(19) TZZ ¥Z_T

(11) EP 2 630 966 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 39/00 (2006.01) 19.04.2017 Bulletin 2017/16

(21) Application number: 13156642.4

(22) Date of filing: 12.10.2008

(54) Vaccine nanotechnology Impfstoffnanotechnologie Nano-technologie de vaccin

(84) Designated Contracting States: •Basto,Pamela AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Cambridge, MA 02139 (US) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • Iannacone, Matteo RO SE SI SK TR 20129 Milano (IT) • Alexis, Frank (30) Priority: 12.10.2007 US 979596 P Greenville, SC 29607-7405 (US)

(43) Date of publication of application: (74) Representative: Potter Clarkson LLP 28.08.2013 Bulletin 2013/35 The Belgrave Centre Talbot Street (62) Document number(s) of the earlier application(s) in Nottingham NG1 5GG (GB) accordance with Art. 76 EPC: 08839738.5 / 2 217 269 (56) References cited: WO-A2-2007/098254 US-A1- 2004 022 840 (73) Proprietors: • Massachusetts Institute of Technology • L. MARTINEZ-POMARES ET AL: "Fc chimeric Cambridge, MA 02139 (US) protein containing the cysteine-rich domain of • President and Fellows of Harvard College the murine mannose receptor binds to Cambridge, MA 02138 (US) macrophages from splenic marginal zone and • The Brigham and Women’s Hospital, Inc. lymph node subcapsular sinus and to germinal Boston, MA 02115 (US) centers", JOURNAL OF EXPERIMENTAL • The Children’s Medical Center Corporation MEDICINE, vol. 184, no. 5, 1 November 1996 Boston, Massachusetts 02115 (US) (1996-11-01), pages 1927-1937, XP55071142, ISSN: 0022-1007, DOI: 10.1084/jem.184.5.1927 (72) Inventors: • P.R. TAYLOR: "Development of a specific system • Von Andrian, Ulrich H for targeting protein to metallophilic Chestnut Hill, MA 02467 (US) macrophages", PROCEEDINGS OF THE • Farokhzad, Omid C NATIONAL ACADEMY OF SCIENCES, vol. 101, Waban, MA 02468 (US) no. 7, 9 February 2004 (2004-02-09), pages • Langer, Robert S 1963-1968, XP55071149, ISSN: 0027-8424, DOI: Newton, MA 02459 (US) 10.1073/pnas.0308490100 • Junt, Tobias 73614 Schorndorf (DE) • Moseman, Elliott Ashley Jamaica Plain, MA 02130 (US) • Zhang, Liangfang San Diego, CA 92130 (US)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 630 966 B1

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• MANOLOVA V ET AL: "Nanoparticles target Remarks: distinct dendritic cell populations according to Thefile contains technical information submitted after their size", EUROPEAN JOURNAL OF the application was filed and not included in this IMMUNOLOGY, WILEY - V C H VERLAG GMBH & specification CO. KGAA, DE, vol. 38, no. 5, 1 May 2008 (2008-05-01), pages 1404-1413, XP002562080, ISSN: 0014-2980, DOI: 10.1002/EJI.200737984 [retrieved on 2008-04-03]

2 1 EP 2 630 966 B1 2

Description Summary of the Invention

Government Support [0005] The invention is as defined in the accompanying claims. The present invention provides a nanocarrier [0001] The United States Government has provided 5 comprising: grant support utilized in the development of the present invention. In particular, National Institutes of Health (con- an immunostimulatory agent, wherein the immunos- tract numbers AI069259, AI072252, CA119349, and timulatory agent is a toll-like receptor agonist; HL56949) and National Institutes of Health/National In- a T cell antigen; and, stitute of Biomedical Imaging and BioEngineering (con- 10 a polymer selected from the group consisting of: tract number EB 003647) have supported development polylactic acid (PLA), poly(glycolic acid) (PGA), and of this invention. The United States Government has cer- poly(lactic acid/glycolic acid) (PLGA); tain rights in the invention. wherein the nanocarrier has a mean geometric di- ameter of between 50 nm and 500 nm; Background of the Invention 15 wherein the immunostimulatory agent is covalently bound to the nanocarrier or the immunostimulatory [0002] Many current vaccines against microbial path- agent is covalently bound to a polymer from which ogens comprise live attenuated or non-virulent strains of the nanocarrier is formed, and the T cell antigen is the causative microorganisms. Many vaccines comprise encapsulated within the nanocarrier or the T cell an- killed or otherwise inactivated microorganisms. Other 20 tigen is covalently bound to the nanocarrier. vaccinesutilize purified componentsof pathogenlysates, such as surface carbohydrates or recombinant patho- The invention also provides a method of preparing the gen-derived proteins. Vaccines that utilize live attenuat- nanocarrier as defined in the claims by nanoprecipitation, ed or inactivated pathogens typically yield a vigorous im- flow focusing using fluidic channels, spray drying, single mune response, but their use has limitations. For exam- 25 or double emulsion solvent evaporation, or solvent ex- ple, live vaccine strains can sometimes cause infectious traction. pathologies, especially when administered to immune- The invention also provides the nanocarrier as defined compromised recipients. Moreover, many pathogens, in the claims for use in medicine. particularly viruses, undergo continuous rapid mutations The present invention provides synthetic nanocarriers for in their genome, which allow them to escape immune 30 modulating the immune system. The synthetic nanocar- responses to antigenically distinct vaccine strains. riers comprise one or more of an immunomodulatory [0003] Given the difficulty of vaccine development, agent, an immunostimulatory agent, and a targeting many vaccines are in extremely short supply. For exam- agent (also referred to herein as "targeting moiety"). The ple, as of October 2007, there are influenza, varicella, immunomodulatory agent induces an immune response and hepatitis A vaccine shortages in the United States. 35 in B and/or T cells. The targeting agent recognizes one In some instances, vaccine shortages occur because not or more targets associated with a particular organ, tissue, enough manufacturers devote their facilities to vaccine cell, and/or subcellular locale. The nanocarriers are use- production to keep up with demand. In some cases, vac- ful in pharmaceutical preparations and kits for the proph- cine shortages are attributed to low potency of the vac- ylaxis and/or treatment of diseases, disorders, or condi- cine, which means a large amount of vaccine product 40 tions susceptible to treatment by immune system mod- must be administered to each individual in order to ulation. Such conditions include those diseases, disor- achieve a prophylactic effect. For example, some vac- ders, or conditions modified by enhancing the immune cines cannot be administered as an intact organism response specifically or nonspecifically, or directing/re- (even if attenuated or killed) because they cause infec- directing the immune response specifically or nonspecif- tious pathologies. Instead, such vaccines usually com- 45 ically. prise purified pathogen components, which typically [0006] As will be recognized by those skilled in the art, leads to a much less potent immune respons WO-A- immune system modulation is useful, among other 2007/098254 discloses polymeric nanoparticles which things, in connection with medical treatments, such as, target APCs in lymph nodes and activate complement. for example, for prophylaxis and/or treatment of infec- [0004] Thus, there is a need in the art for systems and 50 tious disease, cancer, allergy, asthma (including allergic methods for producing highly immunogenic, effective asthma), autoimmune disease (including rheumatoid ar- vaccines. There is also a need for improved vaccine com- thritis), immunization against addictive substances, and positions that can potently induce long-lasting immune immunization against biohazards and other toxic sub- responses. For the treatment and prevention of infectious stances. Immune system modulation also is useful as a diseases, there is a need for improved vaccine compo- 55 tool in industrial and academic research settings, such sitions that are highly immunogenic but do not cause dis- as, for example, to immunize an animal to produce anti- ease. bodies. [0007] One aspect of the invention is the provision of

3 3 EP 2 630 966 B1 4 vaccines. A vaccine according to the invention contains nanocarrier (e.g., one that targets a specific organ, tis- an antigen. In one embodiment, the antigen is physically sue, cell, or subcellular locale) is provided. In some em- ’bound’ to the nanocarrier by covalent means. Such na- bodiments, the nanocarrier targets one or more second- nocarriers which themselves carry an antigen are includ- ary lymphoid tissues or organs. In some embodiments, ed in the category referred to below as vaccine nanocar- 5 the secondary lymphoid tissue or organ is the lymph riers. The nanocarrier has bound to it an immunostimu- nodes, spleen, Peyer’s patches, appendix, or tonsils. latory agent for enhancing, directing, or redirecting an [0011] The scaffold of the nanocarrier comprises pol- immune response, preferably to an antigen. In this case, ymer molecules. The nanocarriers are polymeric nano- the antigen may be mixed with the preparation of agent particles and/or nanoparticles that are developed using bound nanocarrier to which the immunostimulatory agent 10 a combination of nanomaterials such as lipid-polymer na- is bound form the vaccine. The antigen, of course may noparticles. also be bound to a nanocarrier, including as discussed [0012] The nanocarrier comprises one or more poly- below, the same nanocarrier to which the immunostim- mers. The biodegradable polymer is polylactic acid ulatory agent is bound. (PLA), poly(glycolic acid)(PGA), or poly(lactic acid/gly- [0008] The preparations of the invention in many in- 15 colic acid) (PLGA). stances will include one or more nanocarriers. In some [0013] In some embodiments, the nanocarrier is embodiments, the preparation includes a nanocarrier formed by self-assembly. Self-assembly refers to the bound to one or more, but not all, of an immunomodula- process of the formation of a nanocarrier using compo- tory agent, an immunostimulatory agent, and a targeting nents that will orient themselves in a predictable manner agent. The preparations likewise may be one of nano- 20 forming nanocarriers predictably and reproducably. In carriers, wherein each nanocarrier has bound to it all of some embodiments, the nanocarriers are formed using an immunomodulatory agent, the immunostimulatory amphiphillic biomaterials which orient themselves with agent, and a targeting agent. In this instance, the nano- respect toone another toform nanocarriersof predictable carriers themselves, apart from the agents they deliver, dimension, constituents, and placement of constituents. may be the same or different. 25 According to the invention, the amphiphillic biomaterials [0009] Important is the discovery that the nanocarriers may have attached to them immunomodulatory agents, of the invention are powerful at stimulating the immune immunostimulatory agents and/or targeting agents such system. Important is the discovery that the nanocarriers that when the nanocarriers self assemble, there is a re- can be fashioned to mimic, and from an immunological producible pattern of localization and density of the standpoint, improve on, what the immune system ’sees’ 30 agents on/in the nanocarrier. when exposed to antigens in nature or in prior vaccine [0014] In some embodiments, the nanocarrier has a technology. In this respect, it has been discovered unex- positive zeta potential. In some embodiments, the nano- pectedly that the activity of adjuvants can be markedly carrier has a net positive charge at neutral pH. In some enhanced if covalently bound to nanocarriers. It also has embodiments, the nanocarrier comprises one or more been discovered unexpectedly that nanocarriers can35 amine moieties at its surface. In some embodiments, the help target an immunostimulatory agent to appropriate amine moiety is a primary, secondary, tertiary, or qua- immune cells even without a targeting agent. ternary amine. In some embodiments, the amine moiety [0010] The systems described herein permit the ma- is an aliphatic amine. In some embodiments, the nano- nipulation ofthe parameters affecting the immune system carrier comprises an amine-containing polymer. In some in a manner which results in improved immune modula- 40 embodiments, the nanocarrier comprises an amine-con- tion. One important aspect of the invention is that the taining lipid. In some embodiments, the nanocarrier com- nanocarriers can be controlled in terms of size, density prises a protein or a peptide that is positively charged at of agent, degree and location of targeting, degradation, neutral pH. In some embodiments, the nanocarrier with release of agent, etc. A variety of aspects of the invention the one or more amine moieties on its surface has a net achieve one or more of these benefits, described in more 45 positive charge at neutral pH. detail below. In particular, below are described immune [0015] The nanocarriers have mean geometric diam- modulating preparations, synthetic nanocarrier compo- eter that is greater than 50 nm but less than 500 nm. In nents of such preparations, specific and preferred nano- some embodiments, the mean geometric diameter of a carriers, specific and preferred immunomodulatory, im- population of nanocarriers is about 60 nm, 75 nm, 100 munstimulatory, and targeting agents, component parts 50 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, and building blocks of nanocarriers of the invention, as 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 well as methods for manufacturing such nanocarriers, nm, 450 nm, or 475 nm. In some embodiments, the mean including a preferred method involving self-assembled geometricdiameter is between100-400 nm, 100-300 nm, nanocarriers. In addition, preparations and systems for 100-250 nm, or 100-200 nm. In some embodiments, the generating robust immune modulation in connection with 55 mean geometric diameter is between 60-400 nm, 60-350 weak antigens and antigens not recognized by T cells nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some em- (e.g., carbohydrate and small molecule antigens) are de- bodiments, the mean geometric diameter is between scribed. In some aspects, a composition comprising a 75-250 nm. In some embodiments, 10%, 20%, 30%,

