nucleic acid strand may [0252] Nucleic acids comprising a variety of different comprise at least 50% unmodified nucleotides, at least nucleotide analogs, modified backbones, or non-natural- 80% unmodified nucleotides, at least 90% unmodified ly occurring internucleoside linkages can be utilized in 10 nucleotides, or 100% unmodified nucleotides. accordance with the present invention. Nucleic acids of [0254] Nucleic acids in accordance with the present the present invention may include natural nucleosides invention may, for example, comprise a modification to (i.e., adenosine, thymidine, guanosine, cytidine, uridine, a sugar, nucleoside, or internucleoside linkage such as deoxyadenosine, deoxythymidine, deoxyguanosine, those described in U.S. Patent Publications and deoxycytidine) or modified nucleosides. Examples 15 2003/0175950, 2004/0192626, 2004/0092470, of modified nucleotides include base modified nucleo- 2005/0020525, and 2005/0032733. The present inven- sides (e.g., aracytidine, inosine, isoguanosine, nebular- tion encompasses the use of any nucleic acid having any ine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, one or more of the modification described therein. For 2-thiothymidine, 3-deaza-5-azacytidine, 2’-deoxyurid- example, a number of terminal conjugates, e.g., lipids ine, 3-nitorpyrrole, 4-methylindole, 4-thiouridine, 4-thi- 20 such as cholesterol, lithocholic acid, aluric acid, or long othymidine, 2-aminoadenosine, 2-thiothymidine, 2- alkyl branched chains have been reported to improve thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6- cellular uptake. Analogs and modifications may be tested azauridine, 6-chloropurine, 7-deazaadenosine, 7-dea- using, e.g., using any appropriate assay known in the zaguanosine, 8-azaadenosine, 8-azidoadenosine, ben- art, for example, to select those that result in improved zimidazole, M1-methyladenosine, pyrrolo-pyrimidine, 2- 25 delivery of a therapeutic agent, improved specific binding amino-6-chloropurine, 3-methyl adenosine, 5-propynyl- of an aptamer to an aptamer target, etc. In some embod- cytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouri- iments, nucleic acids in accordance with the present in- dine, 5-methylcytidine, 7-deazaadenosine, 7-deazagua- vention may comprise one or more non-natural nucleo- nosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methyl- side linkages. In some embodiments, one or more inter- guanine, and 2-thiocytidine), chemically or biologically 30 nal nucleotides at the 3’-end, 5’-end, or both 3’- and 5’- modified bases (e.g., methylated bases), modified sug- ends of the aptamer are inverted to yield a linkage such ars (e.g., 2’-fluororibose, 2’-aminoribose, 2’-azidoribose, as a 3’-3’ linkage or a 5’-5’ linkage. 2’-O-methylribose, L-enantiomeric nucleosides arab- [0255] In some embodiments, nucleic acids in accord- inose, and hexose), modified phosphate groups (e.g., ance with the present invention are not synthetic, but are phosphorothioates and 5’-N-phosphoramidite linkages), 35 naturally-occurring entities that have been isolated from and combinations thereof. Natural and modified nucle- their natural environments. otide monomers for the chemical synthesis of nucleic [0256] Small Molecule Targeting Moieties. In some acids are readily available. In some cases, nucleic acids embodiments, a targeting moiety in accordance with the comprising such modifications display improved proper- present invention may be a small molecule. In certain ties relative to nucleic acids consisting only of naturally 40 embodiments, small molecules are less than about 2000 occurring nucleotides. In some embodiments, nucleic ac- g/mol in size. In some embodiments, small molecules id modifications described herein are utilized to reduce are less than about 1500 g/mol or less than about 1000 and/or prevent digestion by nucleases (e.g. exonucleas- g/mol. In some embodiments, small molecules are less es, endonucleases, etc.). For example, the structure of than about 800 g/mol or less than about 500 g/mol. a nucleic acid may be stabilized by including nucleotide 45 [0257] In certain embodiments, a small molecule is ol- analogs at the 3’ end of one or both strands order to igomeric. In certain embodiments, a small molecule is reduce digestion. non-oligomeric. In certain embodiments, a small mole- [0253] Modified nucleic acids need not be uniformly cule is a natural product or a natural product-like com- modified along the entire length of the molecule. Different pound having a partial structure (e.g., a substructure) nucleotide modifications and/or backbone structures 50 based on the full structure of a natural product. In certain may exist at various positions in the nucleic acid. One of embodiments, a small molecule is a synthetic product. ordinary skill in the art will appreciate that the nucleotide In some embodiments, a small molecule may be from a analogs or other modification(s) may be located at any chemical library. In some embodiments, a small molecule position(s) of a nucleic acid such that the function of the may be from a pharmaceutical company historical library. nucleic acid is not substantially affected. To give but one 55 In certain embodiments, a small molecule is a drug ap- example, modifications may be located at any position proved by the U.S. Food and Drug Administration as pro- of an aptamer such that the ability of the aptamer to spe- vided in the U.S. Code of Federal Regulations (C.F.R.). cifically bind to the aptamer target is not substantially [0258] One of ordinary skill in the art will appreciate

35 69 EP 2 394 657 A1 70 that any small molecule that specifically binds to a de- duced from a gene encoding the partial antibody se- sired target, as described herein, can be used in accord- quence. Alternatively or additionally, an antibody frag- ance with the present invention. ment may be wholly or partially synthetically produced. [0259] Protein Targeting Moieties. In some embodi- An antibody fragment may optionally comprise a single ments, a targeting moiety in accordance with the present 5 chain antibody fragment. Alternatively or additionally, an invention may be a protein or peptide. In certain embod- antibody fragment may comprise multiple chains which iments, peptides range from about 5 to about 100, from are linked together, for example, by disulfide linkages. about 5 to about 50, from about 10 to about 75, from An antibody fragment may optionally comprise a multi- about 15 to about 50, or from about 20 to about 25 amino molecular complex. A functional antibody fragment will acids in size. In some embodiments, a peptide sequence 10 typically comprise at least about 50 amino acids and can be based on the sequence of a protein. In some more typically will comprise at least about 200 amino embodiments, a peptide sequence can be a random ar- acids. rangement of amino acids. [0266] In some embodiments, antibodies may include [0260] The terms "polypeptide" and "peptide" are used chimeric (e.g. "humanized") and single chain (recom- interchangeably herein, with "peptide" typically referring 15 binant) antibodies. In some embodiments, antibodies to a polypeptide having a length of less than about 100 may have reduced effector functions and/or bispecific amino acids. Polypeptides may contain L-amino acids, molecules. In some embodiments, antibodies may in- D-amino acids, or both and may contain any of a variety clude fragments produced by a Fab expression library. of amino acid modifications or analogs known in the art. [0267] Single-chain Fvs (scFvs) are recombinant an- Useful modifications include, e.g., terminal acetylation, 20 tibody fragments consisting of only the variable light amidation, lipidation, phosphorylation, glycosylation, chain (VL) and variable heavy chain (VH) covalently con- acylation, farnesylation, sulfation, etc. nected to one another by a polypeptide linker. Either VL [0261] . Exemplary proteins that may be used as tar- or VH may comprise the NH2-terminal domain. The geting moieties in accordance with the present invention polypeptide linker may be of variable length and compo- include, but are not limited to, antibodies, receptors, cy- 25 sition so long as the two variable domains are bridged tokines, peptide hormones, glycoproteins, glycopep- without significant steric interference. Typically, linkers tides, proteoglycans, proteins derived from combinatorial primarily comprise stretches of glycine and serine resi- libraries (e.g., Avimers™, Affibodies®, etc.), and charac- dues with some glutamic acid or lysine residues inter- teristic portions thereof. Synthetic binding proteins such spersed for solubility. as Nanobodies™, AdNectins™, etc., can be used. In 30 [0268] Diabodies are dimeric scFvs. Diabodies typi- some embodiments, protein targeting moieties can be cally have shorter peptide linkers than most scFvs, and peptides. they often show a preference for associating as dimers. [0262] One of ordinary skill in the art will appreciate [0269] An Fv fragment is an antibody fragment which that any protein and/or peptide that specifically binds to consists of one VH and one VL domain held together by a desired target, as described herein, can be used in 35 noncovalent interactions. The term "dsFv" as used herein accordance with the present invention. refers to an Fv with an engineered intermolecular di- [0263] In some embodiments, a targeting moiety may sulfide bond to stabilize the VH-VL pair. be an antibody and/or characteristic portion thereof. The [0270] An F(ab’)2 fragment is an antibody fragment term "antibody" refers to any immunoglobulin, whether essentially equivalent to that obtained from immunoglob- natural or wholly or partially synthetically produced and 40 ulins by digestion with an enzyme pepsin at pH 4.0-4.5. to derivatives thereof and characteristic portions thereof. The fragment may be recombinantly produced. An antibody may be monoclonal or polyclonal. An anti- [0271] A Fab’ fragment is an antibody fragment essen- body may be a member of any immunoglobulin class, tially equivalent to that obtained by reduction of the di- including any of the human classes: IgG, IgM, IgA, IgD, sulfide bridge or bridges joining the two heavy chain piec- and IgE. 45 es in the F(ab’)2 fragment. The Fab’ fragment may be [0264] As used herein, an antibody fragment (i.e. char- recombinantly produced. acteristic portion of an antibody) refers to any derivative [0272] A Fab fragment is an antibody fragment essen- of an antibody which is less than full-length. In some em- tially equivalent to that obtained by digestion of immu- bodiments, an antibody fragment retains at least a sig- noglobulins with an enzyme (e.g., papain). The Fab frag- nificant portion of the full-length antibody’s specific bind- 50 ment may be recombinantly produced. The heavy chain ing ability. Examples of such antibody fragments include, segment of the Fab fragment is the Fd piece. but are not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv [0273] Carbohydrate Targeting Moieties. In some em- diabody, and Fd fragments. Antibody fragments also in- bodiments, a targeting moiety in accordance with the clude, but are not limited, to Fc fragments. present invention may comprise a carbohydrate. In some [0265] An antibody fragment may be produced by any 55 embodiments, a carbohydrate may be a polysaccharide means. For example, an antibody fragment may be en- comprising simple sugars (or their derivatives) connect- zymatically or chemically produced by fragmentation of ed by glycosidic bonds, as known in the art. Such sugars an intact antibody and/or it may be recombinantly pro- may include, but are not limited to, glucose, fructose,

36 71 EP 2 394 657 A1 72 galactose, ribose, lactose, sucrose, maltose, trehalose, tify, and/or isolate novel targeting moieties. For example, cellbiose, mannose, xylose, arabinose, glucoronic acid, standard techniques utilizing libraries of molecules and galactoronic acid, mannuronic acid, glucosamine, gala- in vitro binding assays can be utilized to identify novel tosamine, and neuramic acid. In some embodiments, a targeting moieties. carbohydrate may be one or more of pullulan, cellulose, 5 [0281] Nucleic acid targeting moieties (e.g. aptamers, microcrystalline cellulose, hydroxypropyl methylcellu- Spiegelmers®) may be designed and/or identified using lose, hydroxycellulose, methylcellulose, dextran, cyclo- any available method. In some embodiments, nucleic ac- dextran, glycogen, starch, hydroxyethylstarch, cara- id targeting moieties are designed and/or identified by geenan, glycon, amylose, chitosan, N,O-carboxylmeth- identifying nucleic acid targeting moieties from a candi- ylchitosan, algin and alginic acid, starch, chitin, heparin, 10 date mixture of nucleic acids. Systemic Evolution of Lig- konjac, glucommannan, pustulan, heparin, hyaluronic ands by Exponential Enrichment (SELEX), or a variation acid, curdlan, and xanthan. thereof, is a commonly used method of identifying nucleic [0274] In some embodiments, the carbohydrate may acid targeting moieties that bind to a target from a can- be aminated, carboxylated, and/or sulfated. In some em- didate mixture of nucleic acids (see, e.g., U.S. Patents bodiments, hydrophilic polysaccharides can be modified 15 6,482,594; 6,458,543; 6,458,539; 6,376,190; 6,344,318; to become hydrophobic by introducing a large number 6,242,246; 6,184,364; 6,001,577; 5,958,691; 5,874,218; of side-chain hydrophobic groups. In some embodi- 5,853,984; 5,843,732; 5,843,653; 5,817,785; 5,789,163; ments, a hydrophobic carbohydrate may include cellu- 5,763,177; 5,696,249; 5,660,985; 5,595,877; 5,567,588; lose acetate, pullulan acetate, konjac acetate, amylose and 5,270,163; each of which is incorporated herein by acetate, and dextran acetate. 20 reference). Alternatively or additionally, Polyplex In Vivo [0275] One of ordinary skill in the art will appreciate Combinatorial Optimization (PICO) is a method that can that any carbohydrate that specifically binds to a desired be used to identify nucleic acid targeting moieties (e.g. target, as described herein, can be used in accordance aptamers) that bind to a target from a candidate mixture with the present invention. of nucleic acids in vivo and/or in vitro and is described [0276] Lipid Targeting Moieties. In some embodi- 25 in co-pending PCT Application US06/47975, entitled ments, a targeting moiety in accordance with the present "System for Screening Particles," filed December 15, invention may comprise one or more fatty acid groups or 2006, which is incorporated herein by reference. salts thereof. In some embodiments, a fatty acid group may comprise digestible, long chain (e.g., C8-C50), sub- Immunostimulatory Agents stituted or unsubstituted hydrocarbons. In some embod- 30 iments, a fatty acid group may be a C10-C20 fatty acid or [0282] In some embodiments, nanocarriers may trans- salt thereof. In some embodiments, a fatty acid group port one or more immunostimulatory agents which can may be a C15-C20 fatty acid or salt thereof. In some em- help stimulate immune responses. In some embodi- bodiments, a fatty acid group may be a C15-C25 fatty acid ments, immunostimulatory agents boost immune re- or salt thereof. In some embodiments, a fatty acid group 35 sponses by activating APCs to enhance their immunos- may be unsaturated. In some embodiments, a fatty acid timulatory capacity. In some embodiments, immunostim- group may be monounsaturated. In some embodiments, ulatory agents boost immune responses by amplifying a fatty acid group may be polyunsaturated. In some em- lymphocyte responses to specific antigens. In some em- bodiments, a double bond of an unsaturated fatty acid bodiments, immunostimulatory agents boost immune re- group may be in the cis conformation. In some embodi- 40 sponses by inducing the local release of mediators, such ments, a double bond of an unsaturated fatty acid may as cytokines from a variety of cell types. In some embod- be in the trans conformation. iments, the immunostimulatory agents suppress or redi- [0277] In some embodiments, a fatty acid group may rect an immune response. In some embodiments, the be one or more of butyric, caproic, caprylic, capric, lauric, immunostimulatory agents induce regulatory T cells. myristic, palmitic, stearic, arachidic, behenic, or lignocer- 45 [0283] In some embodiments, all of the immunostim- ic acid. In some embodiments, a fatty acid group may be ulatory agents of a vaccine nanocarrier are identical to one or more of palmitoleic, oleic, vaccenic, linoleic, alpha- one another. In some embodiments, a vaccine nanocar- linoleic, gamma-linoleic, arachidonic, gadoleic, arachi- rier comprises a number of different types of immunos- donic, eicosapentaenoic, docosahexaenoic, or erucic timulatory agents. In some embodiments, a vaccine na- acid. 50 nocarrier comprises multiple individual immunostimula- [0278] One of ordinary skill in the art will appreciate tory agents, all of which are identical to one another. In that any fatty acid group that specifically binds to a de- some embodiments, a vaccine nanocarrier comprises sired target, as described herein, can be used in accord- exactly one type of immunostimulatory agent. In some ance with the present invention. embodiments, a vaccine nanocarrier comprises exactly [0279] Novel Targeting Moieties 55 two distinct types of immunostimulatory agents. In some [0280] Any novel targeting moiety can be utilized in the embodiments, a vaccine nanocarrier comprises greater nanocarriers in accordance with the present invention. than two distinct types of immunostimulatory agents. Any method known in the art can be used to design, iden- [0284] In some embodiments, a vaccine nanocarrier

37 73 EP 2 394 657 A1 74 comprises a single type of immunostimulatory agent that iments, a first type of immunostimulatory agent and a stimulates both B cells and T cells. In some embodi- second type of immunostimulatory agent may be in the ments, a vaccine nanocarrier comprises two types of im- same locale of a vaccine nanocarrier (e.g., they may both munostimulatory agents, wherein first type of immunos- be associated with the exterior surface of a vaccine na- timulatory agent stimulates B cells, and the second type 5 nocarrier; they may both be encapsulated within the vac- of immunostimulatory agent stimulates T cells. In some cine nanocarrier; etc.). One of ordinary skill in the art will embodiments, a vaccine nanocarrier comprises greater recognize that the preceding examples are only repre- than two types of immunostimulatory agents, wherein sentative of the many different ways in which multiple one or more types of immunostimulatory agents stimu- immunostimulatory agents may be associated with dif- late B cells, and one or more types of immunostimulatory 10 ferent locales of vaccine nanocarriers. Multiple immu- agents stimulate T cells. nostimulatory agents may be located at any combination [0285] In some embodiments, a vaccine nanocarrier of locales of vaccine nanocarriers. comprises at least one type of immunostimulatory agent [0287] In certain embodiments, immunostimutatory that is associated with the exterior surface of the vaccine agents may be interleukins, interferon, cytokines, etc. In nanocarrier. In some embodiments, the association is 15 specific embodiments, an immunostimulatory agent may covalent. In some embodiments, the covalent associa- be a natural or synthetic agonist for a Toll-like receptor tion is mediated by one or more linkers. In some embod- (TLR). In specific embodiments, vaccine nanocarriers in- iments, the association is non-covalent. In some embod- corporate a ligand for toll-like receptor (TLR)-7, such as iments, the non-covalent association is mediated by CpGs, which induce type I interferon production. In spe- charge interactions, affinity interactions, metal coordina- 20 cific embodiments, an immunostimulatory agent may be tion, physical adsorption, host-guest interactions, hydro- an agonist for the DC surface molecule CD40. In certain phobic interactions, TT stacking interactions, hydrogen embodiments, to stimulate immunity rather than toler- bonding interactions, van der Waals interactions, mag- ance, a nanocarrier incorporates an immunostimulatory netic interactions, electrostatic interactions, dipole-di- agent that promotes DC maturation (needed for priming pole interactions, and/or combinations thereof. Associa- 25 of naive T cells) and the production of cytokines, such tion of immunostimulatory agents with vaccine nanocar- as type I interferons, which promote antibody responses riers is described in further detail below, in the section and anti-viral immunity. In some embodiments, an im- entitled "Production of Vaccine Nanocarriers." munomodulatory agent may be a TLR-4 agonist, such [0286] In some embodiments, a vaccine nanocarrier as bacterial lipopolysacharide (LPS), VSV-G, and/or HM- comprises a lipid membrane (e.g., lipid bilayer, lipid mon- 30 GB-1. In some embodiments, immunomodulatory agents olayer, etc.), wherein at least one type of immunostimu- are cytokines, which are small proteins or biological fac- latory agent is associated with the lipid membrane. In tors (in the range of 5 kD  20 kD) that are released by some embodiments, at least one type of immunostimu- cells and have specific effects on cell-cell interaction, latory agent is embedded within the lipid membrane. In communication and behavior of other cells. In some em- some embodiments, at least one type of immunostimu- 35 bodiments, immunostimulatory agents may be proin- latory agent is embedded within the lumen of a lipid bi- flammatory stimuli released from necrotic cells (e.g., layer. In some embodiments, a vaccine nanocarrier com- urate crystals). In some embodiments, immunostimula- prises at least one type of immunostimulatory agent that tory agents may be activated components of the com- is associated with the interior surface of the lipid mem- plement cascade (e.g., CD21, CD35, etc.). In some em- brane. In some embodiments, at least one type of immu- 40 bodiments, immunostimulatory agents may be activated nostimulatory agent is encapsulated with the lipid mem- components of immune complexes. The immunostimu- brane of a vaccine nanocarrier. In some embodiments, latory agents include TLR-1, TLR-2, TLR-3, TLR-4, TLR- at least one type of immunostimulatory agent may be 5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR-10 agonists. located at multiple locations of a vaccine nanocarrier. For The immunostimulatory agents also include complement example, a first type of immunostimulatory agent may be 45 receptor agonists, such as a molecule that binds to CD21 embedded within a lipid membrane, and a second type or CD35. In some embodiments, the complement recep- of immunostimulatory agent may be encapsulated within tor agonist induces endogenous complement opsoniza- the lipid membrane of a vaccine nanocarrier. To give an- tion of the nanocarrier. Immunostimulatory agents also other example, a first type of immunostimulatory agent include cytokine receptor agonists, such as a cytokine. may be associated with the exterior surface of a lipid 50 In some embodiments, the cytokine receptor agonist is membrane, and a second type of immunostimulatory a small molecule, antibody, fusion protein, or aptamer. agent may be associated with the interior surface of the [0288] In some embodiments, there are more than one lipid membrane of a vaccine nanocarrier. In some em- type of immunostimulatory agent. In some embodiments, bodiments, a first type of immunostimulatory agent may the different immunostimulatory agents each act on a be embedded within the lumen of a lipid bilayer of a vac- 55 different pathway. The immunostimulatory agents, there- cine nanocarrier, and the lipid bilayer may encapsulate fore, can be different Toll-like receptors, a Toll-like re- a polymeric matrix throughout which a second type of ceptor and CD40, a Toll-like receptor and a component immunostimulatory agent is distributed. In some embod- of the inflammasome, etc.

38 75 EP 2 394 657 A1 76

[0289] In some embodiments, the present invention out at varying densities, along with antigen or mitogen, provides pharmaceutical compositions comprising vac- and then placed in a humidified 37˚C CO2 incubator for cine nanocarriers formulated with one or more adjuvants. a specified period of time. Cytokine secreted by activated The term "adjuvant", as used herein, refers to an agent cells is captured locally by the coated antibody on the that does not constitute a specific antigen, but boosts the 5 high surface area PVDF membrane. After washing the immune response to the administered antigen. wells to remove cells, debris, and media components, a [0290] In some embodiments, vaccine nanocarriers secondary antibody (e.g., a biotinylated polyclonal anti- are formulated with one or more adjuvants such as gel- body) specific for the cytokine is added to the wells. This type adjuvants (e.g., aluminum hydroxide, aluminum antibody is reactive with a distinct epitope of the target phosphate, calcium phosphate, etc.), microbial adju- 10 cytokine and thus is employed to detect the captured vants (e.g., immunomodulatory DNA sequences that in- cytokine. Following a wash to remove any unbound bi- clude CpG motifs; endotoxins such as monophosphoryl otinylated antibody, the detected cytokine is then visual- lipid A; exotoxins such as cholera toxin, E. coli heat labile ized using an avidin-HRP, and a precipitating substrate toxin, and pertussis toxin; muramyl dipeptide, etc.); oil- (e.g., AEC, BCIP/NBT). The colored end product (a spot, emulsion and emulsifier-based adjuvants (e.g., Freund’s 15 usually a blackish blue) typically represents an individual Adjuvant, MF59 [Novartis], SAF, etc.); particulate adju- cytokine-producing cell. Spots can be counted manually vants (e.g., liposomes, biodegradable microspheres, sa- (e.g., with a dissecting microscope) or using an automat- ponins, etc.); synthetic adjuvants (e.g., nonionic block ed reader to capture the microwell images and to analyze copolymers, muramyl peptide analogues, polyphosp- spot number and size. In some embodiments, each spot hazene, synthetic polynucleotides, etc.), and/or combi- 20 correlates to a single cytokine-producing cell. nations thereof. Other exemplary adjuvants include [0293] In some embodiments, an immune response in some polymers (e.g., polyphosphazenes, described in T cells is said to be stimulated if between about 1% and U.S. Patent 5,500,161, which is incorporated herein by about 100% of antigen-specific T cells produce cy- reference), QS21, squalene, tetrachlorodecaoxide, etc. tokines. In some embodiments, an immune response in 25 T cells is said to be stimulated if at least about 1%, at Assays for T Cell Activation least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about [0291] In some embodiments, various assays can be 90%, at least about 95%, at least about 99%, or about utilized in order to determine whether an immune re- 100% of antigen-specific T cells produce cytokines. sponse has been stimulated in a T cell or group of T cells 30 [0294] In some embodiments, an immune response in (i.e., whether a T cell or group of T cells has become T cells is said to be stimulated if immunized subjects "activated"). In some embodiments, stimulation of an im- comprise at least about 10-fold, at least about 50-fold, mune response in T cells can be determined by meas- at least about 100-fold, at least about 500-fold, at least uring antigen-induced production of cytokines by T cells. about 1000-fold, at least about 5000-fold, at least about In some embodiments, stimulation of an immune re- 35 10,000-fold, at least about 50,000-fold, at least about sponse in T cells can be determined by measuring anti- 100,000-fold, or greater than at least about 100,000-fold gen-induced production of IFNγ, IL-4, IL-2, IL-10, IL-I7 more cytokine-producing cells than do naive controls. and/or TNFα by T cells. In some embodiments, antigen- [0295] In some embodiments, stimulation of an im- produced production of cytokines by T cells can be meas- mune response in T cells can be determined by meas- ured by intracellular cytokine staining followed by flow 40 uring antigen-induced proliferation of T cells. In some cytometry. In some embodiments, antigen-induced pro- embodiments, antigen-induced proliferation may be duction of cytokines by T cells can be measured by sur- measured as uptake of H3-thymidine in dividing T cells face capture staining followed by flow cytometry. In some (sometimes referred to as "lymphocyte transformation embodiments, antigen-induced production of cytokines test, or "LTT"). In some embodiments, antigen-induced by T cells can be measured by determining cytokine con- 45 proliferation is said to have occurred if H3-thymidine up- centration in supernatants of activated T cell cultures. In take (given as number of counts from a γ counter) is at some embodiments, this can be measured by ELISA. least about 5-fold, at least about 10-fold, at least about [0292] In some embodiments, antigen-produced pro- 20-fold, at least about 50-fold, at least about 100-fold, at duction of cytokines by T cells can be measured by ELIS- least about 500-fold, at least about 1000-fold, at least POT assay. In general, ELISPOT assays employ a tech- 50 about 5000-fold, at least about 10,000-fold, or greater nique very similar to the sandwich enzyme-linked immu- than at least about 10,000-fold higher than a naive con- nosorbent assay (ELISA) technique. An antibody (e.g. trol. monoclonal antibody, polyclonal antibody, etc.) is coated [0296] In some embodiments, antigen-induced prolif- aseptically onto a PVDF (polyvinylidene fluoride) eration may be measured by flow cytometry. In some -backed microplate. Antibodies are chosen for their spe- 55 embodiments, antigen-induced proliferation may be cificity for the cytokine in question. The plate is blocked measured by a carboxyfluorescein succinimidyl ester (e.g. with a serum protein that is non-reactive with any (CFSE) dilution assay. CFSE is a non-toxic, fluorescent, of the antibodies in the assay). Cells of interest are plated membrane-permeating dye that binds the amino groups

39 77 EP 2 394 657 A1 78 of cytoplasmic proteins with its succinimidyl-reactive mechanisms. As described above, T cells recognize an- group (e.g. T cell proteins). When cells divide, CFSE- tigen in a processed form (e.g., as a peptide fragment labeled proteins are equally distributed between the presented by an APC’s MHC molecule to the T cell re- daughter cells, thus halving cell fluorescence with each ceptor). B cells recognize antigens in their native form. division. Consequently, antigen-specific T cells lose their 5 B cells recognize free (e.g., soluble) antigen in blood or fluorescence after culture in the presence of the respec- lymph using B cell receptors (BCRs) and/or membrane tive antigen (CFSElow) and are distinguishable from other bound-immunoglobulins. cells in culture (CFSEhigh). In some embodiments, anti- [0302] The immunomodulatory agent can be a B cell gen-induced proliferation is said to have occurred if antigen. B cell antigens include, but are not limited to CFSE dilution (given as the percentage of CFSElow cells 10 proteins, peptides, small molecules, and carbohydrates. out of all CFSE+ cells) is at least about 5%, at least about In some embodiments, the B cell antigen is a non-protein 10%, at least about 25%, at least about 50%, at least antigen (i.e., not a protein or peptide antigen). In some about 75%, at least about 90%, at least about 95%, or embodiments, the B cell antigen is a carbohydrate asso- at least about 100%. ciated with an infectious agent. In some embodiments, [0297] In some embodiments, an immune response in 15 the B cell antigen is a glycoprotein or glycopeptide as- T cells is said to be stimulated if cellular markers of T cell sociated with an infectious agent. The infectious agent activation are expressed at different levels (e.g. higher can be a bacterium, virus, fungus, protozoan, or parasite. or lower levels) relative to unstimulated cells. In some In some embodiments, the B cell antigen is a poorly im- embodiments, CD11a CD27, CD25, CD40L, CD44, munogenic antigen. In some embodiments, the B cell CD45RO, and/or CD69 are more highly expressed in ac- 20 antigen is an abused substance or a portion thereof. In tivated T cells than in unstimulated T cells. In some em- some embodiments, the B cell antigen is an addictive bodiments, L-selectin (CD62L), CD45RA, and/or CCR7 substance or a portion thereof. Addictive substances in- are less highly expressed in activated T cells than in un- clude, but are not limited to, nicotine, a narcotic, a cough stimulated T cells. suppressant, a tranquilizer, and a sedative. Examples of [0298] In some embodiments, an immune response in 25 addictive substances include those provided elsewhere T cells is measured by assaying cytotoxicity by effector herein. CD8+ T cells against antigen-pulsed target cells. For ex- [0303] In some embodiments, the B cell antigen is a ample, a 51chromium (51Cr) release assay can be per- toxin, such as a toxin from a chemical weapon. In some formed. In this assay, effector CD8+ T cells bind infected embodiments, the toxin from a chemical weapon is bot- cells presenting virus peptide on class I MHC and signal 30 ulinum toxin or phosphene. Toxins from a chemical the infected cells to undergo apoptosis. If the cells are weapon include, but are not limited to, O-Alkyl (

40 79 EP 2 394 657 A1 80 gen chloride, Hydrogen cyanide and Chloropicrin: Targeting Moieties Trichloronitromethane. [0304] The B cell antigen may also be a hazardous [0309] As discussed above, inventive nanocarriers environmental agent. Hazardous environmental agents comprise one or more targeting moieties. For a discus- include, but are not limited to, arsenic, lead, mercury, 5 sion of general and specific properties of targeting moi- vinyl chloride, polychlorinated biphenyls, benzene, poly- eties in accordance with the present invention, see the cyclic aromatic hydrocarbons, cadmium, benzo(a) subheading entitled "Targeting Moieties" in the section pyrene, benzo(b)fluoranthene, chloroform, DDT, P,P’-, above entitled "T Cells." In some embodiments, targeting aroclor 1254, aroclor 1260, dibenzo(a,h)anthracene, moieties target particular cell types. In certain embodi- trichloroethylene, dieldrin, chromium hexavalent, and 10 ments, a target is a B cell marker. In some embodiments, DDE, P,P’. Examples of such agents include those pro- a B cell target is an antigen that is expressed in B cells vided elsewhere herein. but not in non-B cells. In some embodiments, a B cell [0305] In some embodiments, the B cell antigen is a target is an antigen that is more prevalent in B cells than self antigen. In other embodiments, the B cell antigen is in non-B cells. an alloantigen, an allergen, a contact sensitizer, a de- 15 [0310] In certain embodiments, a target is a SCS-Mph generative disease antigen, a hapten, an infectious dis- marker. In some embodiments, an SCS-Mph target is an ease antigen, a cancer antigen, an atopic disease anti- antigen that is expressed in SCS-Mph but not in non- gen, an autoimmune disease antigen, an addictive sub- SCS-Mph. In some embodiments, an SCS-Mph target is stance, a xenoantigen, or a metabolic disease enzyme an antigen that is more prevalent in SCS-Mph than in or enzymatic product thereof. Examples of such antigens 20 non-SCS-Mph. Exemplary SCS-Mph markers are listed include those provided elsewhere herein. below in the section entitled "Subcapsular Sinus Macro- [0306] As described above, the present invention pro- phage Cells" and include those provided elsewhere here- vides vaccine nanocarriers comprising, for example, one in. or more immunomodulatory agents. In some embodi- [0311] In certain embodiments, a target is a FDC mark- ments, inventive nanocarriers comprising one or more 25 er. In some embodiments, an FDC target is an antigen immunomodulatory agents are used as vaccines. In that is expressed in FDCs but not in non-FDCs. In some some embodiments, antigen presentation to B cells can embodiments, an FDC target is an antigen that is more be optimized by presenting structurally intact immu- prevalent in FDCs than in non-FDCs. Exemplary FDC nomodulatory agents on the surface of nanocarriers. In markers are listed below in the section entitled "Follicular some embodiments, structurally intact immunomodula- 30 Dendritic Cells" and include those provided elsewhere tory agents are presented on the surface of vaccine na- herein. nocarriers at high copy number and/or density. [0312] In some embodiments, a target is preferentially [0307] In some embodiments, an immunomodulatory expressed in particular cell types. For example, expres- agent may comprise isolated and/or recombinant pro- sion of an SCS-Mph, FDC, and/or B cell target in SCS- teins or peptides, inactivated organisms and viruses, 35 Mph, FDCs, and/or B cells is at least 2-fold, at least 3- dead organisms and virus, genetically altered organisms fold, at least 4-fold, at least 5-fold, at least 10-fold, at or viruses, and cell extracts. In some embodiments, an least 20-fold, at least 50-fold, at least 100-fold, at least immunomodulatory agent may comprise nucleic acids, 500-fold, or at least 1000-fold overexpressed in SCS- carbohydrates, lipids, and/or small molecules. In some Mph, FDCs, and/or B cells relative to a reference popu- embodiments, an immunomodulatory agent is one that 40 lation. In some embodiments, a reference population elicits an immune response. In some embodiments, an may comprise non-SCS-Mph, FDCs, and/or B cells. immunomodulatory agent is an antigen. In some embod- [0313] In some embodiments, expression of an SCS- iments, an immunomodulatory agent is used for vac- Mph, FDC, and/or B cell target in activated SCS-Mph, cines. Further description of immunomodulatory agents FDCs, and/or B cells is at least 2-fold, at least 3-fold, at can be found in the section above entitled "B Cells." 45 least 4-fold, at least 5-fold, at least 10-fold, at least 20- [0308] As discussed above, a vaccine nanocarrier may fold, at least 50-fold, at least 100-fold, at least 500-fold, comprise a single type of immunomodulatory agent that or at least 1000-fold overexpressed in activated SCS- stimulates both B cells and T cells. In some embodi- Mph, FDCs, and/or B cells relative to a reference popu- ments, a vaccine nanocarrier comprises two types of im- lation. In some embodiments, a reference population munomodulatory agents, wherein first type of immu- 50 may comprise non-activated SCS-Mph, FDCs, and/or B nomodulatory agent stimulates B cells, and the second cells. type of immunomodulatory agent stimulates T cells. In [0314] Subcapsular Sinus Macrophage Cells some embodiments, a vaccine nanocarrier comprises [0315] The present invention encompasses the recog- greater than two types of immunomodulatory agents, nition that targeting of antigens to subcapsular sinus wherein one or more types of immunomodulatory agents 55 macrophages (SCS-Mph) is involved in efficient early stimulate B cells, and one or more types of immunomod- presentation of lymph-borne pathogens, such as viruses, ulatory agents stimulate T cells. to follicular B cells (Figure 2). As described in Example 1, following subcutaneous injection of vesicular stomati-

41 81 EP 2 394 657 A1 82 tis virus (VSV) or adenovirus (AdV) into the footpad of CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophillin); mice, viral particles were efficiently and selectively re- CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1, tained by CD169+ SCS-Mph in the draining popliteal OX-45); CD49a (VLA-1α, α1 Integrin); CD49b (VLA-2α, lymph nodes. VSV-specific B cell receptor (BCR) trans- gpla, α2 Integrin); CD49c (VLA-3α, α3 Integrin); CD49e genic B cells in these lymph nodes were rapidly activated 5 (VLA-5α, α5 Integrin); CD49f (VLA-6α, α6 Integrin, gplc); and generated extremely high antibody titers upon this CD50 (ICAM-3); CD51 (Integrin α, VNR-α, Vitronectin- viral challenge. Depletion of SCS-Mph by injection of li- Rα); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54 posomes laden with clodronate (which is toxic for Mph) (ICAM-1); CD55 (DAF); CD58 (LFA-3); CD59 (lF5Ag, abolished early B cell activation, indicating that SCS-Mph H19, Protectin, MACIF, MIRL, P-18); CD60a (GD3); are essential for the presentation of lymph-borne partic- 10 CD60b (9-O-acetyl GD3); CD61 (GP IIIa, β3 Integrin); ulate antigens to B cells. CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, [0316] B cells are more potently activated by polyva- TQ1); CD63 (LIMP, MLA1, gp55, NGA, LAMP-3, lent antigens that are presented to them on a fixed sur- ME491); CD64 (FcγRI); CD65 (Ceramide, VIM-2); face, rather than in solution. While not wishing to be CD65s (Sialylated-CD65, VIM2); CD72 (Ly-19.2, Ly- bound by any one theory, the present invention suggests 15 32.2, Lyb-2); CD74 (Ii, invariant chain); CD75 (sialo- a reason why many enveloped viruses (such as VSV) masked Lactosamine); CD75S (α2,6 sialylated Lactos- elicit potent neutralizing antibody responses to their en- amine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 velope glycoprotein: the antigenic protein is presented (4F9, C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a at a very high density on the surface of the viral particles, (ILT5, LIR2, HL9); CD85d (ILT4, LIR2, MIR10); CD85j and the viral particles are presented to B cells in a rela- 20 (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86 tively immotile manner, i.e., bound to the plasma mem- (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc brane of SCS-Mph. The present invention encompasses receptor, FcαR); CD91 (α2M-R, LRP); CDw92 (p70); the recognition that vaccine carriers that mimic viral par- CDw93 (GR11); CD95 (APO-1, FAS, TNFRSF6); CD97 ticles by targeting SCS-Mph upon subcutaneous injec- (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99 tion and presenting polyvalent conformationally intact an- 25 (MIC2, E2); CD99R (CD99 Mab restricted); CD100 tigens on their surface can stimulate a potent B cell re- (SEMA4D); CD101 (IGSF2, P126, V7); CD102 (ICAM- sponse. 2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); [0317] In some embodiments, SCS-Mph targeting is CD112 (HveB, PRR2, PVRL2, Nectin2); CD114 (CSF3R, accomplished by moieties that bind CD 169 (i.e., siaload- G-CSRF, HG-CSFR); CD115 (c-fms, CSF-1R, M-CS- hesin), CD 11b (i. e., CD 11b/CD 18, Mac-1, CR3 or 30 FR); CD116 (GM-CSFRα); CDw119 (IFNγR, IFNγRA); αMβ2 integrin), and/or the mannose receptor (i.e., a mul- CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFR ti-valent lectin), proteins which are all prominently ex- p80); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); CD 123 pressed on SCS-Mph. Examples of such moieties in- (IL-3Rα); CD 124 (IL-4Rα); CD 127 (p90, IL-7R, IL-7Rα); clude those provided elsewhere herein. CD128a (IL-8Ra, CXCR1, (Tentatively renamed as [0318] In some embodiments, SCS-Mph targeting can 35 CD181)); CD128b (IL-8Rb, CSCR2, (Tentatively re- be accomplished by any targeting moiety that specifically named as CD182)); CD130 (gp130); CD131 (Common binds to any entity (e.g., protein, lipid, carbohydrate, β subunit); CD132 (Common γ chain, IL-2Rγ); CDw136 small molecule, etc.) that is prominently expressed (MSP-R, RON, p158-ron); CDw137 (4-1BB, ILA); and/or present on macrophages (i.e., SCS-Mph mark- CD139; CD141 (Thrombomodulin, Fetomodulin); CD147 ers). Exemplary SCS-Mph markers include, but are not 40 (Basigin, EMMPRIN, M6, OX47); CD148 (HPTP-η, p260, limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, DEP-1); CD155 (PVR); CD156a (CD156, ADAM8, MS2); MRP-1); CD11a (LFA-1α, α L Integrin chain); CD 11b CD156b (TACE, ADAM17, cSVP); CDw156C (αM Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c (αX (ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); Integrin, p150, 95, AXb2); CDwl2 (p90-120); CD13 (APN, CD164 (MGC-24, MUC-24); CD165 (AD2, gp37); CD168 gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, 45 (RHAMM, IHABP, HMMR); CD 169 (Sialoadhesin, Si- Lewis, X, SSEA-1, 3-FAL); CD15s (Sialyl Lewis X); glec-1); CD 170 (Siglec 5); CD 171 (L1CAM, NILE); CD15u (3’ sulpho Lewis X); CD15su (6 sulpho-sialyl CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb); CDw17 (RP105, Bgp95, Ly64); CD181 (CXCR1, (Formerly (Lactosylceramide, LacCer); CD 18 (Integrin β2, CD11a, known as CD128a)); CD182 (CXCR2, (Formerly known b,c β-subunit); CD26 (DPP IV ectoeneyme, ADA binding 50 as CD128b)); CD184 (CXCR4, NPY3 R); CD191 protein); CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (CCR1); CD192 (CCR2); CD 195 (CCR5); CDw197 (PECAM-1, Endocam); CD32 (FCγRII); CD33 (gp67); (CCR7 (was CDw197)); CDw198 (CCR8); CD204 CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV, (MSR); CD205 (DEC-25); CD206 (MMR); CD207 (Lang- PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, erin); CDw210 (CK); CD213a (CK); CDw217 (CK); T10); CD39 (ATPdehydrogenase, NTPdehydrogenase- 55 CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, 1); CD40 (Bp50); CD43 (Sialophorin, Leukosialin); CD44 IGFII-R); CD224 (GGT); CD226 (DNAM-1, PTA1); (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, CD230 (Prion Protein (PrP)); CD232 (VESP-R); CD244 Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); (2B4, P38, NAIL); CD245 (p220/240); CD256 (APRIL,