4 5 EP 2 630 966 B1 6

40%, 50%, 60%, 70%, 80%, 90%, or more of the nano- alently associated with the nanocarrier. In some embod- carriers of a population of nanocarriers have a diameter iments, the antigen is a degenerative disease antigen, that is greater than 50 nm but less than 500 nm. In some an infectious disease antigen, a cancer antigen, an atopic embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, disease antigen, an autoimmune disease antigen, an al- 80%, 90%, or more of the nanocarriers of a population 5 loantigen, a xenoantigen, an allergen, an addictive sub- of nanocarriers have a diameter of about 60 nm, 75 nm, stance, or a metabolic disease enzyme or enzymatic 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 product. In some embodiments the T cell antigen is a nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, ’universal’ T cell antigen (i.e., one which can be used with 425 nm, 450 nm, or 475 nm. In some embodiments, 10%, an unrelated B cell antigen, including a carbohydrate, to 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more 10 stimulate T cell help). In some embodiments, the nano- of the nanocarriers of a population of nanocarriers have carrier further comprises a targeting moiety. a diameter that is between 100-400 nm, 100-300 nm, [0020] In some embodiments, the nanocarrier com- 100-250 nm, or 100-200 nm. In some embodiments, prises both a B cell antigen and a T cell antigen. In some 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or embodiments, the B cell antigen and the T cell antigen more of the nanocarriers of a population of nanocarriers 15 are different antigens. In some embodiments, the B cell have a diameter that is between 60-400 nm, 60-350 nm, antigen and the T cell antigen are the same antigen. In 60-300 nm, 60-250 nm, or 60-200 nm. some embodiments, the B cell antigen is on the surface [0016] The nanocarrier provided herein can be used of the nanocarrier (e.g., covalently or non-covalently as- to modulate an immune response (e.g., enhance, direct, sociated) or is both on the surface of the nanocarrier (e.g., or redirect) and further comprise an immunomodulatory 20 covalently or non-covalently associated) and encapsu- agent and/or a targeting agent. lated within the nanocarrier (e.g., covalently or non-cov- [0017] In some embodiments, the nanocarrier further alently associated), while the T cell antigen is on the sur- comprises a B cell antigen. The B cell antigen may be face of the nanocarrier (e.g., covalently associated), is on the surface of the nanocarrier, encapsulated within encapsulated within the nanocarrier, or is both on the the nanocarrier, or both. In some embodiments, the B 25 surface of the nanocarrier and encapsulated within the cell antigen is on the surface of the nanocarrier at a den- nanocarrier. sity which activates B cell receptors. In some embodi- [0021] In some embodiments, the immunostimulatory ments, the B cell antigen is associated with the nanocar- agent is on the surface of the nanocarrier and is encap- rier. In some embodiments, the B cell antigen is cova- sulated within the nanocarrier. lently associated with the nanocarrier. In some embodi- 30 [0022] In some embodiments, where a nanocarrier ments, the B cell antigen is non-covalently associated comprises both a B cell antigen and a T cell antigen, the with the nanocarrier. In some embodiments, the nano- nanocarrier further comprises targeting agent. In some carrier further comprises a targeting moiety. In some em- embodiments, the targeting agent is on the surface of bodiments, the B cell antigen is a poorly immunogenic the nanocarrier. In some embodiments, the targeting antigen. In some embodiments, the B cell antigen is a 35 agent is associated with the nanocarrier. In some em- small molecule. In some embodiments, the B cell antigen bodiments, the targeting agent is covalently associated is an addictive substance. Is some embodiments, the B with the nanocarrier. In some embodiments, the targeting cell antigen is a toxin. In some embodiments, the toxin agent is non-covalently associated with the nanocarrier. for inclusion in a nanocarrier is the complete molecule or [0023] In some embodiments, the immunostimulatory a portion thereof. In some embodiments the B cell antigen 40 agent is both on the surface of the nanocarrier and en- is not a T cell antigen. In some embodiments, the B cell capsulated within the nanocarrier. In some embodi- antigen is a carbohydrate. In some embodiments, the B ments, the immunostimulatory agent on the surface of cell antigen is a degenerative disease antigen, an infec- the nanocarrier is different from the immunostimulatory tious disease antigen, a cancer antigen, an atopic dis- agent encapsulated within the nanocarrier. In some em- ease antigen, an autoimmune disease antigen, an al- 45 bodiments, the immunostimulatory agent on the surface loantigen, a xenoantigen, an allergen, an addictive sub- of and encapsulated within the nanocarrier is the same. stance, or a metabolic disease enzyme or enzymatic [0024] In some embodiments, the nanocarrier com- product. prises more than one species of immunostimulatory [0018] An allergen refers to a substance (antigen) that agents, in which case the immunostimulatory agents are can induce an allergic response in a susceptible subject. 50 different. The list of allergens is enormous and includes pollens, [0025] In some embodiments the nanocarrier compris- insect venoms, animal dander dust, fungal spores and es an immunostimulatory agent a B cell antigen and a T drugs (e.g. penicillin). Allergens also include food aller- cell antigen. In some embodiments, the B cell antigen is gens. a poorly immunogenic antigen. In some embodiments, [0019] The nanocarrier comprises a T cell antigen. In 55 the B cell antigen is a small molecule. In some embodi- some embodiments, the T cell antigen is on the surface ments, the B cell antigen is a carbohydrate. In some em- of the nanocarrier, encapsulated within the nanocarrier, bodiments, the B cell antigen is an addictive substance. or both. In some embodiments, the T cell antigen is cov- Is some embodiments, the B cell antigen is a toxin. In

5 7 EP 2 630 966 B1 8 some embodiments, the T cell antigen is a degenerative is a covalent or non-covalent conjugate. The antigen is disease antigen, an infectious disease antigen, a cancer a T cell antigen. In some embodiments, the nanocarrier antigen, an atopic disease antigen, an autoimmune dis- further comprises a conjugate of a polymer and a B cell ease antigen, an alloantigen, a xenoantigen, an allergen, antigen. In some embodiments, such a conjugate is a an addictive substance, or a metabolic disease enzyme 5 covalent or non-covalent conjugate. or enzymatic product. In some embodiments, the T cell [0029] In some aspects, a composition comprising a antigen is a universal T cell antigen. In some embodi- nanocarrier comprising a molecule or molecules of the ments, the nanocarrier further comprises a targeting following formula X-L1-Y-L2-Z, wherein X is a biodegrad- agent. able polymer, Y is a water soluble, non-adhesive poly- [0026] The nanocarrier, in some embodiments, can be 10 mer, Z is a targeting moiety, an immunomodulatory used to induce or enhance an immune response to a agent, an immunostimulatory agent, or a pharmaceutical poorly immunogenic antigen (e.g., a small molecule or agent, and L1 and L2 are bonds or linking molecules, carbohydrate) in a subject. In some embodiments, the wherein either Y or Z, but not both Y and Z, can be absent, nanocarrier can be used to induce or enhance an immune is provided. The nanocarrier comprises an antigen and response to an addictive substance in a subject. In some 15 an immunostimulatory agent. Antigens include a degen- embodiments, the nanocarrier can be used to induce or erative disease antigen, an infectious disease antigen, a enhance an immune response to a toxin in a subject. The cancer antigen, an atopic disease antigen, an autoim- nanocarrier, in some embodiments, can be used to treat mune disease antigen, an alloantigen, a xenoantigen, an a subject that has or is susceptible to an addiction. The allergen, an addictive substance, or a metabolic disease nanocarrier, in some embodiments, can be used to treat 20 enzyme or enzymatic product. Z may be any antigen de- a subject that has been or will be exposed to a toxin. In scribed herein. In some embodiments, Z is a targeting some embodiments, the nanocarrier can be used to treat moiety. In some embodiments, Z is a targeting moiety and/or prevent infectious disease, cancer, allergy, asth- that binds a receptor expressed on the surface of a cell. ma (including allergic asthma), or autoimmune disease In some embodiments, Z is a targeting moiety that binds (including rheumatoid arthritis). In other embodiments, 25 a soluble receptor. In some embodiments, the soluble the nanocarriers can be used for immune suppression in receptor is a complement protein or a pre-existing anti- connection with transplants to ameliorate transplant re- body. In some embodiments, the targeting moiety is for jection. delivery of the nanocarrier to antigen presenting cells, T [0027] In some embodiments, the nanocarrier com- cells or B cells. In some embodiments, the antigen pre- prises a targeting moiety. In some embodiments, the tar- 30 senting cells are dendritic cells (DCs), follicular dendritic getingmoiety is on the surface of thenanocarrier. In some cells (FDCs), or macrophages. In some embodiments, embodiments, the targeting moiety is associated with the the macrophages are subcapsular sinus macrophages nanocarrier. In some embodiments, the targeting moiety (SCS-Mphs). X is PLGA, PLA or PGA. In some embod- is covalently associated with the nanocarrier. In some iments, Z is absent. embodiments, the targeting moiety is non-covalently as- 35 [0030] In some aspects, a composition comprising a sociated with the nanocarrier. nanocarrier comprising an immunostimulatory agent is [0028] In some aspects a composition comprising a provided. In some embodiments, the composition further nanocarrier comprising (a) a conjugate of a polymer and comprises an antigen and/or a targeting moiety. an antigen, (b) a conjugate of a polymer and an immu- [0031] In some aspects, a composition comprising a nostimulatory agent, and (c) a conjugate of a polymer 40 nanocarrier comprising a small molecule, an immunos- and a targeting moiety is provided. In some embodi- timulatory agent, and a T cell antigen is provided. In some ments, the nanocarrier comprises a conjugate of a poly- embodiments, the small molecule is on the surface of the mer and an antigen and a conjugate of a polymer and an nanocarrier or is both on the surface of the nanocarrier immunostimulatory agent.In some embodiments, thena- and encapsulated within the nanocarrier. In some em- nocarrier comprises a conjugate of a polymer and an45 bodiments, the small molecule is an addictive substance. antigen and a conjugate of a polymer and a targeting In some embodiments, the addictive substance is nico- moiety. In some embodiments, the nanocarrier compris- tine. In some embodiments, the small molecule is a toxin. es a conjugate of a polymer and an immunostimulatory In some embodiments, the toxin is from a chemical weap- agent and a conjugate of a polymer and a targeting moi- on, an agent of biowarfare, or a hazardous environmental ety. In some embodiments, the nanocarrier comprises a 50 agent. In some embodiments, the small molecule is con- conjugate of a polymer and an antigen, a conjugate of a jugated to a polymer. In some embodiments, the nano- polymer and an immunostimulatory agent and a conju- carrier further comprises a targeting moiety. In some em- gate of a polymer and a targeting moiety. In some em- bodiments, the targeting moiety is conjugated to a poly- bodiments, the conjugate or conjugates is/are covalent mer. conjugate/conjugates or non-covalent conjugate/conju- 55 [0032] In some embodiments, a composition compris- gates or any combination thereof. In some embodiments, ing a nanocarrier comprising nicotine, an immunostimu- the nanocarrier comprises a conjugate of a polymer and latory agent, a T cell antigen, and a targeting moiety is a T cell antigen. In some embodiments, such a conjugate provided. In some embodiments, the immunostimulatory

6 9 EP 2 630 966 B1 10 agent is a TLR 7/8 agonist. In some embodiments, the [0037] In some aspects, a composition, such as a phar- immunostimulatory agent is R848 or CL097 or imiqui- maceutical composition, comprising an immunostimula- mod. In some embodiments, the nicotine is on the surface tory agent and a nanocarrier is provided. of the nanocarrier or is both on the surface of the nano- [0038] In some embodiments, the B cell antigen is a carrier and encapsulated within the nanocarrier. In some 5 protein or peptide. In some embodiments, the B cell an- embodiments, the nicotine is conjugated to a polymer, tigen is a non-protein antigen (i.e., not a protein or pep- preferably covalently conjugated. In some embodiments, tide). In some embodiments, the protein or peptide is the immunostimulatory agent is on the surface of the na- from an infectious agent. In some embodiments, the in- nocarrier or is both on the surface of the nanocarrier and fectious agent is a bacterium, fungus, virus, protozoan, encapsulated within the nanocarrier. The immunostimu- 10 or parasite. In some embodiments, the virus is a pox vi- latory agent is conjugated to a polymer. In some embod- rus, smallpox virus, ebola virus, marburg virus, dengue iments, targeting moiety is conjugated to a biocompatible fever virus, influenza virus, parainfluenza virus, respira- polymer. The biodegradable polymer is PLGA, PLA, or tory syncytial virus, rubeola virus, human immunodefi- PGA. ciency virus, human papillomavirus, varicella-zoster vi- [0033] In some of these embodiments, the immunos- 15 rus, herpes simplex virus, cytomegalovirus, Epstein-Barr timulatory agent is R848, a TLR9 agonist (e.g., a virus, JC virus, rhabdovirus, rotavirus, rhinovirus, aden- CpG/CpG-containing nucleic acid). Such nanocarriers, ovirus, papillomavirus, parvovirus, picornavirus, poliovi- in some embodiments, may be used to activate CD4 T rus, virus that causes mumps, virus that causes rabies, cells and/or CD8 T cells. In some embodiments, the im- reovirus, rubella virus, togavirus, orthomyxovirus, retro- munostimulatory agent, e.g., R848 or TLR9 agonist, is 20 virus, hepadnavirus, coxsackievirus, equine encephalitis conjugated to a polymer. The conjugation is covalent. In virus, Japanese encephalitis virus, yellow fever virus, Rift some embodiments, the polymer is PLA. The nanocarrier Valley fever virus, hepatitis A virus, hepatitis B virus, hep- comprises a T cell antigen. atitis C virus, hepatitis D virus, or hepatitis E virus. [0034] In some aspects, a composition comprising a [0039] In some embodiments, the B cell antigen is a nanocarrier comprising a poorly immunogenic antigen, 25 small molecule. In some embodiments, the small mole- an immunostimulatory agent, and a T cell antigen is pro- cule is an abused substance, an addictive substance, or vided. In some embodiments, the poorly immunogenic a toxin. antigen is on the surface of the nanocarrier or is both on [0040] In some embodiments, the B cell antigen is an the surface of the nanocarrier and encapsulated within addictive substance. In some embodiments, the addic- the nanocarrier. In some embodiments, the poorly immu- 30 tive substance is nicotine, a narcotic, a hallucinogen, a nogenic antigen is a small molecule or a carbohydrate. stimulant, a cough suppressant, a tranquilizer, or a sed- In some embodiments, the poorly immunogenic antigen ative. In some embodiments, the B cell antigen is an opi- is an addictive substance. In some embodiments, the od or benzodiazepine. poorly immunogenic antigen is a toxin. In some embod- [0041] In some embodiments, the B cell antigen is a iments, the poorly immunogenic antigen is covalently 35 toxin. In some embodiments, the toxin is from a chemical conjugated to a polymer. In some embodiments, the na- weapon. In some embodiments, the toxin from a chem- nocarrier further comprises a targeting moiety. In some ical weapon is botulinum toxin or phosphene. Toxins from embodiments, the targeting moiety is covalently conju- a chemical weapon also include, but are not limited to, gated to a polymer. O-Alkyl (

7 11 EP 2 630 966 B1 12 rovinyl)arsine, Nitrogen mustards: HN1: Bis(2-chloroe- the LDL associated with atherosclerosis is oxidized or thyl)ethylamine, HN2: Bis(2-chloroethyl)methylamine, minimally modified. In some embodiments, the corecep- HN3: Tris(2-chloroethyl)amine, Saxitoxin, Ricin, Amiton: tor for HIV-1 is CCR5. In some embodiments, the self O,O-Diethyl S-(2-(diethylamino)ethyl)phosphorothiolate antigen is an autoimmune disease antigen. and corresponding alkylated or protonated salts, PFIB: 5 [0045] In some embodiments, the B cell antigen is a 1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene, 3- degenerative disease antigen, an infectious disease an- Quinuclidinyl benzilate (BZ), Phosgene: Carbonyl dichlo- tigen, a cancer antigen, an atopic disease antigen, an ride, Cyanogen chloride, Hydrogen cyanide and Chlo- autoimmune disease antigen, or a metabolic disease en- ropicrin: Trichloronitromethane. In some embodiments, zyme or enzymatic product thereof. the toxin for inclusion in a nanocarrier is a complete mol- 10 [0046] In some embodiments, the antigen is a cancer ecule of any of the foregoing or a portion thereof. antigen. Is some embodiments, the cancer antigen is Me- [0042] In some embodiments, the B cell antigen is a lan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adeno- biohazard or hazardous environmental agent. In some sine deaminase-binding protein (ADAbp), cyclophilin b, embodiments, the hazardous environmental agent is ar- Colorectal associated antigen (CRC)--C017-1A/GA733, senic, lead, mercury, vinyl chloride, polychlorinated bi- 15 Carcinoembryonic Antigen (CEA) and its immunogenic phenyls, benzene, polycyclic aromatic hydrocarbons, epitopesCAP-1 and CAP-2, etv6,aml1, Prostate Specific cadmium, benzo(a)pyrene, benzo(b)fluoranthene, chlo- Antigen (PSA) and its immunogenic epitopes PSA-1, roform, dichlor-diphenyl-trichlorethylene (DDT), P,P’-, PSA-2, and PSA-3, prostate-specific membrane antigen aroclor 1254, aroclor 1260, dibenzo(a,h)anthracene, (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family trichloroethylene, dieldrin, chromium hexavalent, or p,p’- 20 of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE- dichlorodiphenyldichloroethene (DDE, P,P’). A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, [0043] In some embodiments, the B cell antigen is a MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE- carbohydrate. In some embodiments, the carbohydrate A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), is from an infectious agent. In some embodiments, the MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE- infectious agent is a bacterium, fungus, virus, protozoan, 25 C3, MAGE-C4, MAGE-C5), GAGE-family of tumor anti- or parasite. In some embodiments, the bacterium is a gens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, Pseudomonas, Pneumococcus, E. coli, Staphylococcus, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, Streptococcus, Treponema, Borrelia, Chlamydia, Hae- RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosi- mophilus, Clostridium, Salmonella, Legionella, Vibrio or nase, p53, MUC family, HER2/neu, p21ras, RCAS1, α- Enterococci bacterium or a Mycobacterium. In some em- 30 fetoprotein, E-cadherin, α-catenin, β-catenin and γ-cat- bodiments, the virus is a pox virus, smallpox virus, ebola enin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, brain virus, marburg virus, dengue fever virus, influenza virus, glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL- parainfluenza virus, respiratory syncytial virus, rubeola 40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adeno- virus, human immunodeficiency virus, human papilloma- matous polyposis coli protein (APC), fodrin, P1A, Con- virus, varicella-zoster virus, herpes simplex virus, cy- 35 nexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 ganglio- tomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, sides, viral products such as human papilloma virus pro- rotavirus, rhinovirus, adenovirus, papillomavirus, parvo- teins, Smad family of tumor antigens, lmp-1, EBV-encod- virus, picornavirus, poliovirus, virus that causes mumps, ed nuclear antigen (EBNA)-1, or c-erbB-2. virus that causes rabies, reovirus, rubella virus, togavi- [0047] In some embodiments, the infectious disease rus, orthomyxovirus, retrovirus, hepadnavirus, coxsack- 40 antigen is a viral antigen. In some embodiments, the viral ievirus, equine encephalitis virus, Japanese encephalitis antigen is an antigen from a pox virus, smallpox virus, virus, yellow fever virus, Rift Valley fever virus, hepatitis ebola virus, marburg virus, dengue fever virus, influenza A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, parainfluenza virus, respiratory syncytial virus, ru- virus, or hepatitis E virus. beola virus, human immunodeficiency virus, human pap- [0044] In some embodiments, the B cell antigen is a 45 illomavirus, varicella-zoster virus, herpes simplex virus, self antigen. In some embodiments, the self antigen is a cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovi- protein or peptide, lipoprotein, lipid, carbohydrate, or a rus, rotavirus, rhinovirus, adenovirus, papillomavirus, nucleic acid. In some embodiments, the self antigen is parvovirus, picornavirus, poliovirus, virus that causes an enzyme, a structural protein, a secreted protein, a cell mumps, virus that causes rabies, reovirus, rubella virus, surface receptor, or a cytokine. In some embodiments, 50 togavirus, orthomyxovirus, retrovirus, hepadnavirus, 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 55 [0048] 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

8 13 EP 2 630 966 B1 14 acarbohydrate or smallmolecule. In some embodiments, igonucleotides (e.g., CpG immunostimulatory oligonu- the poorly immunogenic antigen is an abused substance, cleotides first described by Krieg et al., Nature 374:546-9, addictive substance, or toxin. In some embodiments, the 1995); incomplete Freund’s adjuvant; complete Freund’s toxin is from a chemical weapon. In some embodiments, adjuvant; montanide; vitamin E and various water-in-oil the poorly immunogenic antigen is a hazardous environ- 5 emulsions prepared from biodegradable oils such as mental agent. In some embodiments, the poorly immu- squalene and/or tocopherol, Quil A, Ribi Detox, CRL- nogenic antigen is a self antigen. 1005, or L-121. [0049] In general, the T cell antigen is a protein or pep- [0053] In specific embodiments, an immunostimulato- tide. In some embodiments, the T cell antigen is a de- ry agent may be a natural or synthetic agonist for a Toll- generative disease antigen, an infectious disease anti- 10 like receptor (TLR). In specific embodiments, an immu- gen, a cancer antigen, an atopic disease antigen, an au- nostimulatory agent may be a ligand for toll-like receptor toimmune disease antigen, an alloantigen, a xenoanti- (TLR)-7, such as CpGs, which induce type I interferon gen, an allergen, a contact sensitizer, a hapten, or a met- production; an agonist for the DC surface molecule abolic disease enzyme or enzymatic product. CD40; an agent that promotes DC maturation; a TLR-4 [0050] In some embodiments, the T cell antigen is from 15 agonist; a cytokine; proinflammatory stimuli released an infectious agent. In some embodiments, the infectious from necrotic cells (e.g. urate crystals); activated com- agent is a bacterium, fungus, virus, protozoan, or para- ponents of the complement cascade (e.g. CD21, CD35, site. In some embodiments, the infectious disease anti- etc.); and so forth. gen is a viral antigen. In some embodiments, the viral [0054] In some embodiments, the targeting moiety antigen is an antigen from a pox virus, smallpox virus, 20 binds a receptor expressed on the surface of a cell. In ebola virus, marburg virus, dengue fever virus, influenza some embodiments, the targeting moiety binds a soluble virus, parainfluenza virus, respiratory syncytial virus, ru- receptor. In some embodiments, the soluble receptor is beola virus, human immunodeficiency virus, human pap- a complement protein or a pre-existing antibody. In some illomavirus, varicella-zoster virus, herpes simplex virus, embodiments, the targeting moiety is for delivery of the cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovi- 25 nanocarrier to antigen presenting cells, T cells, or B cells. rus, rotavirus, rhinovirus, adenovirus, papillomavirus, In some embodiments, the antigen presenting cells are parvovirus, picornavirus, poliovirus, virus that causes macrophages. In some embodiments, the macrophages mumps, virus that causes rabies, reovirus, rubella virus, are subcapsular sinus macrophages. In some embodi- togavirus, orthomyxovirus, retrovirus, hepadnavirus, ments, the antigen presenting cells are dendritic cells. In coxsackievirus, equine encephalitis virus, Japanese en- 30 some embodiments, the antigen presenting cells are fol- cephalitis virus, yellow fever virus, Rift Valley fever virus, licular dendritic cells. hepatitis A virus, hepatitis B virus, hepatitis C virus, hep- [0055] In some embodiments, the targeting moiety is atitis D virus, or hepatitis E virus. a molecule that binds to CD11b, CD169, mannose re- [0051] In some embodiments, T cell antigen is a uni- ceptor, DEC-205, CD11c, CD21/CD35, CX3CR1, or an versal T cell antigen. In some embodiments, the universal 35 Fc receptor. In some embodiments, the targeting moiety T cell antigen is one or more peptides derived from tet- is a molecule that binds to CD169, CX3CR1, or an Fc anus toxoid, Epstein-Barr virus, or influenza virus. receptor. In some embodiments, the molecule that binds [0052] Immunostimulatory agents include interleukins, to CD169 is an anti-CD169 antibody. In some embodi- interferon, cytokines, etc. Immunostimulatory agents are ments, the molecule that binds CX3CR1 is CX3CL1 (frac- toll-like receptor (TLR) agonist. Immunostimulatory40 talkine). In some embodiments, the targeting moiety agents also include a CD40 agonist, cytokine receptor comprises the Fc portion of an immunoglobulin. In some agonist, Fc receptor agonist, CpG-containing immunos- embodiments, the targeting moiety comprises the Fc por- timulatory nucleic acid, complement receptor agonist, or tion of an IgG. In some embodiments, the Fc portion of an adjuvant. In some embodiments, the TLR agonist is an immunoglobulin is a human Fc portion of an immu- a TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, 45 noglobulin. In some embodiments, the Fc portion of an TLR-8, TLR-9, or TLR-10 agonist. Fc receptor agonists IgG is a human Fc portion of an IgG. In some embodi- include Fc-gamma receptor agonists. A complement re- ments, the targeting moiety is the soluble receptor, CRFc. ceptor agonist may bind to CD21 or CD35. A complement In some embodiments, CRFc can be used to target mac- receptor agonist may induce endogenous complement rophages in the subcapsular sinus but not macrophages opsonization of the nanocarrier. Cytokine receptor ago- 50 of the medulla. In some embodiments, the targeting moi- nists include a cytokine (such as a small molecule, anti- ety is one or more amine moieties. body, fusion protein, or aptamer). Immunostimulatory [0056] In some aspects, the compositions provided agents include an adjuvant. The adjuvant may induce herein are immunogenic. cytokine biosynthesis. Adjuvants include alum, MF59, [0057] Some aspects comprise administering any of R848, cholera toxin, squalene, phosphate adjuvants, or 55 the compositions provided herein to a subject in an tetrachlorodecaoxide. Adjuvants include monophospho- amount effective to modulate an immune response. In ryl lipid A (MPL, SmithKline Beecham); saponins includ- some embodiments, the composition is in an amount ef- ing QS21 (SmithKline Beecham); immunostimulatory ol- fective to induce or enhance an immune response. In