42 83 EP 2 394 657 A1 84

TALL2, TNF (ligand) superfamily, member 13); CD257 binds to any entity (e.g., protein, lipid, carbohydrate, (BLYS, TALL1, TNF (ligand) superfamily, member 13b); small molecule, etc.) that is prominently expressed CD261 (TRAIL-R1, TNF-R superfamily, member 10a); and/or present on B cells (i.e., B cell marker). Exemplary CD262 (TRAIL-R2, TNF-R superfamily, member 10b); B cell markers include, but are not limited to, CD1c CD263 (TRAIL-R3, TNBF-R superfamily, member 10c); 5 (M241, R7); CD1d (R3); CD2 (E-rosette R, T11, LFA-2); CD264 (TRAIL-R4, TNF-R superfamily, member 10d); CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9 (p24, CD265 (TRANCE-R, TNF-R superfamily, member 11a); DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD277 (BT3.1, B7 family: Butyrophilin 3); CD280 CD11b (αM Integrin chain, CR3, Mo1, C3niR, Mac-1); (TEM22, ENDO180); CD281 (TLR1, TOLL-like receptor CD11c (αX Integrin, P150, 95, AXb2); CDw17 (Lactos- 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4, 10 ylceramide, LacCer); CD18 (Integrin β2, CD11a, b, c β- TOLL-like receptor 4); CD295 (LEPR); CD298 (ATP1B3, subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, Na K ATPase, β3 subunit); CD300a (CMRF-35H); EBV-R, C3dR); CD22 (BL-CAM, Lyb8, Siglec-2); CD23 CD300c (CMRF-35A); CD300e (CMRF-35L1); CD302 (FceRII, B6, BLAST-2, Leu-20); CD24 (BBA-1, HSA); (DCL1); CD305 (LAIR1); CD312 (EMR2); CD315 CD25 (Tac antigen, IL-2Rα, p55); CD26 (DPP IV ec- (CD9P1); CD317 (BST2); CD321 (JAM1); CD322 15 toeneyme, ADA binding protein); CD27 (T14, S152); (JAM2); CDw328 (Siglec7); CDw329 (Siglec9); CD68 CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM- (gp 110, Macrosialin); and/or mannose receptor; wherein 1, Endocam); CD32 (FCγRII); CD35 (CR1, C3b/C4b re- the names listed in parentheses represent alternative ceptor); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, names. Examples of such markers include those provid- T10); CD39 (ATPdehydrogenase, NTPdehydrogenase- ed elsewhere herein. 20 1); CD40 (Bp50); CD44 (ECMRII, H-CAM, Pgp-1); CD45 [0319] In some embodiments, SCS-Mph targeting can (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; be accomplished by any targeting moiety that specifically CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, binds to any entity (e.g., protein, lipid, carbohydrate, OA3, Neurophilin); CD47R (MEM-133); CD48 (Blast-1, small molecule, etc.) that is prominently expressed Hulym3, BCM-1, OX-45); CD49b (VLA-2α, gpla, α2 In- and/or present on macrophages upon activation (i.e., ac- 25 tegrin); CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, tivated SCS-Mph marker). Exemplary activated SCS- α4 Integrin); CD50 (ICAM-3); CD52 (CAMPATH-1, Mph markers include, but are not limited to, CD1a (R4, HES); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD44R CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM- (CD44v, CD44v9); CD49d (VLA-4α, α4 Integrin); CD69 1, LECAM-1, MEL-14, Leu8, TQ1); CD72 (Ly-19.2, Ly- (AIM, EA 1, MLR3, gp34/28, VEA); CD105 (Endoglin); 30 32.2, Lyb-2); CD73 (Ecto-5’-nuciotidase); CD74 (Ii, in- CD142 (Tissue factor, Thromboplastin, F3); CD 143 variant chain); CD75 (sialo-masked Lactosamine); (ACE, Peptidyl dipeptidase A, Kininase II); CD153 CD75S (α2, 6 sialytated Lactosamine); CD77 (Pk anti- (CD30L, TNSF8); CD163 (M130, GHI/61, RM3/1); gen, BLA, CTH/Gb3); CD79a (Igα, MB1); CD79b (Igβ, CD166 (ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); B29); CD80; CD81 (TAPA-1); CD82 (4F9, C33, IA4, CD227 (MUC1, PUM, PEM, EMA); CD253 (TRAIL, TNF 35 KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j (ligand) superfamily, member 10); CD273 (B7DC, (ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, PDL2); CD274 (B7H1,PDL1); CD275 (B7H2, ICOSL); TNFRSF6); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, CD276 (B7H3); CD297 (ART4, ADP-ribosyltransferase E2); CD100 (SEMA4D); CD102 (ICAM-2); CD108 4; and Dombrock blood group glycoprotein; wherein the (SEMA7A, JMH blood group antigen); CDw119 (IFNγR, names listed in parentheses represent alternative 40 IFNγRa); CD120a (TNFRI, p55); CD120b (TNFRII, p75, names. Examples of such markers include those provid- TNFR p80); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); ed elsewhere herein. CD124 (IL-4Rα); CD130 (gp130); CD132 (Common γ [0320] B Cell Targeting Moieties chain, IL-2Rγ); CDw137 (4-1BB, ILA); CD139; CD147 [0321] In some embodiments, B cell targeting can be (Basigin, EMMPRIN, M6, OX47); CD150 (SLAM, IPO- accomplished by moieties that bind the complement re- 45 3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); ceptors, CR1 (i.e., CD35) or CR2 (i.e., CD21), proteins CD166 (ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); which are expressed on B cells as well as FDCs. In some CD167a (DDR1, trkE, cak); CD171 (L1CMA, NILE); embodiments, B cell targeting can be accomplished by CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95, B cell markers such as CD19, CD20, and/or CD22. In Ly64); CD184 (CXCR4, NPY3R); CD185 (CXCR5); some embodiments, B cell targeting can be accom- 50 CD192 (CCR2); CD 196 (CCR6); CD 197 (CCR7 (was plished by B cell markers such as CD40, CD52, CD80, CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CXCR5, VLA-4, class II MHC, surface IgM or IgD, APRL, CD205 (DEC-205); CDw210 (CK); CD213a (CK); and/or BAFF-R. The present invention encompasses the CDw217 (CK); CDw218a (IL18Rα); CDw218b (IL18Rβ); recognition that simultaneous targeting of B cells by moi- CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, eties specific for complement receptors or other APC- 55 IGFII-R); CD224 (GGT); CD225 (Leul3); CD226 (DNAM- associated molecules boosts humoral responses. 1, PTA1); CD227 (MUC1, PUM, PEM, EMA); CD229 [0322] In some embodiments, B cell targeting can be (Ly9); CD230 (Prion Protein (Prp)); CD232 (VESP-R); accomplished by any targeting moiety that specifically CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261

43 85 EP 2 394 657 A1 86

(TRAIL-R1, TNF-R superfamily, member 10a); CD262 surface for long periods of time are predicted to boost (TRAIL-R2, TNF-R superfamily, member 10b); CD263 GC reactions in response to vaccination and improve the (TRAIL-R3, TNF-R superfamily, member 10c); CD264 affinity and longevity of desired humoral immune re- (TRAIL-R4, TNF-R superfamily, member 10d); CD265 sponses. (TRANCE-R, TNF-R superfamily, member 11a); CD267 5 [0326] In some embodiments, FDC targeting can be (TACI, TNF-R superfamily, member 13B); CD268 (BAF- accomplished by moieties that bind the complement re- FR, TNF-R superfamily, member 13C); CD269 (BCMA, ceptors, CR1 (i.e., CD35) or CR2 (i.e., CD21), proteins TNF-R superfamily, member 16); CD275 (B7H2, which are expressed on FDCs as well as B cells. Exam- ICOSL); CD277 (BT3.1.B7 family: Butyrophilin 3); ples of moieties include those provided elsewhere here- CD295 (LEPR); CD298 (ATP1B3 Na K ATPase β3 sub- 10 in. unit); CD300a (CMRF-35H); CD300c (CMRF-35A); [0327] Vaccine Nanocarriers Comprising Multiple CD305 (LAIR1); CD307 (IRTA2); CD315 (CD9P1); Targeting Moieties CD316 (EW12); CD317 (BST2); CD319 (CRACC, [0328] GC reactions and B cell survival not only require SLAMF7); CD321 (JAM1); CD322 (JAM2); CDw327 FDC, but also are dependent on help provided by acti- (Siglec6, CD33L); CD68 (gp 100, Macrosialin); CXCR5; 15 vated CD4 T cells. Help is most efficiently provided when VLA-4; class II MHC; surface IgM; surface IgD; APRL; a CD4 T cell is first stimulated by a DC that presents a and/or BAFF-R; wherein the names listed in parentheses cognate peptide in MHC class II (pMHC) to achieve a represent alternative names. Examples of markers in- follicular helper (TFH) phenotype. The newly generated clude those provided elsewhere herein. TFH cell then migrates toward the B follicle and provides [0323] In some embodiments, B cell targeting can be 20 help to those B cells that present them with the same accomplished by any targeting moiety that specifically pMHC complex. For this, B cells first acquire antigenic binds to any entity (e.g., protein, lipid, carbohydrate, material (e.g., virus or virus-like vaccine), internalize and small molecule, etc.) that is prominently expressed process it (i.e., extract peptide that is loaded into MHC and/or present on B cells upon activation (i.e., activated class II), and then present the pMHC to a TFH cell. B cell marker). Exemplary activated B cell markers in- 25 [0329] Thus, the present invention encompasses the clude, but are not limited to, CD1a (R4, T6, HTA-1); CD1b recognition that a vaccine that stimulates optimal humor- (R1); CD15s (Sialyl Lewis X); CD15u (3’ sulpho Lewis al immunity can combine several features and compo- X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki- nents (Figure 1): (a) antigenic material for CD4 T cells 1); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki- that is targeted to and presented by DCs; (b) high density 24, CD27 ligand); CD80 (B7, B7-1, BB1); CD86 30 surface antigens that can be presented in their native (B7-2/B70); CD97 (BL-KDD/F12); CD125 (IL-5Rα); form by SCS-Mph to antigen-specific follicular B cells; CD126 (IL-6Rα); CD138 (Syndecan-1, Heparan sulfate (c) the capacity to be acquired and processed by follicular proteoglycan); CD152 (CTLA-4); CD252 (OX40L, TNF B cells for presentation to TFH cells (the present invention (ligand) superfamily, member 4); CD253 (TRAIL, TNF encompasses the recognition that B cells readily acquire (ligand) superfamily, member 10); CD279 (PD1); CD289 35 and internalize particulate matter from SCS-Mph); (d) the (TLR9, TOLL-like receptor 9); and CD312 (EMR2); ability to reach FDC and be retained on FDC in intact wherein the names listed in parentheses represent alter- form and for long periods of time; and (e) adjuvant activity native names. Examples of markers include those pro- to render APC fully immunogenic and to avoid or over- vided elsewhere herein. come tolerance. [0324] Follicular Dendritic Cells 40 [0330] In some embodiments, a vaccine nanocarrier [0325] B cells that initially detect a previously unknown comprises at least one targeting moiety. In some embod- antigen typically express a B cell receptor (BCR, i.e., an iments, all of the targeting moieties of a vaccine nano- antibody with a transmembrane domain) with suboptimal carrier are identical to one another. In some embodi- binding affinity for that antigen. However, B cells can in- ments, a vaccine nanocarrier a number of different types crease by several orders of magnitude the affinity of the 45 of targeting moieties. In some embodiments, a vaccine antibodies they make when they enter into a germinal nanocarrier comprises multiple individual targeting moi- center (GC) reaction. This event, which typically lasts eties, all of which are identical to one another. In some several weeks, depends on FDC that accumulate, retain embodiments, a vaccine nanocarrier comprises exactly and present antigenic material to the activated B cells. one type of targeting moiety. In some embodiments, a B cells, while proliferating vigorously, repeatedly mutate 50 vaccine nanocarrier comprises exactly two distinct types the genomic sequences that encode the antigen binding of targeting moieties. In some embodiments, a vaccine site of their antibody and undergo class-switch recombi- nanocarrier comprises greater than two distinct types of nation to form secreted high-affinity antibodies, mostly targeting moieties. of the IgG isotype. GC reactions also stimulate the gen- [0331] In some embodiments, a vaccine nanocarrier eration of long-lived memory B cells and plasma cells 55 comprises a single type of targeting moiety that directs that maintain high protective antibody titers, often for delivery of the vaccine nanocarrier to a single cell type many years. Vaccine carriers that target FDC upon sub- (e.g., delivery to SCS-Mph only). In some embodiments, cutaneous injection and that are retained on the FDC a vaccine nanocarrier comprises a single type of target-

44 87 EP 2 394 657 A1 88 ing moiety that directs delivery of the vaccine nanocarrier ments, a vaccine nanocarrier comprises two types of im- to multiple cell types (e.g., delivery to both SCS-Mph and munostimulatory agents, wherein first type of immunos- FDCs). In some embodiments, a vaccine nanocarrier timulatory agent stimulates B cells, and the second type comprises two types of targeting moieties, wherein the of immunostimulatory agent stimulates T cells. In some first type of targeting moiety directs delivery of the vac- 5 embodiments, a vaccine nanocarrier comprises greater cine nanocarrier to one cell type, and the second type of than two types of immunostimulatory agents, wherein targeting moiety directs delivery of the vaccine nanocar- one or more types of immunostimulatory agents stimu- rier to a second cell type. In some embodiments, a vac- late B cells, and one or more types of immunostimulatory cine nanocarrier comprises greater than two types of tar- agents stimulate T cells. See the section above for a geting moieties, wherein one or more types of targeting 10 more detailed description of immunostimulatory agents moieties direct delivery of the vaccine nanocarrier to one that can be used in accordance with the present inven- cell type, and one or more types of targeting moieties tion. direct delivery of the vaccine nanocarrier to a second cell type. To give but one example, a vaccine nanocarrier Assays for B Cell Activation may comprise two types of targeting moieties, wherein 15 the first type of targeting moiety directs delivery of the [0335] In some embodiments, various assays can be vaccine nanocarrier to DCs, and the second type of tar- utilized in order to determine whether an immune re- geting moiety directs delivery of the vaccine nanocarrier sponse has been stimulated in a B cell or group of B cells to SCS-Mph. (i.e., whether a B cell or group of B cells has become [0332] In some embodiments, a vaccine nanocarrier 20 "activated"). In some embodiments, stimulation of an im- comprises at least one targeting moiety that is associated mune response in B cells can be determined by meas- with the exterior surface of the vaccine nanocarrier. In uring antibody titers. In general, "antibody titer" refers to some embodiments, the association is covalent. In some the ability of antibodies to bind and neutralize antigens embodiments, the covalent association is mediated by at particular dilutions. For example, a high antibody titer one or more linkers. In some embodiments, the associ- 25 refers to the ability of antibodies to bind and neutralize ation is non-covalent. In some embodiments, the non- antigens even at high dilutions. In some embodiments, covalent association is mediated by charge interactions, an immune response in B cells is said to be stimulated affinity interactions, metal coordination, physical adsorp- if antibody titers are measured to be positive at dilutions tion, host-guest interactions, hydrophobic interactions, at least about 5-fold greater, at least about 10-fold great- TT stacking interactions, hydrogen bonding interactions, 30 er, at least about 20-fold greater, at least about 50-fold van der Waals interactions, magnetic interactions, elec- greater, at least about 100-fold greater, at least about trostatic interactions, dipole-dipole interactions, and/or 500-fold greater, at least about 1000 fold greater, or more combinations thereof. than about 1000-fold greater than in non-immunized in- [0333] In some embodiments, a vaccine nanocarrier dividuals or pre-immune serum. comprises a lipid membrane (e.g., lipid bilayer, lipid mon- 35 [0336] In some embodiments, stimulation of an im- olayer, etc.), wherein at least one targeting moiety is as- mune response in B cells can be determined by meas- sociated with the lipid membrane. In some embodiments, uring antibody affinity. In particular, an immune response at least one targeting moiety is embedded within the lipid in B cells is said to be stimulated if an antibody has an -7 membrane. In some embodiments, at least one targeting equilibrium dissociation constant (Kd) less than 10 M, moiety is embedded within the lumen of a lipid bilayer. 40 less than 10-8 M, less than 10-9 M, less than 10-10 M, In some embodiments, at least one targeting moiety may less than 10-11 M, less than 10-12 M, or less. be located at multiple locations of a vaccine nanocarrier. [0337] In some embodiments, a T cell-dependent im- For example, a first targeting moiety may be embedded mune response in B cells is said to be stimulated if class- within a lipid membrane, and a second immunostimula- switch recombination has occurred. In particular, a switch tory agent may be associated with the exterior surface 45 from IgM to an IgG isotype or to IgA or to a mixture of of a vaccine nanocarrier. To give another example, a first these isotypes is indicative of a T cell dependent immune targeting moiety and a second targeting moiety may both response in B cells. be associated with the exterior surface of a vaccine na- [0338] In some embodiments, an immune response in nocarrier. B cells is determined by measuring affinity maturation of 50 antigen-specific antibodies. Affinity maturation occurs Immunostimulatory Agents during the germinal center reaction whereby activated B cells repeatedly mutate a region of the immunoglobulin [0334] As described above, in some embodiments, gene that encodes the antigen-binding region. B cells vaccine nanocarriers may transport one or more immu- producing mutated antibodies which have a higher affin- nostimulatory agents which can help stimulate immune 55 ity for antigen are preferentially allowed to survive and responses. In some embodiments, a vaccine nanocarrier proliferate. Thus, over time, the antibodies made by B comprises a single type of immunostimulatory agent that cells in GCs acquire incrementally higher affinities. In stimulates both B cells and T cells. In some embodi- some embodiments, the readout of this process is the

45 89 EP 2 394 657 A1 90 presence of high antibody titer (e.g. high affinity IgG an- protection of non-immune mice from lethal infection. tibodies that bind and neutralize antigens even at high [0344] One of ordinary skill in the art will recognize that dilutions). the assays described above are only exemplary methods [0339] In some embodiments, an immune response in which could be utilized in order to determine whether B B cells is said to be stimulated if memory B cells and/or 5 cell activation has occurred. Any assay known to one of long-lived plasma cells that can produce large amounts skill in the art which can be used to determine whether of high-affinity antibodies for extended periods of time B cell activation has occurred falls within the scope of have formed. In some embodiments, antibody titers are this invention. The assays described herein as well as measured after different time intervals (e.g. 2 weeks, 1 additional assays that could be used to determine wheth- month, 2 months, 6 months, 1 year, 2 years, 5 years, 10 10 er B cell activation has occurred are described in Current years, 15 years, 20 years, 25 years, or longer) after vac- Protocols in Immunology (John Wiley & Sons, Hoboken, cination in order to test for the presence of memory B NY, 2007; incorporated herein by reference). cells and/or long-lived plasma cells that can produce large amounts of high-affinity antibodies for extended pe- Vaccine Nanocarriers riods of time. In some embodiments, memory B cells 15 and/or long-lived plasma cells that can produce large [0345] In general, a vaccine nanocarrier is an entity amounts of high-affinity antibodies for extended periods that comprises, for example, at least one immunomodu- of time are said to be present by measuring humoral re- latory agent which is capable of stimulating an immune sponses (e.g., if humoral responses are markedly more response in both B cells and T cells. Any vaccine nano- rapid and result in higher titers after a later booster vac- 20 carrier can be used in accordance with the present in- cination than during the initial sensitization). vention. In some embodiments, nanocarriers are biode- [0340] In some embodiments, an immune response in gradable and biocompatible. In general, a biocompatible B cells is said to be stimulated if a vigorous germinal substance is not toxic to cells. In some embodiments, a center reaction occurs. In some embodiments, a vigor- substance is considered to be biocompatible if its addition ous germinal center reaction can be assessed visually 25 to cells results in less than a certain threshhold of cell by performing histology experiments. In some embodi- death (e.g. less than 50%, 20%, 10%, 5%, or less cell ments, vigorous germinal center reaction can be assayed death). In some embodiments, a substance is considered by performing immunohistochemistry of antigen-contain- to be biocompatible if its addition to cells does not induce ing lymphoid tissues (e.g., vaccine-draining lymph adverse effects. In general, a biodegradable substance nodes, spleen, etc.). In some embodiments, immunohis- 30 is one that undergoes breakdown under physiological tochemistry is followed by flow cytometry. conditions over the course of a therapeutically relevant [0341] In some embodiments, stimulation of an im- time period (e.g., weeks, months, or years). In some em- mune response in B cells can be determined by identi- bodiments, a biodegradable substance is a substance fying antibody isotypes (e.g., IgG, IgA, IgE, IgM). In cer- that can be broken down by cellular machinery. In some tain embodiments, production of IgG isotype antibodies 35 embodiments, a biodegradable substance is a substance by B cells is a desirable immune response in a B cell. that can be broken down by chemical processes. In some [0342] In some embodiments, an immune response in embodiments, a nanocarrier is a substance that is both B cells is determined by analyzing antibody function in biocompatible and biodegradable. In some embodi- neutralization assays. In particular, the ability of a micro- ments, a nanocarrier is a substance that is biocompatible, organism (e.g., virus, bacterium, fungus, protozoan, par- 40 but not biodegradable. In some embodiments, a nano- asite, etc.) to infect a susceptible cell line in vitro in the carrier is a substance that is biodegradable, but not bio- absence of serum is compared to conditions when dif- compatible. ferent dilutions of immune and non-immune serum are [0346] In general, a nanocarrier in accordance with the added to the culture medium in which the cells are grown. present invention is any entity having a greatest dimen- In certain embodiments, an immune response in a B cell 45 sion (e.g., diameter) of less than 100 microns (mm). In is said to be stimulated if infection of a microorganism is some embodiments, inventive nanocarriers have a great- neutralized at a dilution of about 1:5, about 1:10, about est dimension of less than 10 mm. In some embodiments, 1:50, about 1:100, about 1:500, about 1:1000, about 1: inventive nanocarriers have a greatest dimension of less 5000, about 1:10,000, or less. than 1000 nanometers (nm). In some embodiments, in- [0343] In some embodiments, the efficacy of vaccines 50 ventive nanocarriers have a greatest dimension of less in animal models may be determined by infecting groups than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, of immunized and non-immunized mice (e.g., 3 or more 300 nm, 200 nm, or 100 nm. Typically, inventive nano- weeks after vaccination) with a dose of a microorganism carriers have a greatest dimension (e.g., diameter) of that is typically lethal. The magnitude and duration of 300 nm or less. In some embodiments, inventive nano- survival of both group is monitored and typically graphed 55 carriers have a greatest dimension (e.g., diameter) of a Kaplan-Meier curves. To assess whether enhanced 250 nm or less. In some embodiments, inventive nano- survival is due to B cell responses, serum from immune carriers have a greatest dimension (e.g., diameter) of mice can be transferred as a "passive vaccine" to assess 200 nm or less. In some embodiments, inventive nano-

46 91 EP 2 394 657 A1 92 carriers have a greatest dimension (e.g., diameter) of in accordance with the present invention. In some em- 150 nm or less. In some embodiments, inventive nano- bodiments, nanocarriers are spheres or spheroids. In carriers have a greatest dimension (e.g., diameter) of some embodiments, nanocarriers are spheres or sphe- 100 nm or less. Smaller nanocarriers, e.g., having a roids. In some embodiments, nanocarriers are flat or greatest dimension of 50 nm or less are used in some 5 plate-shaped. In some embodiments, nanocarriers are embodiments of the invention. In some embodiments, cubes or cuboids. In some embodiments, nanocarriers inventive nanocarriers have a greatest dimension rang- are ovals or ellipses. In some embodiments, nanocarriers ing between 25 nm and 200 nm. In some embodiments, are cylinders, cones, or pyramids. inventive nanocarriers have a greatest dimension rang- [0351] Nanocarriers can be solid or hollow and can ing between 20 nm and 100 nm. 10 comprise one or more layers. In some embodiments, [0347] In some embodiments, nanocarriers have a di- each layer has a unique composition and unique prop- ameter of less than 1000 nm. In some embodiments, erties relative to the other layer(s). To give but one ex- nanocarriers have a diameter of approximately 750 nm. ample, nanocarriers may have a core/shell structure, In some embodiments, nanocarriers have a diameter of wherein the core is one layer (e.g. a polymeric core) and approximately 500 nm. In some embodiments, nanocar- 15 the shell is a second layer (e.g. a lipid bilayer or monol- riers have a diameter of approximately 450 nm. In some ayer). Nanocarriers may comprise a plurality of different embodiments, nanocarriers have a diameter of approx- layers. In some embodiments, one layer may be sub- imately 400 nm. In some embodiments, nanocarriers stantially cross-linked, a second layer is not substantially have a diameter of approximately 350 nm. In some em- cross-linked, and so forth. In some embodiments, one, bodiments, nanocarriers have a diameter of approxi- 20 a few, or all of the different layers may comprise one or mately 300 nm. In some embodiments, nanocarriers more immunomodulatory agents, targeting moieties, im- have a diameter of approximately 275 nm. In some em- munostimulatory agents, and/or combinations thereof. In bodiments, nanocarriers have a diameter of approxi- some embodiments, one layer comprises an immu- mately 250 nm. In some embodiments, nanocarriers nomodulatory agent, targeting moiety, and/or immunos- have a diameter of approximately 225 nm. In some em- 25 timulatory agent, a second layer does not comprise an bodiments, nanocarriers have a diameter of approxi- immunomodulatory agent, targeting moiety, and/or im- mately 200 nm. In some embodiments, nanocarriers munostimulatory agent, and so forth. In some embodi- have a diameter of approximately 175 nm. In some em- ments, each individual layer comprises a different immu- bodiments, nanocarriers have a diameter of approxi- nomodulatory agent, targeting moiety, immunostimula- mately 150 nm. In some embodiments, nanocarriers 30 tory agent, and/or combination thereof. have a diameter of approximately 125 nm. In some em- bodiments, nanocarriers have a diameter of approxi- Lipid Vaccine Nanocarriers mately 100 nm. In some embodiments, nanocarriers have a diameter of approximately 75 nm. In some em- [0352] In some embodiments, nanocarriers may op- bodiments, nanocarriers have a diameter of approxi- 35 tionally comprise one or more lipids. In some embodi- mately 50 nm. In some embodiments, nanocarriers have ments, a nanocarrier may comprise a liposome. In some a diameter of approximately 25 nm. embodiments, a nanocarrier may comprise a lipid bilayer. [0348] In certain embodiments, nanocarriers are In some embodiments, a nanocarrier may comprise a greater in size than the renal excretion limit (e.g., nano- lipid monolayer. In some embodiments, a nanocarrier carriers having diameters of greater than 6 nm). In certain 40 may comprise a micelle. In some embodiments, a nano- embodiments, nanocarriers are small enough to avoid carrier may comprise a core comprising a polymeric ma- clearance of nanocarriers from the bloodstream by the trix surrounded by a lipid layer (e.g., lipid bilayer, lipid liver (e.g., nanocarriers having diameters of less than monolayer, etc.). In some embodiments, a nanocarrier 1000 nm). In general, physiochemical features of nano- may comprise a non-polymeric core (e.g., metal particle, carriers should allow a nanocarrier to circulate longer in 45 quantum dot, ceramic particle, bone particle, viral parti- plasma by decreasing renal excretion and liver clear- cle, etc.) surrounded by a lipid layer (e.g., lipid bilayer, ance. lipid monolayer, etc.). [0349] It is often desirable to use a population of na- [0353] In some embodiments, nanocarriers may com- nocarriers that is relatively uniform in terms of size, prise a lipid bilayer oriented such that the interior and shape, and/or composition so that each nanocarrier has 50 exterior of the nanocarrier are hydrophilic, and the lumen similar properties. For example, at least 80%, at least of the lipid bilayer is hydrophobic. Examples of vaccine 90%, or at least 95% of the nanocarriers may have a nanocarriers comprising lipid bilayers are described in diameter or greatest dimension that falls within 5%, 10%, Example 2 and shown in Figures 3-8. In some embodi- or 20% of the average diameter or greatest dimension. ments, hydrophobic immunomodulatory agents, target- In some embodiments, a population of nanocarriers may 55 ing moieties, and/or immunostimulatory agents may be be heterogeneous with respect to size, shape, and/or associated with (e.g., embedded within) the lumen of the composition. lipid bilayer. In some embodiments, hydrophilic immu- [0350] A variety of different nanocarriers can be used nomodulatory agents, targeting moieties, and/or immu-

47 93 EP 2 394 657 A1 94 nostimulatory agents may be associated with (e.g., cov- 10% by weight, approximately 15% by weight, approxi- alently or non-covalently associated with, encapsulated mately 20% by weight, approximately 25% by weight, or within, etc.) the interior and/or exterior of the nanocarrier. approximately 30% by weight. In some embodiments, hydrophilic immunomodulatory [0357] In some embodiments, lipids are oils. In gener- agents, targeting moieties, and/or immunostimulatory 5 al, any oil known in the art can be included in nanocarri- agents may be associated with (e.g., covalently or non- ers. In some embodiments, an oil may comprise one or covalently associated with, encapsulated within, etc.) the more fatty acid groups or salts thereof. In some embod- interior and/or exterior surface of the lipid bilayer. In some iments, a fatty acid group may comprise digestible, long embodiments, the interior, hydrophilic surface of the lipid chain (e.g.,C8-C50), substituted or unsubstituted hydro- bilayer is associated with an amphiphilic entity. In some 10 carbons. In some embodiments, a fatty acid group may embodiments, the amphiphilic entity is oriented such that be a C10-C20 fatty acid or salt thereof. In some embodi- the hydrophilic end of the amphiphilic entity is associated ments, a fatty acid group may be a C15-C20 fatty acid or with the interior surface of the lipid bilayer, and the hy- salt thereof. In some embodiments, a fatty acid group drophobic end of the amphiphilic entity is oriented toward may be a C15-C25 fatty acid or salt thereof. In some em- the interior of the nanocarrier, producing a hydrophobic 15 bodiments, a fatty acid group may be unsaturated. In environment within the nanocarrier interior. some embodiments, a fatty acid group may be monoun- [0354] In some embodiments, nanocarriers may com- saturated. In some embodiments, a fatty acid group may prise a lipid monolayer oriented such that the interior of be polyunsaturated. In some embodiments, a double the nanocarrier is hydrophobic, and the exterior of the bond of an unsaturated fatty acid group may be in the nanocarrier is hydrophilic. Examples of vaccine nanocar- 20 cis conformation. In some embodiments, a double bond riers comprising lipid monolayers are described in Ex- of an unsaturated fatty acid may be in the trans confor- ample 2 and shown in Figures 9 and 10. In some em- mation. bodiments, hydrophobic immunomodulatory agents, tar- [0358] In some embodiments, a fatty acid group may geting moieties, and/or immunostimulatory agents may be one or more of butyric, caproic, caprylic, capric, lauric, be associated with (e.g., covalently or non-covalently as- 25 myristic, palmitic, stearic, arachidic, behenic, or lignocer- sociated with, encapsulated within, etc.) the interior of ic acid. In some embodiments, a fatty acid group may be the nanocarrier and/or the interior surface of the lipid one or more of palmitoleic, oleic, vaccenic, linoleic, alpha- monolayer. In some embodiments, hydrophilic immu- linolenic, gamma-linoleic, arachidonic, gadoleic, arachi- nomodulatory agents, targeting moieties, and/or immu- donic, eicosapentaenoic, docosahexaenoic, or erucic nostimulatory agents may be associated with (e.g., cov- 30 acid. alently or non-covalently associated with, encapsulated [0359] In some embodiments, the oil is a liquid triglyc- within, etc.) the exterior of the nanocarrier and/or the ex- eride. terior surface of the lipid monolayer. In some embodi- [0360] Suitable oils for use with the present invention ments, the interior, hydrophobic surface of the lipid bi- include, but are not limited to, almond, apricot kernel, layer is associated with an amphiphilic entity. In some 35 avocado, babassu, bergamot, black current seed, bor- embodiments, the amphiphilic entity is oriented such that age, cade, camomile, canola, caraway, carnauba, cas- the hydrophobic end of the amphiphilic entity is associ- tor, cinnamon, cocoa butter, coconut, cod liver, coffee, ated with the interior surface of the lipid bilayer, and the corn, cotton seed, emu, eucalyptus, evening primrose, hydrophilic end of the amphiphilic entity is oriented to- fish, flaxseed, geraniol, gourd, grape seed, hazel nut, ward the interior of the nanocarrier, producing a hy- 40 hyssop, jojoba, kukui nut, lavandin, lavender, lemon, lits- drophilic environment within the nanocarrier interior. ea cubeba, macademia nut, mallow, mango seed, mead- [0355] In some embodiments, a nanocarrier may com- owfoam seed, mink, nutmeg, olive, orange, orange prise one or more nanoparticles associated with the ex- roughy, palm, palm kernel, peach kernel, peanut, poppy terior surface of the nariocarrier. Examples of vaccine seed, púmpkin seed, rapeseed, rice bran, rosemary, saf- nanocarriers comprising nanoparticles associated with 45 flower, sandalwood, sasquana, savoury, sea buckthorn, the exterior surface of the nanocarrier are described in sesame, shea butter, silicone, soybean, sunflower, tea Example 2 and shown in Figures 4, 6, and 8. tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils, [0356] The percent of lipid in nanocarriers can range and combinations thereof. Suitable oils for use with the from 0% to 99% by weight, from 10% to 99% by weight, present invention include, but are not limited to, butyl from 25% to 99% by weight, from 50% to 99% by weight, 50 stearate, caprylic triglyceride, capric triglyceride, cyclom- or from 75% to 99% by weight. In some embodiments, ethicone, diethyl sebacate, dimethicone 360, isopropyl the percent of lipid in nanocarriers can range from 0% to myristate, mineral oil, octyldodecanol, oleyl alcohol, sili- 75% by weight, from 0% to 50% by weight, from 0% to cone oil, and combinations thereof. 25% by weight, or from 0% to 10% by weight. In some In some embodiments, a lipid is a hormone (e.g. estro- embodiments, the percent of lipid in nanocarriers can be 55 gen, testosterone), steroid (e.g., cholesterol, bile acid), approximately 1% by weight, approximately 2% by vitamin (e.g. vitamin E), phospholipid (e.g. phosphatidyl weight, approximately 3% by weight, approximately 4% choline), sphingolipid (e.g. ceramides), or lipoprotein by weight, approximately 5% by weight, approximately (e.g. apolipoprotein).

48 95 EP 2 394 657 A1 96

Nanocarriers Comprising a Polymeric Matrix acrylates; and polycyanoacrylates. [0366] In some embodiments, polymers can be hy- [0361] In some embodiments, nanocarriers can com- drophilic. For example, polymers may comprise anionic prise one or more polymers. In some embodiments, a groups (e.g., phosphate group, sulphate group, carbox- polymeric matrix can be surrounded by a coating layer 5 ylate group); cationic groups (e.g., quaternary amine (e.g., liposome, lipid monolayer, micelle, etc.). In some group); or polar groups (e.g., hydroxyl group, thiol group, embodiments, an immunomodulatory agent, targeting amine group). In some embodiments, a nanocarrier com- moiety, and/or immunostimulatory agent can be associ- prising a hydrophilic polymeric matrix generates a hy- ated with the polymeric matrix. In such embodiments, the drophilic environment within the nanocarrier. In some immunomodulatory agent, targeting moiety, and/or im- 10 embodiments, hydrophilic immunomodulatory agents, munostimulatory agent is effectively encapsulated within targeting moieties, and/or immunostimulatory agents the nanocarrier. may be associated with hydrophilic polymeric matrices. [0362] In some embodiments, an immunomodulatory [0367] In some embodiments, polymers can be hydro- agent, targeting moiety, and/or immunostimulatory agent phobic. In some embodiments, a nanocarrier comprising can be covalently associated with a polymeric matrix. In 15 a hydrophobic polymeric matrix generates a hydrophobic some embodiments, covalent association is mediated by environment within the nanocarrier. In some embodi- a linker. In some embodiments, an immunomodulatory ments, hydrophobic immunomodulatory agents, target- agent, targeting moiety, and/or immunostimulatory agent ing moieties, and/or immunostimulatory agents may be can be non-covalently associated with a polymeric ma- associated with hydrophobic polymeric matrices. trix. For example, in some embodiments, an immu- 20 [0368] In some embodiments, polymers may be mod- nomodulatory agent, targeting moiety, and/or immunos- ified with one or more moieties and/or functional groups. timulatory agent can be encapsulated within, surrounded Any moiety or functional group can be used in accord- by, and/or dispersed throughout a polymeric matrix. Al- ance with the present invention. In some embodiments, ternatively or additionally, an immunomodulatory agent, polymers may be modified with polyethylene glycol targeting moiety, and/or immunostimulatory agent can 25 (PEG), with a carbohydrate, and/or with acyclic polya- be associated with a polymeric matrix by hydrophobic cetals derived from polysaccharides (Papisov, 2001, interactions, charge interactions, van der Waals forces, ACS Symposium Series, 786:301; incorporated herein etc. by reference). [0363] A wide variety of polymers and methods for [0369] In some embodiments, polymers may be mod- forming polymeric matrices therefrom are known in the 30 ified with a lipid or fatty acid group, properties of which art of drug delivery. In general, a polymeric matrix com- are described in further detail below. In some embodi- prises one or more polymers. Any polymer may be used ments, a fatty acid group may be one or more of butyric, in accordance with the present invention. Polymers may caproic, caprylic, capric, lauric, myristic, palmitic, stearic, be natural or unnatural (synthetic) polymers. Polymers arachidic, behenic, or lignoceric acid. In some embodi- may be homopolymers or copolymers comprising two or 35 ments, a fatty acid group may be one or more of palmi- more monomers. In terms of sequence, copolymers may toleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-li- be random, block, or comprise a combination of random noleic, arachidonic, gadoleic, arachidonic, eicosapen- and block sequences. Typically, polymers in accordance taenoic, docosahexaenoic, or erucic acid. with the present invention are organic polymers. [0370] In some embodiments, polymers may be poly- [0364] Examples of polymers include polyethylenes, 40 esters, including copolymers comprising lactic acid and polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhy- glycolic acid units, such as poly(lactic acid-co-glycolic drides (e.g. poly(sebacic anhydride)), polyhydroxyacids acid) and poly(lactide-co-glycolide), collectively referred (e.g. poly(β-hydroxyalkanoate)), polypropylfumerates, to herein as "PLGA"; and homopolymers comprising gly- polycaprolactones, polyamides (e.g. polycaprolactam), colic acid units, referred to herein as "PGA," and lactic polyacetals, polyethers, polyesters (e.g., polylactide, pol- 45 acid units, such as poly-L-lactic acid, poly-D-lactic acid, yglycolide), poly(orthoesters), polycyanoacrylates, poly- poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and vinyl alcohols, polyurethanes, polyphosphazenes, poly- poly-D,L-lactide, collectively referred to herein as "PLA." acrylates, polymethacrylates, polyureas, polystyrenes, In some embodiments, exemplary polyesters include, for and polyamines. example, polyhydroxyacids; PEG copolymers and copol- [0365] In some embodiments, polymers in accordance 50 ymers of lactide and glycolide (e.g., PLA-PEG copoly- with the present invention include polymers which have mers, PGA-PEG copolymers, PLGA-PEG copolymers, been approved for use in humans by the U.S. Food and and derivatives thereof. In some embodiments, polyes- Drug Administration (FDA) under 21 C.F.R. § 177.2600, ters include, for example, polyanhydrides, poly(ortho es- including but not limited to polyesters (e.g., polylactic ac- ter), poly(ortho ester)-PEG copolymers, poly(caprolac- id, poly(lactic-co-glycolic acid), polycaprolactone, poly- 55 tone), poly(caprolactone)-PEG copolymers, polylysine, valerolactone, poly(1,3-dioxan-2one)); polyanhydrides polylysine-PEG copolymers, poly(ethylene imine), poly (e.g., poly(sebacic anhydride)); polyethers (e.g., polyeth- (ethylene imine)-PEG copolymers, poly(L-lactide-co-L- ylene glycol); polyurethanes; polymethacrylates; poly- lysine), poly(serine ester), poly(4-hydroxy-L-proline es-

49 97 EP 2 394 657 A1 98 ter), poly[α-(4-aminobutyl)-L-glycolic acid], and deriva- ine ester) (Zhou et al., 1990, Macromolecules, 23:3399; tives thereof. incorporated herein by reference), poly(4-hydroxy-L-pro- [0371] In some embodiments, a polymer may be line ester) (Putnam et al., 1999, Macromolecules, 32: PLGA. PLGA is a biocompatible and biodegradable co- 3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633; polymer of lactic acid and glycolic acid, and various forms 5 both of which are incorporated herein by reference), and of PLGA are characterized by the ratio of lactic acid:gly- poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, colic acid. Lactic acid can be L-lactic acid, D-lactic acid, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. or D,L-lactic acid. The degradation rate of PLGA can be Chem. Soc., 121:5633; both of which are incorporated adjusted by altering the lactic acid:glycolic acid ratio. In herein by reference). some embodiments, PLGA to be used in accordance with 10 [0375] In some embodiments, polymers in accordance the present invention is characterized by a lactic acid: with the present invention may be carbohydrates, prop- glycolic acid ratio of approximately 85:15, approximately erties of which are described in further detail below. In 75:25, approximately 60:40, approximately 50:50, ap- some embodiments, a carbohydrate may be a polysac- proximately 40:60, approximately 25:75, or approximate- charide comprising simple sugars (or their derivatives) ly 15:85. 15 connected by glycosidic bonds, as known in the art. In [0372] In some embodiments, polymers may be one some embodiments, a carbohydrate may be one or more or more acrylic polymers. In certain embodiments, acrylic of pullulan, cellulose, microcrystalline cellulose, hydrox- polymers include, for example, acrylic acid and meth- ypropyl methylcellulose, hydroxycellulose, methylcellu- acrylic acid copolymers, methyl methacrylate copoly- lose, dextran, cyclodextran, glycogen, starch, hydrox- mers, ethoxyethyl methacrylates, cyanoethyl methacr- 20 yethylstarch, carageenan, glycon, amylose, chitosan, N, ylate, aminoalkyl methacrylate copolymer, poly(acrylic O-carboxylmethylchitosan, algin and alginic acid, starch, acid), poly(methacrylic acid), methacrylic acid alkyla- chitin, heparin, konjac, glucommannan, pustulan, mide copolymer, poly(methyl methacrylate), poly(meth- heparin, hyaluronic acid, curdlan, and xanthan. acrylic acid anhydride), methyl methacrylate, polymeth- [0376] In some embodiments, a polymer in accord- acrylate, poly(methyl methacrylate) copolymer, polyacr- 25 ance with the present invention may be a protein or pep- ylamide, aminoalkyl methacrylate copolymer, glycidyl tide, properties of which are described in further detail methacrylate copolymers, polycyanoacrylates, and com- below. Exemplary proteins that may be used in accord- binations comprising one or more of the foregoing poly- ance with the present invention include, but are not lim- mers. The acrylic polymer may comprise fully-polymer- ited to, albumin, collagen, a poly(amino acid) (e.g., poly- ized copolymers of acrylic and methacrylic acid esters 30 lysine), an antibody, etc. with a low content of quaternary ammonium groups. [0377] In some embodiments, a polymer in accord- [0373] In some embodiments, polymers can be cati- ance with the present invention may be a nucleic acid onic polymers. In general, cationic polymers are able to (i.e., polynucleotide), properties of which are described condense and/or protect negatively charged strands of in further detail below. Exemplary polynucleotides that nucleic acids (e.g. DNA, RNA, or derivatives thereof). 35 may be used in accordance with the present invention Amine-containing polymers such as poly(lysine) (Zauner include, but are not limited to, DNA, RNA, etc. et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et [0378] The properties of these and other polymers and al., 1995, Bioconjugate Chem., 6:7; both of which are methods for preparing them are well known in the art incorporated herein by reference), poly(ethylene imine) (see, for example, U.S. Patents 6,123,727; 5,804,178; (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 40 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 1995, 92:7297; incorporated herein by reference), and 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; poly(amidoamine) dendrimers (Kukowska-Latallo et al., 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123: 1996, Bioconjugate Chem., 7:703; and Haensler et al., 9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; 1993, Bioconjugate Chem., 4:372; all of which are incor- 45 Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. porated herein by reference) are positively-charged at Control. Release, 62:7; and Uhrich et al., 1999, Chem. physiological pH, form ion pairs with nucleic acids, and Rev., 99:3181; all of which are incorporated herein by mediate transfection in a variety of cell lines. reference). More generally, a variety of methods for syn- [0374] In some embodiments, polymers can be degra- thesizing suitable polymers are described in Concise En- dable polyesters bearing cationic side chains (Putnam 50 cyclopedia of Polymer Science and Polymeric Amines et al., 1999, Macromolecules, 32:3658; Barrera et al., and Ammonium Salts, Ed. by Goethals, Pergamon 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989, Press, 1980; Principles of Polymerization by Odian, John Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Wiley & Sons, Fourth Edition, 2004; Contemporary Pol- Soc., 121:5633; and Zhou et al., 1990, Macromolecules, ymer Chemistry by Allcock et al., Prentice-Hall, 1981; 23:3399; all of which are incorporated herein by refer- 55 Deming et al., 1997, Nature, 390:386; and in U.S. Patents ence). Examples of these polyesters include poly(L-lac- 6,506,577, 6,632,922, 6,686,446, and 6,818,732; all of tide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., which are incorporated herein by reference. 115:11010; incorporated herein by reference), poly(ser- [0379] In some embodiments, polymers can be linear

50 99 EP 2 394 657 A1 100 or branched polymers. In some embodiments, polymers lated within the lipid nanocarrier is hydrophilic. However, can be dendrimers. In some embodiments, polymers can if an amphiphilic entity is associated with the interior sur- be substantially cross-linked to one another. In some em- face of the hydrophilic lipid membrane such that the hy- bodiments, polymers can be substantially free of cross- drophilic end of the amphiphilic entity is associated with links. In some embodiments, polymers can be used in 5 the interior surface of the hydrophilic lipid membrane and accordance with the present invention without undergo- the hydrophobic end of the amphiphilic entity is associ- ing a cross-linking step. ated with the interior of the nanocarrier, the space en- [0380] It is further to be understood that inventive na- capsulated within the nanocarrier is hydrophobic. nocarriers may comprise block copolymers, graft copol- [0386] The percent of amphiphilic entity in nanocarri- ymers, blends, mixtures, and/or adducts of any of the 10 ers can range from 0% to 99% by weight, from 10% to foregoing and other polymers. 99% by weight, from 25% to 99% by weight, from 50% [0381] Those skilled in the art will recognize that the to 99% by weight, or from 75% to 99% by weight. In some polymers listed herein represent an exemplary, not com- embodiments, the percent of amphiphilic entity in nano- prehensive, list of polymers that can be of use in accord- carriers can range from 0% to 75% by weight, from 0% ance with the present invention. 15 to 50% by weight, from 0% to 25% by weight, or from 0% [0382] In some embodiments, vaccine nanocarriers to 10% by weight. In some embodiments, the percent of comprise immunomodulatory agents embedded within amphiphilic entity in nanocarriers can be approximately reverse micelles. To give but one example, a liposome 1% by weight, approximately 2% by weight, approximate- nanocarrier may comprise hydrophobic immunomodula- ly 3% by weight, approximately 4% by weight, approxi- tory agents embedded within the liposome membrane, 20 mately 5% by weight, approximately 10% by weight, ap- and hydrophilic immunomodulatory agents embedded proximately 15% by weight, approximately 20% by with reverse micelles found in the interior of the liposomal weight, approximately 25% by weight, or approximately nanocarrier. 30% by weight. [0387] Any amphiphilic entity known in the art is suit- Non-Polymeric Nanocarriers 25 able for use in making nanocarriers in accordance with the present invention. Such amphiphilic entities include, [0383] In some embodiments, nanocarriers may not but are not limited to, phosphoglycerides; phosphatidyl- comprise a polymeric component. In some embodi- cholines; dipalmitoyl phosphatidylcholine (DPPC); dio- ments, nanocarriers may comprise metal particles, quan- leylphosphatidyl ethanolamine (DOPE); dioleyloxypro- tum dots, ceramic particles, bone particles, viral particles, 30 pyltriethylammonium (DOTMA); dioleoylphosphatidyl- etc. In some embodiments, an immunomodulatory choline; cholesterol; cholesterol ester; diacylglycerol; di- agent, targeting moiety, and/or immunostimulatory agent acylglycerolsuccinate; diphosphatidyl glycerol (DPPG); can be associated with the surface of such a non-poly- hexanedecanol; fatty alcohols such as polyethylene gly- meric nanocarrier. In some embodiments, a non-poly- col (PEG); polyoxyethylene-9-lauryl ether; a surface ac- meric nanocarrier is an aggregate of non-polymeric com- 35 tive fatty acid, such as palmitic acid or oleic acid; fatty ponents, such as an aggregate of metal atoms (e.g., gold acids; fatty acid monoglycerides; fatty acid diglycerides; atoms). In some embodiments, an immunomodulatory fatty acid amides; sorbitan trioleate (Span®85) glycocho- agent, targeting moiety, and/or immunostimulatory agent late; sorbitan monolaurate (Span®20); polysorbate 20 can be associated with the surface of, encapsulated with- (Tween®20); polysorbate 60 (Tween®60); polysorbate in, surrounded by, and/or dispersed throughout an ag- 40 65 (Tween®65); polysorbate 80 (Tween®80); polysorb- gregate of non-polymeric components. ate 85 (Tween®85); polyoxyethylene monostearate; sur- [0384] In certain embodiments of the invention, non- factin; a poloxomer; a sorbitan fatty acid ester such as polymeric nanocarriers comprise gradient or homogene- sorbitan trioleate; lecithin; lysolecithin; phosphatidylser- ous alloys. In certain embodiments of the invention, na- ine; phosphatidylinositol; sphingomyelin; phosphati- nocarriers comprise particles which possess optically 45 dylethanolamine (cephalin); cardiolipin; phosphatidic ac- and/or magnetically detectable properties. id; cerebrosides; dicetylphosphate; dipalmitoylphos- phatidylglycerol; stearylamine; dodecylamine; hexade- Nanocarriers Comprising Amphiphilic Entities cyl-amine; acetyl palmitate; glycerol ricinoleate; hexade- cyl sterate; isopropyl myristate; tyloxapol; poly(ethylene [0385] In some embodiments, nanocarriers may op- 50 glycol)5000-phosphatidylethanolamine; poly(ethylene tionally comprise one or more amphiphilic entities. In glycol)400-monostearate; phospholipids; synthetic some embodiments, an amphiphilic entity can promote and/or natural detergents having high surfactant proper- the production of nanocarriers with increased stability, ties; deoxycholates; cyclodextrins; chaotropic salts; ion improved uniformity, or increased viscosity. In some em- pairing agents; and combinations thereof. An amphiphilic bodiments, amphiphilic entities can be associated with 55 entity component may be a mixture of different am- the interior surface of a lipid membrane (e.g., lipid bilayer, phiphilic entities. These amphiphilic entities may be ex- lipid monolayer, etc.). For example, if the interior surface tracted and purified from a natural source or may be pre- of a lipid membrane is hydrophilic, the space encapsu- pared synthetically in a laboratory. In certain specific em-

51 101 EP 2 394 657 A1 102 bodiments, amphiphilic entities are commercially availa- associated with vaccine nanocarriers via covalent link- ble. age. In some embodiments, particles may be associated [0388] Those skilled in the art will recognize that this with vaccine nanocarriers via non-covalent interactions is an exemplary, not comprehensive, list of substances (e.g., charge interactions, affinity interactions, metal co- with surfactant activity. Any amphiphilic entity may be 5 ordination, physical adsorption, host-guest interactions, used in the production of nanocarriers to be used in ac- hydrophobic interactions, TT stacking interactions, hy- cordance with the present invention. drogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole- Vaccine Nanocarriers Comprising Carbohydrates dipole interactions, and/or combinations thereof). In 10 some embodiments, vaccine nanocarriers comprise one [0389] In some embodiments, nanocarriers may op- or more particles encapsulated within the nanocarrier. In tionally comprise one or more carbohydrates. The per- some embodiments, vaccine nanocarriers comprise one cent of carbohydrate in nanocarriers can range from 0% or more particles embedded within the surface of the na- to 99% by weight, from 10% to 99% by weight, from 25% nocarrier (e.g., embedded within a lipid bilayer). In some to 99% by weight, from 50% to 99% by weight, or from 15 embodiments, particles associated with a nanocarrier al- 75% to 99% by weight. In some embodiments, the per- low for tunable membrane rigidity and controllable lipo- cent of carbohydrate in nanocarriers can range from 0% some stability. to 75% by weight, from 0% to 50% by weight, from 0% [0392] In some embodiments, particles to be associ- to 25% by weight, or from 0% to 10% by weight. In some ated with a vaccine nanocarrier may comprise a poly- embodiments, the percent of carbohydrate in nanocarri- 20 meric matrix, as described above. In some embodiments, ers can be approximately 1% by weight, approximately particles to be associated with a vaccine nanocarrier may 2% by weight, approximately 3% by weight, approximate- comprise non-polymeric components (e.g., metal parti- ly 4% by weight, approximately 5% by weight, approxi- cles, quantum dots, ceramic particles, bone particles, vi- mately 10% by weight, approximately 15% by weight, ral particles, etc.), as described above. approximately 20% by weight, approximately 25% by 25 [0393] In some embodiments, a particle to be associ- weight, or approximately 30% by weight. ated with a vaccine nanocarrier may have a negative [0390] Carbohydrates may be natural or synthetic. A charge. In some embodiments, a particle to be associ- carbohydrate may be a derivatized natural carbohydrate. ated with a vaccine nanocarrier may have a positive In certain embodiments, a carbohydrate is a monosac- charge. In some embodiments, a particle to be associ- charide, including but not limited to glucose, fructose, 30 ated with a vaccine nanocarrier may be electrically neu- galactose, ribose, lactose, sucrose, maltose, trehalose, tral. cellbiose, mannose, xylose, arabinose, glucoronic acid, [0394] In some embodiments, the particle has one or galactoronic acid, mannuronic acid, glucosamine, gala- more amine moieties on its surface. The amine moieties tosamine, and neuramic acid. In certain embodiments, can be, for example, aliphatic amine moieties. In certain a carbohydrate is a disaccharide, including but not limited 35 embodiments, the amine is a primary, secondary, terti- to lactose, sucrose, maltose, trehalose, and cellobiose. ary, or quaternary amine. In certain embodiments, the In certain embodiments, a carbohydrate is a polysaccha- particle comprises an amine-containing polymer. In cer- ride, including but not limited to pullulan, cellulose, mi- tain embodiments, the particle comprises an amine-con- crocrystalline cellulose, hydroxypropyl methylcellulose taining lipid. In certain embodiments, the particles com- (HPMC), hydroxycellulose (HC), methylcellulose (MC), 40 prises a protein or a peptide that is positively charged at dextran, cyclodextran, glycogen, starch, hydroxyethyl- neutrol pH. In some embodiments, the particle with the starch, carageenan, glycon, amylose, chitosan, N,O-car- one or more amine moieties on its surface has a net pos- boxylmethylchitosan, algin and alginic acid, starch, chi- itive charge at neutral pH. Other chemical moieties that tin, heparin, konjac, glucommannan, pustulan, heparin, provide a positive charge at neutrol pH may also be used hyaluronic acid, curdlan, and xanthan. In certain embod- 45 in the inventive particles. iments, the carbohydrate is a sugar alcohol, including but [0395] Zeta potential is a measurement of surface po- not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, tential of a particle. In some embodiments, the particle and lactitol. has a positive zeta potential. In some embodiments, par- ticles have a zeta potential ranging between -50 mV and Particles Associated with Vaccine Nanocarriers 50 +50 mV. In some embodiments, particles have a zeta potential ranging between -25 mV and +25 mV. In some [0391] In some embodiments, vaccine nanocarriers in embodiments, particles have a zeta potential ranging be- accordance with the present invention may comprise one tween -10 mV and +10 mV. In some embodiments, par- or more particles. In some embodiments, one or more ticles have a zeta potential ranging between -5 mV and particles are associated with a vaccine nanocarrier. In 55 +5 mV. In some embodiments, particles have a zeta po- some embodiments, vaccine nanocarriers comprise one tential ranging between 0 mV and +50 mV. In some em- or more particles associated with the outside surface of bodiments, particles have a zeta potential ranging be- the nanocarrier. In some embodiments, particles may be tween 0 mV and +25 mV. In some embodiments, particles

52 103 EP 2 394 657 A1 104 have a zeta potential ranging between 0 mV and +10 to any particle having a diameter of less than 1000 nm. mV. In some embodiments, particles have a zeta poten- In some embodiments, particles are picoparticles (e.g., tial ranging between 0 mV and +5 mV. In some embod- picospheres). In general, a "picoparticle" refers to any iments, particles have a zeta potential ranging between particle having a diameter of less than 1 nm. In some -50 mV and 0 mV. In some embodiments, particles have 5 embodiments, particles are liposomes. In some embod- a zeta potential ranging between -25 mV and 0 mV. In iments, particles are micelles. some embodiments, particles have a zeta potential rang- [0399] A variety of different particles can be used in ing between -10 mV and 0 mV. In some embodiments, accordance with the present invention. In some embod- particles have a zeta potential ranging between -5 mV iments, particles are spheres or spheroids. In some em- and 0 mV. In some embodiments, particles have a sub- 10 bodiments, particles are spheres or spheroids. In some stantially neutral zeta potential (i.e. approximately 0 mV). embodiments, particles are flat or plate-shaped. In some [0396] In general, particles to be associated with a vac- embodiments, particles are cubes or cuboids. In some cine nanocarrier have a greatest dimension (e.g., diam- embodiments, particles are ovals or ellipses. In some eter) of less than 10 microns (mm). In some embodi- embodiments, particles are cylinders, cones, or pyra- ments, particles have a greatest dimension of less than 15 mids. 1000 nanometers (nm). In some embodiments, particles [0400] Particles (e.g., nanoparticles, microparticles) have a greatest dimension of less than 900 nm, 800 nm, may be prepared using any method known in the art. For 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or example, particulate formulations can be formed by 100 nm. Typically, particles have a greatest dimension methods as nanoprecipitation, flow focusing using fluidic (e.g., diameter) of 300 nm or less. In some embodiments, 20 channels, spray drying, single and double emulsion sol- particles have a greatest dimension (e.g., diameter) of vent evaporation, solvent extraction, phase separation, 200 nm or less. In some embodiments, particles have a milling, microemulsion procedures, microfabrication, na- greatest dimension (e.g., diameter) of 100 nm or less. nofabrication, sacrificial layers, simple and complex Smaller particles, e.g., having a greatest dimension of coacervation, and other methods well known to those of 50 nm or less are used in some embodiments of the in- 25 ordinary skill in the art. Alternatively or additionally, aque- vention. In some embodiments, particles have a greatest ous and organic solvent syntheses for monodisperse dimension ranging between 25 nm and 200 nm. In some semiconductor, conductive, magnetic, organic, and other embodiments, particles have a greatest dimension rang- nanoparticles have been described (Pellegrino et al., ing between 1 nm and 100 nm. In some embodiments, 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. particles have a greatest dimension ranging between 1 30 Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13: nm and 30 nm. 3843; all of which are incorporated herein by reference). [0397] In some embodiments, particles have a diam- [0401] In certain embodiments, particles are prepared eter of approximately 1000 nm. In some embodiments, by the nanoprecipitation process or spray drying. Condi- particles have a diameter of approximately 750 nm. In tions used in preparing particles may be altered to yield some embodiments, particles have a diameter of approx- 35 particles of a desired size or property (e.g., hydrophobic- imately 500 nm. In some embodiments, particles have a ity, hydrophilicity, external morphology, "stickiness," diameter of approximately 400 nm. In some embodi- shape, etc.). The method of preparing the particle and ments, particles have a diameter of approximately 300 the conditions (e.g., solvent, temperature, concentration, nm. In some embodiments, particles have a diameter of air flow rate, etc.) used may depend on the therapeutic approximately 200 nm. In some embodiments, particles 40 agent to be delivered and/or the composition of the pol- have a diameter of approximately 100 nm. In some em- ymer matrix. bodiments, particles have a diameter of approximately [0402] Methods for making microparticles for delivery 75 nm. In some embodiments, particles have a diameter of encapsulated agents are described in the literature of approximately 50 nm. In some embodiments, particles (see, e.g., Doubrow, Ed., "Microcapsules and Nanopar- have a diameter of approximately 30 nm. In some em- 45 ticles in Medicine and Pharmacy," CRC Press, Boca Ra- bodiments, particles have a diameter of approximately ton, 1992; Mathiowitz et al., 1987, J. Control. Release, 25 nm. In some embodiments, particles have a diameter 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; of approximately 20 nm. In some embodiments, particles and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35: have a diameter of approximately 15 nm. In some em- 755; all of which are incorporated herein by reference). bodiments, particles have a diameter of approximately 50 [0403] If particles prepared by any of the above meth- 10 nm. In some embodiments, particles have a diameter ods have a size range outside of the desired range, par- of approximately 5 nm. In some embodiments, particles ticles can be sized, for example, using a sieve. have a diameter of approximately 1 nm. [0398] In some embodiments, particles are micropar- Production of Vaccine Nanocarriers ticles (e.g., microspheres). In general, a "microparticle" 55 refers to any particle having a diameter of less than 1000 [0404] Vaccine nanocarriers may be prepared using mm. In some embodiments, particles are nanoparticles any method known in the art. For example, particulate (e.g., nanospheres). In general, a "nanoparticle" refers nanocarrier formulations can be formed by methods as

53 105 EP 2 394 657 A1 106 nanoprecipitation, flow focusing using fluidic channels, it is able to reach specific cells in the body. spray drying, single and double emulsion solvent evap- [0409] In some embodiments, immunomodulatory oration, solvent extraction, phase separation, milling, mi- agents, targeting moieties, immunostimulatory agents, croemulsion procedures, microfabrication, nanofabrica- and/or nanoparticles are not covalently associated with tion, sacrificial layers, simple and complex coacervation, 5 a vaccine nanocarrier. For example, vaccine nanocarri- and other methods well known to those of ordinary skill ers may comprise a polymeric matrix, and immunomod- in the art. Alternatively or additionally, aqueous and or- ulatory agents, targeting moieties, immunostimulatory ganic solvent syntheses for monodisperse semiconduc- agents, and/or nanoparticles may be associated with the tor, conductive, magnetic, organic, and other nanoparti- surface of, encapsulated within, and/or distributed cles have been described (Pellegrino et al., 2005, Small, 10 throughout the polymeric matrix of an inventive vaccine 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; nanocarrier. Immunomodulatory agents are released by and Trindade et al., 2001, Chem. Mat., 13:3843; all of diffusion, degradation of the vaccine nanocarrier, and/or which are incorporated herein by reference). combination thereof. In some embodiments, polymers [0405] In certain embodiments, vaccine nanocarriers degrade by bulk erosion. In some embodiments, poly- are prepared by the nanoprecipitation process or spray 15 mers degrade by surface erosion. drying. Conditions used in preparing nanocarriers may [0410] In some embodiments, immunomodulatory be altered to yield particles of a desired size or property agents, targeting moieties, immunostimulatory agents, (e.g., hydrophobicity, hydrophilicity, external morpholo- and/or nanoparticles are covalently associated with a gy, "stickiness," shape, etc.). The method of preparing vaccine nanocarrier. For such vaccine nanocarriers, re- the nanocarrier and the conditions (e.g., solvent, tem- 20 lease and delivery of the immunomodulatory agent to a perature, concentration, air flow rate, etc.) used may de- target site occurs by disrupting the association. For ex- pend on the composition and/or resulting architecture of ample, if an immunomodulatory agent is associated with the vaccine nanocarrier. a nanocarrier by a cleavable linker, the immunomodula- [0406] Methods for making microparticles for delivery tory agent is released and delivered to the target site of encapsulated agents are described in the literature 25 upon cleavage of the linker. (see, e.g., Doubrow, Ed., "Microcapsules and Nanopar- [0411] In some embodiments, immunomodulatory ticles in Medicine and Pharmacy," CRC Press, Boca Ra- agents, targeting moieties, immunostimulatory agents, ton, 1992; Mathiowitz et al., 1987, J. Control. Release, and/or nanoparticles are not covalently associated with 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; a vaccine nanocarrier. For example, vaccine nanocarri- and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35: 30 ers may comprise polymers, and immunomodulatory 755; all of which are incorporated herein by reference). agents, targeting moieties, immunostimulatory agents, [0407] In some embodiments, inventive vaccine nano- and/or nanoparticles may be associated with the surface carriers comprise at least one immunomodulatory agent of, encapsulated within, surrounded by, and/or distribut- and, optionally, a lipid membrane, a polymeric matrix, ed throughout the polymer of an inventive vaccine nano- and/or a non-polymeric particle. In certain embodiments, 35 carrier. In some embodiments, immunomodulatory inventive vaccine nanocarriers comprise at least one im- agents, targeting moieties, immunostimulatory agents, munomodulatory agent; a lipid membrane, a polymeric and/or nanoparticles are physically associated with a matrix, and/or a non-polymeric particle; and at least one vaccine nanocarrier. targeting moiety. In certain embodiments, inventive vac- [0412] Physical association can be achieved in a va- cine nanocarriers comprise at least one immunomodu- 40 riety of different ways. Physical association may be cov- latory agent; a lipid membrane, a polymeric matrix, and/or alent or non-covalent. The vaccine nanocarrier, immu- a non-polymeric particle; at least one targeting moiety; nomodulatory agent, targeting moiety, immunostimula- and at least one immunostimulatory agent. In certain em- tory agent, and/or nanoparticle may be directly associ- bodiments, inventive vaccine nanocarriers comprise at ated with one another, e.g., by one or more covalent least one immunomodulatory agent; a lipid membrane, 45 bonds, or may be associated by means of one or more a polymeric matrix, and/or a non-polymeric particle; at linkers. In one embodiment, a linker forms one or more least one targeting moiety; at least one immunostimula- covalent or non-covalent bonds with the immunomodu- tory agent; and at least one nanoparticle. latory agent, targeting moiety, immunostimulatory agent, [0408] Inventive vaccine nanocarriers may be manu- and/or nanoparticle and one or more covalent or non- factured using any available method. It is desirable to 50 covalent bonds with the immunomodulatory agent, tar- associate immunomodulatory agents, targeting moie- geting moiety, immunostimulatory agent, and/or nano- ties, and/or immunostimulatory agents to vaccine nano- particle, thereby attaching them to one another. In some carriers without adversely affecting the 3-dimensional embodiments, a first linker forms a covalent or non-cov- characteristic and conformation of the immunomodula- alent bond with the vaccine nanocarrier and a second tory agents, targeting moieties, and/or immunostimula- 55 linker forms a covalent or non-covalent bond with the tory agents. It is desirable that the vaccine nanocarrier immunomodulatory agent, targeting moiety, immunos- should be able to avoid uptake by the mononuclear timulatory agent, and/or nanoparticle. The two linkers phagocytic system after systemic administration so that form one or more covalent or non-covalent bond(s) with

54 107 EP 2 394 657 A1 108 each other. N-hydroxy-succinimide ester (SMCC), succinimidyl-[(N- [0413] Any suitable linker can be used in accordance maleimidopropionamido)-dodecaethyleneglycol] ester with the present invention. Linkers may be used to form (NHS-PEO12), etc. For example, carbodiimide-mediat- amide linkages, ester linkages, disulfide linkages, etc. ed amide formation and active ester maleimide-mediated Linkers may contain carbon atoms or heteroatoms (e.g., 5 amine and sulfhydryl coupling are widely used approach- nitrogen, oxygen, sulfur, etc.). Typically, linkers are 1 to es. 50 atoms long, 1 to 40 atoms long, 1 to 25 atoms long, [0418] In some embodiments, a vaccine nanocarrier 1 to 20 atoms long, 1 to 15 atoms long, 1 to 10 atoms can be formed by coupling an amine group on one mol- long, or 1 to 10 atoms long. Linkers may be substituted ecule to a thiol group on a second molecule, sometimes with various substituents including, but not limited to, hy- 10 by a two- or three-step reaction sequence. A thiol-con- drogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, taining molecule may be reacted with an amine-contain- dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, ing molecule using a heterobifunctional cross-linking re- aryl, heterocyclic, aromatic heterocyclic, cyano, amide, agent, e.g., a reagent containing both a succinimidyl es- carbamoyl, carboxylic acid, ester, thioether, alkylth- ter and either a maleimide, a pyridyldisulfide, or an io- ioether, thiol, and ureido groups. As would be appreciat- 15 doacetamide. Amine-carboxylic acid and thiol-carboxylic ed by one of skill in this art, each of these groups may in acid cross-linking, maleimide-sulfhydryl coupling chem- turn be substituted. istries (e.g., the maleimidobenzoyl-N-hydroxysuccinim- [0414] In some embodiments, a linker is an aliphatic ide ester (MBS) method), etc., may be used. Polypep- or heteroaliphatic linker. In some embodiments, the linker tides can conveniently be attached to particles via amine is a polyalkyl linker. In certain embodiments, the linker is 20 or thiol groups in lysine or cysteine side chains respec- a polyether linker. In certain embodiments, the linker is tively, or by an N-terminal amino group. Nucleic acids a polyethylene linker. In certain specific embodiments, such as RNAs can be synthesized with a terminal amino the linker is a polyethylene glycol (PEG) linker. group. A variety of coupling reagents (e.g., succinimidyl [0415] In some embodiments, the linker is a cleavable 3-(2-pyridyldithio)propionate (SPDP) and sulfosuccinim- linker. To give but a few examples, cleavable linkers in- 25 idyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate clude protease cleavable peptide linkers, nuclease sen- (sulfo-SMCC) may be used to associate the various com- sitive nucleic acid linkers, lipase sensitive lipid linkers, ponents of vaccine nanocarriers. Vaccine nanocarriers glycosidase sensitive carbohydrate linkers, pH sensitive can be prepared with functional groups, e.g., amine or linkers, hypoxia sensitive linkers, photo-cleavable link- carboxyl groups, available at the surface to facilitate as- ers, heat-labile linkers, enzyme cleavable linkers (e.g. 30 sociation with a biomolecule. esterase cleavable linker), ultrasound-sensitive linkers, [0419] Non-covalent specific binding interactions can x-ray cleavable linkers, etc. In some embodiments, the be employed. For example, either a particle or a biomol- linker is not a cleavable linker. ecule can be functionalized with biotin with the other be- [0416] Any of a variety of methods can be used to as- ing functionalized with streptavidin. These two moieties sociate a linker with a vaccine nanocarrier. General strat- 35 specifically bind to each other non-covalently and with a egies include passive adsorption (e.g., via electrostatic high affinity, thereby associating the particle and the bi- interactions), multivalent chelation, high affinity non-cov- omolecule. Other specific binding pairs could be similarly alent binding between members of a specific binding pair, used. Alternately, histidine-tagged biomolecules can be covalent bond formation, etc. (Gao et al., 2005, Curr. Op. associated with particles conjugated to nickel-nitrolot- Biotechnol., 16:63; incorporated herein by reference). In 40 riaceteic acid (Ni-NTA). some embodiments, click chemistry can be used to as- [0420] Any biomolecule to be attached to a particle, sociate a linker with a particle (e.g. Diels-Alder reaction, targeting moiety, and/or therapeutic agent. The spacer Huigsen 1,3-dipolar cycloaddition, nucleophilic substitu- can be, for example, a short peptide chain, e.g., between tion, carbonyl chemistry, epoxidation, dihydroxylation, 1 and 10 amino acids in length, e.g., 1, 2, 3, 4, or 5 amino etc.). 45 acids in length, a nucleic acid, an alkyl chain, etc. [0417] A bifunctional cross-linking reagent can be em- [0421] For additional general information on associa- ployed. Such reagents contain two reactive groups, tion and/or conjugation methods and cross-linkers, see thereby providing a means of covalently associating two the journal Bioconjugate Chemistry, published by the target groups. The reactive groups in a chemical cross- American Chemical Society, Columbus OH, PO Box linking reagent typically belong to various classes of func- 50 3337, Columbus, OH, 43210; "Cross-Linking," Pierce tional groups such as succinimidyl esters, maleimides, Chemical Technical Library, available at the Pierce web and pyridyldisulfides. Exemplary cross-linking agents in- site and originally published in the 1994-95 Pierce Cat- clude, e.g., carbodiimides, N-hydroxysuccinimidyl-4-azi- alog, and references cited therein; Wong SS, Chemistry dosalicylic acid (NHS-ASA), dimethyl pimelimidate dihy- of Protein Conjugation and Cross-linking, CRC Press drochloride (DMP), dimethylsuberimidate (DMS), 3,3’- 55 Publishers, Boca Raton, 1991; and Hermanson, G. T., dithiobispropionimidate (DTBP), N-Succinimidyl 3-[2-py- Bioconjugate Techniques, Academic Press, Inc., San Di- ridyldithio]-propionamido (SPDP), succimidyl α-methyl- ego, 1996. butanoate, biotinamidohexanoyl-6-aminohexanoic acid [0422] Alternatively or additionally, vaccine nanocarri-