9 15 EP 2 630 966 B1 16 some embodiments, the composition is in an amount ef- ments, immunomodulatory agents presented on nano- fective to suppress an immune response. In some em- carrier surfaces stimulate B cells, and immunomodula- bodiments, the composition is in an amount effective to tory agents encapsulated within the nanocarriers are direct or redirect an immune response. In some embod- processed and presented to T cells. In some embodi- iments, the nanocarrier is for prophylaxis and/or treat- 5 ments, vaccine nanocarriers comprise at least one tar- ment of the conditions identified herein. geting moiety that is useful for selective delivery of the [0058] In some embodiments, where the nanocarrier vaccine nanocarrier to specific antigen-presenting cells is to induce or enhance an immune response, the subject (APCs). has or is susceptible to having cancer, an infectious dis- [0063] In some embodiments, an immunomodulatory ease, a non-autoimmune metabolic or degenerative dis- 10 agent may comprise isolated and/or recombinant pro- ease, an atopic disease, an allergic disease, or an ad- teins or peptides, carbohydrates, glycoproteins, glyco- diction. In some embodiments, the subject has been ex- peptides, proteoglycans, inactivated organisms and vi- posed to or may be exposed to a toxin. In some embod- ruses, dead organisms and virus, genetically altered or- iments, the subject has been exposed to or may be ex- ganisms or viruses, and cell extracts. In some embodi- posed to a toxin from a chemical weapon. In some em- 15 ments, an immunomodulatory agent may comprise nu- bodiments, the subject has been exposed to or may be cleic acids, carbohydrates, lipids, and/or small mole- exposed to a toxin from a hazardous environmental sub- cules. In some embodiments, an immunomodulatory stance. In some embodiments, the nanocarrier compris- agent is one that elicits an immune response. In some es a B-cell antigen, an immunostimulatory agent, and a embodiments, an immunomodulatory agent is an anti- T cell antigen, such as a universal T cell antigen. In some 20 gen. In some embodiments, an immunomodulatory embodiments, the nanocarrier further comprises a tar- agent is used for vaccines. geting moiety. [0064] In some embodiments, an immunomodulatory [0059] In some embodiments, where the nanocarrier agent is any protein and/or other antigen derived from a is for treating or preventing an addiction (or for treating pathogen. The pathogen may be a virus, bacterium, fun- a subject exposed to or who may be exposed to a toxin), 25 gus, protozoan, parasite, etc. In some embodiments, an thenanocarrier comprises the addictive substance or tox- immunomodulatory agent may be in the form of whole in, an adjuvant, and a T cell. In some embodiments, the killed organisms, peptides, proteins, glycoproteins, glyc- nanocarrier raises high titer antibodies that bind and neu- opeptides, proteoglycans, carbohydrates, or combina- tralize the offending agent before it reaches its effector tions thereof. site (e.g., the brain). In some embodiments, the addictive 30 [0065] In some embodiments, all of the immunomod- substance or toxin is at a high density on the surface of ulatory agents of a vaccine nanocarrier are identical to the nanocarrier. one another. In some embodiments, all of the immu- [0060] In some embodiments, the infectious disease nomodulatory agents of a vaccine nanocarrier are differ- is a chronic viral infection. In some embodiments, the ent. In some embodiments, a vaccine nanocarrier com- chronicviral infection is HIV,HPV, HBV, or HCV infection. 35 prises exactly one distinct type (i.e., species) of immu- In some embodiments, the infectious disease is or is nomodulatory agent. For example, when the immu- caused by a bacterial infection. In some embodiments, nomodulatory agent is an antigen, all of the antigens that the subject has or is susceptible to havingPseu- a are in the vaccine nanocarrier are the same. In some domonas infection, a Pneumococcus infection, tubercu- embodiments, a vaccine nanocarrier comprises exactly losis, malaria, leishmaniasis, H. pylori, a Staphylococcus 40 two distinct types of immunomodulatory agents. In some infection, or a Salmonella infection. In some embodi- embodiments, a vaccine nanocarrier comprises greater ments, the infectious disease is or is caused by a fungal than two distinct types of immunomodulatory agents. infection. In some embodiments, the infectious disease [0066] In some embodiments, a vaccine nanocarrier is or is caused by a parasitic infection. In some embod- comprises a single type of immunomodulatory agent that iments, the infectious disease is or is caused by a proto- 45 stimulates an immune response in B cells. In some em- zoan infection. In some embodiments, the subject has or bodiments, a vaccine nanocarrier comprises a single is susceptible to having influenza. type of immunomodulatory agent that stimulates an im- [0061] In some embodiments, the composition is ad- mune response in T cells. In some embodiments, a vac- ministered in an amount effective to modify an immune cine nanocarrier comprises two types of immunomodu- response (e.g., from a Th2 to a Th1 immune response). 50 latory agents, wherein the first immunomodulatory agent In some embodiments, the subject has or is susceptible stimulates B cells, and the second immunomodulatory to having an allergic disease. agent stimulates T cells. In certain embodiments, any of [0062] In some aspects, vaccine nanocarriers for de- 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, 55 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- greater than two types of immunomodulatory agents,

10 17 EP 2 630 966 B1 18 wherein one or more types of immunomodulatory agents ments, a target can comprise a protein, a carbohydrate, stimulate B cells, and one or more types of immunomod- a lipid, and/or a nucleic acid. In some embodiments, a ulatory agents stimulate T cells. target is a tumor marker. In some embodiments, a target [0067] In some embodiments, a vaccine nanocarrier is an APC marker. In certain embodiments, a target is a includes a lipid membrane (e.g. lipid bilayer, lipid mon- 5 T cell marker. In some embodiments, the targeting moi- olayer, etc.). At least one immunomodulatory agent may eties target secondary lymphoid tissues or organs. Sec- be associated with the lipid membrane. In some embod- ondary lymphoid tissues or organs include lymph nodes, iments, at least one immunomodulatory agent is embed- the spleen, Peyer’s patches, the appendix, or tonsils. ded within the lipid membrane, embedded within the lu- [0072] In certain embodiments, a target is a dendritic men of a lipid bilayer, associated with the interior surface 10 cell marker. In some embodiments, DC markers include of the lipid membrane, and/or encapsulated with the lipid DC-205, CD11c, class II MHC, CD80, CD86, DC-SIGN, membrane of a vaccine nanocarrier. CD11b, BDCA-1, BDCA-2, BDCA-4, Siglec-H, CX3CR1, [0068] The vaccine nanocarrier includes a polymer and/or Langerin. Examples of such markers are provided (e.g. a polymeric core). The immunomodulatory agent elsewhere herein. may be associated with the polymer, and in some em- 15 [0073] In certain embodiments, a target is a subcap- bodiments, at least one type of immunomodulatory agent sular sinus macrophage marker. In some embodiments, is associated with the polymer. In some embodiments, SCS-Mph markers include CD169 i.e. ( sialoadhesin), theimmunomodulatory agent is embedded within thepol- CD11b (i.e. CD11b/CD18,Mac-1, CR3 or αMβ2integrin), ymer, associated with the interior surface of the polymer, Fc receptor, and/or the mannose receptor i.e.( a multi- and/or encapsulated within the polymer of a vaccine na- 20 valent lectin), proteins which are all prominently ex- nocarrier, and, in some embodiments, at least one type pressed on SCS-Mph. Examples of such markers are of immunomodulatory agent is embedded within the pol- provided elsewhere herein. ymer, associated with the interior surface of the polymer, [0074] In certain embodiments, a target is a B cell and/or encapsulated within the polymer of a vaccine na- marker. In some embodiments, B cell markers may in- nocarrier. 25 clude complement receptors, CR1 i.e.( CD35) or CR2 [0069] In some embodiments, inventive vaccine nano- (i.e. CD21), proteins which are expressed on B cells. In carriers comprise less than less than 90% by weight, less some embodiments, B cell targeting can be accom- than 75% by weight, less than 50% by weight, less than plished by B cell markers such as CD19, CD20, and/or 40% by weight, less than 30% by weight, less than 20% CD22. In some embodiments, B cell targeting can be by weight, less than 15% by weight, less than 10% by 30 accomplished by B cell markers such as CD40, CD52, weight, less than 5% by weight, less than 1% by weight, CD80, CXCR5, VLA-4, class II MHC, surface IgM or IgD, or less than 0.5% by weight of the immunomodulatory APRL, and/or BAFF-R. Examples of such markers are agent. provided elsewhere herein. [0070] In some embodiments, vaccine nanocarriers [0075] In certain embodiments, a target is a FDC mark- are associated with at least one targeting moiety. In some 35 er. In some embodiments, FDC markers include comple- embodiments, a targeting moiety may be a nucleic acid, ment receptors, CR1 ( i.e. CD35) or CR2 ( i.e. CD21), pro- polypeptide, peptide, glycoprotein, glycopeptide, prote- teins which are expressed on FDCs. Examples of such oglycan, carbohydrate, lipid, small molecule, etc. For ex- markers are provided elsewhere herein. ample, a targeting moiety can be a nucleic acid targeting [0076] In some embodiments, a vaccine nanocarrier moiety (e.g. an aptamer, Spiegelmer ®, etc.) that binds to 40 comprises a single type of targeting moiety that directs a cell type specific marker. In some embodiments, a tar- delivery of the vaccine nanocarrier to a single cell type geting moiety may be a naturally occurring or synthetic (e.g. delivery to SCS-Mph only). In some embodiments, ligand for a cell surface protein, e.g., DEC-205, CD169, a vaccine nanocarrier comprises a single type of target- CD11b, etc. Examples of targeting moieties also include ing moiety that directs delivery of the vaccine nanocarrier those provided elsewhere herein, such as those de-45 to multiple cell types (e.g. delivery to both SCS-Mphs and scribed above. FDCs, or to both SCS-Mphs and DCs). In some embod- [0071] In accordance with the present invention, a tar- iments, a vaccine nanocarrier comprises two types of geting moiety recognizes one or more "targets" or "mark- targeting moieties, wherein the first type of targeting moi- ers" associatedwith a particular organ, tissue,cell, and/or ety directs delivery of the vaccine nanocarrier to one cell subcellular locale. In some embodiments, a target may 50 type, and the second type of targeting moiety directs de- be a marker that is exclusively or primarily associated livery of the vaccine nanocarrier to a second cell type. with one or a few cell types, with one or a few diseases, For example, in some embodiments, the first type of tar- and/or with one or a few developmental stages. Exam- geting moiety directs delivery to SCS-Mphs, and the sec- ples of cells that are targeted include antigen presenting ond type of targeting moiety directs delivery to DCs. As cells (APCs), such as dendritic cells, follicular dendritic 55 another example, the first type of targeting moiety directs cells, and macrophages. One example of a macrophage delivery to SCS-Mphs, and the second type of targeting is a subcapsular sinus macrophage. Other cells that are moiety directs delivery to FDCs. targeted include T cells and B cells. In some embodi- [0077] In some embodiments, inventive vaccine nano-

11 19 EP 2 630 966 B1 20 carriers comprise less than 50% by weight, less than 40% reactions, and/or antibody performance in neutralization by weight, less than 30% by weight, less than 20% by assays. weight, less than 15% by weight, less than 10% by [0083] A vaccine nanocarrier is an entity that compris- weight, less than 5% by weight, less than 1% by weight, es, for example, at least one immunomodulatory agent or less than 0.5% by weight of the targeting moiety. 5 which is capable of stimulating an immune response in [0078] In some embodiments, vaccine nanocarriers B cells and/or T cells. may transport one or more types of immunostimulatory [0084] A variety of different nanocarriers can be used agents which can help stimulate immune responses. In in accordance with the present invention. In some em- some embodiments, immunostimulatory agents boost bodiments, nanocarriers are spheres or spheroids. In immune responses by activating APCs to enhance their 10 some embodiments, nanocarriers are flat or plate- immunostimulatory capacity. In some embodiments, im- shaped. In some embodiments, nanocarriers are cubes munostimulatory agents boost immune responses by or cuboids. In some embodiments, nanocarriers are amplifying lymphocyte responses to specific antigens. In ovals or ellipses. In some embodiments, nanocarriers some embodiments, immunostimulatory agents boost are cylinders, cones, or pyramids. Nanocarriers may be immune responses by inducing the local release of me- 15 hollow and may comprise one or more layers. In some diators, such as cytokines from a variety of cell types. embodiments, each layer has a unique composition and [0079] In some embodiments, a vaccine nanocarrier unique properties relative to the other layer(s). To give comprises a single type of immunostimulatory agent that but one example, nanocarriers may have a core/shell stimulates both B cells and T cells. In some embodi- structure, wherein the core is one layer ( e.g. a polymeric ments, a vaccine nanocarrier comprises two types of im- 20 core) and the shell is a second layer (e.g. a lipid bilayer munostimulatory agents, wherein the first type of immu- or monolayer). Nanocarriers may comprise a plurality of nostimulatory agent stimulates B cells, and the second different layers. In some embodiments, one layer may type of immunostimulatory agent stimulates T cells. In be substantially cross-linked, a second layer is not sub- some embodiments, a vaccine nanocarrier comprises stantially cross-linked, and so forth. In some embodi- greater than two types of immunostimulatory agents,25 ments, one, a few, or all of the different layers may com- wherein one or more types of immunostimulatory agents prise one or more immunomodulatory agents, targeting stimulate B cells, and one or more types of immunostim- moieties, immunostimulatory agents, and/or combina- ulatory agents stimulate T cells. tionsthereof. In some embodiments,one layer comprises [0080] In some embodiments, various assays can be an immunomodulatory agent, targeting moiety, and/or utilized in order to determine whether an immune re-30 immunostimulatory agent, a second layer does not com- sponse has been modulated in a B cell or group of B cells prise an immunomodulatory agent, targeting moiety, or in a T cell or group of T cells. In some embodiments, and/or immunostimulatory agent, and so forth. In some the assay assesses whether or not the cell or group of embodiments, each individual layer comprises a different cells has/have become "activated". immunomodulatory agent, targeting moiety, immunos- [0081] In some embodiments, various assays can be 35 timulatory agent, and/or combination thereof. utilized in order to determine whether an immune re- [0085] In some embodiments, nanocarriers may op- sponse has been stimulated in a T cell or group of T cells. tionally comprise one or more lipids. In some embodi- In some embodiments, stimulation of an immune re- ments, a nanocarrier comprises a lipid bilayer. In some sponse in T cells can be determined by measuring anti- embodiments, a nanocarrier comprises a lipid monolay- gen-induced production of cytokines by T cells. In some 40 er. In some embodiments, a nanocarrier comprises a embodiments, stimulation of an immune response in T core of a polymeric matrix surrounded by a lipid layer cells can be determined by measuring antigen-induced (e.g. lipid bilayer, lipid monolayer, etc.). proliferation of T cells. In some embodiments, an immune [0086] The nanocarrier comprises one or more poly- response in T cells is determined to be stimulated if cel- mers. In some embodiments, a polymeric matrix can be lularmarkers ofT cell activationare expressed atdifferent 45 surrounded by a coating layer (e.g. liposome, lipid mon- levels( e.g. higher or lower levels) relative to unstimulated olayer, micelle, etc.). In some embodiments, an immu- cells. nomodulatory agent, targeting moiety, and/or immunos- [0082] In some embodiments, various assays can be timulatory agent can be associated with the polymeric utilized in order to determine whether an immune re- matrix. In such embodiments, the immunomodulatory sponse has been stimulated in a B cell or group of B cells. 50 agent, targeting moiety, and/or immunostimulatory agent In some embodiments, stimulation of an immune re- is effectively encapsulated within the nanocarrier. sponse in B cells can be determined by measuring anti- [0087] In some embodiments, an immunomodulatory body titers, antibody affinities, antibody performance in agent, targeting moiety, and/or immunostimulatory agent neutralization assays, class-switch recombination, affin- can be covalently associated with a nanocarrier. In some ity maturation of antigen-specific antibodies, develop- 55 embodiments, covalent association is mediated by a link- ment of memory B cells, development of long-lived plas- er. In some embodiments, an immunomodulatory agent, ma cells that can produce large amounts of high-affinity targeting moiety, and/or immunostimulatory agent is non- antibodies for extended periods of time, germinal center covalently associated with a nanocarrier. For example,

12 21 EP 2 630 966 B1 22 in some embodiments, an immunomodulatory agent, tar- throughout the polymeric matrix of an inventive nanocar- geting moiety, and/or immunostimulatory agent is encap- rier. Immunomodulatory agents may be released by dif- sulated within, surrounded by, and/or dispersed through- fusion, degradation of the nanocarrier, and/or a combi- out a polymeric matrix, a lipid membrane, etc. Alterna- nation thereof. In some embodiments, polymer(s) of the tively or additionally, an immunomodulatory agent, tar- 5 nanocarrier degrade by bulk erosion. In some embodi- geting moiety, and/or immunostimulatory agent may be ments, polymer(s) of the nanocarrier degrade by surface associated with a polymeric matrix, a lipid membrane, erosion. etc. by hydrophobic interactions, charge interactions, van [0094] In some embodiments, immunomodulatory der Waals forces, etc. agents, targeting moieties, and/or immunostimulatory [0088] A wide variety of polymers and methods for10 agents are covalently associated with a particle. In some forming polymeric matrices therefrom are known in the embodiments, covalent association is mediated by one art of drug delivery. In general, a polymeric matrix com- or more linkers. Any suitable linker can be used in ac- prises one or more polymers. Polymers may be natural cordance with the present invention. In some embodi- or unnatural (synthetic) polymers. Polymers may be ments, the linker is a cleavable linker (e.g., an ester link- homopolymers or copolymers comprising two or more 15 age, an amide linkage, a disulfide linkage, etc.). monomers. In terms of sequence, copolymers may be [0095] In some embodiments, nanocarriers are made random, block, or comprise a combination of random and by self-assembly. As a comparative example, lipids are block sequences. In some embodiments, the polymers mixed with a lipophilic immunomodulatory agent, and are dendritic polymers or blends of polymers. then formed into thin films on a solid surface. A hy- [0089] Examples of polymers include polyethylenes, 20 drophilic immunomodulatory agent is dissolved in an polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhy- aqueous solution, which is added to the lipid films to hy- drides (e.g. poly(sebacic anhydride)), polyhydroxyacids drolyze lipids under vortex. Liposomes with lipophilic im- (e.g. poly(β-hydroxyalkanoate)), polypropylfumerates, munomodulatory agents incorporated into the bilayer polycaprolactones, polyamides (e.g. polycaprolactam), wall and hydrophilic immunomodulatory agents inside polyacetals, polyethers, polyesters ( e.g. polylactide, pol- 25 the liposome lumen are spontaneously assembled. In yglycolide), poly(orthoesters), polycyanoacrylates, poly- certain embodiments, pre-formulated polymeric nano- vinyl alcohols, polyurethanes, polyphosphazenes, poly- particles are mixed with small liposomes under gentle acrylates, polymethacrylates, polyureas, polystyrenes, vortex to induce liposome fusion onto polymeric nano- and polyamines. particle surface. [0090] In some embodiments, nanocarriers may op- 30 [0096] As another comparative example, a hydrophilic tionally comprise one or more amphiphilic entities (i.e., immunomodulatory agent to be encapsulated is first in- entities that possess both hydrophilic and hydrophobic corporated into reverse micelles by mixing with naturally properties). In some embodiments, an amphiphilic entity derived and nontoxic amphiphilic entities in a volatile, can promote the production of nanocarriers with in- water-miscible organic solvent. In some embodiments, creased stability, improved uniformity, or increased vis- 35 a biodegradable polymer is added after reverse micelle cosity. formation is complete. The resulting biodegradable pol- [0091] Insome embodiments, a nanocarrier comprises ymer-reverse micelle mixture is combined with a poly- one or more nanoparticles associated with the exterior mer-insoluble hydrophilic non-solvent to form nanoparti- surface of and/or encapsulated within the nanocarrier. cles by the rapid diffusion of the solvent into the non- [0092] Nanocarriers may be prepared using any meth- 40 solvent and evaporation of the organic solvent. od known in the art. For example, particulate nanocarrier [0097] In some embodiments, lipid monolayer stabi- formulations can be formed by methods such as nano- lized polymeric nanocarriers are used to deliver one or precipitation, flow focusing using fluidic channels, spray a plurality of immunomodulatory agents. In certain em- drying, single and double emulsion solvent evaporation, bodiments, a hydrophilic immunomodulatory molecule is solvent extraction, phase separation, milling, microemul- 45 first chemically conjugated to the polar headgroup of a sion procedures, microfabrication, nanofabrication, sac- lipid. The conjugate is mixed with a certain ratio of un- rificial layers, simple and complex coacervation, as well conjugated lipid molecules in an aqueous solution con- as other methods well known to those of ordinary skill in taining one or more water-miscible solvents. A biode- the art. Alternatively or additionally, aqueous and organic gradable polymeric material is mixed with the hydropho- solvent syntheses for monodisperse semiconductor,50 bic immunomodulatory agents to be encapsulated in a conductive, magnetic, organic, and other nanoparticles water miscible or partially water miscible organic solvent. may be utilized. The resulting polymer solution is added to the aqueous [0093] In some embodiments, immunomodulatory solution of conjugated and unconjugated lipid to yield na- agents and/or targeting moieties are not covalently as- noparticles by the rapid diffusion of the organic solvent sociated with a nanocarrier. For example, nanocarriers 55 into the water and evaporation of the organic solvent. may comprise a polymeric matrix, and immunomodula- [0098] Thecompositions described herein can be used tory agents and/or targeting moieties are associated with for the prophylaxis and/or treatment of any infectious dis- the surface of, encapsulated within, and/or distributed ease, disorder, and/or condition. Examples of other dis-