55 109 EP 2 394 657 A1 110 ers can be attached to immunomodulatory agents, tar- agent, targeting moiety, immunostimulatory agent, geting moieties, immunostimulatory agents, and/or nan- and/or nanoparticle to the vaccine nanocarrier. oparticles directly or indirectly via non-covalent interac- [0426] In some embodiments, a vaccine nanocarrier tions. Non-covalent interactions include but are not lim- may be associated with an immunomodulatory agent, ited to charge interactions, affinity interactions, metal co- 5 targeting moiety, immunostimulatory agent, and/or nan- ordination, physical adsorption, host-guest interactions, oparticle via physical adsorption. For example, a hydro- hydrophobic interactions, TT stacking interactions, hy- phobic tail, such as polymethacrylate or an alkyl group drogen bonding interactions, van der Waals interactions, having at least about 10 carbons, may be attached to magnetic interactions, electrostatic interactions, dipole- one end of the immunomodulatory agent, targeting moi- dipole interactions, and/or combinations thereof. 10 ety, immunostimulatory agent, and/or nanoparticle. The [0423] In some embodiments, a vaccine nanocarrier hydrophobic tail will adsorb onto the surface of a hydro- may be associated with an immunomodulatory agent, phobic vaccine nanocarrier, thereby binding the immu- targeting moiety, immunostimulatory agent, and/or nan- nomodulatory agent, targeting moiety, immunostimula- oparticle via charge interactions. For example, a vaccine tory agent, and/or nanoparticle to the vaccine nanocar- nanocarrier may have a cationic surface or may be re- 15 rier. acted with a cationic polymer, such as poly(lysine) or poly [0427] In some embodiments, a vaccine nanocarrier (ethylene imine), to provide a cationic surface. The vac- may be associated with an immunomodulatory agent, cine nanocarrier surface can then bind via charge inter- targeting moiety, immunostimulatory agent, and/or nan- actions with a negatively charged immunomodulatory oparticle via host-guest interactions. For example, a agent, targeting moiety, immunostimulatory agent, 20 macrocyclic host, such as cucurbituril or cyclodextrin, and/or nanoparticle. One end of the immunomodulatory may be attached to the surface of the vaccine nanocarrier agent, targeting moiety, immunostimulatory agent, and a guest group, such as an alkyl group, a polyethylene and/or nanoparticle is, typically, attached to a negatively glycol, or a diaminoalkyl group, may be attached to the charged polymer (e.g., a poly(carboxylic acid)) or an ad- immunomodulatory agent, targeting moiety, immunos- ditional oligonucleotide sequence that can interact with 25 timulatory agent, and/or nanoparticle; or conversely, the the cationic polymer surface without disrupting the func- host group may be attached to the immunomodulatory tion of the immunomodulatory agent, targeting moiety, agent, targeting moiety, immunostimulatory agent, immunostimulatory agent, and/or nanoparticle. and/or nanoparticle and the guest group may be attached [0424] In some embodiments, a vaccine nanocarrier to the surface of the vaccine nanocarrier. In some em- may be associated with an immunomodulatory agent, 30 bodiments, the host and/or the guest molecule may be targeting moiety, immunostimulatory agent, and/or nan- attached to the immunomodulatory agent, targeting moi- oparticle via affinity interactions. For example, biotin may ety, immunostimulatory agent, and/or nanoparticle or the be attached to the surface of the vaccine nanocarrier and vaccine nanocarrier via a linker, such as an alkylene link- streptavidin may be attached to the immunomodulatory er or a polyether linker. agent, targeting moiety, immunostimulatory agent, 35 [0428] In some embodiments, a vaccine nanocarrier and/or nanoparticle; or conversely, biotin may be at- may be associated with an immunomodulatory agent, tached to the immunomodulatory agent, targeting moie- targeting moiety, immunostimulatory agent, and/or nan- ty, immunostimulatory agent, and/or nanoparticle and the oparticle via hydrogen bonding interactions. For exam- streptavidin may be attached to the surface of the vaccine ple, an oligonucleotide having a particular sequence may nanocarrier. The biotin group and streptavidin may be 40 be attached to the surface of the vaccine nanocarrier, attached to the vaccine nanocarrier or to the immu- and an essentially complementary sequence may be at- nomodulatory agent, targeting moiety, immunostimula- tached to one or both ends of the immunomodulatory tory agent, and/or nanoparticle via a linker, such as an agent, targeting moiety, immunostimulatory agent, alkylene linker or a polyether linker. Biotin and strepta- and/or nanoparticle such that it does not disrupt the func- vidin bind via affinity interactions, thereby binding the 45 tion of the immunomodulatory agent, targeting moiety, vaccine nanocarrier to the immunomodulatory agent, tar- immunostimulatory agent, and/or nanoparticle. The im- geting moiety, immunostimulatory agent, and/or nano- munomodulatory agent, targeting moiety, immunostim- particle. ulatory agent, and/or nanoparticle then binds to the vac- [0425] In some embodiments, a vaccine nanocarrier cine nanocarrier via complementary base pairing with may be associated with an immunomodulatory agent, 50 the oligonucleotide attached to the vaccine nanocarrier. targeting moiety, immunostimulatory agent, and/or nan- Two oligonucleotides are essentially complimentary if oparticle via metal coordination. For example, a polyhis- about 80% of the nucleic acid bases on one oligonucle- tidine may be attached to one end of the immunomodu- otide form hydrogen bonds via an oligonucleotide base latory agent, targeting moiety, immunostimulatory agent, pairing system, such as Watson-Crick base pairing, re- and/or nanoparticle, and a nitrilotriacetic acid can be at- 55 verse Watson-Crick base pairing, Hoogsten base pair- tached to the surface of the vaccine nanocarrier. A metal, ing, etc., with a base on the second oligonucleotide. Typ- such as Ni2+, will chelate the polyhistidine and the nitrilo- ically, it is desirable for an oligonucleotide sequence at- triacetic acid, thereby binding the immunomodulatory tached to the vaccine nanocarrier to form at least about

56 111 EP 2 394 657 A1 112

6 complementary base pairs with a complementary oli- brane rigidity and controllable liposome stability. When gonucleotide attached to the immunomodulatory agent, small charged nanoparticles approach the surface of li- targeting moiety, immunostimulatory agent, and/or nan- posomes carrying either opposite charge or no net oparticle. charge, electrostatic or charge-dipole interaction be- [0429] In some embodiments, vaccine nanocarriers 5 tween nanoparticles and membrane attracts the nano- are made by self-assembly. For a detailed example of particles to stay on the membrane surface, being partially self-assembly of vaccine nanocarriers, see Examples 1 wrapped by lipid membrane. This induces local mem- and 2. In certain embodiments, small liposomes (10 nm brane bending and globule surface tension of liposomes, - 1000 nm) are manufactured and employed to deliver both of which enable tuning of membrane rigidity. This one or multiple immunomodulatory agents to cells of the 10 aspect is significant for vaccine delivery using liposomes immune system (Figure 3). In general, liposomes are ar- to mimic viruses whose stiffness depends on the com- tificially-constructed spherical lipid vesicles, whose con- position of other biological components within virus mem- trollable diameter from tens to thousands of nm signifies brane. Moreover, adsorbed nanoparticles form a that individual liposomes comprise biocompatible com- charged shell which protects liposomes against fusion, partments with volume from zeptoliters (10-21 L) to fem- 15 thereby enhancing liposome stability. In certain embod- toliters (10-15 L) that can be used to encapsulate and iments, small nanoparticles are mixed with liposomes un- store various cargoes such as proteins, enzymes, DNA der gentle vortex, and the nanoparticles stick to liposome and drug molecules. Liposomes may comprise a lipid surface spontaneously. In specific embodiments, small bilayer which has an amphiphilic property: both interior nanoparticles can be, but are not limited to, polymeric and exterior surfaces of the bilayer are hydrophilic, and 20 nanoparticles, metallic nanoparticles, inorganic or organ- the bilayer lumen is hydrophobic. Lipophilic molecules ic nanoparticles, hybrids thereof, and/or combinations can spontaneously embed themselves into liposome thereof. membrane and retain their hydrophilic domains outside, [0433] In some embodiments, liposome-polymer na- and hydrophilic molecules can be chemically conjugated nocarriers are used to deliver one or a plurality of immu- to the outer surface of liposome taking advantage of 25 nomodulatory agents to cells of the immune system (Fig- membrane biofunctionality. ure 5). Instead of keeping the liposome interior hollow, [0430] In certain embodiments, lipids are mixed with hydrophilic immunomodulatory agents can be encapsu- a lipophilic immunomodulatory agent, and then formed lated. Figure 3 shows liposomes that are loaded with di- into thin films on a solid surface. A hydrophilic immu- block copolymer nanoparticles to form liposome-coated nomodulatory agent is dissolved in an aqueous solution, 30 polymeric nanocarriers, which have the merits of both which is added to the lipid films to hydrolyze lipids under liposomes and polymeric nanoparticles, while excluding vortex. Liposomes with lipophilic immunomodulatory some of their limitations. In some embodiments, the li- agents incorporated into the bilayer wall and hydrophilic posome shell can be used to carry lipophilic or conjugate immunomodulatory agents inside the liposome lumen hydrophilic immunomodulatory agents, and the polymer- are spontaneously assembled. 35 ic core can be used to deliver hydrophobic immunomod- [0431] In certain embodiments, a lipid to be used in ulatory agents. liposomes can be, but is not limited to, one or a plurality [0434] In certain embodiments, pre-formulated poly- of the following: phosphatidylcholine, lipid A, cholesterol, meric nanoparticles (40 nm - 1000 nm) are mixed with dolichol, sphingosine, sphingomyelin, ceramide, glyco- small liposomes (20 nm - 100 nm) under gentle vortex sylceramide, cerebroside, sulfatide, phytosphingosine, 40 to induce liposome fusion onto polymeric nanoparticle phosphatidyl-ethanolamine, phosphatidylglycerol, phos- surface. In specific embodiments, di-block copolymer na- phatidylinositol, phosphatidylserine, cardiolipin, phos- noparticles can be, but are not limited to, one or a plurality phatidic acid, and lyso-phophatides. In certain embodi- of following: poly(D,Llactic acid)-block-poly(ethylene gly- ments, an immunomodulatory agent can be conjugated col) (PLA-b-PEG), poly(D,Lglycolic acid)-block-poly(eth- 45 to the surface of a liposome. In some embodiments, the ylene glycol) (PLG-b-PEG), poly(D,Llactic-co-glycolic ac- liposome carries an identical or a non-identical immu- id)-block-poly(ethylene glycol) (PLGA-b-PEG), and poly nomodulatory agent inside. In some embodiments, the (ε-caprolactone)-block-poly(ethylene glycol) (PCL-b- liposome surface membrane can be modified with tar- PEG). geting moieties that can selectively deliver the immu- [0435] In some embodiments, nanoparticle-stabilized nomodulatory agent(s) to specific antigen expressing 50 liposome-polymer nanocarriers are used to deliver one cells. or a plurality of immunomodulatory agents (Figure 6). By [0432] In some embodiments, nanoparticle-stabilized adsorbing small nanoparticles (1 nm - 30 nm) to the li- liposomes are used to deliver one or a plurality of immu- posome-polymer nanocarrier surface, the nanocarrier nomodulatory agents to cells of the immune system (Fig- has not only the merit of both aforementioned nanopar- ure 4). By allowing small charged nanoparticles (1 nm - 55 ticle-stabilized liposomes (Figure 4) and aforementioned 30 nm) to adsorb on liposome surface, liposome-nano- liposome-polymer nanoparticles (Figure 5), but also tun- particle complexes have not only the merits of aforemen- able membrane rigidity and controllable liposome stabil- tioned bare liposomes (Figure 3), but also tunable mem- ity.

57 113 EP 2 394 657 A1 114

[0436] In some embodiments, liposome-polymer na- drophilic segments. Lipid-stabilized polymeric nanocar- nocarriers containing reverse micelles are used to deliver riers can be formed within one single step instead of for- one or a plurality of immunomodulatory agents (Figure mulating polymeric nanoparticle and liposome separate- 7). Since the aforementioned liposome-polymer nano- ly followed by fusing them together. carriers (Figures 5 and 6) are limited to carry hydrophobic 5 [0440] In certain embodiments, a hydrophilic immu- immunomodulatory agents within polymeric nanoparti- nomodulatory molecule is first chemically conjugated to cles, here small reverse micelles (1 nm - 20 nm) are for- lipid headgroup. The conjugate is mixed with a certain mulated to encapsulate hydrophilic immunomodulatory ratio of unconjugated lipid molecules in an aqueous so- agents and then mixed with the di-block copolymers to lution containing one or more water-miscible solvents. In formulate polymeric core of liposomes. 10 certain embodiments, the amphiphilic entity can be, but [0437] In certain embodiments, a hydrophilic immu- is not limited to, one or a plurality of the following: phos- nomodulatory agent to be encapsulated is first incorpo- phatidylcholine, lipid A, cholesterol, dolichol, shingosine, rated into reverse micelles by mixing with naturally de- sphingomyelin, ceramide, cerebroside, sulfatide, phyt- rived and non-toxic amphiphilic entities in a volatile, wa- osphingosine, phosphatidylethanolamine, glycosylcera- ter-miscible organic solvent. In certain embodiments, the 15 mide, phosphatidylglycerol, phosphatidylinositol, phos- amphiphilic entity can be, but is not limited to, one or a phatidylserine, cardiolipin, phosphatidic acid, and lyso- plurality of the following: phosphatidylcholine, lipid A, phosphatides. In some embodiments, the water miscible cholesterol, dolichol, shingosine, sphingomyelin, cera- solvent can be, but is not limited to: acetone, ethanol, mide, glycosylceramide, cerebroside, sulfatide, phyt- methanol, and isopropyl alcohol. A biodegradable poly- osphingosine, phosphatidylethanolamine, phosphati- 20 meric material is mixed with the hydrophobic immu- dylglycerol, phosphatidylinositol, phosphatidylserine, nomodulatory agents to be encapsulated in a water mis- cardiolipin, phosphatidic acid, and lysophophatides. In cible or partially water miscible organic solvent. In spe- some embodiments, the volatile, water-miscible organic cific embodiments, the biodegradable polymer can be, solvent can be, but is not limited to: tetrahydrofuran, ac- but is not limited to one or a plurality of the following: poly etone, acetonitrile, or dimethylformamide. In some em- 25 (D,L-lactic acid), poly(D,L-glycolic acid), poly(ε-caprolac- bodiments, a biodegradable polymer is added to this mix- tone), or their copolymers at various molar ratios. In some ture after reverse micelle formation is complete. The re- embodiments, the water miscible organic solvent can be sulting biodegradable polymer-reverse micelle mixture but is not limited to: acetone, ethanol, methanol, or iso- is combined with a polymer-insoluble hydrophilic non- propyl alcohol. In some embodiments, the partially water solvent to form nanoparticles by the rapid diffusion of the 30 miscible organic solvent can be, but is not limited to: ac- solvent into the non-solvent and evaporation of the or- etonitrile, tetrahydrofuran, ethyl acetate, isopropyl alco- ganic solvent. In certain embodiments, the polymer-in- hol, isopropyl acetate, or dimethylformamide. The result- soluble hydrophilic non-solvent can be, but is not limited ing polymer solution is added to the aqueous solution of to one or a plurality of the following: water, ethanol, meth- conjugated and unconjugated lipid to yield nanoparticles anol, and mixtures thereof. Reverse micelle contained 35 by the rapid diffusion of the organic solvent into the water polymeric nanoparticles are mixed with lipid molecules and evaporation of the organic solvent. to form the aforementioned liposome-polymer complex [0441] In some embodiments, lipid monolayer stabi- structure (Figure 5). lized polymeric nanoparticles comprising reverse mi- [0438] In some embodiments, nanoparticle-stabilized celles are used to deliver one or a plurality of immu- liposome-polymer nanocarriers containing reverse mi- 40 nomodulatory agents (Figure 10). Since the aforemen- celles are used to deliver one or a plurality of immu- tioned lipid-stabilized polymeric nanocarriers (Figure 9) nomodulatory agents (Figure 8). By adsorbing small na- are limited to carry hydrophobic immunomodulatory noparticles (1 nm - 30 nm) to a liposome-polymer nano- agents, here, small reverse micelles (1 nm - 20 nm) are carrier surface, the nanocarrier has not only the merit of formulated to encapsulate hydrophilic immunomodula- both aforementioned nanoparticle-stabilized liposomes 45 tory agents and mixed with biodegradable polymers to (Figure 4) and aforementioned reverse micelle contained form polymeric nanocarrier core. liposome-polymer nanoparticles (Figure 7), but also tun- [0442] It is to be understood that the compositions of able membrane rigidity and controllable liposome stabil- the invention can be made in any suitable manner, and ity. the invention is in no way limited to compositions that [0439] In some embodiments, lipid monolayer stabi- 50 can be produced using the methods described herein. lized polymeric nanocarriers are used to deliver one or Selection of an appropriate method may require attention a plurality of immunomodulatory agents (Figure 9). As to the properties of the particular moieties being associ- compared to aforementioned liposome-polymer nano- ated. carrier (Figures 5-8), this system has the merit of sim- [0443] If desired, various methods may be used to sep- plicity in terms to both agents and manufacturing. In some 55 arate vaccine nanocarriers with an attached immu- embodiments, a hydrophobic homopolymer can form the nomodulatory agent, targeting moiety, immunostimula- polymeric core in contrast to the di-block copolymer used tory agent, and/or nanoparticle from vaccine nanocarri- in Figures 5-8, which has both hydrophobic and hy- ers to which the immunomodulatory agent, targeting moi-

58 115 EP 2 394 657 A1 116 ety, immunostimulatory agent, and/or nanoparticle has sitic infection, etc.). not become attached, or to separate vaccine nanocarri- [0448] In one aspect of the invention, a method for the ers having different numbers of immunomodulatory prophylaxis and/or treatment of a disease, disorder, or agents, targeting moieties, immunostimulatory agents, condition (e.g., a microbial infection) is provided. In some and/or nanoparticles attached thereto. For example, size 5 embodiments, the prophylaxis and/or treatment of the exclusion chromatography, agarose gel electrophoresis, disease, disorder, or condition comprises administering or filtration can be used to separate populations of vac- a therapeutically effective amount of inventive vaccine cine nanocarriers having different numbers of entities at- nanocarriers to a subject in need thereof, in such tached thereto and/or to separate vaccine nanocarriers amounts and for such time as is necessary to achieve from other entities. Some methods include size-exclu- 10 the desired result. In certain embodiments of the present sion or anion-exchange chromatography. invention a "therapeutically effective amount" of an in- [0444] In some embodiments, inventive vaccine nano- ventive vaccine nanocarrier is that amount effective for carriers are manufactured under sterile conditions. This treating, alleviating, ameliorating, relieving, delaying on- can ensure that resulting vaccines are sterile and non- set of, inhibiting progression of, reducing severity of, infectious, thus improving safety when compared to live 15 and/or reducing incidence of one or more symptoms or vaccines. This provides a valuable safety measure, es- features of microbial infection. In some embodiments, a pecially when subjects receiving vaccine have immune "therapeutically effective amount" is an amount effective defects, are suffering from infection, and/or are suscep- to modulate the immune system. Such an amount may tible to infection. be an immunogenic amount, i.e., an amount sufficient to [0445] In some embodiments, inventive vaccine nano- 20 elicit a detectable immune response in a subject, e.g., a carriers may be lyophilized and stored in suspension or detectable antibody response and/or detectable T cell as lyophilized powder depending on the formulation strat- response. egy for extended periods without losing activity. [0449] Inventive prophylactic and/or therapeutic pro- tocols involve administering a therapeutically effective Applications 25 amount of one or more inventive vaccine nanocarriers to a healthy subject (e.g., a subject who does not display [0446] The compositions and methods described any symptoms of microbial infection and/or who has not herein can be used to induce, enhance, suppress, direct, been diagnosed with microbial infection; a subject who or redirect an immune response. The compositions and has not yet been exposed to a toxin, a subject who has methods described herein can be used for the prophy- 30 not yet ingested an abused or addictive substance, etc.). laxis and/or treatment of any cancer, infectious disease, For example, healthy individuals may be vaccinated us- metabolic disease, degenerative disease, autoimmune ing inventive vaccine nanocarrier(s) prior to development disease, allergic disease, inflammatory disease, immu- of microbial infection, exposure to the toxin, abused sub- nological disease, or other disorder and/or condition. The stance, addictive substance, etc. and/or onset of symp- compositions and methods described herein can also be 35 toms related thereto; at risk individuals (e.g., patients ex- used for the treatment of an addiction, such as an addic- posed to individuals suffering from microbial infection, tion to any of the addictive substances described herein. traveling to locations where microbes/toxins are preva- The compositions and methods described herein can al- lent; etc.) can be treated substantially contemporaneous- so be used for the prophylaxis and/or treatment of a con- ly with (e.g., within 48 hours, within 24 hours, or within dition resulting from the exposure to a toxin, hazardous 40 12 hours of) the onset of symptoms of and/or exposure/ substance, environmental toxin, or other harmful agent. ingestion. Of course individuals known to have microbial Subjects include, but are not limited to, humans and/or infection, have been exposed to a toxin, or ingested an other primates; mammals, including commercially rele- abused or additive substance may receive treatment at vant mammals such as cattle, pigs, horses, sheep, cats, any time. and/or dogs; and/or birds, including commercially rele- 45 [0450] In some embodiments, inventive prophylactic vant birds such as chickens, ducks, geese, and/or tur- and/or therapeutic protocols involve administering a ther- keys. apeutically effective amount of one or more inventive vac- [0447] In some embodiments, vaccine nanocarriers in cine nanocarriers to a subject such that an immune re- accordance with the present invention may be used to sponse is stimulated in both T cells and B cells. treat, alleviate, ameliorate, relieve, delay onset of, inhibit 50 [0451] In some embodiments, by combining selected progression of, reduce severity of, and/or reduce inci- immunomodulatory agents with targeting moieties and dence of one or more symptoms or features of a disease, immunostimulatory agents for different APCs, immune disorder, and/or condition. In some embodiments, inven- responses (e.g. effector responses) can be tailored to tive vaccine nanocarriers may be used to treat, alleviate, preferentially elicit the most desirable type of immune ameliorate, relieve, delay onset of, inhibit progression of, 55 response for a given indication, e.g., humoral response, reduce severity of, and/or reduce incidence of one or type 1 T cell response, type 2 T cell response, cytotoxic more symptoms or features of microbial infection (e.g. T cell, response, and/or a combination of these respons- bacterial infection, fungal infection, viral infection, para- es. Thus, the same platform may be used for a broad

59 117 EP 2 394 657 A1 118 range of different clinical applications, including prophy- tions comprising a therapeutically effective amount of lactic vaccines to a host of pathogens as well as immu- one or more vaccine nanocarriers and one or more phar- notherapy of existing diseases, such as infections, aller- maceutically acceptable excipients. In some embodi- gies, autoimmune diseases, and/or cancer. ments, the present invention provides for pharmaceutical [0452] Cancers include but are not limited to biliary 5 compositions comprising inventive vaccine nanocarriers tract cancer; brain cancer; breast cancer; cervical can- and/or any of the compositions thereof described herein. cer; choriocarcinoma; colon cancer; endometrial cancer; Such pharmaceutical compositions may optionally com- esophageal cancer; gastric cancer; intraepithelial neo- prise one or more additional therapeutically-active sub- plasms; lymphomas; liver cancer; lung cancer (e.g., stances. In accordance with some embodiments, a meth- small cell and non-small cell); melanoma; neuroblasto- 10 od of administering a pharmaceutical composition com- mas; oral cancer; ovarian cancer; pancreatic cancer; prising inventive compositions to a subject in need there- prostate cancer; rectal cancer; sarcomas; skin cancer; of is provided. In some embodiments, inventive compo- testicular cancer; thyroid cancer; and renal cancer, as sitions are administered to humans. For the purposes of well as other carcinomas and sarcomas. the present invention, the phrase "active ingredient" gen- [0453] Autoimmune diseases include, but are not lim- 15 erally refers to an inventive vaccine nanocarrier compris- ited to, rheumatoid arthritis, rheumatic fever, ulcerative ing at least one immunomodulatory agent and optionally colitis, celiac disease, Crohn’s disease, inflammatory comprising one or more targeting moieties, immunostim- bowel disease, insulin-dependent diabetes mellitus, di- ulatory agents, and/or nanoparticles. abetes mellitus, juvenile diabetes, spontaneous autoim- [0457] Although the descriptions of pharmaceutical mune diabetes, gastritis, autoimmune atrophic gastritis, 20 compositions provided herein are principally directed to autoimmune hepatitis, thyroiditis, Hashimoto’s thyroidi- pharmaceutical compositions which are suitable for ad- tis, autoimmune thyroiditis, insulitis, oophoritis, orchitis, ministration to humans, it will be understood by the skilled uveitis, phacogenic uveitis, multiple sclerosis, myasthe- artisan that such compositions are generally suitable for nia gravis, primary myxoedema, thyrotoxicosis, perni- administration to animals of all sorts. Modification of phar- cious anemia, autoimmune haemolytic anemia, Addi- 25 maceutical compositions suitable for administration to son’s disease, scleroderma, Goodpasture’s syndrome, humans in order to render the compositions suitable for Guillain-Barre syndrome, Graves’ disease, glomerulone- administration to various animals is well understood, and phritis, psoriasis, pemphigus vulgaris, pemphigoid, sym- the ordinarily skilled veterinary pharmacologist can de- pathetic opthalmia, idiopathic thrombocylopenic purpu- sign and/or perform such modification with merely ordi- ra, idiopathic feucopenia, Siogren’s syndrome, Wegen- 30 nary, if any, experimentation. Subjects to which admin- er’s granulomatosis, poly/dermatomyositis or systemic istration of the pharmaceutical compositions of the in- lupus erythematosus. vention is contemplated include, but are not limited to, [0454] Allergic diseases include, but are not limited to, humans and/or other primates; mammals, including com- eczema, allergic rhinitis or coryza, hay fever, conjuncti- mercially relevant mammals such as cattle, pigs, horses, vitis, asthma, urticaria (hives), topic allergic reactions, 35 sheep, cats, and/or dogs; and/or birds, including com- food allergies, anaphylaxis, atopic dermatitis, hypersen- mercially relevant birds such as chickens, ducks, geese, sitivity reactions, and other allergic conditions. The aller- and/or turkeys. gic reaction may be the result of an immune reaction to [0458] The formulations of the pharmaceutical compo- any allergen including but not limited to, common dust, sitions described herein may be prepared by any method pollen, plants, animal dander, drugs, food allergens, in- 40 known or hereafter developed in the art of pharmaceu- sect venom, viruses, or bacteria. tics. In general, such preparatory methods include the [0455] Inflammatory disease/disorders include, for ex- step of bringing the active ingredient into association with ample, cardiovascular disease, chronic obstructive pul- one or more excipients and/or one or more other acces- monary disease (COPD), bronchiectasis, chronic chole- sory ingredients, and then, if necessary and/or desirable, cystitis, tuberculosis, Hashimoto’s thyroiditis, sepsis, 45 shaping and/or packaging the product into a desired sin- sarcoidosis, silicosis and other pneumoconioses, and an gle- or multi-dose unit. implanted foreign body in a wound, but are not so limited. [0459] A pharmaceutical composition of the invention As used herein, the term "sepsis" refers to a well-recog- may be prepared, packaged, and/or sold in bulk, as a nized clinical syndrome associated with a host’s systemic single unit dose, and/or as a plurality of single unit doses. inflammatory response to microbial invasion. The term 50 As used herein, a "unit dose" is discrete amount of the "sepsis" as used herein refers to a condition that is typ- pharmaceutical composition comprising a predeter- ically signaled by fever or hypothermia, tachycardia, and mined amount of the active ingredient. The amount of tachypnea, and in severe instances can progress to hy- the active ingredient is generally equal to the dosage of potension, organ dysfunction, and even death. the active ingredient which would be administered to a 55 subject and/or a convenient fraction of such a dosage Pharmaceutical Compositions such as, for example, one-half or one-third of such a dos- age. [0456] The present invention provides novel composi- [0460] The relative amounts of the active ingredient,

60 119 EP 2 394 657 A1 120 the pharmaceutically acceptable excipient(s), and/or any [0465] Exemplary granulating and/or dispersing additional ingredients in a pharmaceutical composition agents include, but are not limited to, potato starch, corn of the invention will vary, depending upon the identity, starch, tapioca starch, sodium starch glycolate, clays, size, and/or condition of the subject treated and further alginic acid, guar gum, citrus pulp, agar, bentonite, cel- depending upon the route by which the composition is to 5 lulose and wood products, natural sponge, cation-ex- be administered. By way of example, the composition change resins, calcium carbonate, silicates, sodium car- may comprise between 0.1 % and 100% (w/w) active bonate, cross-linked poly(vinylpyrrolidone) (crospovi- ingredient. done), sodium carboxymethyl starch (sodium starch gly- [0461] Pharmaceutical formulations of the present in- colate), carboxymethyl cellulose, cross-linked sodium vention may additionally comprise a pharmaceutically 10 carboxymethyl cellulose (croscarmellose), methylcellu- acceptable excipient, which, as used herein, includes lose, pregelatinized starch (starch 1500), microcrystal- any and all solvents, dispersion media, diluents, or other line starch, water insoluble starch, calcium carboxyme- liquid vehicles, dispersion or suspension aids, surface thyl cellulose, magnesium aluminum silicate (Veegum), active agents, isotonic agents, thickening or emulsifying sodium lauryl sulfate, quaternary ammonium com- agents, preservatives, solid binders, lubricants and the 15 pounds, etc., and combinations thereof. like, as suited to the particular dosage form desired. Rem- [0466] Exemplary surface active agents and/or emul- ington’s The Science and Practice of Pharmacy, 21st sifiers include, but are not limited to, natural emulsifiers Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, (e.g. acacia, agar, alginic acid, sodium alginate, traga- Baltimore, MD, 2006; incorporated herein by reference) canth, chondrux, cholesterol, xanthan, pectin, gelatin, discloses various excipients used in formulating pharma- 20 egg yolk, casein, wool fat, cholesterol, wax, and lecithin), ceutical compositions and known techniques for the colloidal clays (e.g. bentonite [aluminum silicate] and preparation thereof. Except insofar as any conventional Veegum [magnesium aluminum silicate]), long chain excipient is incompatible with a substance or its deriva- amino acid derivatives, high molecular weight alcohols tives, such as by producing any undesirable biological (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin effect or otherwise interacting in a deleterious manner 25 monostearate, ethylene glycol distearate, glyceryl mon- with any other component(s) of the pharmaceutical com- ostearate, and propylene glycol monostearate, polyvinyl position, its use is contemplated to be within the scope alcohol), carbomers (e.g. carboxy polymethylene, poly- of this invention. acrylic acid, acrylic acid polymer, and carboxyvinyl pol- [0462] In some embodiments, the pharmaceutically ymer), carrageenan, cellulosic derivatives (e.g. car- acceptable excipient is at least 95%, 96%, 97%, 98%, 30 boxymethylcellulose sodium, powdered cellulose, hy- 99%, or 100% pure. In some embodiments, the excipient droxymethyl cellulose, hydroxypropyl cellulose, hydrox- is approved for use in humans and for veterinary use. In ypropyl methylcellulose, methylcellulose), sorbitan fatty some embodiments, the excipient is approved by United acid esters (e.g. polyoxyethylene sorbitan monolaurate States Food and Drug Administration. In some embodi- [Tween®20], polyoxyethylene sorbitan [Tween®60], ments, the excipient is pharmaceutical grade. In some 35 polyoxyethylene sorbitan monooleate [Tween®80], sorb- embodiments, the excipient meets the standards of the itan monopalmitate [Span®40], sorbitan monostearate United States Pharmacopoeia (USP), the European [Span®60], sorbitan tristearate [Span®65], glyceryl mo- Pharmacopoeia (EP), the British Pharmacopoeia, and/or nooleate, sorbitan monooleate [Span®80]), polyoxyeth- the International Pharmacopoeia. ylene esters (e.g. polyoxyethylene monostearate [0463] Pharmaceutically acceptable excipients used 40 [Myrj®45], polyoxyethylene hydrogenated castor oil, pol- in the manufacture of pharmaceutical compositions in- yethoxylated castor oil, polyoxymethylene stearate, and clude, but are not limited to, inert diluents, dispersing Solutol), sucrose fatty acid esters, polyethylene glycol and/or granulating agents, surface active agents and/or fatty acid esters (e.g. Cremophor®), polyoxyethylene emulsifiers, disintegrating agents, binding agents, pre- ethers, (e.g. polyoxyethylene lauryl ether [Brij®30]), poly servatives, buffering agents, lubricating agents, and/or 45 (vinyl-pyrrolidone), diethylene glycol monolaurate, trieth- oils. Such excipients may optionally be included in the anolamine oleate, sodium oleate, potassium oleate, ethyl inventive formulations. Excipients such as cocoa butter oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, and suppository waxes, coloring agents, coating agents, Pluronic F 68, Poloxamer 188, cetrimonium bromide, ce- sweetening, flavoring, and perfuming agents can be tylpyridinium chloride, benzalkonium chloride, docusate present in the composition, according to the judgment of 50 sodium, etc. and/or combinations thereof. the formulator. [0467] Exemplary binding agents include, but are not [0464] Exemplary diluents include, but are not limited limited to, starch (e.g. cornstarch and starch paste); gel- to, calcium carbonate, sodium carbonate, calcium phos- atin; sugars (e.g. sucrose, glucose, dextrose, dextrin, phate, dicalcium phosphate, calcium sulfate, calcium hy- molasses, lactose, lactitol, mannitol,); natural and syn- drogen phosphate, sodium phosphate lactose, sucrose, 55 thetic gums (e.g. acacia, sodium alginate, extract of Irish cellulose, microcrystalline cellulose, kaolin, mannitol, moss, panwar gum, ghatti gum, mucilage of isapol husks, sorbitol, inositol, sodium chloride, dry starch, cornstarch, carboxymethylcellulose, methylcellulose, ethylcellulose, powdered sugar, etc., and combinations thereof hydroxyethylcellulose, hydroxypropyl cellulose, hydrox-

61 121 EP 2 394 657 A1 122 ypropyl methylcellulose, microcrystalline cellulose, cel- cium phosphate, calcium hydroxide phosphate, potassi- lulose acetate, poly(vinylpyrrolidone), magnesium alumi- um acetate, potassium chloride, potassium gluconate, num silicate (Veegum), and larch arabogalactan); algi- potassium mixtures, dibasic potassium phosphate, nates; polyethylene oxide; polyethylene glycol; inorganic monobasic potassium phosphate, potassium phosphate calcium salts; silicic acid; polymethacrylates; waxes; wa- 5 mixtures, sodium acetate, sodium bicarbonate, sodium ter; alcohol; etc.; and combinations thereof. chloride, sodium citrate, sodium lactate, dibasic sodium [0468] Exemplary preservatives may include antioxi- phosphate, monobasic sodium phosphate, sodium phos- dants, chelating agents, antimicrobial preservatives, an- phate mixtures, tromethamine, magnesium hydroxide, tifungal preservatives, alcohol preservatives, acidic pre- aluminum hydroxide, alginic acid, pyrogen-free water, servatives, and other preservatives. Exemplary antioxi- 10 isotonic saline, Ringer’s solution, ethyl alcohol, etc., and dants include, but are not limited to, alpha tocopherol, combinations thereof. ascorbic acid, acorbyl palmitate, butylated hydroxy- [0470] Exemplary lubricating agents include, but are anisole, butylated hydroxytoluene, monothioglycerol, po- not limited to, magnesium stearate, calcium stearate, tassium metabisulfite, propionic acid, propyl gallate, so- stearic acid, silica, talc, malt, glyceryl behanate, hydro- dium ascorbate, sodium bisulfite, sodium metabisulfite, 15 genated vegetable oils, polyethylene glycol, sodium ben- and sodium sulfite. Exemplary chelating agents include zoate, sodium acetate, sodium chloride, leucine, mag- ethylenediaminetetraacetic acid (EDTA), citric acid nesium lauryl sulfate, sodium lauryl sulfate, etc., and monohydrate, disodium edetate, dipotassium edetate, combinations thereof. edetic acid, fumaric acid, malic acid, phosphoric acid, [0471] Exemplary oils include, but are not limited to, sodium edetate, tartaric acid, and trisodium edetate. Ex- 20 almond, apricot kernel, avocado, babassu, bergamot, emplary antimicrobial preservatives include, but are not black current seed, borage, cade, camomile, canola, car- limited to, benzalkonium chloride, benzethonium chlo- away, carnauba, castor, cinnamon, cocoa butter, coco- ride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium nut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, chloride, chlorhexidine, chlorobutanol, chlorocresol, evening primrose, fish, flaxseed, geraniol, gourd, grape chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, 25 seed, hazel nut, hyssop, isopropyl myristate, jojoba, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, kukui nut, lavandin, lavender, lemon, litsea cubeba, phenylmercuric nitrate, propylene glycol, and thimerosal. macademia nut, mallow, mango seed, meadowfoam Exemplary antifungal preservatives include, but are not seed, mink, nutmeg, olive, orange, orange roughy, palm, limited to, butyl paraben, methyl paraben, ethyl paraben, palm kernel, peach kernel, peanut, poppy seed, pumpkin propyl paraben, benzoic acid, hydroxybenzoic acid, po- 30 seed, rapeseed, rice bran, rosemary, safflower, sandal- tassium benzoate, potassium sorbate, sodium benzoate, wood, sasquana, savoury, sea buckthorn, sesame, shea sodium propionate, and sorbic acid. Exemplary alcohol butter, silicone, soybean, sunflower, tea tree, thistle, preservatives include, but are not limited to, ethanol, pol- tsubaki, vetiver, walnut, and wheat germ oils. Exemplary yethylene glycol, phenol, phenolic compounds, bisphe- oils include, but are not limited to, butyl stearate, caprylic nol, chlorobutanol, hydroxybenzoate, and phenylethyl al- 35 triglyceride, capric triglyceride, cyclomethicone, diethyl cohol. Exemplary acidic preservatives include, but are sebacate, dimethicone 360, isopropyl myristate, mineral not limited to, vitamin A, vitamin C, vitamin E, beta-car- oil, octyldodecanol, oleyl alcohol, silicone oil, and com- otene, citric acid, acetic acid, dehydroacetic acid, ascor- binations thereof. bic acid, sorbic acid, and phytic acid. Other preservatives [0472] Liquid dosage forms for oral and parenteral ad- include, but are not limited to, tocopherol, tocopherol ac- 40 ministration include, but are not limited to, pharmaceuti- etate, deteroxime mesylate, cetrimide, butylated hy- cally acceptable emulsions, microemulsions, solutions, droxyanisol (BHA), butylated hydroxytoluened (BHT), suspensions, syrups and elixirs. In addition to the active ethylenediamine, sodium lauryl sulfate (SLS), sodium ingredients, the liquid dosage forms may comprise inert lauryl ether sulfate (SLES), sodium bisulfite, sodium me- diluents commonly used in the art such as, for example, tabisulfite, potassium sulfite, potassium metabisulfite, 45 water or other solvents, solubilizing agents and emulsi- Glydant Plus®, Phenonip® , methylparaben, Germall fiers such as ethyl alcohol, isopropyl alcohol, ethyl car- 115, Germaben II, Neolone™, Kathon™, and Euxyl®.In bonate, ethyl acetate, benzyl alcohol, benzyl benzoate, certain embodiments, the preservative is an antioxidant. propylene glycol, 1,3-butylene glycol, dimethylforma- In other embodiments, the preservative is a chelating mide, oils (in particular, cottonseed, groundnut, corn, agent. 50 germ, olive, castor, and sesame oils), glycerol, tetrahy- [0469] Exemplary buffering agents include, but are not drofurfuryl alcohol, polyethylene glycols and fatty acid limited to, citrate buffer solutions, acetate buffer solu- esters of sorbitan, and mixtures thereof. Besides inert tions, phosphate buffer solutions, ammonium chloride, diluents, the oral compositions can include adjuvants calcium carbonate, calcium chloride, calcium citrate, cal- such as wetting agents, emulsifying and suspending cium glubionate, calcium gluceptate, calcium gluconate, 55 agents, sweetening, flavoring, and perfuming agents. In D-gluconic acid, calcium glycerophosphate, calcium lac- certain embodiments for parenteral administration, vac- tate, propanoic acid, calcium levulinate, pentanoic acid, cine nanocarriers of the invention are mixed with solubi- dibasic calcium phosphate, phosphoric acid, tribasic cal- lizing agents such as Cremophor®, alcohols, oils, modi-