13 23 EP 2 630 966 B1 24 eases, disorders, and/or conditions are provided else- certain embodiments, a therapeutic amount of an inven- where herein. In some embodiments, vaccine nanocar- tive vaccine nanocarrier composition is administered to riers in accordance with the present invention may be a patient and/or animal prior to exposure to an addictive used to treat, alleviate, ameliorate, relieve, delay onset substance or a toxin. In certain embodiments, a thera- of, inhibit progression of, reduce severity of, and/or re- 5 peutic amount of an inventive vaccine nanocarrier com- duce incidence of one or more symptoms or features of position is administered to a patient and/or animal after a disease, disorder, and/or condition. In some embodi- exposure to an addictive substance or a toxin. ments, inventive vaccine nanocarriers may be used to [0102] In some embodiments, the pharmaceutical treat, alleviate, ameliorate, relieve, delay onset of, inhibit compositions of the present invention are administered progression of, reduce severity of, and/or reduce inci- 10 by a variety of routes, including oral, intravenous, intra- dence of one or more symptoms or features of microbial muscular, intra-arterial, intramedullary, intrathecal, sub- infection (e.g. bacterial infection, fungal infection, viral cutaneous, intraventricular, transdermal, interdermal, infection, parasitic infection, etc.). In some embodiments, rectal, intravaginal, intraperitoneal, topical (as by pow- the prophylaxis and/or treatment of microbial infection ders, ointments, creams, and/or drops), transdermal, comprises administering a therapeutically effective15 mucosal, nasal, buccal, enteral, sublingual; by intratra- amount of inventive vaccine nanocarriers to a subject in cheal instillation, bronchial instillation, and/or inhalation; need thereof, in such amounts and for such time as is and/or as an oral spray, nasal spray, and/or aerosol. In necessary to achieve the desired result. In certain em- certain embodiments, the composition is administered bodiments of the present invention, a "therapeutically ef- orally. In certain embodiments, the composition is admin- fective amount" of an inventive vaccine nanocarrier is 20 istered parenterally. In certain embodiments, the com- that amount effective for treating, alleviating, ameliorat- position is administered via intramuscular injection. ing, relieving, delaying onset of, inhibiting progression of, [0103] In certain embodiments, vaccine nanocarriers reducing severity of, and/or reducing incidence of one or which delay the onset and/or progression of a disease, more symptoms or features of disease, disorder, and/or disorder, and/or condition (e.g., a particular microbial in- condition provided herein. 25 fection) may be administered in combination with one or [0099] Prophylactic and/or therapeutic protocols in- more additional therapeutic agents which treat the symp- volve administering a therapeutically effective amount of toms of the disease, disorder, and/or condition. For ex- one or more inventive vaccine nanocarriers to a subject ample, the vaccine nanocarriers may be combined with such that an immune response is modulated (e.g., stim- the use of an anti-cancer agent, anti-inflammatory agent, ulated in both T cells and/or B cells). 30 antibiotic, or antiviral agent. [0100] The present invention provides compositions [0104] The invention provides a variety of kits compris- comprising a therapeutically effective amount of one or ing one or more of the nanocarriers of the invention. For more vaccine nanocarriers and one or more pharmaceu- example, the invention provides a kit comprising an in- tically acceptable excipients. In some embodiments, the ventive nanocarrier and instructions for use. A kit may present invention provides for pharmaceutical composi- 35 comprise multiple different nanocarriers. A kit may com- tions comprising inventive vaccine nanocarriers. The prise any of a number of additional components or rea- composition may include more than one type of nano- gents in any combination. carrier, each type having different constituents (e.g., im- [0105] In some embodiments, the kit comprises an in- munomodulatory agents, targeting agents, immunostim- ventive nanocarrier and instructions for mixing. ulatory agents,excipients, etc.). Inaccordance with some 40 [0106] In any of the foregoing embodiments described embodiments, administering a pharmaceutical composi- above, the word conjugated means covalently or non- tion comprising inventive compositions to a subject (e.g. covalently conjugated, unless the context clearly indi- human) in need thereof is provided. cates otherwise. In any of the foregoing embodiments [0101] In some embodiments, a therapeutically effec- described above, the word encapsulated means physi- tive amount of an inventive vaccine nanocarrier compo- 45 cally trapped within, whether by admixture, by a shell sition is delivered to a patient and/or animal prior to, si- surrounding a core, by covalent bonding internal of the multaneously with, and/or after diagnosis with a disease, surface of the nanocarrier, and the like. disorder, and/or condition. In some embodiments, a ther- apeutic amount of an inventive vaccine nanocarrier com- Brief Description of the Drawing position is delivered to a patient and/or animal prior to, 50 simultaneously with, and/or after onset of symptoms of [0107] a disease, disorder, and/or condition. In certain embod- iments, a therapeutic amount of an inventive vaccine na- Figure 1: Combined vaccine targeting strategy for nocarrier composition is administered to a patient and/or optimal humoral and cellular immune response. The animal prior to exposure to an infectious agent. In certain 55 composite vaccine carries internal T cell antigens, embodiments, a therapeutic amount of an inventive vac- adjuvants (not shown) and targeting moieties for cine nanocarrier composition is administered to a patient DCs, FDC and SCS-Mph together with surface an- and/or animal after exposure to an infectious agent. In tigen for B cell recognition. Upon s.c. or i.m. injection,

14 25 EP 2 630 966 B1 26 the material reaches lymph nodes via draining lymph Figure 7: A comparative exemplary liposome-poly- vessels and accumulates on each APC (for clarity, mer nanocarrier containing reverse micelles with a only APC-specific targeting moieties are shown, but lipophilic immunomodulatory agent incorporated in- each APC acquires the entire complex). DCs inter- to the membrane, and a hydrophilic immunomodu- nalize and digest the complex and present antigenic 5 latory agent encapsulated within the reverse mi- peptides in MHC class I and class II to CD8 and CD4 celles. T cells, respectively. The activated T cells differen- Figure8 :A comparativeexemplary nanoparticle-sta- tiate into effector/memory (T Eff/Mem) cells that medi- bilized liposome-polymer nanocarrier containing re- ate cellular immune responses. TFH cells provide verse micelles with a lipophilic immunomodulatory help to B cells that were initially stimulated by antigen 10 agent incorporated into the membrane, and a hy- on SCS-Mph and in the process have acquired and drophilic immunomodulatory agent encapsulated in- processed T cell antigens for restimulation of TFH. side the liposome. The help provided by TFH cells allows the develop- Figure 9: A comparative exemplary lipid-stabilized ment of a GC reaction during which B cells proliferate polymeric nanocarrier with a hydrophilic immu- and generate high-affinity antibodies. 15 nomodulatory agent conjugated to the lipid monol- Figure 2: SCS-Mph bind lymph-borne viral particles ayer, and a hydrophobic immunomodulatory agent and present them to follicular B cells. (A) Immuno- encapsulated inside the polymer core. histochemical staining of the cortex of a mouse pop- Figure 10: A comparative exemplary lipid-stabilized liteal lymph node stained with anti-CD169 and coun- polymeric nanocarrier containing reverse micelles ter-stained with wheat germ agglutinin. The lymph 20 with a hydrophilic immunomodulatory agent conju- node was harvested 30 minutes after footpad injec- gated to the lipid monolayer, and a hydrophilic im- tion of red fluorescent vesicular stomatitis virus munomodulatory agent encapsulated inside the pol- (VSV). In the subcapsular sinus of the draining lymph ymer core. node, the red virus colocalized exclusively with Figure 11: Capture of lymph-borne VSV by SCS CD169+ macrophages. (B) Electron micrograph of a 25 macrophages. (A) MP-IVM micrographs of VSV in a lymph node macrophage (Mph) and a follicular B cell popliteal LN (numbers: minutes after footpad injec- (B1) below the floor of the subcapsular sinus (SCSf) tion; scale bar: 100 mm). (B) VSV accumulation in a 30 minutes after VSV injection shows VSV at the C57BL/6→Act(EGFP) recipient 3 hours after injec- surface and within a phagolysosome of the Mph and tion (scale bar: 50 mm). (C) Electron micrographs of at the interface between Mph and B cells (arrow-30 VSV in LN 5 minutes after injection. Center micro- heads). (C) Injection of VSV into the footpad of un- graph is shown schematically (left) and at higher treated mice (B6) results in rapid downregulation of magnification (right). Arrowheads identify VSV par- surface-expressed IgM on virus-specific B cells, a ticles (scale bars: 2 mm). (D) Confocal micrographs sign of B cell activation. Depletion of SCS-Mph after of VSV-draining LN (30 minutes). Scale bars: 100 footpad injection of clodronate liposomes (CLL)35 mm (left), 15 mm (right). (E) VSV titers in popliteal abolished B cell activation, indicating that SCS-Mph LNs 2 hours after injection into wildtype, C3-deficient are essential to present particulate antigen to B cells. or CLL-depleted mice. ***: p < 0.001 (two-way ANO- Figure 3: A comparative exemplary liposome nano- VA, Bonferroni’s post-test). (F) VSV capture in DH- carrier with a lipophilic immunomodulatory agent in- LMP2a mice. *: p < 0.05 (unpaired t-test). (G) VSV corporated in the membrane, and a hydrophilic im- 40 titers after footpad injection in untreated and CLL- munomodulatory agent encapsulated within the li- treated mice (one of two similar experiments; n = 3). posome. ProxLN: inguinal, paraaortic LNs; BrachLN: brachial Figure4 :A comparative exemplary nanoparticle-sta- LN. (H) Viral titers in lymph, spleen and blood after bilized liposome nanocarrier with a lipophilic immu- TD cannulation; *: p < 0.05 (unpaired t-test). Hori- nomodulatory agent incorporated into the mem-45 zontal bars in (E-H) indicate means. brane, and a hydrophilic immunomodulating agent Figure 12: Characterization of CD169+ macrophag- encapsulated within the liposome. es in peripheral LNs. (A-C) Lineage marker expres- Figure 5: A comparative exemplary liposome-poly- sion analysis of pooled mononuclear cells from LNs mer nanocarrier with a lipophilic immunomodulatory of naive C57BL/6 mice. (A) After gating on the agent incorporated into the membrane, and a hydro- 50 CD169+ population (middle panel), cells were ana- phobic immunomodulating agent encapsulated with- lyzed for expression of the two macrophage-associ- in the polymeric nanoparticle. ated surface markers, I-Ab (MHC class II) and Figure6 :A comparative exemplary nanoparticle-sta- CD11b (bottom panel). Staining with an isotype con- bilized liposome-polymer nanocarrier with a li- trol for anti-CD169 is shown in the top panel. (B) pophilic immunomodulatory agent incorporated into 55 CD169+I-Ab+CD11b+ cells were further analyzed for the membrane, and a hydrophobic immunomodulat- expression of CD68, F4/80, CD11c, and Gr-1. Gates ing agent encapsulated within the polymeric nano- were drawn to identify marker+ cells, except for particle. CD11c staining where the marker was positioned to

15 27 EP 2 630 966 B1 28 identify conventional CD11c high dendritic cells (over- dulla where viruses were not only bound by CD169 + lay). Numbers indicate percentage of CD169+I- macrophages, but also by LYVE-1+ lymphatic en- Ab+CD11b+ cells under the histogram gate. Data are dothelial cells. Scale bars indicate 125 mm (left pan- representative of 3-5 experiments with similar re- el) and 25 mm (right panel). (E) Confocal micrograph sults. (C) Quantitative analyses of data in panel (B), 5 of a popliteal LN 30 minutes after hind footpad injec- error bars represent SEM. (D-G) Confocal micro- tion of Alexa-568 labeled VSV and approximately graphs of popliteal LNs from naive C57BL/6 mice 1011 Crimson Fluospheres (200 nm diameter). Fro- showing co-expression of selected markers on zen LN sections were counter-stained with FITC-α- CD169+ cells (arrowheads). Scale bars: 125 mm in CD169. Note that the Latex beads, unlike VSV, were the left column and 20 mm in all other columns. 10 poorly retained in draining LNs. Scale bar: 125 mm. Figure 13: Morphological changes in popliteal LNs Figure 15: Effect of CLL footpad injection on VSV following CLL treatment. (A) Confocal micrographs distribution in draining LNs. Confocal micrographs of popliteal LNs (top three rows) and spleens (bottom show the localization of fluorescent VSV particles in row) of untreated control mice (-CLL, left column) poplitealLNs without (A)or 7 daysafter (B)CLL treat- and animalsthat hadreceived CLL footpad injections 15 ment. B follicles were identified by FITC-α-B220 6-10 days earlier. CLL treatment depleted CD169+ staining. In the medulla (boxed area), VSV was macrophages in the LN (top row), but not in spleens; bound by LYVE-1+ cells that were not affected by Lyve-1+ medullary lymphatic endothelial cells (sec- CLL treatment. Scale bars: 125 mm (left column) and ond row) and cortical CD11c high dendritic cells (third 25 mm (right column). row) were not affected. (B) Cellular subset frequency 20 Figure 16: SCS macrophages present lymph-de- in popliteal LNs with and without CLL treatment, data rived AdV to follicular B lymphocytes. (A) Confocal are from n = 3 mice and shown as mean 6 SEM; *: micrograph of CD169+ macrophages in the SCS p < 0.05, **: p < 0.01; unpaired student’s t-test. (C) above a B follicle in a popliteal LN. Frozen sections Frequency of different I-Ab +CD1 1b+ leukocyte sub- were counterstained with wheatgerm agglutinin sets in popliteal LNs at 6-10 days after footpad in- 25 (WGA) to identify extracellular matrix and withα - jection of 50 ml CLL. Each symbol represents pooled B220 to detect B cells. Note that some B cells reside popliteal LNs from one mouse. Subset frequencies in the SCS, and one B cell appears to migrate be- among total mononuclear cells in popliteal LNs were tween the follicle and the SCS (arrowhead). Scale assessed by flow cytometry after gating on I- bar: 25 mm. (B) Electron micrograph and (C) sche- Ab+CD11b+ cells as shown in Figure 12A. (D) Im- 30 matic drawing of a SCS macrophage and surround- munohistochemical analysis of popliteal LNs without ing cells in a popliteal LN 30 minutes after footpad treatment (-CLL) or 7 days after footpad injection of injection of AdV. Scale bar: 2 mm. The boxes drawn CLL (+CLL). Scale bars: 300 mm. (E) Ultrastructure in (C) indicate areas of higher magnification shown of the SCS in a representative popliteal LN 7 days in panels (D) and (E). These panels show two ex- after CLL treatment and 5 minutes after footpad in- 35 amples of AdV particles at the interface between the jection of 20 mg VSV-IND. Note the complete ab- SCS macrophage and B cells (arrowheads). Aster- sence of SCS macrophages and viral particles. isks denote other macrophage-associated AdV par- Scale bar: 2 mm. ticles. Scale bars: 500 nm. Figure 14: Retention of fluorescent viruses and latex Figure 17: Macrophage-mediated transfer of lymph- nanoparticles in popliteal LNs. (A) Confocal micro- 40 borne VSV across the SCS floor alters virus-specific graphs of popliteal LNs 30 minutes after footpad in- B cell behavior. (A) Electron micrographs and sche- jection of Alexa-568-labeled adenovirus (AdV). Fro- matic drawing (middle) showing a macrophage pen- zen sections were stained with FITC-α-CD169 and etrating the SCS floor of a popliteal LN 30 minutes Alexa-647-α-B220 to identify B cells. Scale bars: 100 after VSV injection. Scale bars: 10 mm (left) and 2 mm (left panel) and 15 mm (right panel). (B) Trans- 45 mm (right). Arrow: vacuole with digested VSV. Ar- mission electron micrographs of AdV particles cap- rowheads: virions in contact zone between macro- tured by a SCS macrophage. The top panel shows phage and B cells. (B) MP-IVM of polyclonal and an annotated schematic drawing of the low magnifi- VI10YEN B cells in popliteal LNs. Scale bars: 50 mm. cation overview (middle panel). The boxed area in (C) Regional ratios of VI10YEN B cells/control B cells the middle panel is enlarged in the lower panel, ar- 50 following VSV injection. Results are from 3 mov- rowheads denote electrondense, spherical AdV par- ies/group. (D,E) Localization of VI10YEN B cells in ticles. Scale bars: 2 mm (top and middle panel) and popliteal LNs relative to the SCS. **: p < 0.01 (one- 1 mm (lower panel). (C-D) Confocal micrographs of way ANOVA with Bonferroni’s post-test). popliteal LNs from C57BL/6 mice 30 minutes after Figure 18: Characteristics of VSV serotypes and footpad injection of 20 mg Alexa-568 labeled UV-in- 55 VSV-IND-specific VI10YEN B cells. (A) SDS-PAGE activated AdV (C) or W (D). Fluorescent viruses ac- gels (12%) of purified VSV lysates. Top: VSV-IND cumulated in the cortical SCS above B follicles iden- and VSV-NJ. The N and P proteins co-migrate in tified by FITC-α-B220 staining and also in the me- VSV-NJ, approximate molecular weights are shown

16 29 EP 2 630 966 B1 30 in parentheses. (B) Binding of Alexa-488 labeled IND injection (0.4 mg). Scale bar: 125m m. (H) VSV-IND (middle row) or VSV-NJ (bottom row) to B VI10YEN B cell frequency at T/B borders and in fol- cells from C57BL/6 mice (left column) or VI10YEN licles 6 hours after VSV-IND injection at indicated mice (right column). The upper row shows control doses. Means 6 SEM; n = 3-4 follicles/2 mice; *: p staining with the anti-idiotypic antibody 35.61 to the 5 < 0.05; **: p < 0.01; ***: p < 0.001 (t-test). VI10YEN BCR (Dang and Rock, 1991, J. Immunol., Figure 21: VI10YEN B cell motility in draining LNs 146:3273). (C) Intracellular calcium flux in CD43 neg following virus injection. Median 3D instantaneous purified, Fluo-LOJO loaded B cells from VI10YEN velocities of wildtype (triangles) and VI10YEN B cells mice (upper row) or C57BL/6 mice (lower row). (circles) in deep follicles and the SCS/superficial fol- Events were collected continuously over time, aster- 10 licle about 5-35 min after VSV footpad injection. Hor- isks indicate the timepoint when antibodies or virus izontal bars represent means; *: p < 0.05; **: p < 0.01 were added. Virus particles were used at 1000/B cell, (one-way ANOVA with Bonferroni’s post test). Note 6 anti-IgM-(Fab)2 at 10 mg/10 B cells. (D) Neutraliza- that specific B cells slow down throughout the entire tion assay for total Ig and IgG in serum of C57BL/6 follicle, likely as a consequence of free VSV-G in our mice 4 and 10 days after immunization by footpad 15 preparation (see Figure 18). Control experiments injection of 10 mg UV-VSV or UV-VSV-AlexaFluor- showed similar B cell motility parameters in CLL- 488-IND. (E) Calcium flux in VI10YEN B cells ex- treated and nontreated popLNs. posed to supernatant from VSV stocks. Supernatant Figure22 : Timecourse of activation marker induction was generated by ultracentrifugation through a su- on VI10YEN B cells in virus-draining and non-drain- crose cushion resulting in approximately 10,000-fold 20 ing LNs following injection of VSV-IND. VI10YEN B reduction in viral titers and was used on B cells either cells were fluorescently tagged with CMTMR and undiluted(top right) or at1:100 dilution (bottom right). transferred to naive mice that were injected 18 hours As a control, VSV stock solution was diluted to equiv- later with 20 mg UV-inactivated VSV-IND (time 0 alent viral titers (MOI; left panels). The results dem- hours). The draining popliteal LN (popLN) and a dis- onstrate the presence of antigenic VSV-G that is not 25 tal brachial LN (brachLN) were harvested after the associated with virus particles in our virus prepara- indicated time intervals to generate single-cell sus- tion. pensions. CD69 and CD86 expression on B cells Figure 19: VSV-induced adhesion of VI10YEN B was assessed by flow cytometry after gating on (A) cells to ICAM-1 and VCAM-1. (A,B) Adhesion of pu- B220+CMTMR+VI10YEN cells or (B) B220+CMT- rified naive and VSV-IND activated (30 minute ex- 30 MR- endogenous control B cells. posure) VI10YEN B cells to plastic plates coated with Figure 23: Confocal (left and middle columns) and the indicated concentrations of recombinant ICAM- MP-IVM micrographs (right column) of popliteal LNs 1-Fc (A) or VCAM-1-Fc (B). Pooled data of two trip- of mice that had received adoptive transfers of a mix- licate experiments are shown. Horizontal bars rep- tureof CMTMR-labeled VI10YENB cells and CMAC- resentmeans. (C, D)Confocal micrographs ofICAM- 35 labeled polyclonal B cells (in the right column). On 1 and VCAM-1 expression in popliteal LNs of the following day, 20 mg UV-inactivated VSV-IND C57BL/6 mice. Scale bars: 50 mm. (E) Adhesion of was injected in a footpad and the draining popliteal purifiednaive wildtype and VI10YENB cells toplastic LNs were either surgically prepared for MP-IVM or dishes coated with the indicated pfu-equivalent con- harvested for confocal analysis of frozen sections at centrations of UV-inactivated VSV-IND. Data repre- 40 the indicated time points. MP-IVM images show that sent means 6 SEM of triplicates. VSV-specific,but notpolyclonal B cells made contact Figure 20: SCS macrophages are required for early with VSV in the SCS as early as 30 minutes after activation of VSV-specific B cells in LNs. (A) Confo- virus injection. VI10YEN B cells relocated to the T/B cal micrograph shows MHC-II colocalization with border at 6 hours following injection. Scale bars: 150 VSV-IND (30 minutes after injection)45 inmmin theleft column and 25 mmin theother columns. VI10YENxMHCII(EGFP) B cells at the SCS (arrow- Figure 24: In vivo targeting of SCS-Mph using Fc head), not the deep follicle (asterisk). Scale bar: 25 fragments from human IgG. (A) The FACS histo- mm. (B) Distance of VSV-associated and VSV-free grams on the left document the binding of fluorescent VI10YENxMHCII(EGFP) B cells to the SCS. Hori- PEG-PLGA nanoparticles ∼ (100 nm diameter) to zontal lines: medians. (C) BCR expression kinetics 50 lymph node macrophages. (B) Fc-nanoparticle (NP) on VI10YEN and (D) polyclonal B cells after VSV- targets SCS-Mph and follicular dendritic cells. IND footpad injection. (E) BCR expression on Figure 25: Identification of the chemokine receptor VI10YEN cells in CLL-treated or untreated popliteal CX3CR1 (fractalkine receptor) on macrophages in LNs after VSV-IND injection (20 mg). Mean fluores- lymph nodesubcapsular sinus(SCS), but notin mac- cence intensities were normalized to virus-free val- 55 rophages in the medulla. The micrograph on the right ues (dashed line). Means6 SEM (3-5 mice). (F) is a 3D projection of a lymph node from a double- Confocal micrograph of VI10YEN B cells in control knockin mouse where green fluorescent protein and (G) CLL-treated popliteal LNs 6 hours after VSV- (GFP) is expressed in the CX3CR1 locus, while red