62 123 EP 2 394 657 A1 124 fied oils, glycols, polysorbates, cyclodextrins, polymers, as kaolin and bentonite clay, and i) lubricants such as and combinations thereof. talc, calcium stearate, magnesium stearate, solid poly- [0473] Injectable formulations, for example, sterile in- ethylene glycols, sodium lauryl sulfate, and mixtures jectable aqueous or oleaginous suspensions may be for- thereof. In the case of capsules, tablets and pills, the mulated according to the known art using suitable dis- 5 dosage form may comprise buffering agents. persing or wetting agents and suspending agents. A ster- [0478] Solid compositions of a similar type may be em- ile injectable preparation may be a sterile injectable so- ployed as fillers in soft and hard-filled gelatin capsules lution, suspension or emulsion in a nontoxic parenterally using such excipients as lactose or milk sugar as well as acceptable diluent or solvent, for example, as a solution high molecular weight polyethylene glycols and the like. in 1,3-butanediol. Among the acceptable vehicles and 10 Solid dosage forms of tablets, dragees, capsules, pills, solvents that may be employed are water, Ringer’s so- and granules can be prepared with coatings and shells lution, U.S.P. and isotonic sodium chloride solution. In such as enteric coatings and other coatings well known addition, sterile, fixed oils are conventionally employed in the pharmaceutical formulating art. They may option- as a solvent or suspending medium. For this purpose ally comprise opacifying agents and can be of a compo- any bland fixed oil can be employed including synthetic 15 sition that they release the active ingredient(s) only, or mono- or diglycerides. In addition, fatty acids such as preferentially, in a certain part of the intestinal tract, op- oleic acid are used in the preparation of injectables. tionally, in a delayed manner. Examples of embedding [0474] Injectable formulations can be sterilized, for ex- compositions which can be used include polymeric sub- ample, by filtration through a bacterial-retaining filter, or stances and waxes. Solid compositions of a similar type by incorporating sterilizing agents in the form of sterile 20 may be employed as fillers in soft and hard-filled gelatin solid compositions which can be dissolved or dispersed capsules using such excipients as lactose or milk sugar in sterile water or other sterile injectable medium prior to as well as high molecular weight polethylene glycols and use. the like. [0475] In order to prolong the effect of a drug, it is often [0479] Active ingredients can be in micro-encapsulat- desirable to slow the absorption of the drug from subcu- 25 ed form with one or more excipients as noted above. taneous or intramuscular injection. This may be accom- Solid dosage forms of tablets, dragees, capsules, pills, plished by the use of a liquid suspension of crystalline or and granules can be prepared with coatings and shells amorphous material with poor water solubility. The rate such as enteric coatings, release controlling coatings and of absorption of the drug then depends upon its rate of other coatings well known in the pharmaceutical formu- dissolution which, in turn, may depend upon crystal size 30 lating art. In such solid dosage forms, active ingredient and crystalline form. Alternatively, delayed absorption of may be admixed with at least one inert diluent such as a parenterally administered drug form may be accom- sucrose, lactose or starch. Such dosage forms may com- plished by dissolving or suspending the drug in an oil prise, as is normal practice, additional substances other vehicle. than inert diluents, e.g., tableting lubricants and other [0476] Compositions for rectal or vaginal administra- 35 tableting aids such a magnesium stearate and microc- tion are typically suppositories which can be prepared by rystalline cellulose. In the case of capsules, tablets and mixing vaccine nanocarriers of this invention with suita- pills, dosage forms may comprise buffering agents. They ble nonirritating excipients such as cocoa butter, poly- may optionally comprise opacifying agents and can be ethylene glycol or a suppository wax which are solid at of a composition that they release the active ingredient ambient temperature but liquid at body temperature and 40 (s) only, or preferentially, in a certain part of the intestinal therefore melt in the rectum or vaginal cavity and release tract, optionally, in a delayed manner. Examples of em- active ingredient. bedding compositions which can be used include poly- [0477] Solid dosage forms for oral administration in- meric substances and waxes. clude capsules, tablets, pills, powders, and granules. In [0480] Dosage forms for topical and/or transdermal such solid dosage forms, the active ingredient is mixed 45 administration of vaccine nanocarriers in accordance with at least one inert, pharmaceutically acceptable ex- with the invention may include ointments, pastes, cipient such as sodium citrate or dicalcium phosphate creams, lotions, gels, powders, solutions, sprays, inha- and/or a) fillers or extenders such as starches, lactose, lants and/or patches. Generally, active ingredient is ad- sucrose, glucose, mannitol, and silicic acid, b) binders mixed under sterile conditions with a pharmaceutically such as, for example, carboxymethylcellulose, alginates, 50 acceptable excipient and/or any needed preservatives gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) and/or buffers as may be required. Additionally, the humectants such as glycerol, d) disintegrating agents present invention contemplates the use of transdermal such as agar, calcium carbonate, potato or tapioca patches, which often have the added advantage of pro- starch, alginic acid, certain silicates, and sodium carbon- viding controlled delivery of an active ingredient to the ate, e) solution retarding agents such as paraffin, f) ab- 55 body. Such dosage forms may be prepared, for example, sorption accelerators such as quaternary ammonium by dissolving and/or dispensing the active ingredient in compounds, g) wetting agents such as, for example, cetyl the proper medium. Alternatively or additionally, the rate alcohol and glycerol monostearate, h) absorbents such may be controlled by either providing a rate controlling

63 125 EP 2 394 657 A1 126 membrane and/or by dispersing the active ingredient in 90% of the particles by number have a diameter less than a polymer matrix and/or gel. 6 mm. Dry powder compositions may include a solid fine [0481] Suitable devices for use in delivering intrader- powder diluent such as sugar and are conveniently pro- mal pharmaceutical compositions described herein in- vided in a unit dose form. clude short needle devices such as those described in 5 [0484] Low boiling propellants generally include liquid U.S. Patents 4,886,499; 5,190,521; 5,328,483; propellants having a boiling point of below 65˚F at atmos- 5,527,288; 4,270,537; 5,015,235; 5,141,496; and pheric pressure. Generally the propellant may constitute 5,417,662. Intradermal compositions may be adminis- 50% to 99.9% (w/w) of the composition, and the active tered by devices which limit the effective penetration ingredient may constitute 0.1% to 20% (w/w) of the com- length of a needle into the skin, such as those described 10 position. The propellant may further comprise additional in PCT publication WO 99/34850 and functional equiva- ingredients such as a liquid non-ionic and/or solid anionic lents thereof. Jet injection devices which deliver liquid surfactant and/or a solid diluent (which may have a par- vaccines to the dermis via a liquid jet injector and/or via ticle size of the same order as particles comprising the a needle which pierces the stratum corneum and pro- active ingredient). duces a jet which reaches the dermis are suitable. Jet 15 [0485] Pharmaceutical compositions of the invention injection devices are described, for example, in U.S. Pat- formulated for pulmonary delivery may provide the active ents 5,480,381; 5,599,302; 5,334,144; 5,993,412; ingredient in the form of droplets of a solution and/or sus- 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; pension. Such formulations may be prepared, packaged, 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; and/or sold as aqueous and/or dilute alcoholic solutions 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; 20 and/or suspensions, optionally sterile, comprising the ac- and PCT publications WO 97/37705 and WO 97/13537. tive ingredient, and may conveniently be administered Ballistic powder/particle delivery devices which use com- using any nebulization and/or atomization device. Such pressed gas to accelerate vaccine in powder form formulations may further comprise one or more additional through the outer layers of the skin to the dermis are ingredients including, but not limited to, a flavoring agent suitable. Alternatively or additionally, conventional sy- 25 such as saccharin sodium, a volatile oil, a buffering agent, ringes may be used in the classical mantoux method of a surface active agent, and/or a preservative such as intradermal administration. methylhydroxybenzoate. The droplets provided by this [0482] Formulations suitable for topical administration route of administration may have an average diameter include, but are not limited to, liquid and/or semi liquid in the range from about 0.1 mm to about 200 mm. preparations such as liniments, lotions, oil in water and/or 30 [0486] The formulations described herein as being water in oil emulsions such as creams, ointments and/or useful for pulmonary delivery are useful for intranasal pastes, and/or solutions and/or suspensions. Topically- delivery of a pharmaceutical composition of the inven- administrable formulations may, for example, comprise tion. Another formulation suitable for intranasal adminis- from about 1% to about 10% (w/w) active ingredient, al- tration is a coarse powder comprising the active ingredi- though the concentration of the active ingredient may be 35 ent and having an average particle from about 0.2 mmto as high as the solubility limit of the active ingredient in about 500 mm. Such a formulation is administered in the the solvent. Formulations for topical administration may manner in which snuff is taken, i.e. by rapid inhalation further comprise one or more of the additional ingredients through the nasal passage from a container of the powder described herein. held close to the nares. [0483] A pharmaceutical composition of the invention 40 [0487] Formulations suitable for nasal administration may be prepared, packaged, and/or sold in a formulation may, for example, comprise from about as little as 0.1% suitable for pulmonary administration via the buccal cav- (w/w) and as much as 100% (w/w) of the active ingredi- ity. Such a formulation may comprise dry particles which ent, and may comprise one or more of the additional in- comprise the active ingredient and which have a diameter gredients described herein. A pharmaceutical composi- in the range from about 0.5 mm to about 7 mm or from 45 tion of the invention may be prepared, packaged, and/or about 1 mm to about 6 mm. Such compositions are con- sold in a formulation suitable for buccal administration. veniently in the form of dry powders for administration Such formulations may, for example, be in the form of using a device comprising a dry powder reservoir to tablets and/or lozenges made using conventional meth- which a stream of propellant may be directed to disperse ods, and may, for example, 0.1 % to 20% (w/w) active the powder and/or using a self propelling solvent/powder 50 ingredient, the balance comprising an orally dissolvable dispensing container such as a device comprising the and/or degradable composition and, optionally, one or active ingredient dissolved and/or suspended in a low- more of the additional ingredients described herein. Al- boiling propellant in a sealed container. Such powders ternately, formulations suitable for buccal administration comprise particles wherein at least 98% of the particles may comprise a powder and/or an aerosolized and/or by weight have a diameter greater than 0.5 mm and at 55 atomized solution and/or suspension comprising the ac- least 95% of the particles by number have a diameter tive ingredient. Such powdered, aerosolized, and/or aer- less than 7 mm. Alternatively, at least 95% of the particles osolized formulations, when dispersed, may have an av- by weight have a diameter greater than 1 mm and at least erage particle and/or droplet size in the range from about

64 127 EP 2 394 657 A1 128

0.1 mm to about 200 mm, and may further comprise one invention are typically formulated in dosage unit form for or more of the additional ingredients described herein. ease of administration and uniformity of dosage. It will [0488] A pharmaceutical composition of the invention be understood, however, that the total daily usage of the may be prepared, packaged, and/or sold in a formulation compositions of the present invention will be decided by suitable for ophthalmic administration. Such formulations 5 the attending physician within the scope of sound medical may, for example, be in the form of eye drops including, judgment. The specific therapeutically effective dose lev- for example, a 0.1%/1.0% (w/w) solution and/or suspen- el for any particular subject or organism will depend upon sion of the active ingredient in an aqueous or oily liquid a variety of factors including the disorder being treated excipient. Such drops may further comprise buffering and the severity of the disorder; the activity of the specific agents, salts, and/or one or more other of the additional 10 active ingredient employed; the specific composition em- ingredients described herein. Other opthalmically-ad- ployed; the age, body weight, general health, sex and ministrable formulations which are useful include those diet of the subject; the time of administration, route of which comprise the active ingredient in microcrystalline administration, and rate of excretion of the specific active form and/or in a liposomal preparation. Ear drops and/or ingredient employed; the duration of the treatment; drugs eye drops are contemplated as being within the scope 15 used in combination or coincidental with the specific ac- of this invention. tive ingredient employed; and like factors well known in [0489] General considerations in the formulation the medical arts. and/or manufacture of pharmaceutical agents may be [0492] The pharmaceutical compositions of the found, for example, in Remington: The Science and Prac- present invention may be administered by any route. In tice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 20 some embodiments, the pharmaceutical compositions of 2005. the present invention are administered by a variety of routes, including oral, intravenous, intramuscular, in- Administration traarterial, intramedullary, intrathecal, subcutaneous, in- traventricular, transdermal, interdermal, rectal, intravag- [0490] In some embodiments, a therapeutically effec- 25 inal, intraperitoneal, topical (as by powders, ointments, tive amount of an inventive vaccine nanocarrier compo- creams, and/or drops), transdermal, mucosal, nasal, sition is delivered to a patient and/or animal prior to, si- buccal, enteral, sublingual; by intratracheal instillation, multaneously with, and/or after diagnosis with a disease, bronchial instillation, and/or inhalation; and/or as an oral disorder, and/or condition. In some embodiments, a ther- spray, nasal spray, and/or aerosol. Specifically contem- apeutic amount of an inventive composition is delivered 30 plated routes are oral administration, intravenous injec- to a patient and/or animal prior to, simultaneously with, tion, intramuscular injection, and/or subcutaneous injec- and/or after onset of symptoms of a disease, disorder, tion. In some embodiments, inventive vaccine nanocar- and/or condition. In some embodiments, the amount of riers are administered parenterally. In some embodi- a vaccine nanocarrier is sufficient to treat, alleviate, amel- ments, inventive vaccine nanocarriers are administered iorate, relieve, delay onset of, inhibit progression of, re- 35 intravenously. In some embodiments, inventive vaccine duce severity of, and/or reduce incidence of one or more nanocarriers are administered orally. symptoms or features of the disease, disorder, and/or [0493] In general the most appropriate route of admin- condition. In some embodiments, the amount of a vac- istration will depend upon a variety of factors including cine nanocarrier is sufficient to elicit a detectable immune the nature of the vaccine nanocarrier (e.g., its stability in response in a subject. In some embodiments, the amount 40 the environment of the gastrointestinal tract), the condi- of a vaccine nanocarrier is sufficient to elicit a detectable tion of the subject (e.g., whether the subject is able to antibody response in a subject. In some embodiments, tolerate oral administration), etc. The invention encom- the amount of a vaccine nanocarrier is sufficient to elicit passes the delivery of the inventive pharmaceutical com- a detectable T cell response in a subject. In some em- position by any appropriate route taking into considera- bodiments, the amount of a vaccine nanocarrier is suffi- 45 tion likely advances in the sciences of drug delivery. cient to elicit a detectable antibody and T cell response [0494] In certain embodiments, the vaccine nanocar- in a subject. In some embodiments, an advantage of the riers of the invention may be administered in amounts nanocarriers provided is that the nanocarriers can elicit ranging from about 0.001 mg/kg to about 100 mg/kg, from potent responses with a much lower concentration of an- about 0.01 mg/kg to about 50 mg/kg, from about 0.1 tigen than required with a conventional vaccine. 50 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about [0491] The compositions, according to the method of 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from the present invention, may be administered using any about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg amount and any route of administration effective for treat- to about 25 mg/kg, of subject body weight per day, one ment. The exact amount required will vary from subject or more times a day, to obtain the desired therapeutic to subject, depending on the species, age, and general 55 effect. The desired dosage may be delivered three times condition of the subject, the severity of the infection, the a day, two times a day, once a day, every other day, particular composition, its mode of administration, its every third day, every week, every two weeks, every mode of activity, and the like. The compositions of the three weeks, or every four weeks. In certain embodi-

65 129 EP 2 394 657 A1 130 ments, the desired dosage may be delivered using mul- regimen will take into account compatibility of the desired tiple administrations (e.g., two, three, four, five, six, sev- therapeutics and/or procedures and/or the desired ther- en, eight, nine, ten, eleven, twelve, thirteen, fourteen, or apeutic effect to be achieved. It will be appreciated that more administrations). the therapies employed may achieve a desired effect for [0495] In some embodiments, the present invention 5 the same disorder (for example, an inventive vaccine na- encompasses "therapeutic cocktails" comprising popu- nocarrier may be administered concurrently with another lations of inventive vaccine nanocarriers. In some em- therapeutic agent used to treat the same disorder), bodiments, all of the vaccine nanocarriers within a pop- and/or they may achieve different effects (e.g., control of ulation of vaccine nanocarriers comprise a single species any adverse effects attributed to the vaccine nanocarri- of targeting moiety which can bind to multiple targets (e.g. 10 er). In some embodiments, vaccine nanocarriers of the can bind to both SCS-Mph and FDCs). In some embod- invention are administered with a second therapeutic iments, different vaccine nanocarriers within a population agent that is approved by the U.S. Food and Drug Ad- of vaccine nanocarriers comprise different targeting moi- ministration. eties, and all of the different targeting moieties can bind [0499] In will further be appreciated that therapeutical- to the same target. In some embodiments, different vac- 15 ly active agents utilized in combination may be adminis- cine nanocarriers comprise different targeting moieties, tered together in a single composition or administered and all of the different targeting moieties can bind to dif- separately in different compositions. ferent targets. In some embodiments, such different tar- [0500] In general, it is expected that agents utilized in gets may be associated with the same cell type. In some combination with be utilized at levels that do not exceed embodiments, such different targets may be associated 20 the levels at which they are utilized individually. In some with different cell types. embodiments, the levels utilized in combination will be lower than those utilized individually. Combination Therapies [0501] In some embodiments, inventive vaccine nano- carriers may be administered in combination with an [0496] It will be appreciated that vaccine nanocarriers 25 agent, including, for example, therapeutic, diagnostic, and pharmaceutical compositions of the present inven- and/or prophylactic agents. Exemplary agents to be de- tion can be employed in combination therapies. The par- livered in accordance with the present invention include, ticular combination of therapies (therapeutics or proce- but are not limited to, small molecules, organometallic dures) to employ in a combination regimen will take into compounds, nucleic acids, proteins (including multimeric account compatibility of the desired therapeutics and/or 30 proteins, protein complexes, etc.), peptides, lipids, car- procedures and the desired therapeutic effect to be bohydrates, hormones, metals, radioactive elements achieved. It will be appreciated that the therapies em- and compounds, drugs, vaccines, immunological ployed may achieve a desired effect for the same pur- agents, etc., and/or combinations thereof. pose (for example, an inventive vaccine nanocarrier use- [0502] In certain embodiments, vaccine nanocarriers ful for vaccinating against a particular type of microbial 35 which delay the onset and/or progression of a particular infection may be administered concurrently with another microbial infection may be administered in combination agent useful for treating the same microbial infection), or with one or more additional therapeutic agents which they may achieve different effects (e.g., control of any treat the symptoms of microbial infection. To give but one adverse effects attributed to the vaccine nanocarrier). example, upon exposure to rabies virus, nanocarriers [0497] In some embodiments, pharmaceutical compo- 40 comprising immunomodulatory agents useful for vacci- sitions of the present invention may be administered ei- nation against rabies virus may be administered in com- ther alone or in combination with one or more other ther- bination with one or more therapeutic agents useful for apeutic agents. By "in combination with," it is not intended treatment of symptoms of rabies virus (e.g. antipsychotic to imply that the agents must be administered at the same agents useful for treatment of paranoia that is sympto- time and/or formulated for delivery together, although 45 matic of rabies virus infection). these methods of delivery are within the scope of the [0503] In some embodiments, pharmaceutical compo- invention. The compositions can be administered con- sitions comprising inventive vaccine nanocarriers com- currently with, prior to, or subsequent to, one or more prise less than 50% by weight, less than 40% by weight, other desired therapeutics or medical procedures. In less than 30% by weight, less than 20% by weight, less general, each agent will be administered at a dose and/or 50 than 15% by weight, less than 10% by weight, less than on a time schedule determined for that agent. Addition- 5% by weight, less than 1% by weight, or less than 0.5% ally, the invention encompasses the delivery of the in- by weight of an agent to be delivered. ventive pharmaceutical compositions in combination [0504] In some embodiments, vaccine nanocarriers with agents that may improve their bioavailability, reduce are administered in combination with one or more small and/or modify their metabolism, inhibit their excretion, 55 molecules and/or organic compounds with pharmaceu- and/or modify their distribution within the body. tical activity. In some embodiments, the agent is a clini- [0498] The particular combination of therapies (thera- cally-used drug. In some embodiments, the drug is an peutics and/or procedures) to employ in a combination anti-cancer agent, antibiotic, anti-viral agent, anti-HIV

66 131 EP 2 394 657 A1 132 agent, anti-parasite agent, anti-protozoal agent, anes- "Protein Targeting Moieties." thetic, anticoagulant, inhibitor of an enzyme, steroidal [0509] In some embodiments, vaccine nanocarriers agent, steroidal or non-steroidal anti-inflammatory agent, are administered in combination with one or more car- antihistamine, immunosuppressant agent, antineoplas- bohydrates, such as a carbohydrate that is associated tic agent, antigen, vaccine, antibody, decongestant, sed- 5 with a protein (e.g. glycoprotein, proteogycan, etc.). A ative, opioid, analgesic, antipyretic, birth control agent, carbohydrate may be natural or synthetic. A carbohy- hormone, prostaglandin, progestational agent, anti-glau- drate may also be a derivatized natural carbohydrate. In coma agent, ophthalmic agent, anti-cholinergic, analge- certain embodiments, a carbohydrate may be a simple sic, anti-depressant, anti-psychotic, neurotoxin, hypnot- or complex sugar. In certain embodiments, a carbohy- ic, tranquilizer, anti-convulsant, muscle relaxant, anti- 10 drate is a monosaccharide, including but not limited to Parkinson agent, anti-spasmodic, muscle contractant, glucose, fructose, galactose, and ribose. In certain em- channel blocker, miotic agent, anti-secretory agent, anti- bodiments, a carbohydrate is a disaccharide, including thrombotic agent, anticoagulant, anti-cholinergic, β- but not limited to lactose, sucrose, maltose, trehalose, adrenergic blocking agent, diuretic, cardiovascular ac- and cellobiose. In certain embodiments, a carbohydrate tive agent, vasoactive agent, vasodilating agent, anti-hy- 15 is a polysaccharide, including but not limited to cellulose, pertensive agent, angiogenic agent, modulators of cell- microcrystalline cellulose, hydroxypropyl methylcellu- extracellular matrix interactions (e.g. cell growth inhibi- lose (HPMC), methylcellulose (MC), dextrose, dextran, tors and anti-adhesion molecules), inhibitors of DNA, glycogen, xanthan gum, gellan gum, starch, and pullulan. RNA, or protein synthesis, etc. In certain embodiments, a carbohydrate is a sugar alco- [0505] In certain embodiments, a small molecule agent 20 hol, including but not limited to mannitol, sorbitol, xylitol, can be any drug. In some embodiments, the drug is one erythritol, malitol, and lactitol. Molecular properties of car- that has already been deemed safe and effective for use bohydrates are described in the section above entitled in humans or animals by the appropriate governmental "Vaccine Nanocarriers Comprising Carbohydrates." agency or regulatory body. For example, drugs approved [0510] In some embodiments, vaccine nanocarriers for human use are listed by the FDA under 21 C.F.R. §§ 25 are administered in combination with one or more lipids, 330.5, 331 through 361, and 440 through 460, incorpo- such as a lipid that is associated with a protein (e.g. li- rated herein by reference; drugs for veterinary use are poprotein). Exemplary lipids that may be used in accord- listed by the FDA under 21 C.F.R. §§ 500 through 589, ance with the present invention include, but are not lim- incorporated herein by reference. All listed drugs are con- ited to, oils, fatty acids, saturated fatty acid, unsaturated sidered acceptable for use in accordance with the 30 fatty acids, essential fatty acids, cis fatty acids, trans fatty present invention. acids, glycerides, monoglycerides, diglycerides, triglyc- [0506] A more complete listing of classes and specific erides, hormones, steroids (e.g., cholesterol, bile acids), drugs suitable for use in the present invention may be vitamins (e.g. vitamin E), phospholipids, sphingolipids, found in Pharmaceutical Drugs: Syntheses, Patents, Ap- and lipoproteins. Molecular properties of lipids are de- plications by Axel Kleemann and Jurgen Engel, Thieme 35 scribed in the section above entitled "Lipid Vaccine Na- Medical Publishing, 1999 and the Merck Index: An En- nocarriers." cyclopedia of Chemicals, Drugs and Biologicals, Ed. by [0511] Those skilled in the art will recognize that this Budavari et al., CRC Press, 1996, both of which are in- is an exemplary, not comprehensive, list of therapeutic, corporated herein by reference. diagnostic, and/or prophylactic agents that can be deliv- [0507] In some embodiments, vaccine nanocarriers 40 ered in combination with the vaccine nanocarriers of the are administered in combination with one or more nucleic present invention. Any therapeutic, diagnostic, and/or acids (e.g. functional RNAs, functional DNAs, etc.) to a prophylactic agent may be administered with vaccine na- specific location such as a tissue, cell, or subcellular lo- nocarriers in accordance with the present invention. cale. For example, inventive vaccine nanocarriers which are used to delay the onset and/or progression of a par- 45 Kits ticular microbial infection may be administered in com- bination with RNAi agents which reduce expression of [0512] The invention provides a variety of kits compris- microbial proteins. Molecular properties of nucleic acids ing one or more of the nanocarriers of the invention. For are described in the section above entitled "Nucleic Acid example, the invention provides a kit comprising an in- Targeting Moieties." 50 ventive vaccine nanocarrier and instructions for use. A [0508] In some embodiments, vaccine nanocarriers kit may comprise multiple different vaccine nanocarriers. are administered in combination with one or more pro- A kit may comprise any of a number of additional com- teins or peptides. In some embodiments, the agent to be ponents or reagents in any combination. All of the various delivered may be a peptide, hormone, erythropoietin, in- combinations are not set forth explicitly but each combi- sulin, cytokine, antigen for vaccination, etc. In some em- 55 nation is included in the scope of the invention. bodiments, the agent to be delivered may be an antibody [0513] According to certain embodiments of the inven- and/or characteristic portion thereof. Molecular proper- tion, a kit may include, for example, (i) a vaccine nano- ties of which are described in the section above entitled carrier comprising at least one immunomodulatory agent,

67 133 EP 2 394 657 A1 134 wherein the at least one immunomodulatory agent is ca- antiviral B cells pable of stimulating both a T cell and B cell response; (ii) instructions for administering the vaccine nanocarrier to Materials and Methods a subject in need thereof. [0514] In certain embodiments, a kit may include, for 5 Method Summary example, (i) a vaccine nanocarrier comprising at least one immunomodulatory agent, wherein the at least one [0518] VSV-IND and VSV-NJ virions were purified immunomodulatory agent is capable of stimulating both from culture supernatants of infected BSRT7 cells and a T cell and B cell response, at least one targeting moiety, used either unmodified or fluorescently labeled with Al- and/or at least one immunomodulatory agent; (ii) instruc- 10 exa-568 (red) or Alexa-488 (green). Fluorescent viruses tions for administering the vaccine nanocarrier to a sub- used for tissue imaging were UV-irradiated to prevent ject in need thereof. generation of non-fluorescent progeny. Fluorescent la- [0515] In certain embodiments, a kit may include, for beling or UV-irradiation of VSV-IND particles did not af- example, (i) at least one immunomodulatory agent, fect their antigenicity or their ability to elicit a calcium flux wherein the at least one immunomodulatory agent is ca- 15 in VI10YEN cells (not shown). Following fluorescent virus pable of stimulating both a T cell and B cell response; (ii) injection into footpads, draining popliteal LNs were har- at least one targeting moiety; (iii) at least one immunos- vested for analysis by electron microscopy or to generate timulatory agent; (iv) a polymeric matrix precursor; (v) frozen sections for immunostaining and confocal micro- lipids and amphiphilic entities; (vi) instructions for assem- scopy. To image adoptively transferred B cells in LNs, bling inventive vaccine nanocarriers from individual com- 20 VI10YEN and wildtype B cells were fluorescently labeled ponents (i)-(v). and co-transferred by i.v. injection into wildtype or mutant [0516] In some embodiments, the kit comprises an in- recipient mice. 18 hours later, when B cells had homed ventive nanocarrier and instructions for mixing. Such kits, to B cell follicles, mice were injected with labeled or un- in some embodiments, also include an immunostimula- labeled VSV in the right footpad. At different time intervals tory agent and/or an immunomodulatory agent (e.g., a B 25 thereafter, the draining popliteal LN was observed by MP- cell, or T cell antigen) The nanocarrier of such kits may IVM or harvested for confocal microscopy or for flow cy- comprise an immunomodulatory agent (e.g., a T cell an- tometry to analyze the activation state of virus-specific tigen, such as a universal T cell antigen) and/or a target- and control B cells. In some experiments, macrophages ing moiety. The T cell antigen and/or the targeting moiety in the popliteal LN were depleted by sc injections of CLL, may be on the surface of the nanocarrier. In some em- 30 and animals were used for experiments 7-10 days later. bodiments, the immunomodulatory agent and the anti- MP-IVM, electron microscopy, immunohistochemistry gen are the same. In some embodiments, they are dif- and flow cytometry for various markers was performed ferent. on LNs with and without prior CLL treatment. VSV prop- [0517] Kits typically include instructions for use of in- agation from the footpad injection site to the blood and ventive vaccine nanocarriers. Instructions may, for ex- 35 other organs was assessed by injecting a defined amount ample, comprise protocols and/or describe conditions for of live VSV into footpads followed by tissue harvest at production of vaccine nanocarriers, administration of two hours or six hours after VSV injection. To measure vaccine nanocarriers to a subject in need thereof, etc. viral titers, tissues were homogenized and used in plaque Kits generally include one or more vessels or containers assays. Some viral propagation experiments were per- so that some or all of the individual components and re- 40 formed after cannulation of the thoracic duct. agents may be separately housed. Kits may also include a means for enclosing individual containers in relatively Mice and Antibodies close confinement for commercial sale, e.g., a plastic box, in which instructions, packaging materials such as [0519] C57BL/6 and BALB/c mice were purchased styrofoam, etc., may be enclosed. An identifier, e.g., a 45 from Taconic Farms (Germantown, NY). VI10YEN bar code, radio frequency identification (ID) tag, etc., may (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA, be present in or on the kit or in or one or more of the 100:12883; incorporated herein by reference), C3-/- vessels or containers included in the kit. An identifier can (Wessels et al., 1995, Proc. Natl. Acad. Sci., USA, 92: be used, e.g., to uniquely identify the kit for purposes of 11490; incorporated herein by reference), MHCII-EGFP quality control, inventory control, tracking, movement be- 50 (Boes et al., 2002, Nature, 418: 983; incorporated herein tween workstations, etc. by reference), Act-EGFP (Wright et al., 2001, Blood, 97: 2278), and DH-LMP2A mice (Casola et al., 2004, Nat. Exemplification Immunol., 5:317; incorporated herein by reference) were bred in barrier animal facilities at Harvard Medical School Example 1: Subcapsular sinus macrophages in lymph 55 and the Immune Disease Institute (IDI). Radiation chi- nodes clear lymph-borne viruses and present them to meras were generated by irradiation of Act(EGFP) mice with two doses of 650 rad and reconstitution with C57BL/ 6 bone marrow, and were allowed to reconstitute for 8

68 135 EP 2 394 657 A1 136 weeks prior to use. In some experiments, SCS macro- gated at a MOI of 0.01 on BSRT7 cells. Supernatants of phages were depleted by footpad injections of 30 ml infected cells were cleared from cell debris by centrifu- clodronate liposomes (CLL), 7-10 days prior to the ex- gation at 2000 x g, filtered through 0.45 mm sterile filters periment. and subjected to ultracentrifugation at 40,000 x g for 90 [0520] Clodronate was a gift of Roche Diagnostics 5 minutes. Pellets were resuspended in PBS and purified GmbH, Mannheim, Germany. Other reagents for prepa- by ultracentrifugation (157,000 x g, 60 minutes) through ration of liposomes were: Phosphatidylcholine (LIPOID a cushion of 10% sucrose in NTE (0.5 mM NaCl, 10 mM E PC, Lipoid GmbH, Ludwigshafen, Germany) and cho- Tris-HClpH 7.5, 5 mM EDTA pH 8). After resuspension lesterol (Sigma-Aldrich). in PBS overnight, virus protein was quantified by BCA [0521] Mice were housed under specific pathogen- 10 assay (Pierce), and infectivity was quantified by plaque free and anti-viral antibody-free conditions in accordance assay. Some batches were labeled with carboxylic acid with National Institutes of Health guidelines. All experi- succinimidyl esters of AlexaFluor-488 or AlexaFluor-568 mental animal procedures were approved by the Institu- (Invitrogen-Molecular Probes) at a 104-105-fold molar tional Animal Committees of Harvard Medical School and excess of Alexa dye over virus particles. Unconjugated the IDI. 15 dye was removed by ultracentrifugation through 10% su- [0522] Antibodies were purchased from BD Bioscienc- crose in NTE, pellets resuspended in PBS and stored es (San Jose, CA), except anti-B220-Alexa647 (Invitro- frozen. Infectivity of VSV preparations was quantified by gen-Caltag), anti-LYVE-1 (Millipore-Upstate), goat-anti- plaque assay on green monkey kidney cells (Vero). VSV rabbit-APC (Invitrogen), goat-anti-GFP-FITC (Rock- titers from organs of infected mice were determined sim- land), anti-FITC-Alexa488 (Invitrogen), and Fab anti- 20 ilarly, after homogenization of the organs with a Potter- IgM-FITC (Jackson Immunoresearch). The following an- Elvejhem homogenizer. When necessary, during viral tibodies were purchased from AbD-Serotec: anti-CD68- preparation, the approximately 4 ml supernatants from Alexa647, anti-CD11b-Alexa647, F4/80-Alexa647, anti- the 157,000 x g ultracentrifugation were collected and CD169-FITC (3D6). The anti-idiotypic antibody 35.61 for concentrated with a 10,000 MWCO Amicon Ultra (Milli- detection of the VI10 BCR in VI10YEN mice (Hangartner 25 pore). In order to account for residual infectivity in con- et al., 2003, Proc. Natl. Acad. Sci., USA, 100:12883; in- centrated supernatants, VSV stocks were diluted to lev- corporated herein by reference) was produced from hy- els of infectivity equal to that of the concentrated super- bridoma supernatants according to standard methods. natants and calcium flux in VI10YEN B cells was com- pared over further 100 fold dilutions of VSV and super- Flow Cytometry 30 natant. UV-inactivated, AlexaFluor-568 labeled Adeno- virus 5 (AdV5) was generated following standard proce- [0523] Flow cytometric analysis of blood samples was dures (Leopold et al., 1998, Human Gene Therapy, 9: performed after retro-orbital phlebotomy of mice and lysis 367; incorporated herein by reference). All infectious of erythrocytes with ACK buffer (0.15 M NH4Cl, 1 mM work was performed in designated BL2+ workspaces, in 35 KHCO3, 0.1 mM EDTA (disodium salt), pH 7.2). Single- accordance with institutional guidelines, and approved cell suspensions of LNs and spleens for flow cytometry by the Harvard Committee on Microbiological Safety. were generated by careful mincing of tissues and sub- sequent digestion at 37˚C for 40 minutes in DMEM (In- VSV Neutralization Assay vitrogen-Gibco) in the presence of 250 mg/ml liberase CI (Roche) plus 50 mg/ml DNase-I (Roche). After 20 minutes 40 [0525] Serum of immunized mice was prediluted 40- of digestion, samples were vigorously passed through fold in MEM containing 2% FCS. Serial two-fold dilutions an 18G needle to ensure complete organ dissociation. were mixed with equal volumes of VSV (500 pfu/ml) and All flow cytometric analyses were performed in FACS incubated for 90 minutes at 37˚C in 5% CO2. 100 mlof buffer containing PBS with 2 mM EDTA and 2% FBS serum-virus mixture was transferred onto Vero cell mon- (Invitrogen-GIBCO) on a FACScalibur (BD Pharmingen), 45 olayers in 96-well plates and incubated for 1 hour at 37˚C. and analyzed by FlowJo software (Treestar Inc., Ash- The monolayers were overlaid with 100 ml DMEM con- land, OR). For calcium flux, cells were labeled with 4 mM taining 1% methylcellulose and incubated for 24 hours Fluo-LOJO (Teflabs) in DMEM containing 10% FCS for at 37˚C. Subsequently, the overlay was discarded, and 90 minutes at 37˚C. Cells were spun through FCS and the monolayer was fixed and stained with 0.5% crystal used immediately. 50 violet. The highest dilution of serum that reduced the number of plaques by 50% was taken as titer. To deter- Viruses and VSV Plaque Assay mine IgG titers, undiluted serum was pretreated with an equal volume of 0.1 mM β-mercaptoethanol in saline. [0524] VSV serotypes Indiana (VSV-IND, Mudd-Sum- mers derived clone, in vitro rescued (Whelan et al., 1995, 55 Adhesion Assays Proc. Natl. Acad. Sci., USA, 92:8388; incorporated here- in by reference) and plaque purified) or New Jersey [0526] 96-well plates (Coming) were coated overnight (VSV-NJ, Pringle Isolate, plaque purified) were propa- with dilutions of recombinant murine VCAM-1-Fc or

69 137 EP 2 394 657 A1 138

ICAM-1-Fc (R&D systems), or purified VSV-IND in PBS 0.1 M sodium cacodylate buffer, pH 7.4. The LNs were in triplicates. Negative control wells were coated with 4% excised and immersed in the same buffer overnight at BSA, positive control wells were coated with 1 mg/ml 4˚C, washed in cacodylate buffer, and osmicated with poly-L-lysine. Plates were blocked for 1-2 h at 4˚C with 1% Osmium tetroxide/1.5% Potassium ferrocyanide (in Hanks Balanced Salt Solution (HBSS)/1% BSA and 5 water) for 1 hour at room temperature in the dark. After washed. Naïve B cells from VI10YEN or C57BL/6 mice washing in water, samples were washed 3-4 times in were negatively selected by magnetic cell separation us- 0.05 M malelate buffer pH 5.15. Samples were counter- ing CD43 magnetic beads (Miltenyi, Bergisch Gladbach, stained for 2 hours in 1% uranyl acetate in maleate buffer Germany) and added to the plates at 3 x 105/well in HBSS and washed three times in water. Samples were dehy- + + 10 with 1% BSA, 1mM Ca2 and 1 mM Mg2 in the presence drated by incubation for 15 minutes in dilutions of ethanol or absence of UV-inactivated VSV-IND (MOI of 1000) for in water (70%-90%-100%), incubated in propylene oxide 30 minutes at 37˚C. After gentle washing (3 times in HB- for 1 hour, and transferred into Epon mixed 1:1 with pro- SS with 1% BSA), plates were fixed for 10 minutes with pylene oxide RT overnight. Samples were moved to em- PBS/10% glutaraldehyde, stained for 45 minutes with bedding mold filled with freshly mixed Epon, and heated 0.5% crystal violet/20% methanol, and washed in water. 15 for 24-48 hours at 60˚C for polymerization. Samples were Dye was eluted by addition of 1% SDS and absorbance analyzed on a Tecnai G2 Spirit BioTWIN electron micro- at 570 nm was spectrophotometrically determined scope at the Harvard Medical School EM facility. (SpectraMax340PC microplate reader and SoftmaxPro 3.1.2 software, Molecular Devices Corporation) after 30 Intravital Multiphoton Microscopy (MP-IVM) of the Pop- minutes. 20 liteal LN

Confocal Microscopy [0529] Naive B cells were negatively selected by mag- netic isolation using CD43 beads (Miltenyi). VI10YEN B [0527] For some analyses, C57BL/6 mice were inject- cells were labeled for 20 minutes at 37˚C with 10 mM ed into both hind footpads with 20 mg AlexaFluor-568 or 25 5-(and 6-)-(((4-chloromethyl)benzoyl) amino)tetrameth- AlexaFluor-488 labeled VSV-IND or VSV-NJ for 30 min- ylrhodamine (CMTMR; Invitrogen), C57BL/6 B cells were utes. For other experiments, mice were transfused with labeled for 25 minutes at 37˚C with 10 mM 7-amino-4- 1 3 107 negatively selected naive B cells from VI10YEN chloromethylcoumarin (CMAC; Invitrogen). In some ex- x MHCII-EGFP mice one day prior to the experiment. At periments, labels were swapped between wildtype and predetermined time points, popliteal LNs were fixed in 30 VI10YEN B cells to exclude unspecific dye effects. 5-6 x situ by footpad injections of phosphate buffered L-lysine 106 B cells of each population were mixed and adoptively with 1% paraformaldehyde/periodate (PLP). After re- transferred by tail vein injection into C57BL/6 recipient moval of popliteal LNs and 3-5 hours incubation in PLP mice one day before analysis. In some experiments, re- at 4˚C, popliteal LNs were washed in 0.1 M PBS, pH 7.2 cipient C57BL/6 mice had received an injection of 30 ml and cryoprotected by an ascending series of 10%, 20%, 35 CLL into the hind footpad 7-10 days before the experi- and 30% sucrose in PBS. Samples were snap-frozen in ment to eliminate SCS macrophages (Delemarre et al., TBS tissue freezing liquid (Triangle Biomedical Scienc- 1990, J. Leukoc. Biol., 47:251; incorporated herein by es, Durham NC) and stored at -80˚C. Sections of 40 mm reference). Eighteen hours following adoptive B cell thickness were mounted on Superfrost Plus slides (Fish- transfer, recipient mice were anaesthetized by intraperi- erbrand) and stained with fluorescent antibodies in a hu- 40 toneal injection on of ketamine (50 mg/kg) and xylazine midified chamber after Fc receptor blockade with 1 mg/ml (10 mg/kg). The right popliteal LN was prepared micro- antibody 2.4G2 (BD Pharmingen). Samples were mount- surgically for MP-IVM and positioned on a custom-built ed in FluorSave reagent solution (EMD-Calbiochem) and microscope stage as described (Mempel et al., 2004, stored at 4˚C until analysis. Images were collected with Nature, 427:154; incorporated herein by reference). Care a BioRad confocal microscopy system using an Olympus 45 was taken to spare blood vessels and afferent lymph ves- BX50WI microscope and 10x/0.4 or 60x/1.2W objectives. sels. The exposed LN was submerged in normal saline Images were analyzed using LaserSharp2000 software and covered with a glass coverslip. A thermocouple was (BioRad Cell Science, Hemel Hempstead, Great Britain) placed next to the LN to monitor local temperature, which and Photoshop CS (Adobe). Quantification of T/B border was maintained at 36-38˚C. MP-IVM was performed on localized B cells was done by counting cells that were 50 a BioRad 2100MP system at an excitation wavelength within 50 mm of the T/B border, as denoted by B220 coun- of 800 nm, from a tunable MaiTai Ti:sapphire laser (Spec- terstain, any cells localized in more central regions were tra-Physics). Fluorescently labeled VSV (20 mgin20ml) considered follicular. was injected through a 31G needle into the right hind footpad of recipient mice concomitant to observation. For Electron Microscopy 55 four-dimensional off-line analysis of cell migration, stacks of 11 optical x-y sections with 4 mm z spacing were ac- [0528] Popliteal LNs were fixed in situ by footpad in- quired every 15 seconds with electronic zooming to 1.8x- jection of 2% formaldehyde and 2.5 % glutaraldehyde in 3x through a 20x/0.95 water immersion objective (Olym-

70 139 EP 2 394 657 A1 140 pus). Emitted fluorescence and second harmonic signals pel, 2003, Nat. Rev. Immunol., 3:867; and Karrer et al., were detected through 400/40 nm, 450/80 nm, 525/50 1997, J. Exp. Med., 185:2157; both of which are incor- nm, and 630/120 nm band-pass filters with non-des- porated herein by reference). It is unclear how virus par- canned detectors to generate three-color images. Se- ticles are cleared from afferent lymph and presented to quences of image stacks were transformed into volume- 5 cognate B cells to induce antibody responses. Here, we rendered, four-dimensional time-lapse movies using Vo- identify a population of CD11 b+CD169+MHCII+ macro- locity software (Improvision). 3D instantaneous veloci- phages on the floor of the subcapsular sinus (SCS) and ties were determined by semi-automated cell tracking in the medulla of LNs that capture viral particles within with Volocity and computational analysis by Matlab minutes after subcutaneous (s.c.) injection. SCS macro- (Mathworks). Accumulation of cells at the SCS was de- 10 phages translocated surface-bound viral particles across termined by manual movie analysis performed by blinded the SCS floor and presented them to migrating B cells in observers. Every 2 minutes, the VI10YEN B cells and the underlying follicles. Selective depletion of these mac- polyclonal B cells were counted at the SCS, in the su- rophages compromised local viral retention, exacerbat- perficial follicle (<50 mm distance from the SCS) and the ed viremia of the host, and impaired local B cell activation. deep follicle (>50 mm distance from the SCS), and ratios 15 These findings indicate that CD 169+ macrophages have of VI10YEN/polyclonal B cells was expressed for each a dual physiological function. They act as innate "flypa- compartment in the entire 30 minute movie. per" by preventing the systemic spread of lymph-borne pathogens and as critical gatekeepers at the lymph-tis- Thoracic Duct Cannulation sue interface that facilitate B cell recognition of particulate 20 antigens and initiate humoral immune responses. [0530] For thoracic duct cannulation, mice received [0532] We have investigated how virus particles that 200 ml olive oil p.o. 30 minutes prior to cannulation to enter peripheral tissues are handled within draining LNs. facilitate visualization of the lymph vessels. Animals were Hind footpads of mice were injected with fluorescently then anesthetized with xylazine (10 mg/kg) and ketamine labeled UV-inactivated vesicular stomatitis virus (VSV), HCl (50 mg/kg). A polyethylene catheter (PE-10) was 25 a cytopathic rhabdovirus that is transmittable by insect inserted into the right jugular vein for continuous infusion bites (Mead et al., 2000, Ann. N.Y. Acad. Sci., 916:437; (2 ml/hour) of Ringer’s lactate (Abbott Laboratories, incorporated herein by reference) and elicits T-independ- North Chicago, IL) containing 1 U/ml heparin (American ent neutralizing B cell responses (Bachmann et al., 1995, Pharmaceutical partners, Los Angeles, CA). Using a dis- Eur. J. Immunol., 25:3445; incorporated herein by refer- secting microscope, the TD was exposed through a left 30 ence). Using multiphoton intravital microscopy (MP-IVM) subcostal incision. Silastic® silicon tubing (0.012" I.D., in popliteal LNs (Mempel et al., 2004, Nature, 427:154; Dow Corning, Midland, USA) was flushed with incorporated herein by reference) draining the injected heparinised (50 U/ml) phosphate-buffered saline (DPBS, footpad, we observed that VSV accumulated in discrete Mediatech, Herndon, VA), inserted into the cisterna chyli patches on the SCS floor within minutes after sc injection, through an approximately 0.3 mm incision and fixed with 35 while the parenchyma and roof of the SCS remained free isobutyl cyanoacrylate monomer (Nexaband®, Abbott of virus (Figure 11A). The viral deposits became progres- Laboratories). The remaining part of the tubing was ex- sively denser forming conspicuous irregular reticular pat- teriorized through the posterior abdominal wall. Subse- terns, which remained fixed in place for hours. quently, the abdominal incision was closed using a 6-0 [0533] To characterize the predilection sites for VSV nonabsorbable running suture (Sofsilk, Tyco Healthcare 40 binding in LNs, we reconstituted irradiated Act(EGFP) Group, Norwalk, CO). Following a 30 minute equilibration mice with wildtype bone marrow. The resulting B6→Act of lymph flow, animals were footpad injected with 108 (EGFP) chimeras expressed EGFP in non-hematopoiet- pfu of VSV-IND and lymph samples were collected on ic cells, presumably lymphatic endothelial cells, on the ice for 6 hours. Lymph and organs were taken after 6 SCS floor and roof. Upon footpad injection of fluorescent hours of thoracic duct lymph collection and plaqued as 45 VSV into C57BL/6→Act(EGFP) chimeras, viral particles described above. Lymph and organs were plaqued as flooded the SCS. Three hours later, unbound lumenal described above. In some experiments the draining pop- VSV had disappeared, but the SCS floor displayed prom- liteal and paraaortic lymph nodes were surgically excised inent patches of VSV that did not colocalize with EGFP+ and the surrounding lymph vessels cauterized to prevent cells, suggesting that VSV was captured by hematopoi- lymph borne viral access to the blood. 50 etic cells (Figure 11B). To characterize the putative VSV- capturing leukocytes, we performed electron microscopy Results and Discussion on popliteal LNs that were harvested 5 min after VSV injection (Figure 11C). Bullet-shaped, electron-dense [0531] Lymph nodes (LNs) prevent systemic dissem- VSV particles were selectively bound to discrete regions ination of pathogens, such as viruses that enter the 55 on the surface of scattered large cells that resided within body’s surfaces, from peripheral sites of infection. They the SCS or just below the SCS floor. VSV-binding cells are also the staging ground of adaptive immune respons- that were located beneath the SCS floor were typically es to pathogen-derived antigens (von Andrian and Mem- in contact with the lymph compartment via protrusions

71 141 EP 2 394 657 A1 142 that extended into the SCS lumen. 7 days after treatment, but other morphological param- [0534] Ultrastructural studies of LNs have shown that eters, e.g. demarcation of the T/B border and SCS ul- the SCS contains many macrophages (Clark, 1962, Am. trastructure remained unaltered (Figures 13 C-E). J. Anat., 110:217; and Farr et al., 1980, Am. J. Anat., [0537] Compared to untreated LNs, we recovered ap- 157:265; both of which are incorporated herein by refer- 5 proximately 10-fold lower viral titers from the draining ence), so we hypothesized that the VSV-retaining cells LNs of CLL-treated mice (Figure 11G), suggesting that belonged to this population. Indeed, confocal microscopy macrophage depletion rendered lymph filtration ineffi- of frozen LN sections obtained thirty minutes after foot- cient. Indeed, VSV titers were dramatically increased in pad injection showed that VSV co-localized in the SCS blood, spleen, and non-draining LNs of CLL-treated with a macrophage marker, CD169/sialoadhesin (Figure 10 mice. Viral dissemination from the injection site to the 11D). Using flow cytometry, we detected CD 169 on ap- blood depended strictly on lymph drainage, because cir- proximately 1%-2% of mononuclear cells (MNCs) in LNs, culating VSV was undetectable when virus was injected which uniformly co-expressed CD 11b and MHC-II, indi- into footpads of mice that carried an occluding catheter cating that the VSV-binding cells are indeed macrophag- in the thoracic duct (TD), even in CLL-treated mice. Viral es (Figure 12). Most CD169+ cells also expressed other 15 titers were low, but detectable in TD lymph fluid of un- macrophage markers, including CD68 and F4/80, while treated mice, but increased significantly in CLL-treated few expressed the granulocyte/monocyte marker Gr-1. animals (Figure 11H). This indicates that the principal CD 169+ cells also expressed CD 11c, but at lower levels conduit for early viral dissemination from peripheral tis- than CD 11chigh conventional dendritic cells (DCs). We sues is the lymph, which is monitored by LN-resident, conclude that intact virions enter the lymph within min- 20 CLL-sensitive macrophages that prevent the systemic utes after transcutaneous deposition and accumulate spread of lymph-borne VSV. rapidly and selectively on macrophages in the medulla [0538] This capture mechanism was not unique to and SCS of draining LNs. VSV; CD169+ SCS macrophages also retained adeno- [0535] To explore mechanisms for virus fixation, live virus (AdV; Figures 14 A-C) and vaccinia virus (VV, Fig- VSV (20 mg containing 2 x 108 pfu) was injected into hind 25 ure 14D), indicating that macrophages act as guardians footpads and viral titers in draining LNs were assessed against many structurally distinct pathogens. In contrast, 2 hours later. There was no defect in VSV retention in virus-sized latex beads (200 nm) were poorly retained in draining LNs of complement C3-deficient mice (Figure the SCS after footpad injection (Figure 14E). Thus, SCS 11E). DH-LMP2a mice, which lack secreted immu- macrophages discriminate between lymph-borne virus- noglobulins, had reduced virus titers in spleen, but not 30 es and other particles of similar size. Fluorescent VSV, in popliteal LNs (Figure 11F). Therefore, VSV fixation in AdV and VV also accumulated in the medulla of draining LNs occurs via a mechanism distinct from that used by LNs, where they were not only bound by CD 169low cells splenic marginal zone macrophages, which require C3 (Figure 11D) but also by CD169-LYVE-1+ lymphatic en- and natural antibodies to capture blood-borne VSV (Och- dothelial cells (Figures 14 C, D). This was corroborated senbein et al., 1999, J. Exp. Med., 190:1165; and Och- 35 in CLL-treated LNs, where VSV accumulated exclusively senbein et al., 1999, Science, 286:2156; both of which on medullary LYVE-1+ cells (Figure 15). are incorporated herein by reference). Conceivably, the [0539] Next, we examined how captured VSV is rec- VSV surface glycoprotein (VSV-G) may be recognized ognized by B cells. Popliteal LNs contain rare B cells in in LNs by macrophage-expressed carbohydrate-binding the SCS lumen (Figure 16A), but we found no evidence scavenger receptors (Taylor et al., 2005, Ann. Rev. Im- 40 for virus-binding lymphocytes within the SCS on electron munol., 23:901; incorporated herein by reference), but micrographs. Instead, viral particles were presented to the precise mechanism will require further investigation. B cells within superficial follicles by macrophages that [0536] What are the consequences of viral capture by extended across the SCS floor. Following injection of ei- macrophages for virus dissemination and anti-viral im- ther VSV (Figure 17A) or AdV (Figures 16 B-E), virions munity? To address this question, we depleted LN-resi- 45 were readily detectable at B cell-macrophage interfaces dent macrophages by footpad injection of clodronate li- for at least 4 hours. This suggested that SCS macrophag- posomes (CLL; Delemarre et al., 1990, J. Leukoc. Biol., es shuttle viral particles across the SCS floor for presen- 47:251; incorporated herein by reference). At the dose tation to B cells. Transcytosis seemed unlikely, because used, sc injected CLL selectively eliminated macrophag- the few vesicles containing VSV in SCS macrophages es in LNs draining the injection site, including the pop- 50 showed evidence of viral degradation. In addition, we did liteal, inguinal and paraortic LNs (Delemarre et al., 1990, not detect substantial motility of virus-binding macro- J. Leukoc. Biol., 47:251; incorporated herein by refer- phages by MP-IVM, at least during the first 6 hours after ence), while macrophages in distal LNs and spleen were challenge. Therefore, viral particles most likely reached spared (Figures13 A, B). Among the different LN-resident the LN parenchyma by moving along the macrophage CD11b+MHCII+ phagocytes, CLL preferentially removed 55 surface. Of note, VSV and other antigens are also pre- the CD169+ subset, whereas LYVE-1+ cells and conven- sented to B cells by DCs immigrating from peripheral tional DCs remained unchanged. CLL-treated popliteal locations (Ludewig et al., 2000, Eur. J. Immunol., 30:185; LNs had increased B cell numbers and enlarged follicles and Qi et al., 2006, Science, 312:1672; both of which are

72 143 EP 2 394 657 A1 144 incorporated herein by reference), but footpad-derived SCS macrophages may provide a substrate for VI10YEN DCs are not likely to play a role during these very early B cell adhesion directly via the BCR. events, because their migration into popliteal LNs takes [0542] To investigate how captured virions are proc- much longer. We conclude that the SCS floor is not un- essed upon detection by B cells, we tested B cells from surmountable for lymph-borne viruses; CD169+ macro- 5 VI10YENxMHCII-EGFP mice, which allowed us to visu- phages appear to act as gatekeepers and facilitators of alize endocytosed VSV co-localizing with endosomal viral translocation and presentation to B cells. MHC-II as an indicator of B cell priming (Vascotto et al., [0540] Next, we explored how naive B cells respond 2007, Curr., Opin., Immunol., 19:93; incorporated herein to viral encounter using two VSV serotypes, Indiana by reference). Within 30 minutes after injection, (VSV-IND) and New Jersey (VSV-NJ) (Figure 18; Roost 10 VI10YENxMHCII-EGFP B cells in the superficial follicle et al., 1996, J. Immunol. Methods, 189:233; incorporated had extensively internalized VSV-IND, but not VSV-NJ herein by reference). We compared wildtype B cells to particles (Figures 20 A, B). Virus-carrying VSV-specific B cells from VI10YEN mice; which express a VSV-IND- B cells were infrequent, but detectable in deep follicles. specific B cell receptor that does not bind VSV-NJ These cells may have acquired virions from rare polyclo- (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA, 15 nal B cells that carried VSV on their surface, or may cor- 100:12883; incorporated herein by reference). By con- respond to VI10YEN cells which failed to arrest at the trast, a small fraction (2%-5%) of wildtype B cells bound SCS after acquiring VSV-IND. both serotypes without being activated. This might reflect [0543] While our histological findings demonstrate that low-affinity reactivity with VSV-G or indirect interactions, intact virions are preferentially detected and acquired by e.g. via complement (Rossbacher and Shlomchik, 2003, 20 B cells in the SCS and superficial follicle, MP-IVM meas- J. Exp. Med., 198:591; incorporated herein by reference). urements of B cell motility revealed broader antigen dis- To assess in vivo responses, differentially labeled semination. After VSV-IND injection, VI10YEN cells ex- wildtype and VI10YEN B cells were adoptively trans- hibited a rapid drop in velocity throughout the entire B ferred and allowed to home to LN follicles. Fluorescent follicle, (Figure 21). This was equally observed in CLL- UV-inactivated virus was then injected into footpads and 25 treated and control LNs, indicating that viral antigen popliteal LNs were recorded by MP-IVM about 5-35 min- reached B cells independent of macrophages. This an- utes later. In virus-free LNs or after injection of VSV-NJ, tigenic material was most likely composed of free viral VI10YEN and control B cells displayed the same distri- protein, an inevitable by-product of natural infections. In- bution (Figures 17 B-C). In contrast, upon VSV-IND in- deed, purified supernatant of our VSV stocks induced a jection VI10YEN cells rapidly accumulated below and at 30 potent calcium flux in VI10YEN B cells (Figure 18E). the SCS floor. There was no difference in baseline B cell Small lymph-borne proteins are known to diffuse rapidly motility and distribution between CLL-treated and un- into follicles and activate cognate B cells (Pape et al., treated LNs, suggesting that VSV-specific B cells are 2007, Immunity, 26:491; incorporated herein by refer- equally likely to probe the SCS in both conditions. How- ence). Accordingly, injection of viral supernatant sup- ever, in CLL-treated LNs, fluorescent virus was not re- 35 pressed the motility of follicular VI10YEN B cells without tained in the SCS and VI10YEN B cells failed to congre- inducing their accumulation at the SCS, indicating that gate in that region, indicating that SCS macrophages are free VSV-G was contained and active within the viral in- essential for both events (Figure 17B). oculum. This can explain the macrophage-independent [0541] To rigorously quantify VI10YEN B cell distribu- pan-follicular effect of VSV-IND injection. tion, LNs were harvested 30 minutes after VSV challenge 40 [0544] To determine the kinetics of VI10YEN B cell and analyzed by confocal microscopy. While the entire activation upon viral encounter, we measured common follicular VI10YEN population retained its overall distri- activation markers (Figure 22). The costimulatory mole- bution (Figure 17D), the subset of cells residing ≤50 mm cule CD86 was first up-regulated 6 hours after VSV-IND below the SCS shifted toward the SCS in VSV-IND, but challenge. CD69 was induced more rapidly, but also on not VSV-NJ containing LNs (Figure 17E). It seems un- 45 polyclonal B cells, presumably by pleiotropic IFN-α sig- likely that VI10YEN B cells redistributed to the SCS be- naling (Barchet et al., 2002, J. Exp. Med., 195:507; and cause of chemoattractant signals, since unresponsive Shiow et al., 2006, Nature, 440:540; both of which are polyclonal B cells express the same chemoattractant re- incorporated herein by reference). Surface IgM (Figures ceptors. More likely, the random contacts of motile 20 C, D) was down-regulated as early as 30 minutes after VI10YEN cells with macrophage-bound VSV-IND trig- 50 challenge reaching a maximum within 2h when >70% of gered a BCR-dependent "stop signal" (Okada et al., VI10YEN cells were BCRlow/neg. Therefore, BCR inter- 2005, PLoS Biol., 3:el50; incorporated herein by refer- nalization provided the earliest specific readout for virus- ence): Short-term exposure to VSV-IND activates LFA- specific B cell activation. Remarkably, VI10YEN B cells 1 and/or α4 integrins (Dang and Rock, 1991, J. Immunol., in CLL-treated LNs failed to downregulate their BCR dur- 146:3273; incorporated herein by reference) on 55 ing the first 2 hours after subcutaneous injection of 20 VI10YEN B cells, resulting in adhesion to the respective mg VSV-IND (Figure 20E), indicating that SCS macro- ligands, ICAM-1 and VCAM-1, which are both expressed phages are necessary for efficient early presentation of in the SCS (Figure 19). Additionally, VSV-IND bound to captured virions to B cells.

73 145 EP 2 394 657 A1 146

[0545] Primed B cells eventually solicit help from CD4+ Nanoparticle-Stabilized Liposome Nanocarriers T cells (Vascotto et al., 2007, Curr., Opin., Immunol., 19: 93; incorporated herein by reference) for class switch [0549] In some embodiments, nanoparticle-stabilized recombination and germinal center formation. To contact liposomes are used to deliver one or a plurality of immu- T cells, newly activated B cells migrate toward the T/B 5 nomodulatory agents to cells of the immune system (Fig- border (Okada et al., 2005, PLoS Biol., 3:el150; and Reif ure 4). When small charged nanoparticles approach the et al., Nature, 416:94; both of which are incorporated surface of liposomes carrying either opposite charge or herein by reference). This mechanism operated efficient- no net charge, electrostatic or charge-dipole interaction ly in macrophage-sufficient mice; most VI10YEN B cells between nanoparticles and membrane attracts the nan- redistributed to the T/B border within 6h after footpad 10 oparticles to stay on the membrane surface, being par- injection of as little as 40 ng VSV-IND (Figures 20 F, H tially wrapped by lipid membrane. This induces local and 23). By contrast, a 100-fold higher viral dose was membrane bending and globule surface tension of lipo- needed to elicit full redistribution of VI10YEN B cells in somes, both of which enable tuning of membrane rigidity. CLL-treated mice (Figures 20 G, H). By 12 hours after This aspect is significant for vaccine delivery using lipo- injection, most VSV-specific cells reached the T-B bor- 15 somes to mimic viruses whose stiffness depends on the der, irrespective of the injected dose. Thus, even without composition of other biological components within virus SCS macrophages follicular B cells are eventually acti- membrane. Moreover, adsorbed nanoparticles form a vated by VSV-derived antigen, albeit less efficiently. charged shell which protects liposomes against fusion, [0546] In conclusion, we demonstrate a dual role for thereby enhancing liposome stability. In certain embod- CD 169+ macrophages in LNs: they capture lymph-borne 20 iments, small nanoparticles are mixed with liposomes un- viruses preventing their systemic dissemination and they der gentle vortex, and the nanoparticles stick to liposome guide captured virions across the SCS floor for efficient surface spontaneously. presentation and activation of follicular B cells. Liposome-Polymer Nanocarrier Example 2: Exemplary Lipid-Based Vaccine Nanotech- 25 nology Architectures [0550] In some embodiments, liposome-polymer na- nocarriers are used to deliver one or a plurality of immu- Liposome Nanocarriers nomodulatory agents to cells of the immune system (Fig- ure 5). Instead of keeping the liposome interior hollow, [0547] In some embodiments, small liposomes (10 nm 30 hydrophilic immunomodulatory agents may be encapsu- - 1000 nm) are manufactured and employed to deliver, lated. Figure 3 shows liposomes that are loaded with di- in some embodiments, one or multiple immunomodula- block copolymer nanoparticles to form liposome-coated tory agents to cells of the immune system (Figure 3). In polymeric nanocarriers, which have the merits of both general, liposomes are artificially-constructed spherical liposomes and polymeric nanoparticles, while excluding lipid vesicles, whose controllable diameter from tens to 35 some of their limitations. In some embodiments, the li- thousands of nm signifies that individual liposomes com- posome shell can be used to carry lipophilic or conjugate prise biocompatible compartments with volume from hydrophilic immunomodulatory agents, and the polymer- zeptoliters (10-21 L) to femtoliters (10-15 L) that can be ic core can be used to deliver hydrophobic immunomod- used to encapsulate and store various cargoes such as ulatory agents. In certain embodiments, pre-formulated proteins, enzymes, DNA and drug molecules. Liposomes 40 polymeric nanoparticles (40 nm - 1000 nm) are mixed may comprise a lipid bilayer which has an amphiphilic with small liposomes (20 nm - 100 nm) under gentle vor- property: both interior and exterior surfaces of the bilayer tex to induce liposome fusion onto polymeric nanoparti- are hydrophilic, and the bilayer lumen is hydrophobic. cle surface. Lipophilic molecules can spontaneously embed them- selves into liposome membrane and retain their hy- 45 Nanoparticle-Stabilized Liposome-Polymer Nanocarri- drophilic domains outside, and hydrophilic molecules can ers be chemically conjugated to the outer surface of liposome taking advantage of membrane biofunctionality. [0551] In some embodiments, nanoparticle-stabilized [0548] In certain embodiments, lipids are mixed with liposome-polymer nanocarriers are used to deliver one a lipophilic immunomodulatory agent, and then formed 50 or a plurality of immunomodulatory agents (Figure 6). By into thin films on a solid surface. A hydrophilic immu- adsorbing small nanoparticles (1 nm - 30 nm) to the li- nomodulatory agent is dissolved in an aqueous solution, posome-polymer nanocarrier surface, the nanocarrier which is added to the lipid films to hydrolyze lipids under has not only the merit of both aforementioned nanopar- vortex. Liposomes with lipophilic immunomodulatory ticle-stabilized liposomes (Figure 4) and aforementioned agents incorporated into the bilayer wall and hydrophilic 55 liposome-polymer nanoparticles (Figure 5), but also tun- immunomodulatory agents inside the liposome lumen able membrane rigidity and controllable liposome stabil- are spontaneously assembled. ity.

74 147 EP 2 394 657 A1 148

Liposome-Polymer Nanocarriers Comprising Reverse a lipid headgroup. The conjugate is mixed with a certain Micelles ratio of unconjugated lipid molecules in an aqueous so- lution containing one or more water-miscible solvents. A [0552] In some embodiments, liposome-polymer na- biodegradable polymeric material is mixed with the hy- nocarriers containing reverse micelles are used to deliver 5 drophobic immunomodulatory agents to be encapsulat- one or a plurality of immunomodulatory agents (Figure ed in a water miscible or partially water miscible organic 7). Since the aforementioned liposome-polymer nano- solvent. The resulting polymer solution is added to the carriers (Figures 5 and 6) are limited to carry hydrophobic aqueous solution of conjugated and unconjugated lipid immunomodulatory agents within polymeric nanoparti- to yield nanoparticles by the rapid diffusion of the organic cles, here small reverse micelles (1 nm - 20 nm) are for- 10 solvent into the water and evaporation of the organic sol- mulated to encapsulate hydrophilic immunomodulatory vent. agents and then mixed with the di-block copolymers to formulate polymeric core of liposomes. Lipid Monolayer-Stabilized Polymeric Nanocarrier Com- [0553] In certain embodiments, a hydrophilic immu- prising Reverse Micelles nomodulatory agent to be encapsulated is first incorpo- 15 rated into reverse micelles by mixing with naturally de- [0557] In some embodiments, lipid monolayer stabi- rived and non-toxic amphiphilic entities in a volatile, wa- lized polymeric nanoparticles comprising reverse mi- ter-miscible organic solvent. The resulting biodegradable celles are used to deliver one or a plurality of immu- polymer-reverse micelle mixture is combined with a pol- nomodulatory agents (Figure 10). Since the aforemen- ymer-insoluble hydrophilic non-solvent to form nanopar- 20 tioned lipid-stabilized polymeric nanocarriers (Figure 9) ticles by the rapid diffusion of the solvent into the non- are limited to carry hydrophobic immunomodulatory solvent and evaporation of the organic solvent. Reverse agents, here, small reverse micelles (1 nm - 20 nm) are micelle contained polymeric nanoparticles are mixed with formulated to encapsulate hydrophilic immunomodula- lipid molecules to form the aforementioned liposome-pol- tory agents and mixed with biodegradable polymers to ymer complex structure (Figure 5). 25 form polymeric nanocarrier core.

Nanoparticle-Stabilized Liposome-Polymer Nanocarri- Example 3: In vivo targeting of SCS-Mph using Fc frag- ers Comprising_Reverse Micelles ments from human IgG

[0554] In some embodiments, nanoparticle-stabilized 30 [0558] Fluorescent unmodified control nanoparticles liposome-polymer nanocarriers containing reverse mi- (top panel, Figure 24A) or Fc surface-conjugated target- celles are used to deliver one or a plurality of immu- ed nanoparticles (middle and lower panel, Figure 24A) nomodulatory agents (Figure 8). By adsorbing small na- were injected into footpads of anesthetized mice, and the noparticles (1 nm - 30 nm) to a liposome-polymer nano- draining popliteal lymph node was excised 1 hour later carrier surface, the nanocarrier has not only the merit of 35 and single-cell suspensions were prepared for flow cy- both aforementioned nanoparticle-stabilized liposomes tometry. Targeted nanoparticles were also injected into (Figure 4) and aforementioned reverse micelle contained mice one week after lymph node macrophages had been liposome-polymer nanoparticles (Figure 7), but also tun- depleted by injection of clodronate-laden liposomes (low- able membrane rigidity and controllable liposome stabil- er panel, Figure 24A). The cell populations in gates were ity. 40 identified as nanoparticle-associated macrophages based on high expression of CD11b. These results indi- Lipid Monolayer-Stabilized Polymeric Nanocarrier cate that (i) nanoparticle binding depends on the pres- ence of clodronate-sensitive macrophages and (ii) tar- [0555] In some embodiments, lipid monolayer stabi- geted nanoparticles are bound to ~ twice as many mac- lized polymeric nanocarriers are used to deliver one or 45 rophages as control nanoparticles. a plurality of immunomodulatory agents (Figure 9). As [0559] The Panels on the right of Figure 24 show flu- compared to aforementioned liposome-polymer nano- orescent micrographs of frozen lymph node sections af- carrier (Figures 5-8), this system has the merit of sim- ter injection of blue fluorescent control (top panel, Figure plicity in terms to both agents and manufacturing. In some 24A) or targeted (middle and lower panels, Figure 24A) embodiments, a hydrophobic homopolymer can form the 50 nanoparticles. Sections were counter-stained with anti- polymeric core in contrast to the di-block copolymer used CD169 and a marker that identifies either the medulla (in in Figures 5-8, which has both hydrophobic and hy- top and bottom panel, Figure 24A) or B cells (in middle drophilic segments. Lipid-stabilized polymeric nanocar- panel, Figure 24A). At one hour after nanoparticle injec- riers can be formed within one single step instead of for- tion most control particles are found in the medulla (top, mulating polymeric nanoparticle and liposome separate- 55 Figure 24A), while targeted nanoparticles colocalise with ly followed by fusing them together. CD169+ SCS-Mph adjacent to B cell follicles (middle, [0556] In certain embodiments, a hydrophilic immu- Figure 24A). At 24 hours after injection, discrete cell- nomodulatory molecule is first chemically conjugated to sized accumulations of targeted nanoparticles are seen

75 149 EP 2 394 657 A1 150 in the cortical region between the SCS and the medulla, Example 5: In vivo T cell activation by immunomodula- suggesting uptake and transport by migratory dendritic tory nanoparticles cells. [0560] Mice were injected i.v. with red fluorescent B [0563] C57BL6J mice were injected i.v. with CFSE- cells and in a footpad with a 1:1 mixture of control and 5 labeled CD4 T cells from OT-II donor mice, which express Fc targeted nanoparticles. 24 hours later, when some of a transgenic TCR specific for chicken ovalbumin (OVA) the transferred B cells had migrated into B cell follicles, presented in MHC class II. Subsequently, immunization the draining popliteal lymph node was excised and sec- experiments were performed by injecting one footpad tioned for confocal microscopy and quantitative image with free OVA or with nanoparticles composed of either analysis of green:blue fluorescent ratios. The subcapsu- 10 PLA or PLGA that encapsulated an equivalent amount lar sinus (SCS) region contained similar levels of blue of OVA as a model antigen. All antigenic mixtures also and green nanoparticles (cells encircled on the right, Fig- contained CpG (a TLR9 agonist) as an adjuvant. The ure 24B), while green fluorescence associated with Fc- animals were injected, sacrificed three days after immu- targeted nanoparticles was about twice higher in the nization, and OT-II T cell activation was assessed by flow SCS. There were also prominent accumulations of green 15 cytometry in single-cell suspensions from different tis- nanoparticles within B follicles delineated by scattered sues. red B cells. These regions have the characteristic size, [0564] Unstimulated 5,6-carboxy-succinimidyl-fluo- shape, and distribution of follicular dendritic cells (FDC), rescein-ester (CFSE)-labeled T cells do not divide and, which like macrophages and dendritic cells are known to therefore, carry an uniformly high concentration of CFSE express abundant Fc receptors. 20 resulting in a single narrow peak of brightly fluorescent cells. By contrast, activated T cells divide and in the proc- Example 4: Antigen-bearing targeted nanoparticles are ess split the fluorescent dye evenly between the two highly immunogenic and induce high antibody titers daughter cells resulting in an incremental decrease in fluorescence intensity upon each successive division. [0561] Groups of mice (5/group) were immunized with: 25 Thus, the greater the left shift in CFSE, fluorescence the UV-inactivated vesicular stomatits virus (VSV, serotype stronger T cells were activated. The results indicate that: Indiana) or with the purified immunogenic envelope glyc- (i) nanoparticle-encapsulated antigen generated a more oprotein (VSV-G) of VSV. VSV-G was either given in potent CD4 T cell response than free antigen in the drain- soluble form mixed with alum or conjugated to non-tar- ing popliteal lymph node (popLN, top row); (ii) only nan- geted or targeted (with surface immobilized human Fc) 30 oparticles, but not free OVA induced local T cell prolifer- PLGA nanoparticles with or without alum as an adjuvant. ation in distal lymphoid tissues, including the brachial The dose of free VSV-G was estimated to be ~10-fold lymph node (middle row) and the spleen (bottom row). higher than the dose of VSV-G delivered with nanopar- In recipients of free OVA, the brachial LN and spleen ticles. Mice received a booster injection at day 55 after contained only undivided cells or cells with very low the primary immunization, and serum was obtained after 35 CFSE content. The latter population does not indicate 10 weeks and tested for neutralization of VSV-mediated local T cell activation but migration of T cells that were plaque formation on Vero cells. Results show titers as activated elsewhere. the highest serum dilution that blocked plaque formation [0565] C57BL6J mice were injected i.v. with CFSE- by at least 50%. Each symbol reflects the neutralizing labeled CD8 T cells from OT-I donor mice, which express anti-VSV titer in one mouse. The group of mice immu- 40 a transgenic T cell receptor (TCR) specific for chicken nized with VSV-G presented on Fc-targeted nanoparti- ovalbumin (OVA) presented in MHC class I. The exper- cles generated significantly higher neutralizing anti-VSV imental protocol was otherwise identical as immediately titers than any other group (the two animals with the high- described above. est titers in that group completely neutralized plaque for- [0566] C57BL6J mice were injected i.v. with CFSE- mation at the highest dilution tested, so actual titers may 45 labeled CD8 T cells from OT-I donor mice as above. How- have been even higher). ever, in this experiment CL097, an imidazoquinoline [0562] The induced immune response elicited by na- compound that activates TLR-7 and TLR-8, was used as noparticle (NP) vaccines confers potent protection from adjuvant and different methods of adjuvant delivery were a lethal dose of VSV. While all vaccinated groups showed tested. T cell activation in this case was assessed by some protection, only the group that received VSV-G 50 counting the total number of OT-I T cells in the draining conjugates to Fc-targeted NPs plus alum showed 100% popliteal lymph node three days after footpad injection protection from lethal infection. Recipients of free VSV- of either free OVA (1 mg or 100 ng) mixed with free ad- G (VSV-G + alum) received ~10-fold more antigen than juvant (160 ng). All animals that received nanoparticles animals that were given VSV-G conjugated to nanopar- were given 100 ng OVA with or without 160 ng CL097. ticles. As a negative control, one group of mice received 55 Material that was encapsulated within nanoparticles, but Fc-targeted nanoparticles (NP-Fc) without VSV-G, which not covalently attached to the PLA polymer is shown in did not confer protection. [ ]. Covalent linkage of CL097 to PLA is identified by hy- phenation. Materials that were mixed in free form within

76 151 EP 2 394 657 A1 152 the same compartment are separated by "+". These re- understood that, in general, where the invention, or as- sults revealed a marked increase in CD8 T cell prolifer- pects of the invention, is/are referred to as comprising ation in animals that received encapsulated OVA in na- particular elements, features, etc., certain embodiments noparticles in which the adjuvant was covalently linked of the invention or aspects of the invention consist, or to the excipient. 5 consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been Equivalents and Scope specifically set forth in haec verba herein. It is noted that the term "comprising" is intended to be open and permits [0567] Those skilled in the art will recognize, or be able the inclusion of additional elements or steps. to ascertain using no more than routine experimentation, 10 [0571] Where ranges are given, endpoints are includ- many equivalents to the specific embodiments of the in- ed. Furthermore, it is to be understood that unless oth- vention, described herein. The scope of the present in- erwise indicated or otherwise evident from the context vention is not intended to be limited to the above Descrip- and understanding of one of ordinary skill in the art, val- tion, but rather is as set forth in the appended claims. ues that are expressed as ranges can assume any spe- [0568] Those skilled in the art will recognize, or be able 15 cific value or subrange within the stated ranges in differ- to ascertain using no more than routine experimentation, ent embodiments of the invention, to the tenth of the unit many equivalents to the specific embodiments of the in- of the lower limit of the range, unless the context clearly vention described herein. The scope of the present in- dictates otherwise. vention is not intended to be limited to the above Descrip- [0572] In addition, it is to be understood that any par- tion, but rather is as set forth in the appended claims. 20 ticular embodiment of the present invention that falls with- [0569] In the claims articles such as "a," "an," and "the" in the prior art may be explicitly excluded from any one may mean one or more than one unless indicated to the or more of the claims. Since such embodiments are contrary or otherwise evident from the context. Thus, for deemed to be known to one of ordinary skill in the art, example, reference to "a nanoparticle" includes a plural- they may be excluded even if the exclusion is not set ity of such nanoparticle, and reference to "the cell" in- 25 forth explicitly herein. Any particular embodiment of the cludes reference to one or more cells known to those compositions of the invention (e.g., any immunomodu- skilled in the art, and so forth. Claims or descriptions that latory agent, any targeting moiety, any immunostimula- include "or" between one or more members of a group tory agent, any antigen presenting cell, any vaccine na- are considered satisfied if one, more than one, or all of nocarrier architecture, any microorganism, any method the group members are present in, employed in, or oth- 30 of administration, any prophylactic and/or therapeutic ap- erwise relevant to a given product or process unless in- plication, etc.) can be excluded from any one or more dicated to the contrary or otherwise evident from the con- claims, for any reason, whether or not related to the ex- text. The invention includes embodiments in which ex- istence of prior art. actly one member of the group is present in, employed [0573] The publications discussed above and through- in, or otherwise relevant to a given product or process. 35 out the text are provided solely for their disclosure prior The invention includes embodiments in which more than to the filing date of the present application. Nothing herein one, or all of the group members are present in, employed is to be construed as an admission that the inventors are in, or otherwise relevant to a given product or process. not entitled to antedate such disclosure by virtue of prior Furthermore, it is to be understood that the invention en- disclosure. compasses all variations, combinations, and permuta- 40 tions in which one or more limitations, elements, clauses, 1. A composition comprising a nanocarrier that tar- descriptive terms, etc., from one or more of the listed gets a specific cell, tissue, or organ and modulates claims is introduced into another claim. For example, any an immune response comprising at least one of a B claim that is dependent on another claim can be modified cell antigen, a T cell antigen and an immunostimu- to include one or more limitations found in any other claim 45 latory agent. that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be under- 2. The composition of paragraph 1, wherein the na- stood that methods of using the composition for any of nocarrier comprises a B cell antigen. the purposes disclosed herein are included, and methods of making the composition according to any of the meth- 50 3. The composition of paragraph 2, wherein the B ods of making disclosed herein or other methods known cell antigen is on the surface of the nanocarrier. in the art are included, unless otherwise indicated or un- less it would be evident to one of ordinary skill in the art 4. The composition of paragraph 3, wherein the B that a contradiction or inconsistency would arise. cell antigen is on the surface of the nanocarrier at a [0570] Where elements are presented as lists, e.g., in 55 density which activates B cell receptors. Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any el- 5. The composition of paragraph 2, wherein the B ement(s) can be removed from the group. It should it be cell antigen is encapsulated within the nanocarrier.

77 153 EP 2 394 657 A1 154

6. The composition of paragraph 3 or 4, wherein the wherein the B cell antigen is a protein or peptide. B cell antigen is on the surface of the nanocarrier and is also encapsulated within the nanocarrier. 21. The composition of any of paragraphs 2-8, wherein the B cell antigen is a carbohydrate. 7. The composition of any of paragraphs 2-5, where- 5 in the B cell antigen is associated with the nanocar- 22. The composition of paragraph 20 or 21, wherein rier. the carbohydrate is from an infectious agent.

8. The composition of paragraph 7, wherein the B 23. The composition of paragraph 22, wherein the cell antigen is covalently associated with the nano- 10 infectious agent is a bacterium, fungus, virus, proto- carrier. zoan, or parasite.

9. The composition of any of paragraphs 2-8, where- 24. The composition of paragraph 23, wherein the in the B cell antigen is a poorly immunogenic antigen. bacterium is a Pseudomonas, Pneumococcus, E. 15 coli, Staphylococcus, Streptococcus, Treponema, 10. The composition of any of paragraphs 2-8, Borrelia, Chlamydia, Haemophilus, Clostridium, wherein the B cell antigen is a protein or peptide. Salmonella, Legionella, Vibrio or Enterococci bac- terium or a Mycobacterium. 11. The composition of any of paragraphs 2-8, wherein the B cell antigen is a small molecule. 20 25. The composition of paragraph 23, wherein the virus is a pox virus, smallpox virus, ebola virus, mar- 12. The composition of any of paragraphs 2-8, burg virus, dengue fever virus, influenza virus, wherein the B cell antigen is an abused substance. parainfluenza virus, respiratory syncytial virus, rube- ola virus, human immunodeficiency virus, human 13. The composition of any of paragraphs 2-8, 25 papillomavirus, varicella-zoster virus, herpes sim- wherein the B cell antigen is an addictive substance. plex virus, cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, 14. The composition of paragraph 13, wherein the papillomavirus, parvovirus, picornavirus, poliovirus, addictive substance is nicotine, a narcotic, a hallu- virus that causes mumps, virus that causes rabies, cinogen, a stimulant, a cough suppressant, a tran- 30 reovirus, rubella virus, togavirus, orthomyxovirus, quilizer, or a sedative. retrovirus, hepadnavirus, coxsackievirus, equine en- cephalitis virus, Japanese encephalitis virus, yellow 15. The composition of any of paragraphs 2-8, fever virus, Rift Valley fever virus, hepatitis A virus, wherein the B cell antigen is a toxin. hepatitis B virus, hepatitis C virus, hepatitis D virus, 35 or hepatitis E virus. 16. The composition of paragraph 15, wherein the toxin is from a chemical weapon or an agent of bio- 26. The composition of any of paragraphs 2-8, warfare. wherein the B cell antigen is a self antigen.

17. The composition of paragraph 16, wherein the 40 27. The composition of paragraph 26, wherein the chemical weapon or agent of biowarfare is mustard self antigen is a protein or peptide, lipoprotein, lipid, gas, ricin, botulinum toxin, saxitoxin, sarin, phos- carbohydrate, or a nucleic acid. phene, chloropicrin, or lewisite. 28. The composition of paragraph 26, wherein the 18. The composition of any of paragraphs 2-8, 45 self antigen is an enzyme, a structural protein, a se- wherein the B cell antigen is a hazardous environ- creted protein, a cell surface receptor, or a cytokine. mental agent. 29. The composition of paragraph 28, wherein the 19. The composition of paragraph 18, wherein the cytokine is TNF, IL-I, or IL-6. hazardous environmental agent is arsenic, lead, 50 mercury, vinyl chloride, polychlorinated biphenyls, 30. The composition of paragraph 26, wherein the benzene, polycyclic aromatic hydrocarbons, cadmi- self antigen is cholesteryl ester transfer protein (CE- um, benzo(a)pyrene, benzo(b)fluoranthene, chloro- TP), the Aβ protein associated with Alzheimer’s, a form, DDT, P, P’-, aroclor 1254, aroclor 1260, diben- proteolytic enzyme that processes the pathological zo(a,h)anthracene, trichloroethylene, dieldrin, chro- 55 form of the Aβ protein, LDL associated with athero- mium hexavalent, or DDE, P,P’. sclerosis, or a coreceptor for HIV-1.

20. The composition of any of paragraphs 2-8, 31. The composition of paragraph 30, wherein the

78 155 EP 2 394 657 A1 156 proteolytic enzyme that processes the pathological enzyme or enzymatic product. form of the Aβ protein is beta-secretase. 42. The composition of paragraph 41, wherein the 32. The composition of paragraph 30, wherein the infectious disease antigen is a viral antigen. LDL associated with atherosclerosis is oxidized or 5 minimally modified. 43. The composition of paragraph 42, wherein the viral antigen is an antigen from a pox virus, smallpox 33. The composition of paragraph 30, wherein the virus, ebola virus, marburg virus, dengue fever virus, coreceptor for HIV-1 is CCR5. influenza virus, parainfluenza virus, respiratory syn- 10 cytial virus, rubeola virus, human immunodeficiency 34. The composition of any of paragraphs 2-8, virus, human papillomavirus, varicella-zoster virus, wherein the B cell antigen is a degenerative disease herpes simplex virus, cytomegalovirus, Epstein-Barr antigen, an infectious disease antigen, a cancer an- virus, JC virus, rhabdovirus, rotavirus, rhinovirus, ad- tigen, an atopic disease antigen, an autoimmune dis- enovirus, papillomavirus, parvovirus, picornavirus, ease antigen, or a metabolic disease enzyme. 15 poliovirus, virus that causes mumps, virus that caus- es rabies, reovirus, rubella virus, togavirus, or- 35. The composition of paragraph 34, wherein the thomyxovirus, retrovirus, hepadnavirus, coxsackie- infectious disease antigen is a viral antigen. virus, equine encephalitis virus, Japanese encepha- litis virus, yellow fever virus, Rift Valley fever virus, 36. The composition of paragraph 35, wherein the 20 hepatitis A virus, hepatitis B virus, hepatitis C virus, viral antigen is an antigen from a pox virus, smallpox hepatitis D virus, or hepatitis E virus. virus, ebola virus, marburg virus, dengue fever virus, influenza virus, parainfluenza virus, respiratory syn- 44. The composition of any of paragraphs 37-43, cytial virus, rubeola virus, human immunodeficiency wherein when the nanocarrier comprises a B cell virus, human papillomavirus, varicella-zoster virus, 25 antigen and T cell antigen, the B cell antigen and the herpes simplex virus, cytomegalovirus, Epstein-Barr T cell antigen are different antigens. virus, JC virus, rhabdovirus, rotavirus, rhinovirus, ad- enovirus, papillomavirus, parvovirus, picornavirus, 45. The composition of paragraph 37, wherein when poliovirus, virus that causes mumps, virus that caus- the nanocarrier comprises a B cell antigen and a T es rabies, reovirus, rubella virus, togavirus, or- 30 cell antigen, the B cell antigen and the T cell antigen thomyxovirus, retrovirus, hepadnavirus, coxsackie- are the same. virus, equine encephalitis virus, Japanese encepha- litis virus, yellow fever virus, Rift Valley fever virus, 46. The composition of any of paragraphs 37-45, hepatitis A virus, hepatitis B virus, hepatitis C virus, wherein the T cell antigen is encapsulated within the hepatitis D virus, or hepatitis E virus. 35 nanocarrier.

37. The composition of any of paragraphs 36, where- 47. The composition of any of paragraphs 37-45, in the nanocarrier comprises a T cell antigen. wherein the T cell antigen is associated with the na- nocarrier. 38. The composition of paragraph 37, wherein when 40 the nanocarrier comprises both a B cell and T cell 48. The composition of paragraph 47, wherein the T antigen, and wherein the B cell antigen is a protein, cell antigen is covalently associated with the nano- peptide, or non-protein antigen. carrier.

39. The composition of paragraph 37, wherein the T 45 49. The composition of any of paragraphs 1-48, cell antigen is a universal T cell antigen. wherein the nanocarrier comprises a immunostimu- latory agent. 40. The composition of paragraph 39, wherein the universal T cell antigen is one or more peptides de- 50. The composition of paragraph 49, wherein the rived from tetanus toxoid, Epstein-Barr virus, or in- 50 immunostimulatory agent is on the surface of the fluenza virus. nanocarrier.

41. The composition of paragraph 37, wherein the T 51. The composition of paragraph 49, wherein the cell antigen is a degenerative disease antigen, an immunostimulatory agent is encapsulated within the infectious disease antigen, a cancer antigen, an at- 55 nanocarrier. opic disease antigen, an autoimmune disease anti- gen, an alloantigen, a xenoantigen, an allergen, a 52. The composition of paragraph 49, wherein the contact sensitizer, a hapten, or a metabolic disease immunostimulatory agent is both on the surface of

79 157 EP 2 394 657 A1 158 the nanocarrier and encapsulated within the nano- the adjuvant is alum, MF59, QS21, cholera toxin, carrier. Freund’s adjuvant, squalene, phosphate adjuvants, or tetrachlorodecaoxide. 53. The composition of paragraph 52, wherein the immunostimulatory agent on the surface of the na- 5 67. The composition of any of paragraphs 1-66, nocarrier is different from the immunostimulatory wherein the nanocarrier further comprises a target- agent encapsulated within the nanocarrier. ing moiety.

54. The composition of paragraph 49, wherein the 68. The composition of paragraph 67, wherein the nanocarrier comprises more than one immunostim- 10 targeting moiety is on the surface of the nanocarrier. ulatory agents. 69. The composition of paragraph 68, wherein the 55. The composition of paragraph 54, wherein the targeting moiety is associated with the nanocarrier. more than one immunostimulatory agents are differ- ent. 15 70. The composition of paragraph 69, wherein the targeting moiety is covalently associated with the na- 56. The composition of any of paragraphs 49-51, nocarrier. wherein the immunostimulatory agent is associated with the nanocarrier. 71. The composition of any of paragraphs 67-70, 20 wherein the targeting moiety binds a receptor ex- 57. The composition of paragraph 56, wherein the pressed on the surface of a cell. immunostimulatory agent is covalently associated with the nanocarrier. 72. The composition of any of paragraphs 67-70, wherein the targeting moiety binds a soluble recep- 58. The composition of any of paragraphs 49-57, 25 tor. wherein the immunostimulatory agent is selected from the group consisting of toll-like receptor (TLR) 73. The composition of paragraph 72, wherein the agonists, cytokine receptor agonists, CD40 ago- soluble receptor is a complement protein or a pre- nists, Fc receptor agonists, CpG-containing nucleic existing antibody. acids, complement receptor agonists, or an adju- 30 vant. 74. The composition of any of paragraphs 67-71, wherein the targeting moiety is for delivery of the 59. The composition of paragraph 58, wherein the nanocarrier to antigen presenting cells, T cells, or B TLR agonist is a TLR-1, TLR-2, TLR-3, TLR-4, TLR- cells. 5, TLR-6, TLR-7, TLR-8, TLR-9, or TLR- 10 agonist. 35 75. The composition of paragraph 74, wherein the 60. The composition of paragraph 58, wherein the antigen presenting cells are macrophages. Fc receptor agonist is a Fc-gamma receptor agonist. 76. The composition of paragraph 75, wherein the 61. The composition of paragraph 58, wherein the 40 macrophages are subcapsular sinus macrophages. complement receptor agonist binds to CD21 or CD35. 77. The composition of paragraph 74, wherein the antigen presenting cells are dendritic cells. 62. The composition of paragraph 61, wherein the complement receptor agonist induces endogenous 45 78. The composition of paragraph 74, wherein the complement opsonization of the nanocarrier. antigen presenting cells are follicular dendritic cells.

63. The composition of paragraph 58, wherein the 79. The composition of any of paragraphs 67-78, cytokine receptor agonist is a cytokine. wherein the targeting moiety is a molecule that binds 50 to CD11b, CD169, mannose receptor, DEC-205, 64. The composition of paragraph 58, wherein the CD11c, CD21/CD35, CX3CR1 , or a Fc receptor. cytokine receptor agonist is a small molecule, anti- body, fusion protein, or aptamer. 80. The composition of paragraph 79, wherein the targeting moiety is a molecule that binds to CD169, 65. The composition of paragraph 58, wherein the 55 CX3CR1, or a Fc receptor. adjuvant induces cytokine biosynthesis. 81. The composition of paragraph 79 or 80, wherein 66. The composition of paragraph 58 or 65, wherein the targeting moiety comprises the Fc portion of an

80 159 EP 2 394 657 A1 160 immunoglobulin. secondary lympoid tissues or organ is/are lymph nodes, spleen, Peyer’s patches, appendix, or tonsils. 82. The composition of paragraph 81, wherein the targeting moiety comprises the Fc portion of an IgG. 98. The composition of any of paragraphs 1-97, 5 wherein the nanocarrier is a microparticle, nanopar- 83. The composition of any of paragraphs 1-66, ticle, or picoparticle. wherein the nanocarrier has a positive zeta potential. 99. The composition of paragraph 98, wherein the 84. The composition of any of paragraphs 1-66, particle is formed by self-assembly. wherein the nanocarrier comprises one or more 10 amine moieties at its surface. 100. A composition comprising a nanocarrier com- prising (a) a conjugate of a polymer and an antigen, 85. The composition of paragraph 84, wherein the (b) a conjugate of a polymer and a immunostimula- one or more amine moieties are one or more aliphatic tory agent, and/or (c) a conjugate of a polymer and amines. 15 a targeting moiety.

86. The composition of any of paragraphs 83-85, 101. The composition of paragraph 100, wherein the wherein the nanocarrier is a latex particle. nanocarrier comprises (a) a conjugate of a polymer and an antigen and (b) a conjugate of a polymer and 87. The composition of paragraphs 83-85, wherein 20 an immunostimulatory agent. the nanocarrier has a net positive charge at neutral pH. 102. The composition of paragraph 100, wherein the nanocarrier comprises (a) a conjugate of a polymer 88. The composition of any of paragraphs 1-87, and an antigen and (c) a conjugate of a polymer and wherein the nanocarrier is composed of one or more 25 a targeting moiety. polymers. 103. The composition of paragraph 100, wherein the 89. The composition of paragraph 88, wherein the nanocarrier comprises (b) a conjugate of a polymer one or more polymers is a water soluble, non-adhe- and an immunostimulatory agent and (c) a conjugate sive polymer. 30 of a polymer and a targeting moiety.

90. The composition of paragraph 89, wherein the 104. The composition of paragraph 100, wherein the water soluble, non-adhesive polymer is PEG, PEO, nanocarrier comprises (a) a conjugate of a polymer polyalkylene glycol, or polyalkylene oxide. and an antigen, (b) a conjugate of a polymer and an 35 immunostimulatory agent and (c) a conjugate of a 91. The composition of paragraph 88, wherein the polymer and a targeting moiety. one or more polymers is a biodegradable polymer. 105. The composition of any of paragraphs 100-104, 92. The composition of paragraph 88, wherein the wherein the conjugate or conjugates is/are a cova- one or more polymers is a biocompatible polymer 40 lent conjugate or covalent conjugates. that is a conjugate of a water soluble, non-adhesive polymer and a biodegradable polymer. 106. The composition of any of paragraphs 100-105, wherein the nanocarrier is a microparticle, nanopar- 93. The composition of paragraph 91 or 92, wherein ticle, or picoparticle formed by self-assembly. the biodegradable polymer is PLA, PGA or PLGA. 45 107. The composition of any of paragraphs 101-102 94. The composition of any of paragraphs 1-93, and 104-106, wherein the antigen is a B cell antigen. wherein the nanocarrier is formed by self- assembly. 108. The composition of paragraph 107, wherein the 95. The composition of any of paragraphs 1-94, 50 nanocarrier further comprises a conjugate of a pol- wherein the immune response is induced, en- ymer and a T cell antigen. hanced, suppressed, directed or redirected. 109. The composition of paragraph 108, wherein the 96. The composition of any of paragraphs 1-95, conjugate is a covalent conjugate. wherein the nanocarrier targets one or more second- 55 ary lymphoid tissues or organ. 110. The composition of any of paragraphs 101-102 and 104-106, wherein the antigen is a T cell antigen. 97. The composition of paragraph 96, wherein the

81 161 EP 2 394 657 A1 162

111. The composition of paragraph 110, wherein the antigen presenting cells are macrophages. nanocarrier further comprises a conjugate of a pol- ymer and a B cell antigen. 123. The composition of paragraph 122, wherein the macrophages are subcapsular sinus macrophages. 112. The composition of paragraph 111, wherein the 5 conjugate is a covalent conjugate. 124. The composition of paragraph 121, wherein the antigen presenting cells are dendritic cells. 113. A composition comprising a nanocarrier com- prising a molecule or molecules of the following for- 125. The composition of paragraph 121, wherein the mula X-L1-Y-L2-Z, wherein X is a biodegradable pol- 10 antigen presenting cells are follicular dendritic cells. ymer, Y is a water soluble, non- adhesive polymer, Z is a targeting moiety, an immunostimulatory agent, 126. The composition of any of paragraphs 113-118, an immunomodulatory agent, or a pharmaceutical wherein the targeting moiety is a molecule that binds agent, and L1 and L2 are bonds or linking molecules, to CD11b, CD169, mannose receptor, DEC-205, wherein either Y or Z, but not both Y and Z, can be 15 CD11c, CD21/CD35, CX3CR1 or a Fc receptor. absent. 127. The composition of paragraph 126, wherein the 114. The composition of paragraph 113, wherein the targeting moiety is a molecule that binds to CD169, nanocarrier encapsulates an antigen, an immunos- CX3CR1, or a Fc receptor. timulatory agent, or both. 20 128. The composition of any of paragraphs 113-127, 115. The composition of paragraph 113 or 114, wherein Y is PEG, PEO, polyalkylene glycol, or poly- wherein Z is an antigen. alkylene oxide.

116. The composition of any of paragraphs 113-115, 25 129. The composition of any of paragraphs 113-128, wherein Z is a degenerative disease antigen, an in- wherein X is PLGA, PLA or PGA. fectious disease antigen, a cancer antigen, an atopic disease antigen, an autoimmune disease antigen, 130. The composition of any of paragraphs 113 and an alloantigen, a xenoantigen, an allergen, an ad- 121-126, wherein Z is absent. dictive substance, or a metabolic disease enzyme 30 or enzymatic product. 131. The composition of any of paragraphs 113-130, wherein the nanocarrier is a microparticle, nanopar- 117. The composition of paragraph 114, wherein the ticle, or picoparticle. antigen is a degenerative disease antigen, an infec- tious disease antigen, a cancer antigen, an atopic 35 132. The composition of paragraph 131, wherein the disease antigen, an autoimmune disease antigen, particle is formed by self-assembly. an alloantigen, a xenoantigen, an allergen, an ad- dictive substance, or a metabolic disease enzyme 133. A composition comprising a nanocarrier com- or enzymatic product. prising an immunostimulatory agent. 40 118. The composition of any of paragraphs 113, 114, 134. The composition of paragraph 133, further com- or 117, wherein Z is a targeting moiety that binds a prising an antigen and/or a targeting moiety. receptor expressed on the surface of a cell. 135. The composition of paragraph 134, wherein at 119. The composition of any of paragraphs 113, 114, 45 least one of the antigen, targeting moiety and immu- or 117, wherein Z is a targeting moiety that binds a nostimulatory agent is conjugated to a water soluble, soluble receptor. non-adhesive polymer.

120. The composition of paragraph 119, wherein the 136. The composition of paragraph 134, wherein at soluble receptor is a complement protein or a pre- 50 least one of the antigen, targeting moiety and immu- existing antibody. nostimulatory agent is conjugated to a biodegrada- ble polymer. 121. The composition of any of paragraphs 113-118, wherein the targeting moiety is for delivery of the 137. The composition of paragraph 134, wherein at nanocarrier to antigen presenting cells, T cells or B 55 least one of the antigen, targeting moiety and immu- cells. nostimulatory agent is conjugated to a biocompatible polymer that is a conjugate of a water soluble, non- 122. The composition of paragraph 121, wherein the adhesive polymer conjugated to a biodegradable

82 163 EP 2 394 657 A1 164 polymer. wherein the particle is formed by self- assembly.

138. The composition of any of paragraphs 134-137, 153. The composition of any of paragraphs 144-150, wherein the antigen is a B cell antigen. wherein the small molecule is an addictive sub- 5 stance, a small molecule from a chemical weapon, 139. The composition of paragraph 138, wherein the or a hazardous environmental agent. B cell antigen is a T cell independent antigen. 154. A composition comprising: a nanocarrier that 140. The composition of paragraph 138 or 139, targets a specific cell or tissue and modulates an wherein the nanocarrier further comprises a T cell 10 immune response comprising a B cell antigen on its antigen. surface at a density that activates B cell receptors and a immunostimulatory agent. 141. The composition of any of paragraphs 134-137, wherein the antigen is a T cell antigen. 155. The composition of paragraph 154, wherein the 15 nanocarrier further comprises a targeting moiety. 142. The composition of any of paragraphs 134-141, wherein the nanocarrier is a microparticle, nanopar- 156. The composition of paragraph 154 or 155, ticle, or picoparticle. wherein the nanocarrier is a microparticle, nanopar- ticle, or picoparticle. 143. The composition of paragraph 142, wherein the 20 particle is formed by self-assembly. 157. The composition of paragraph 156, where the particle is formed by self-assembly. 144. A composition comprising a microparticle, na- noparticle, or picoparticle comprising a small mole- 158. The composition of any of paragraphs 1-157, cule, a targeting moiety, a immunostimulatory agent, 25 wherein the composition is a pharmaceutical com- and a T cell antigen. position and further comprises a pharmaceutically acceptable carrier. 145. The composition of paragraph 144, wherein the small molecule is conjugated to a polymer. 159. The composition of paragraph 158, wherein the 30 pharmaceutical composition is a vaccine composi- 146. The composition of paragraph 145, wherein the tion. polymer is a biocompatible polymer that is a conju- gate of a water soluble, non-adhesive polymer and 160. A pharmaceutical composition comprising a a biodegradable polymer. conjugate of an antigen presenting cell-targeting 35 moiety and a nanocarrier. 147. The composition of any of paragraphs 144-146, wherein the targeting moiety is conjugated to a pol- 161. The pharmaceutical composition of paragraph ymer. 160, wherein the conjugate is a covalent conjugate.

148. The composition of paragraph 147, wherein the 40 162. The pharmaceutical composition of paragraph polymer is a biocompatible polymer that is a conju- 160 or 161, wherein the antigen presenting cell-tar- gate of a water soluble, non-adhesive polymer and geting moiety targets macrophages. a biodegradable polymer. 163. The pharmaceutical composition of paragraph 149. The composition of any of paragraphs 144-148, 45 162, wherein the macrophages are subcapsular si- wherein the immunostimulatory agent is conjugated nus macrophages. to a biodegradable polymer. 164. The pharmaceutical composition of paragraph 150. The composition of any of paragraphs 146, 148, 160 or 161, wherein the antigen presenting cell-tar- or 149, wherein the biodegradable polymer is PLGA, 50 geting moiety targets dendritic cells. PLA or PGA. 165. The pharmaceutical composition of paragraph 151. The composition of paragraph 146 or 148, 160 or 161, wherein the antigen presenting cell-tar- wherein the water soluble, non-adhesive polymer is geting moiety targets follicular dendritic cells. PEG, PEO, polyalkylene glycol, or polyalkylene ox- 55 ide. 166. The pharmaceutical composition of any of par- agraphs 160-165, wherein the nanocarrier is a mi- 152. The composition of any of paragraphs 144-150, croparticle, nanoparticle, or picoparticle.

83 165 EP 2 394 657 A1 166

167. The pharmaceutical composition of paragraph presenting cells, T cells or B cells. 166, wherein the particle is formed by self- assembly. 180. The composition of paragraph 179, wherein the 168. A pharmaceutical composition comprising a antigen presenting cells are macrophages. conjugate of an immunostimulatory agent and a na- 5 nocarrier. 181. The composition of paragraph 180, wherein the macrophages are subcapsular sinus macrophages. 169. The pharmaceutical composition of paragraph 168, wherein the conjugate is a covalent conjugate. 182. The composition of paragraph 179, wherein the 10 antigen presenting cells are dendritic cells. 170. The pharmaceutical composition of paragraph 168 or 169, wherein the nanocarrier is a microparti- 183. The composition of paragraph 179, wherein the cle, nanoparticle, or picoparticle. antigen presenting cells are follicular dendritic cells.

171. The pharmaceutical composition of paragraph 15 184. The composition of any of paragraphs 175 - 170, wherein the particle is formed by self- assembly. 176, wherein the targeting moiety is a molecule that specifically binds to CD11b, CD169, mannose re- 172. A composition comprising a molecule with the ceptor, DEC-205, CD11c, CD21/CD35, CX3CR1, or formula X-L1-Y-L2-Z, wherein X is a biodegradable a Fc receptor. polymer, Y is a water soluble, non-adhesive polymer, 20 Z is a targeting moiety, an immunostimulatory agent, 185. The composition of paragraph 184, wherein the an immunomodulatory agent, or a pharmaceutical targeting moiety is CD169, CX3CR1, or a Fc recep- agent, and L1 and L2 are bonds or linking molecules, tor. wherein either Y or Z, but not both Y and Z, can be absent. 25 186. The composition of any of paragraphs 175-188, wherein Y is PEG, PEO, polyalkylene glycol, or poly- 173. A composition comprising a molecules with the alkylene oxide. formula T-L1-X-L2-Y-L3-Z, where T is a T cell anti- gen, X is a biodegradable polymer, Y is a water sol- 187. The composition of any of paragraphs 175- 186, uble, non-adhesive polymer, Z is a B cell antigen, 30 wherein X is PLGA, PGA, or PLA. wherein L1, L2, and L3 are bonds or linking mole- cules, and wherein any one or two of T, Y, and Z, 188. The composition of any of paragraphs 175 and but not all three of T, Y, and Z, can be absent. 186-188, wherein Z is absent.

174. The composition of paragraph 172, wherein Z 35 189. The composition of any of paragraphs 175 and is an antigen. 186- 187, wherein Y is absent.

175. The composition of any of paragraphs 172-174, 190. A pharmaceutical composition comprising a wherein Z is a degenerative disease antigen, an in- conjugate of an immunostimulatory agent and a pol- fectious disease antigen, a cancer antigen, an atopic 40 ymer. disease antigen, an autoimmune disease antigen, an alloantigen, a xenoantigen, an allergen, a hapten, 191. The composition of paragraph 190, wherein the an addictive substance, or a metabolic disease en- conjugate is a covalent conjugate. zyme or enzymatic product. 45 192. The pharmaceutical composition of paragraph 176. The composition of paragraph 175, wherein Z 190 or 191, wherein the polymer is a biocompatible is a targeting moiety that binds a receptor expressed polymer. on the surface of a cell. 193. The pharmaceutical composition of paragraph 177. The composition of paragraph 175, wherein Z 50 192, wherein the biocompatible polymer is a biode- is a targeting moiety that binds a soluble receptor. gradable polymer or a water soluble, non-adhesive polymer. 178. The composition of paragraph 177, wherein the soluble receptor is complement or a preexisting an- 194. The pharmaceutical composition of paragraph tibody. 55 193, wherein the biodegradable polymer is synthetic.

179. The composition of any of paragraphs 175-176, 195. The pharmaceutical composition of any of par- wherein the targeting moiety is for targeting antigen agraphs 190-194, wherein the pharmaceutical com-

84 167 EP 2 394 657 A1 168 position comprises nanocarriers wherein the conju- 211. The method of paragraph 210, wherein the gate is a component of the nanocarriers. composition is in an amount effective to induce or enhance an immune response. 196. The pharmaceutical composition of paragraph 195, wherein the nanocarriers are microparticles, 5 212. The method of paragraph 211, wherein the sub- nanoparticles, or picoparticles. ject has or is susceptible to having cancer, an infec- tious disease, a non-autoimmune metabolic or de- 197. The pharmaceutical composition of any of par- generative disease, or an addiction. agraphs 190-196, further comprising an antigen. 10 213. The method of paragraph 212, wherein the in- 198. The pharmaceutical composition of any of par- fectious disease is a chronic viral infection. agraphs 190-196, wherein the pharmaceutical com- position does not comprise an antigen. 214. The method of paragraph 213, wherein the chronic viral infection is HIV, HPV, HBV, or HCV in- 199. The pharmaceutical composition of any of par- 15 fection. agraphs 190-198, further comprising a targeting agent. 215. The method of paragraph 212, wherein the in- fectious disease is or is caused by a bacterial infec- 200. A vaccine composition comprising a conjugate tion. of an immunostimulatory agent and a polymer. 20 216. The method of paragraph 212, wherein the sub- 201. The vaccine composition of paragraph 200, ject has or is susceptible to having a Pseudomonas wherein the conjugate is a covalent conjugate. infection, a Pneumococcus infection, tuberculosis, malaria, leishmaniasis, H. pylori, a Staphylococcus 202. The vaccine composition of paragraph 200 or 25 infection, or a Salmonella infection. 201, wherein the polymer is a water soluble, non- adhesive polymer. 217. The method of paragraph 212, wherein the in- fectious disease is or is caused by a fungal infection. 203. The vaccine composition of paragraph 202, wherein the polymer is synthetic. 30 218. The method of paragraph 212, wherein the in- fectious disease is or is caused by a parasitic infec- 204. The vaccine composition of paragraph 202 or tion. 203 wherein the water soluble, non-adhesive poly- mer is polyethylene glycol. 219. The method of paragraph 212, wherein the in- 35 fectious disease is or is caused by a protozoan in- 205. The vaccine composition of any of paragraphs fection. 200-204, wherein the vaccine composition compris- es nanocarriers wherein the conjugate is a compo- 220. The method of paragraph 212, wherein the sub- nent of the nanocarriers. ject has or is susceptible to having influenza. 40 206. The vaccine composition of paragraph 205, 221. The method of paragraph 210, wherein the wherein the nanocarriers are microparticles, nano- composition is administered in an amount effective particles, or picoparticles. to suppress an immune response.

207. The vaccine composition of any of paragraphs 45 222. The method of paragraph 210, wherein the 200-206, further comprising an antigen. composition is administered in an amount effective to modify an immune response. 208. The vaccine composition of any of paragraphs 200-206, wherein the vaccine composition does not 223. The method of paragraph 221 or 222, wherein comprise an antigen. 50 the subject has an autoimmune disease, an allergy, or asthma. 209. The vaccine composition of any of paragraphs 200-208, further comprising a targeting agent. 224. The method of paragraph 223, wherein the au- toimmune disease is disease is lupus, multiple scle- 210. A method comprising administering the com- 55 rosis, rheumatoid arthritis, diabetes mellitus type I, position of any one of paragraphs 1-209 to a subject inflammatory bowel disease, thyroiditis, or celiac dis- in an amount effective to modulate an immune re- ease. sponse.

85 169 EP 2 394 657 A1 170

225. A nanocarrier comprising an immunomodula- 236. A nanocarrier comprising an immunomodula- tory agent that is capable of stimulating an immune tory agent that is capable of stimulating an immune response in T cells and in B cells; a targeting moiety response in T cells and in B cells; a targeting moiety, for delivery of the nanocarrier to antigen presenting wherein the targeting moiety specifically binds to cells, T cells, or B cells; and an excipient. 5 CD11 c; and an excipient.

226. The nanocarrier of paragraph 225, wherein the 237. A nanocarrier comprising an immunomodula- antigen presenting cells are subcapsular sinus mac- tory agent that is capable of stimulating an immune rophages. response in T cells and in B cells; a targeting moiety, 10 wherein the targeting moiety specifically binds to 227. The nanocarrier of paragraph 225, wherein the CD21/CD35; and an excipient. antigen presenting cells are dendritic cells. 238. A nanocarrier comprising an immunomodula- 228. The nanocarrier of paragraph 225, wherein the tory agent that is capable of stimulating an immune antigen presenting cells are follicular dendritic cells. 15 response in T cells and in B cells in regional lymph nodes; a targeting moiety for delivery of the nano- 229. A nanocarrier comprising an immunomodula- carrier to antigen presenting cells, T cells, or B cells; tory agent that is capable of stimulating an immune and an excipient. response in T cells and in B cells; a targeting moiety for delivery of the nanocarrier to subcapsular sinus 20 239. A nanocarrier comprising a first immunomodu- macrophages; and an excipient. latory agent, wherein the first immunomodulatory agent is on the surface of the nanocarrier and is ca- 230. A nanocarrier comprising an immunomodula- pable of stimulating B cells or antigen- presenting tory agent that is capable of stimulating an immune cells; a second immunomodulatory agent, wherein response in T cells and in B cells; a targeting moiety 25 the second immunomodulatory agent is either on the for delivery of the nanocairier to dendritic cells; and surface or is encapsulated within the nanocarrier and an excipient. is capable of being processed by antigen presenting cells and presented to T cells; and an excipient, 231. A nanocarrier comprising an immunomodula- tory agent that is capable of stimulating an immune 30 240. The nanocarrier of paragraph 239, wherein the response in T cells and in B cells; a targeting moiety stimulation of an immune response in B cells is de- for delivery of the nanocarrier to follicular dendritic termined by comparing antibody titers of an immu- cells; and an excipient. nized subject to antibody titers of a non-immunized subject. 232. A nanocarrier comprising an immunomodula- 35 tory agent that is capable of stimulating an immune 241. The nanocarrier of paragraph 240, wherein an response in T cells and in B cells; a targeting moiety, immune response in B cells is said to be stimulated wherein the targeting moiety specifically binds to if antibody titers are measured to be about 10-fold CD11b; and an excipient. greater in the immunized subject than in the non- 40 immunized subject. 233. A nanocarrier comprising an immunomodula- tory agent that is capable of stimulating an immune 242. The nanocarrier of paragraph 240, wherein an response in T cells and in B cells; a targeting moiety, immune response in B cells is said to be stimulated wherein the targeting moiety specifically binds to if antibody titers are measured to be about 100-fold CD169; and an excipient. 45 greater in the immunized subject than in the non- immunized subject. 234. A nanocarrier comprising an immunomodula- tory agent that is capable of stimulating an immune 243. The nanocarrier of paragraph 240, wherein an response in T cells and in B cells; a targeting moiety, immune response in B cells is said to be stimulated wherein the targeting moiety specifically binds to the 50 if antibody titers are measured to be about 1000-fold mannose receptor; and an excipient. greater in the immunized subject than in the non- immunized subject. 235. A nanocarrier comprising an immunomodula- tory agent that is capable of stimulating an immune 244. The nanocarrier of paragraph 239, wherein the response in T cells and in B cells; a targeting moiety, 55 stimulation of an immune response in B cells is de- wherein the targeting moiety specifically binds to termined by measuring antibody affinity. DEC-205; and an excipient. 245. The nanocarrier of paragraph 244, wherein an-

86 171 EP 2 394 657 A1 172 tibody affinity is determined by measuring the equi- vigorous germinal center reaction is determined to librium dissociation constant of the antibody. have occurred by performing histology analyses.

246. The nanocarrier of paragraph 245, wherein an 258. The nanocarrier of paragraph 239, wherein a immune response in B cells is said to be stimulated 5 vigorous germinal center reaction is determined to if an antibody has an equilibrium dissociation con- have occurred by performing immunofluorescence stant that is less than 10-7 M. of lymphoid tissues.

247. The nanocarrier of paragraph 245, wherein an 259. The nanocarrier of paragraph 239, wherein the immune response in B cells is said to be stimulated 10 stimulation of an immune response in B cells is de- if an antibody has an equilibrium dissociation con- termined by identifying antibody isotypes. stant that is less than 10-8 M. 260. The nanocarrier of paragraph 259, wherein an 248. The nanocarrier of paragraph 245, wherein an immune response in a B cell is said to be stimulated immune response in B cells is said to be stimulated 15 if a B cell is identified that produces IgG isotype an- if an antibody has an equilibrium dissociation con- tibodies. stant that is less than 10-9 M. 261. The nanocarrier of paragraph 239, wherein the 249. The nanocarrier of paragraph 245, wherein an stimulation of an immune response in B cells is de- immune response in B cells is said to be stimulated 20 termined by analyzing antibody function in neutrali- if an antibody has an equilibrium dissociation con- zation assays. stant that is less than 10-10 M. 262. The nanocarrier of paragraph 261, wherein an 250. The nanocarrier of paragraph 245, wherein an immune response in B cells is said to be stimulated immune response in B cells is said to be stimulated 25 if infection of a microorganism is neutralized at a di- if an antibody has an equilibrium dissociation con- lution of about 1:10. stant that is less than 10-11 M. 263. The nanocarrier of paragraph 261, wherein an 251. The nanocarrier of paragraph 245, wherein an immune response in B cells is said to be stimulated immune response in B cells is said to be stimulated 30 if infection of a microorganism is neutralized at a di- if an antibody has an equilibrium dissociation con- lution of about 1:100. stant that is less than 10-12 M. 264. The nanocarrier of paragraph 261, wherein an 252. The nanocarrier of paragraph 239, wherein an immune response in B cells is said to be stimulated immune response in B cells is said to be stimulated 35 if infection of a microorganism is neutralized at a di- if class-switch recombination in B cells has occurred. lution of about 1 : 1000.

253. The nanocarrier of paragraph 252, wherein 265. The nanocarrier of paragraph 261, wherein an class-switch recombination in B cells causes the an- immune response in B cells is said to be stimulated tibody isotype produced by the B cells to switch from 40 if infection of a microorganism is neutralized at a di- IgM to IgG. lution of about 1:5000.

254. The nanocarrier of paragraph 239, wherein the 266. The nanocarrier of paragraph 261, wherein an stimulation of an immune response in B cells is de- immune response in B cells is said to be stimulated termined by measuring affinity maturation of antigen- 45 if infection of a microorganism is neutralized at a di- specific antibodies. lution of about 1 :10,000.

255. The nanocarrier of paragraph 239, wherein an 267. The nanocarrier of paragraph 239, wherein the immune response in B cells is said to be stimulated stimulation of an immune response in T cells is de- if memory B cells or long-lived plasma cells that can 50 termined by measuring antigen-induced production produce large amounts of high-affinity antibodies for of cytokines by T cells. extended periods of time have formed. 268. The nanocarrier of paragraph 267, wherein the

256. The nanocarrier of paragraph 239, wherein an cytokine is IFNγ, IL-4, IL-2, or TNFα. immune response in B cells is said to be stimulated 55 if a vigorous germinal center reaction occurs. 269. The nanocarrier of paragraph 267, wherein an- tigen-induced production of cytokines by T cells is 257. The nanocarrier of paragraph 239, wherein a measured by intracellular cytokine staining followed

87 173 EP 2 394 657 A1 174 by flow cytometry. immune response in T cells is said to be stimulated if immunized subjects comprise about 10,000-fold 270. The nanocarrier of paragraph 267, wherein an- more cytokine-producing cells than do naive con- tigen-induced production of cytokines by T cells is trols. measured by surface capture cytokine staining fol- 5 lowed by flow cytometry. 282. The nanocarrier of paragraph 277, wherein an immune response in T cells is said to be stimulated 271. The nanocarrier of paragraph 267, wherein an- if immunized subjects comprise about 100,000-fold tigen-induced production of cytokines by T cells is more cytokine-producing cells than do naive con- measured by determining cytokine concentration in 10 trols. culture supernatant by ELISA. 283. The nanocarrier of paragraph 267, wherein an- 272. The nanocarrier of paragraph 269, wherein an tigen-induced production of cytokines by T cells is immune response in T cells is said to be stimulated measured by determining antigen-induced prolifer- if at least 1% of antigen-specific T cells produce cy- 15 ation of T cells. tokines. 284. The nanocarrier of paragraph 283, wherein an- 273. The nanocarrier of paragraph 269, wherein an tigen-induced proliferation of T cells is measured by immune response in T cells is said to be stimulated comparing uptake of H3-thymidine in a test sample if at least 25% of antigen-specific T cells produce 20 to H3-thymidine uptake in a naïve control. cytokines. 285. The nanocarrier of paragraph 284, wherein an 274. The nanocarrier of paragraph 269, wherein an immune response in T cells is said to be stimulated immune response in T cells is said to be stimulated if H3-thymidine uptake is about 10-fold higher in the if at least 50% of antigen-specific T cells produce 25 test sample than in the naïve control. cytokines. 286. The nanocarrier of paragraph 284, wherein an 275. The nanocarrier of paragraph 269, wherein an immune response in T cells is said to be stimulated immune response in T cells is said to be stimulated if H3-thymidine uptake is about 50-fold higher in the if at least 90% of antigen-specific T cells produce 30 test sample than in the naïve control. cytokines. 287. The nanocarrier of paragraph 284, wherein an 276. The nanocarrier of paragraph 269, wherein an immune response in T cells is said to be stimulated immune response in T cells is said to be stimulated if H3-thymidine uptake is about 100-fold higher in the if substantially 100% of antigen-specific T cells pro- 35 test sample than in the naïve control. duce cytokines. 288. The nanocarrier of paragraph 284, wherein an 277. The nanocarrier of paragraph 267, wherein an- immune response in T cells is said to be stimulated tigen -induced production of cytokines by T cells is if H3-thymidine uptake is about 1000-fold higher in measured by ELISPOT assay. 40 the test sample than in the naïve control.

278. The nanocarrier of paragraph 277, wherein an 289. The nanocarrier of paragraph 284, wherein an immune response in T cells is said to be stimulated immune response in T cells is said to be stimulated if immunized subjects comprise about 10-fold more if H3-thymidine uptake is about 10-fold higher in the cytokine-producing cells than do naive controls. 45 test sample than in the naïve control.

279. The nanocarrier of paragraph 277, wherein an 290. The nanocarrier of paragraph 283, wherein an- immune response in T cells is said to be stimulated tigen-induced proliferation of T cells is measured by if immunized subjects comprise about 100-fold more performing a carboxyfluorescein succinimidyl ester cytokine-producing cells than do naive controls. 50 (CFSE) dilution assay.

280. The nanocarrier of paragraph 277, wherein an 291. The nanocarrier of paragraph 290, wherein an immune response in T cells is said to be stimulated immune response in T cells is said to be stimulated if immunized subjects comprise about 1000-fold if CFSE dilution is about 10%. more cytokine-producing cells than do naive con- 55 trols. 292. The nanocarrier of paragraph 290, wherein an immune response in T cells is said to be stimulated 281. The nanocarrier of paragraph 277, wherein an if CFSE dilution is about 25%.

88 175 EP 2 394 657 A1 176

293. The nanocarrier of paragraph 290, wherein an second immunomodulatory agent is capable of stim- immune response in T cells is said to be stimulated ulating an immune response in T cells. if CFSE dilution is about 50%. 306. The nanocarrier of paragraph 225, wherein the 294. The nanocarrier of paragraph 290, wherein an 5 second immunomodulatory agent is capable of stim- immune response in T cells is said to be stimulated ulating an immune response in B cells. if CFSE dilution is about 75%. 307. The nanocarrier of paragraph 225, wherein the 295. The nanocarrier of paragraph 291, wherein an second immunomodulatory agent is capable of stim- immune response in T cells is said to be stimulated 10 ulating an immune response in antigen presenting if CFSE dilution is about 100%. cells.

296. The nanocarrier of paragraph 267, wherein the 308. The nanocarrier of paragraph 225, further com- stimulation of an immune response in T cells is de- prising a targeting moiety. termined by measuring cellular markers of T cell ac- 15 tivation. 309. The nanocarrier of paragraph 225, further com- prising a plurality of targeting moieties. 297. The nanocarrier of paragraph 296, wherein T cells are said to be stimulated if they express elevat- 310. The nanocarrier of paragraph 309, wherein all ed levels of CD11a CD27, CD25, CD40L, CD44, 20 of the individual targeting moieties of the plurality of CD45RO, or CD69 relative to naïve control cells. targeting moieties are identical to one another.

298. The nanocarrier of paragraph 296, wherein T 311. The nanocarrier of paragraph 309, wherein all cells are said to be stimulated if they express lower of the individual targeting moieties of the plurality of levels of L-selectin (CD62L), CD45RA, or CCR7 rel- 25 targeting moieties are not identical to one another. ative to naïve control cells. 312. The nanocarrier of paragraph 308, wherein the 299. The nanocarrier of paragraph 267, wherein the targeting moiety is selected from the group consist- stimulation of an immune response in T cells is de- ing of antibodies, fragments of antibodies, proteins, termined by assaying cytotoxicity by effector CD8+ 30 peptides, glycoproteins, aptamers, oligonucle- T cells against antigen-pulsed target cells. otides, carbohydrates, lipid, glycosamiooglycan, af- fibody, adnectin, spiegelmer, avimer, nanobody, and 300. The nanocarrier of paragraph 299, wherein as- small molecules. saying cytotoxicity by effector CD8+ T cells against antigen-pulsed target cells is performed using 35 313. The nanocarrier of paragraph 308, wherein the a 51chromium (51Cr) release assay. targeting moiety targets an antigen presenting cell.

301. The nanocarrier of paragraph 225, wherein the 314. The nanocarrier of paragraph 308, wherein the immunomodulatory agent is covalently associated targeting moiety targets an antigen presenting cell with the nanocarrier. 40 selected from the group consisting of dendritic cells (DC), subcapsular sinus macrophages (SCS-Mph), 302. The nanocarrier of paragraph 225, wherein the B cells, and follicular dendritic cells (FDC). immunomodulatory agent is non-covalently associ- ated with the nanocarrier. 315. The nanocarrier of paragraph 308, wherein the 45 targeting moiety targets a dendritic cells. 303. The nanocarrier of paragraph 302, wherein the non-covalent association is mediated by charge in- 316. The nanocarrier of paragraph 308, wherein the teractions, affinity interactions, metal coordination, targeting moiety targets DEC-205 (CD205). physical adsorption, host-guest interactions, hydro- phobic interactions, TT stacking interactions, hydro- 50 317. The nanocarrier of paragraph 308, wherein the gen bonding interactions, van der Waals interac- targeting moiety targets CD169. tions, magnetic interactions, electrostatic interac- tions, or dipole-dipole interactions. 318. The nanocarrier of paragraph 308, wherein the targeting moiety targets CD11b. 304. The nanocarrier of paragraph 225, further com- 55 prising a second immunomodulatory agent. 319. The nanocarrier of paragraph 308, wherein the targeting moiety targets mannose receptor. 305. The nanocarrier of paragraph 225, wherein the

89 177 EP 2 394 657 A1 178

320. The nanocarrier of paragraph 308, wherein the face of the liposome. targeting moiety targets CD21/CD35 (CR2/CR1). 338. The nanocarrier of paragraph 336, wherein the 321. The nanocarrier of paragraph 225, further com- amphiphilic entity is selected from the group consist- prising an immunostimulatory agent. 5 ing of phosphatidylcholine, lipid A, cholesterol, doli- chol, shingosine, sphingomyelin, ceramide, cerebro- 322. The nanocarrier of paragraph 321, wherein the side, sulfatide, phytosphingosine, phosphatidyleth- immunostimulatory agent is selected from the group anolamine, glycosylceramide, phosphatidylglycerol, consisting of toll-like receptor (TLR) agonists, cy- phosphatidylinositol, phosphatidylserine, cardioli- tokine receptor agonists, CD40 agonists, Fc receptor 10 pin, phosphatidic acid, and lysophosphatides. agonists, and complement receptor agonists. 339. The nanocarrier of paragraph 225, wherein the 323. The nanocarrier of paragraph 225, further com- nanocarrier is a liposome-polymer-based nanocar- prising a TLR-7 agonist. rier. 15 324. The nanocarrier of paragraph 225, further com- 340. The nanocarrier of paragraph 225, wherein the prising CpGs. nanocarrier is a lipid monolayer-based nanocarrier.

325. The nanocarrier of paragraph 225, further com- 341. The nanocarrier of paragraph 225, wherein the prising a TLR-4 agonist. 20 second immunomodulatory agent is located within a reverse micelle. 326. The nanocarrier of paragraph 225, further com- prising bacterial lipopolysaccharide (LPS). 342. The nanocarrier of paragraph 225, wherein the excipient is a polymer selected from the group con- 327. The nanocarrier of paragraph 225, further com- 25 sisting of polyethylenes, polycarbonates, polyanhy- prising a CD40 agonist. drides, polyhydroxyacids, polypropylfumarates, polycaprolactones, polyamides, polyacetals, poly- 328. The nanocarrier of paragraph 225, further com- ethers, polyesters, poly(orthoesters), polycyanoacr- prising a TLR-2 agonist. ylates, polyvinyl alcohols, polyurethanes, polyphos- 30 phazenes, polyacrylates, polymethacrylates, polyu- 329. The nanocarrier of paragraph 225, further com- rias, polystyrenes, polyamines, derivatives thereof, prising an Fc-gamma receptor agonist. combinations thereof, and copolymers thereof.

330. The nanocarrier of paragraph 225, further com- 343. The nanocarrier of paragraph 225, wherein the prising a complement receptor agonist. 35 excipient is a polymer selected from the group con- sisting of polyethylene glycol (PEG); poly(lactic acid- 331. The nanocarrier of paragraph 324, wherein the co-glycolic acid); copolymers of poly(lactic acid-co- complement receptor agonist binds to CD21 or glycolic acid) and PEG; poly(lactide-co-glycolide); CD35. copolymers of poly(lactide-co-glycolide) and PEG; 40 polyglycolic acid; copolymers of polyglycolic acid 332. The nanocarrier of paragraph 225, further com- and PEG; poly-L-lactic acid; copolymers of poly-L- prising a cytokine. lactic acid and PEG; poly-D-lactic acid; copolymers of poly-D-lactic acid and PEG; poly-D,L-lactic acid; 333. The nanocarrier of paragraph 225, wherein the copolymers of poly-D,L-lactic acid and PEG; poly-L- nanocarrier comprises a lipid membrane. 45 lactide; copolymers of poly-L-lactide and PEG; poly- D-lactide; copolymers of poly-D-lactide and PEG; 334. The nanocarrier of paragraph 225, wherein the poly-D,L-lactide; copolymers of poly-D-lactide and nanocarrier comprises a polymeric matrix. PEG; poly(ortho ester); copolymers of poly(ortho es- ter) and PEG; poly(caprolactone); copolymers of po- 335. The nanocarrier of paragraph 225, wherein the 50 ly(caprolactone) and PEG; poly lysine; copolymers nanocarrier comprises a metallic particle. of polylysine and PEG; poly (ethylene imine); copol- ymers of poly(ethylene imine) and PEG; polyhy- 336. The nanocarrier of paragraph 225, wherein the droxyacids; polyanhydrides; poly(L-lactide-co-L- nanocarrier is a liposome comprising an amphiphilic lysine); poly(serine ester); poly(4-hydroXy-L-proline entity. 55 ester); poly[α-(4-aminobutyl)-L-glycolic acid]; deriv- atives thereof; combinations thereof; and copoly- 337. The nanocarrier of paragraph 336, wherein the mers thereof. amphiphilic entity is located within the interior sur-

90 179 EP 2 394 657 A1 180

344. The nanocarrier of paragraph 225, wherein the second immunomodulatory agent is a collection of excipient is a copolymer of polyethylene glycol and peptides. poly(lactide-co-glycolide). 359. The nanocarrier of paragraph 225, wherein the 345. The nanocarrier of paragraph 225, wherein the 5 first immunomodulatory agent and the second im- excipient is poly(lactic-co-glycolic acid) (PLGA). munomodulatory agent are identical to one another.

346. The nanocarrier of paragraph 225, wherein the 360. The nanocarrier of paragraph 225, wherein the excipient comprises poly(lactic-coglycolic acid) first immunomodulatory agent and the second im- (PLGA), lipid, and polyethylene glycol. 10 munomodulatory agent are not identical to one an- other. 347. The nanocarrier of paragraph 225, wherein the excipient comprises a lipid. 361. A pharmaceutical composition comprising a therapeutically effective amount of the nanocarrier 348. The nanocarrier of paragraph 225, wherein the 15 of paragraph 225 and a pharmaceutically acceptable lipid is selected from the group consisting of phos- excipient. phatidylcholine, lipid A, cholesterol, dolichol, shingo- sine, sphingomyelin, ceramide, glycosylceramide, 362. A pharmaceutical composition comprising a cerebroside, sulfatide, phytosphingosine, phos- therapeutically effective amount of the nanocarrier phatidylethanolamine, phosphatidylglycerol, phos- 20 of paragraph 225 and an adjuvant. phatidylinositol, phosphatidylsefine, cardiolipin, phosphatidic acid, and lysophosphatides. 363. The pharmaceutical composition of paragraph 362, wherein the adjuvant is alum, MF59 (Novartis), 349. The nanocarrier of paragraph 225, wherein the QS21, cholera toxin, Freund’s adjuvant, squalene, nanocarrier ranges from approximately 10 nm to ap- 25 phosphate adjuvants, or tetrachlorodecaoxide. proximately 200 nm in diameter. 364. A method of preparing a nanocarrier of para- 350. The nanocarrier of paragraph 225, wherein the graph 225, the method comprising steps of: nanocarrier ranges from approximately 50 nm to ap- proximately 200 nm in diameter. 30 providing:

351. The nanocarrier of paragraph 225, wherein the an immunomodulatory agent; nanocarrier ranges from approximately 10 nm to ap- a polymer; proximately 150 nm in diameter. a lipid; and 35 352. The nanocarrier of paragraph 225, wherein the mixing the immunomodulatory agent, targeting nanocarrier ranges from approximately 50 nm to ap- moiety, polymer, and lipid; proximately 150 nm in diameter. and allowing self-assembly of the nanocarrier to occur. 353. The nanocarrier of paragraph 225, wherein the 40 first immunomodulatory agent is a B cell antigen. 365. A method of preparing a nanocarrier of para- graph 225, the method comprising steps of: 354. The nanocarrier of paragraph 225, wherein the first immunomodulatory agent is a pathogen-derived providing: protein. 45 an immunomodulatory agent; 355. The nanocarrier of paragraph 225, wherein the a particle; first immunomodulatory agent is a collection of pep- a lipid; and tides. 50 mixing the immunomodulatory agent, targeting 356. The nanocarrier of paragraph 225, wherein the moiety, particle, and lipid; second immunomodulatory agent is a T cell antigen. and allowing self-assembly of the nanocarrier to occur. 357. The nanocarrier of paragraph 225, wherein the second immunomodulatory agent is a pathogen-de- 55 366. The method of paragraph 364 or 365, further rived protein. comprising a step of providing a targeting moiety.

358. The nanocarrier of paragraph 225, wherein the 367. A method of preparing a nanocarrier of para-

91 181 EP 2 394 657 A1 182 graph 225, the method comprising steps of: 375. The nanocarrier of paragraph 369, wherein the outer portion is lipid. providing a first mixture comprising: 376. The nanocarrier of paragraph 375, wherein the a hydrophobic immunomodulatory agent; 5 outer lipid portion comprises a lipid bilayer. and a lipid; and 377. The nanocarrier of paragraph 375, wherein the outer lipid portion comprises a lipid monolayer. providing a second mixture comprising: 10 378. The nanocarrier of paragraph 375, wherein the a hydrophilic immunomodulatory agent; immunomodulatory agent is associated with the ex- and terior surface of the lipid outer portion. an aqueous solution; and 379. The nanocarrier of paragraph 375, wherein the combining the first mixture and the second mix- 15 immunomodulatory agent is associated with the in- ture, wherein combining the first mixture and the terior surface of the lipid outer portion. second mixture results in formation of a nano- carrier by self- assembly. 380. The nanocarrier of paragraph 375, wherein the immunomodulatory agent is embedded within the li- 368. A method of vaccinating a subject, the method 20 pid outer portion. comprising steps of administering a therapeutically effective amount of nanocarriers of paragraph 225 381. The nanocarrier of paragraph 376, wherein the to a subject. immunomodulatory agent is embedded within the lu- men of the lipid bilayer. 369. A nanocarrier comprising an outer portion com- 25 prising an immunomodulatory agent, wherein the im- 382. The nanocarrier of paragraph 375, wherein the munomodulatory agent is capable of stimulating B interior surface of the lipid outer portion is associated cells and T cells; an inner portion; and an excipient. with an amphiphilic entity.

370. The nanocarrier of paragraph 369, wherein the 30 383. The nanocarrier of paragraph 369, wherein the inner portion comprises an immunomodulatory inner portion comprises a polymeric matrix. agent, wherein the immunomodulatory agent of the inner portion is capable of being processed by anti- 384. The nanocarrier of paragraph 383, wherein the gen presenting cells and presented to T cells. polymeric matrix comprises a crosslinked polymer 35 network. 371. A nanocarrier comprising an outer portion com- prising an immunomodulatory agent, wherein the im- 385. The nanocarrier of paragraph 383, wherein a munomodulatory agent is capable of stimulating B second immunomodulatory agent is distributed cells and antigen presenting cells; an inner portion; throughout the polymeric matrix. and an excipient. 40 386. The nanocarrier of paragraph 383, wherein at 372. The nanocarrier of paragraph 371, wherein the least one immunomodulatory agent is encapsulated immunomodulatory agent is capable of being proc- within reverse micelles, and wherein the reverse mi- essed by antigen presenting cells and presented to celles are contained within the inner polymeric por- T cells. 45 tion.

373. The nanocarrier of paragraph 371, wherein the outer portion comprises a second immunomodula- Claims tory agent, wherein the second immunomodulatory agent of the outer portion is capable of being proc- 50 1. Nanoparticles formed of polymer or lipid having a essed by antigen presenting cells and presented to mean geometric diameter of between 40 nm and 400 T cells. nm, the nanoparticles comprising a T cell antigen, such 374. The nanocarrier of paragraph 371, wherein the as a self antigen, a cancer antigen or an allergenic inner portion comprises an immunomodulatory 55 peptide, optionally wherein the nanoparticles further agent, wherein the immunomodulatory agent of the comprise inner portion is capable of being processed by anti- gen presenting cells and presented to T cells. i) a B cell antigen on the surface of the nanopar-

92 183 EP 2 394 657 A1 184

ticles or encapsulated within the nanoparticles, lipoproteins, lipids and nucleic acids, optionally preferably in a density activating B cell recep- wherein the B cell antigen is a small molecule ad- tors, and/or dictive substance or toxin. ii) an immunostimulatory agent, such as a CpG- containing immunostimulatory nucleic acid, 5 7. The nanoparticles of claim 6 wherein the small mol- cholera toxin, squalene, a phosphate adjuvant, ecule addictive substance is nicotine, the immunos- tetrachlorodecaoxide, monophosphoryl lipid A, timulatory agent is a toll-like receptor agonist, and a saponin, a CD40 agonist, R848, a cytokine, the nanoparticles are formed of polymer, wherein or a complement receptor agonist, and wherein the nicotine is optionally conjugated to the polymer. one or more of the antigens or the immunostim- 10 ulatory agent may be covalently bound to the 8. The nanoparticles of any preceding claim comprising nanoparticles or polymers forming the nanopar- a polymer having incorporated therein T cell antigen ticles in combination with an immunostimulatory agent, and having B cell antigen on the surface of the na- wherein the B and/or T cell antigen is preferably a 15 noparticles. degenerative disease antigen, an infectious disease antigen, a cancer antigen, an atopic disease antigen, 9. The nanoparticles of any preceding claim formed of an autoimmune disease antigen, an alloantigen, a a hydrophobic polymer or having a hydrophobic xenoantigen, an allergen, an addictive substance, core, optionally wherein the nanoparticles comprise or a metabolic disease enzyme or enzymatic prod- 20 hydrophilic polymer or lipid on the surface, preferably uct, optionally wherein the addictive substance is conjugated to the hydrophobic polymer within the nicotine. nanoparticles.

2. The nanoparticles of claim 1, having bound to or 10. The nanoparticles of any preceding claim formed of present on the surface, an agent or material targeting 25 a biodegradable polymer optionally wherein the pol- the nanoparticles to secondary lymphoid tissue or ymer is selected from the group consisting of poly- cells therein, such as dendritic cells, macrophages mers of lactic acid, glycolic acid, ethylene glycol, eth- (e.g. lymph node macrophages), T cells or B cells, ylene oxide, copolymers and block copolymers wherein the targeting agent is preferably for a mol- thereof, and wherein the nanoparticles may have a ecule selected from the group consisting of CD11b, 30 positive zeta potential. CD169, mannose receptor, DEC-205, CD11c, and CD21/35, and the targeting agent may be covalently 11. The nanoparticles of any preceding claim comprising bound to the nanoparticles or polymers forming the polymeric nanoparticles, liposomes or emulsions. nanoparticles. 35 12. The nanoparticles of any preceding claim wherein 3. The nanoparticles of any preceding claim comprising the nanoparticle forms by self-assembly. a specific or non-specific immunomodulatory agent or a specific or non-specific immunosuppressant 13. A method of making the nanoparticles of any of agent. claims 1-12 comprising forming the nanoparticle with 40 the T cell antigen incorporated therein, optionally in 4. The nanoparticles of any preceding claim wherein combination with an immunostimulatory agent, the immunostimulatory agent non-selectively targets and/or a B cell antigen on the surface. the nanoparticles to secondary lymphoid tissue or cells therein and wherein the immunostimulatory 14. The nanoparticles of any of claims 1-12 for use in agent is selected from the group consisting of an 45 therapeutically or prophylactically treating an indi- adjuvant, a hydrophilic polymer, like polyethylene vidual in need thereof by eliciting an immune re- glycol, an amphiphilic polymer, an emulsion and a sponse, optionally wherein the individual is in need liposome associated with the nanoparticles. of vaccination or where the individual has or is at risk of developing a degenerative disease, an infectious 5. The nanoparticles of any preceding claim, wherein 50 disease, cancer, an atopic disease, an autoimmune the immunostimulatory agent comprises a Toll-like disease, an allergy, an addiction, or a metabolic dis- receptor agonist, cyclosporin, a steroid, methotrex- ease. ate, TGF-beta, rapamycin, retinoic acid, or any agent that interferes with T cell activation. 55 6. The nanoparticles of any preceding claim wherein the B and/or T cell antigen is selected from the group consisting of proteins, carbohydrates, glycoproteins,

93 EP 2 394 657 A1

94 EP 2 394 657 A1

95 EP 2 394 657 A1

96 EP 2 394 657 A1

97 EP 2 394 657 A1

98 EP 2 394 657 A1

99 EP 2 394 657 A1

100 EP 2 394 657 A1

101 EP 2 394 657 A1

102 EP 2 394 657 A1

103 EP 2 394 657 A1

104 EP 2 394 657 A1

105 EP 2 394 657 A1

106 EP 2 394 657 A1

107 EP 2 394 657 A1

108 EP 2 394 657 A1

109 EP 2 394 657 A1

110 EP 2 394 657 A1

111 EP 2 394 657 A1

112 EP 2 394 657 A1

113 EP 2 394 657 A1

114 EP 2 394 657 A1

115 EP 2 394 657 A1

116 EP 2 394 657 A1

117 EP 2 394 657 A1

118 EP 2 394 657 A1

119 EP 2 394 657 A1

120 EP 2 394 657 A1

121 EP 2 394 657 A1

122 EP 2 394 657 A1

123 EP 2 394 657 A1

124 EP 2 394 657 A1

125 EP 2 394 657 A1

126 EP 2 394 657 A1

127 EP 2 394 657 A1

128 EP 2 394 657 A1

129 EP 2 394 657 A1

130 EP 2 394 657 A1

131 EP 2 394 657 A1

132 EP 2 394 657 A1

133 EP 2 394 657 A1

134 EP 2 394 657 A1

135 EP 2 394 657 A1

136 EP 2 394 657 A1

137 EP 2 394 657 A1

138 EP 2 394 657 A1

139 EP 2 394 657 A1

140 EP 2 394 657 A1

141 EP 2 394 657 A1

142 EP 2 394 657 A1

143 EP 2 394 657 A1

144 EP 2 394 657 A1

145 EP 2 394 657 A1

146 EP 2 394 657 A1

147 EP 2 394 657 A1

148 EP 2 394 657 A1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 97959607 P [0001] • US 5696175 A [0378] • US 6403779 B [0251] • US 5514378 A [0378] • US 6399754 B [0251] • US 5512600 A [0378] • US 6225460 B [0251] • US 5399665 A [0378] • US 6127533 B [0251] • US 5019379 A [0378] • US 6031086 B [0251] • US 5010167 A [0378] • US 6005087 B [0251] • US 4806621 A [0378] • US 5977089 B [0251] • US 4638045 A [0378] • US 20030175950 A [0254] • US 4946929 A [0378] • US 20040192626 A [0254] • US 6506577 B [0378] • US 20040092470 A [0254] • US 6632922 B [0378] • US 20050020525 A [0254] • US 6686446 B [0378] • US 20050032733 A [0254] • US 6818732 B [0378] • US 6482594 B [0281] • US 4886499 A [0481] • US 6458543 B [0281] • US 5190521 A [0481] • US 6458539 B [0281] • US 5328483 A [0481] • US 6376190 B [0281] • US 5527288 A [0481] • US 6344318 B [0281] • US 4270537 A [0481] • US 6242246 B [0281] • US 5015235 A [0481] • US 6184364 B [0281] • US 5141496 A [0481] • US 6001577 B [0281] • US 5417662 A [0481] • US 5958691 B [0281] • WO 9934850 A [0481] • US 5874218 B [0281] • US 5480381 A [0481] • US 5853984 B [0281] • US 5599302 A [0481] • US 5843732 B [0281] • US 5334144 A [0481] • US 5843653 B [0281] • US 5993412 A [0481] • US 5817785 B [0281] • US 5649912 A [0481] • US 5789163 B [0281] • US 5569189 A [0481] • US 5763177 B [0281] • US 5704911 A [0481] • US 5696249 B [0281] • US 5383851 A [0481] • US 5660985 B [0281] • US 5893397 A [0481] • US 5595877 B [0281] • US 5466220 A [0481] • US 5567588 B [0281] • US 5339163 A [0481] • US 5270163 B [0281] • US 5312335 A [0481] • US 0647975 W [0281] • US 5503627 A [0481] • US 5500161 A [0290] • US 5064413 A [0481] • US 6123727 A [0378] • US 5520639 A [0481] • US 5804178 A [0378] • US 4596556 A [0481] • US 5770417 A [0378] • US 4790824 A [0481] • US 5736372 A [0378] • US 4941880 A [0481] • US 5716404 A [0378] • US 4940460 A [0481] • US 6095148 A [0378] • WO 9737705 A [0481] • US 5837752 A [0378] • WO 9713537 A [0481] • US 5902599 A [0378]

Non-patent literature cited in the description

• KREIG et al. Nature, 1995, vol. 374, 546-9 [0066]

149 EP 2 394 657 A1

• DANG ; ROCK. J. Immunol., 1991, vol. 146, 3273 • ODIAN. Principles of Polymerization. John Wiley & [0144] [0541] Sons, 2004 [0378] • LINDBLAD. Immunol. Cell. Biol., 2004, vol. 82, 497 • ALLCOCK et al. Contemporary Polymer Chemistry. [0195] Prentice-Hall, 1981 [0378] • JORDAN et al. Science, 2004, vol. 304, 1808 [0195] • DEMING et al. Nature, 1997, vol. 390, 386 [0378] • BABA et al. Nat. Med., 2000, vol. 6, 200 [0196] • PELLEGRINO et al. Small, 2005, vol. 1, 48 [0400] • HAWIGER et al. J. Exp. Med., 2001, vol. 194, 769 [0404] [0239] • MURRAY et al. Ann. Rev. Mat. Sci., 2000, vol. 30, • BONIFAZ et al. J. Exp. Med., 2002, vol. 196, 1627 545 [0400] [0404] [0239] • TRINDADE et al. Chem. Mat., 2001, vol. 13, 3843 • BOZZACCO et al. Proc. Natl. Acad. Sci., USA, 2007, [0400] [0404] vol. 104, 1289 [0239] • Microcapsules and Nanoparticles in Medicine and • Oligonucleotide synthesis: α practical approach. IRL Pharmacy. CRC Press, 1992 [0402] [0406] Press, 1984 [0249] • MATHIOWITZ et al. J. Control. Release, 1987, vol. • Oligonucleotide synthesis: methods and applica- 5, 13 [0402] tions. Methods in molecular biology. Humana Press, • MATHIOWITZ et al. Reactive Polymers, vol. 6, 275 2005, vol. 288 [0249] [0402] • Antisense Drug Technology: Principles, Strategies, • MATHIOWITZ et al. J. Appl. Polymer Sci., 1988, vol. and Applications. Marcel Dekker, 2001 [0251] 35, 755 [0402] • Current Protocols in Immunology. John Wiley & Sons, • MATHIOWITZ et al. J. Control. Release. 1987, vol. 2007 [0299] [0344] 5, 13 [0406] • PAPISOV. ACS Symposium Series, 2001, vol. 786, • MATHIOWITZ et al. Reactive Polymers, 1987, vol. 301 [0368] 6, 275 [0406] • ZAUNER et al. Adv. Drug Del. Rev., 1998, vol. 30, • MATHIOWITZ et al. J. Appl. Polymer Sci., 1998, vol. 97 [0373] 35, 755 [0406] • KABANOV et al. Bioconjugate Chem., 1995, vol. 6, • GAO et al. Curr. Op. Biotechnol., 2005, vol. 16, 63 7 [0373] [0416] • BOUSSIF et al. Proc. Natl. Acad. Sci., USA, 1995, • journal Bioconjugate Chemistry. American Chemical vol. 92, 7297 [0373] Society [0421] • KUKOWSKA-LATALLO et al. Proc. Natl. Acad. • Cross-Linking. Pierce Chemical Technical Library Sci., USA, 1996, vol. 93, 4897 [0373] [0421] • TANG et al. Bioconjugate Chem., 1996, vol. 7, 703 • WONG SS. Chemistry of Protein Conjugation and [0373] Cross-linking. CRC Press, 1991 [0421] • HAENSLER et al. Bioconjugate Chem., 1993, vol. • HERMANSON, G. T. Bioconjugate Techniques. Ac- 4, 372 [0373] ademic Press, Inc, 1996 [0421] • PUTNAM et al. Macromolecules, 1999, vol. 32, 3658 • Remington’s The Science and Practice of Pharmacy. [0374] Lippincott, Williams & Wilkins, 2006 [0461] • BARRERA et al. J. Am. Chem. Soc., 1993, vol. 115, • Remington: The Science and Practice of Pharmacy. 11010 [0374] Lippincott Williams & Wilkins, 2005 [0489] • KWON et al. Macromolecules, 1989, vol. 22, 3250 • AXEL KLEEMANN ; JURGEN ENGEL. Pharma- [0374] ceutical Drugs: Syntheses, Patents, Applications. • LIM et al. J. Am. Chem. Soc., 1999, vol. 121, 5633 Thieme Medical Publishing, 1999 [0506] [0374] • Merck Index: An Encyclopedia of Chemicals, Drugs • ZHOU et al. Macromolecules, 1990, vol. 23, 3399 and Biologicals. CRC Press, 1996 [0506] [0374] • HANGARTNER et al. Proc. Natl. Acad. Sci., USA, • WANG et al. J. Am. Chem. Soc., vol. 123, 9480 2003, vol. 100, 12883 [0519] [0522] [0540] [0378] • WESSELS et al. Proc. Natl. Acad. Sci., USA, 1995, • LIM et al. J. Am. Chem. Soc., 2001, vol. 123, 2460 vol. 92, 11490 [0519] [0378] • BOES et al. Nature, 2002, vol. 418, 983 [0519] • LANGER. Acc. Chem. Res., 2000, vol. 33, 94 [0378] • WRIGHT et al. Blood, 2001, vol. 97, 2278 [0519] • LANGER. J. Control. Release, 1999, vol. 62, 7 • CASOLA et al. Nat. Immunol., 2004, vol. 5, 317 [0378] [0519] • UHRICH et al. Chem. Rev., 1999, vol. 99, 3181 • WHELAN et al. Proc. Natl. Acad. Sci., USA, 1995, [0378] vol. 92, 8388 [0524] • Concise Encyclopedia of Polymer Science and Pol- • LEOPOLD et al. Human Gene Therapy, 1998, vol. ymeric Amines and Ammonium Salts. Pergamon 9, 367 [0524] Press, 1980 [0378] • DELEMARRE et al. J. Leukoc. Biol., 1990, vol. 47, 251 [0529] [0536]

150 EP 2 394 657 A1

• MEMPEL et al. Nature, 2004, vol. 427, 154 [0529] • LUDEWIG et al. Eur. J. Immunol., 2000, vol. 30, 185 [0532] [0539] • ANDRIAN ; MEMPEL. Nat. Rev. Immunol., 2003, • QI et al. Science, 2006, vol. 312, 1672 [0539] vol. 3, 867 [0531] • ROOST et al. J. Immunol. Methods, 1996, vol. 189, • KARRER et al. J. Exp. Med., 1997, vol. 185, 2157 233 [0540] [0531] • ROSSBACHER ; SHLOMCHIK. J. Exp. Med., 2003, • MEAD et al. Ann. N.Y. Acad. Sci., 2000, vol. 916, vol. 198, 591 [0540] 437 [0532] • OKADA et al. PLoS Biol., 2005, vol. 3, el50 [0541] • BACHMANN et al. Eur. J. Immunol., 1995, vol. 25, • VASCOTTO et al. Curr., Opin., Immunol., 2007, vol. 3445 [0532] 19, 93 [0542] [0545] • CLARK. Am. J. Anat., 1962, vol. 110, 217 [0534] • PAPE et al. Immunity, 2007, vol. 26, 491 [0543] • FARR et al. Am. J. Anat., 1980, vol. 157, 265 [0534] • BARCHET et al. J. Exp. Med., 2002, vol. 195, 507 • OCHSENBEIN et al. J. Exp. Med., 1999, vol. 190, [0544] 1165 [0535] • SHIOW et al. Nature, 2006, vol. 440, 540 [0544] • OCHSENBEIN et al. Science, 1999, vol. 286, 2156 • OKADA et al. PLoS Biol., 2005, vol. 3, el150 [0545] [0535] • REIF et al. Nature, vol. 416, 94 [0545] • TAYLOR et al. Ann. Rev. Immunol., 2005, vol. 23, 901 [0535]

151