17 31 EP 2 630 966 B1 32

fluorescent protein (RFP) reports the expression of altering effects, and, therefore, includes inhalants and another chemokine receptor, CCR2. SCS-Mph are solvents. In other embodiments, the abused substance readily identifiable by their prominent green fluores- is one that has no mood-altering effects or intoxication cence, while medullary macrophages express pri- properties, and, therefore, includes anabolic steroids. marily RFP. 5 Abused substances include, but are not limited to, can- Figure 26: SCS-Mph express the chemokine recep- nabinoids (e.g., hashish, marijuana), depressants (e.g., tor CX3CR1. The graph shows a FACS plot of a sin- barbituates, benodiazepines, flunitrazepam (Rohypnol), gle cell suspension from a lymph node of a knockin GHB, methaqualone (quaaludes)), dissociative anes- mouse that was genetically engineered to express thetics (e.g., ketamine, PCP), hallucinogens (e.g, LSD, GFP from the CX3CR1 locus. SCS-Mphs are iden- 10 mescaline, psilocybin), opioids and morphine derivatives tified by staining with a soluble receptor, CRFc, (e.g., codeine, fentanyl, heroin, morphine, opium), stim- which binds macrophages in the SCS, but not the ulants (amphetamine, cocaine, Ecstacy (MDMA), meth- medulla. The CRFc negative CX3CR1-expressing amphetamine, methylphenidate (Ritalin), nicotine), ana- (i.e., GFP-high) cells are conventional dendritic cells bolic steroids, and inhalants. In some embodiments, the that express this chemokine receptor. 15 abused substance for inclusion in a nanocarrier is the Figure 27: Fluorescent micrographs of frozen sec- complete molecule or a portion thereof. tions from mouse popliteal lymph nodes 24h after [0109] Addictive substance: As used herein, the term footpad injection of 0.2 mm diameter Latex beads "addictive substance" is a substance that causes obses- surface modified with either amine (left and middle sion, compulsion, or physical dependence or psycholog- panel) or carboxy moieties (right panel). Both sets 20 ical dependence. In some embodiments, the addictive of beads were purchased from Invitrogen (Cat. no. substance is an illegal drug. In other embodiment, the F8763 and F8805). Sections on left and right were addictive substance is an over-the-counter drug. In still counterstained with anti-CD169. Images are orient- other embodiments, the addictive substance is a pre- ed so that the medulla (weak, diffuse staining with scription drug. Addictive substances include, but are not anti-CD169) faces to the right and the subcapsular 25 limited to, cocaine, heroin, marijuana, methampheta- sinus (SCS) region (bright anti-CD169) faces to the mines, and nicotine. In some embodiments, the addictive left. Note that the red amine modified particles prom- substance for inclusion in a nanocarrier is the complete inently localize to the SCS, while blue carboxy mod- molecule or a portion thereof. ified beads are primarily retained in the medulla. [0110] Amino acid: As used herein, term "amino acid," Figure 28: (A) Antigen-bearing targeted nanoparti- 30 in its broadest sense, refers to any compound and/or cles are highly immunogenic and induce high anti- substance that can be incorporated into a polypeptide body titers. (B) The induced immune response elic- chain. In some embodiments, an amino acid has the gen- ited by nanoparticle vaccines confers potent protec- eral structure H2N-C(H)(R)-COOH. In some embodi- tion from a lethal dose of VSV. ments, an amino acid is a naturally-occurring amino acid. Figure 29: In vivo T cell activation by immunomod- 35 Insome embodiments, an amino acidis a synthetic amino ulatory nanoparticles. (A) Effect of NPs on CD4 T acid; in some embodiments, an amino acid is a D-amino cell activation. (B) Effect of NPs on CD8 T cell re- acid; in some embodiments, an amino acid is an L-amino sponse mixed with CpG adjuvant (TLR9 agonist). acid. "Standard amino acid" or "natural amino acid" refers (C) Effect of co-encapsulated adjuvant on CD8 T cell to any of the twenty standard L-amino acids commonly activation. 40 found in naturally occurring peptides. "Nonstandard ami- Figure 30: Shows an exemplary nicotine conjugation no acid" refers to any amino acid, other than the standard strategy. amino acids, regardless of whether it is prepared syn- Figure 31: Shows an exemplary R848 conjugation theticallyor obtained from a natural source. As used here- strategy. in, "non-natural amino acid" encompasses chemically 45 produced or modified amino acids, including but not lim- Definitions ited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- [0108] Abused substance: As used herein, the term and/or amino-terminal amino acids in peptides, can be "abused substance" is any substance taken by a subject modified by methylation, amidation, acetylation, and/or (e.g., a human) for purposes other than those for which 50 substitution with other chemical groups that can change it is indicated or in a manner or in quantities other than the peptide’s circulating half-life without adversely affect- directed by a physician. In some embodiments, the ing their activity. Amino acids may participate in a di- abused substance is a drug, such as an illegal drug. In sulfide bond. The term "amino acid" is used interchange- certain embodiments, the abused substance is an over- ably with "amino acid residue," and may refer to a free the-counter drug. In some embodiments, the abused55 amino acid and/or to an amino acid residue of a peptide. substance is a prescription drug. The abused substance, It will be apparent from the context in which the term is in some embodiments, is an addictive substance. In used whether it refers to a free amino acid or a residue some embodiments, the abused substance has mood- of a peptide.

18 33 EP 2 630 966 B1 34

[0111] Animal: As used herein, the term "animal" refers which are linked by a direct or indirect covalent or non- to any member of the animal kingdom. In some embod- covalent interaction. In some embodiments, an associa- iments, "animal" refers to humans, at any stage of devel- tion is covalent. In some embodiments, a covalent asso- opment. In some embodiments, "animal" refers to non- ciation is mediated by a linker moiety. In some embodi- human animals, at any stage of development. In certain 5 ments, an association is non-covalent (e.g., charge in- embodiments, the non-human animal is a mammal (e.g., teractions, affinity interactions, metal coordination, phys- a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a ical adsorption, host-guest interactions, hydrophobic in- cat, a sheep, cattle, a primate, and/or a pig). In some teractions, TT stacking interactions, hydrogen bonding embodiments, animals include, but are not limited to, interactions, van der Waals interactions, magnetic inter- mammals, birds, reptiles, amphibians, fish, and/or10 actions, electrostatic interactions, dipole-dipole interac- worms. In some embodiments, an animal may be a trans- tions, etc.). For example, in some embodiments, an entity genic animal, genetically-engineered animal, and/or a (e.g., immunomodulatory agent, targeting moiety, immu- clone. nostimulatory agent, nanoparticle, etc.) may be covalent- [0112] Antibody: As used herein, the term "antibody" ly associated with a vaccine nanocarrier. In some em- refers to any immunoglobulin, whether natural or wholly 15 bodiments, an entity ( e.g., immunomodulatory agent, tar- or partially synthetically produced. All derivatives thereof geting moiety, immunostimulatory agent, nanoparticle, which maintain specific binding ability are also included etc.) may be non-covalently associated with a vaccine in the term. The term also covers any protein having a nanocarrier. For example, the entity may be associated binding domain which is homologous or largely homolo- with the surface of, encapsulated within, surrounded by, gous to an immunoglobulin binding domain. Such pro- 20 and/or distributed throughout a lipid bilayer, lipid monol- teins may be derived from natural sources, or partly or ayer, polymeric matrix, etc. of an inventive vaccine na- wholly synthetically produced. An antibody may be mon- nocarrier. oclonal or polyclonal. An antibody may be a member of [0115] Biocompatible: As used herein, the term "bio- any immunoglobulin class, including any of the human compatible" refers to substances that are not toxic to classes: IgG, IgM, IgA, IgD, and IgE. As used herein, the 25 cells. In some embodiments, a substance is considered terms "antibody fragment" or "characteristic portion of an to be "biocompatible" if its addition to cells in vivo does antibody" are used interchangeably and refer to any de- not induce inflammation and/or other adverse effects in rivative of an antibody which is less than full-length. An vivo. In some embodiments, a substance is considered antibody fragment can retain at least a significant portion to be "biocompatible" if its addition to cells in vitro or in of the full-length antibody’s specific binding ability. Ex- 30 vivo results in less than or equal to about 50%, about amples of such antibody fragments include, but are not 45%, about 40%, about 35%, about 30%, about 25%, limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv diabody, about 20%, about 15%, about 10%, about 5%, or less and Fd fragments. Antibody fragment also include Fc than about 5% cell death. fragments. An antibody fragment may be produced by [0116] Biodegradable: As used herein, the term "bio- any means. For example, an antibody fragment may be 35 degradable" refers to substances that are degraded un- enzymatically or chemically produced by fragmentation der physiological conditions. In some embodiments, a of an intact antibody and/or it may be recombinantly pro- biodegradable substance is a substance that is broken duced from a gene encoding the partial antibody se- down by cellular machinery. In some embodiments, a quence. Alternatively or additionally, an antibody frag- biodegradable substance is a substance that is broken ment may be wholly or partially synthetically produced. 40 down by chemical processes. An antibody fragment may optionally comprise a single [0117] B cell antigen: As used herein, the term "B cell chain antibody fragment. Alternatively or additionally, an antigen" refers to any antigen that is recognized by and antibody fragment may comprise multiple chains which triggers an immune response in a B cell. In some em- are linked together, for example, by disulfide linkages. bodiments, an antigen that is a B cell antigen is also a T An antibody fragment may optionally comprise a multi- 45 cell antigen. In certain embodiments, the B cell antigen molecular complex. A functional antibody fragment will is not also a T cell antigen. In certain embodiments, when typicallycomprise atleast about 50amino acidsand more a nanocarrier, as provided herein, comprises both a B typically will comprise at least about 200 amino acids. cell antigen and a T cell antigen, the B cell antigen and [0113] Approximately: As used herein, the terms "ap- T cell antigen are not the same antigen, although each proximately" or "about" in reference to a number are gen- 50 of the B cell and T cell antigens may be, in some embod- erally taken to include numbers that fall within a range of iments, both a B cell antigen and a T cell antigen. In other 5%, 10%, 15%, or 20% in either direction (greater than embodiments, the B cell antigen and T cell antigen of the or less than) of the number unless otherwise stated or nanocarrier are the same. otherwise evident from the context (except where such [0118] Cell type: As used herein, the term "cell type" number would be less than 0% or exceed 100% of a55 refers to a form of cell having a distinct set of morpho- possible value). logical, biochemical, and/or functional characteristics [0114] Associated with: As used herein, the term "as- that define the cell type. One of skill in the art will recog- sociated with" refers to the state of two or more entities nize that a cell type can be defined with varying levels of

19 35 EP 2 630 966 B1 36 specificity. For example, T cells and B cells are distinct agent. In some embodiments, the more than one type of cell types, which can be distinguished from one another immunostimulatory agent act on different pathways. Ex- but share certain features that are characteristic of the amples of immunostimulatory agents include those pro- broader "lymphocyte" cell type of which both are mem- vided elsewhere herein. bers. Typically, cells of different types may be distin-5 [0123] 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 10 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 residuese.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 15 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" [0119] Hazardous environmental agent : As used here- encompasses RNA as well as single and/or double- in, the term "hazardous environmental agent" refers to stranded DNA and/or cDNA. Furthermore, the terms "nu- any hazardous substance found in the environment.20 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 they "peptide nucleic acids," which are known in the art and may not actually pose a risk. Hazardous environmental have peptide bonds instead of phosphodiester bonds in agents include, but are not limited to, arsenic, lead, mer- 25 the backbone, are considered within the scope of the cury, vinyl chloride, polychlorinated biphenyls, benzene, present invention. The term "nucleotide sequence en- polycyclic aromatic hydrocarbons, cadmium, ben- coding an amino acid sequence" includes all nucleotide zo(a)pyrene, benzo(b)fluoranthene, chloroform, DDT, sequences that are degenerate versions of each other P,P’-, aroclor 1254, aroclor 1260, dibenzo(a,h)anthra- and/or encode the same amino acid sequence. Nucle- cene, trichloroethylene, dieldrin, chromium hexavalent, 30 otide sequences that encode proteins and/or RNA may and DDE, P,P’. In some embodiments, the hazardous include introns. Nucleic acids can be purified from natural environmental agent for inclusion in a nanocarrier is the sources, produced using recombinant expression sys- complete molecule or a portion thereof. tems and optionally purified, chemically synthesized, etc. [0120] In vitro: As used herein, the term "in vitro" refers Where appropriate, e.g., in the case of chemically syn- to events that occur in an artificial environment, e.g., in 35 thesized molecules, nucleic acids can comprise nucleo- a test tube or reaction vessel, in cell culture, etc., rather side analogs suchas analogs having chemicallymodified than within an organism (e.g., animal, plant, and/or mi- bases or sugars, backbone modifications, etc. A nucleic crobe). acid sequence is presented in the 5’ to 3’ direction unless [0121] In vivo: As used herein, the term "in vivo" refers otherwise indicated. The term "nucleic acid segment" is to events that occur within an organism e.g( ., animal, 40 used herein to refer to a nucleic acid sequence that is a plant, and/or microbe). portion of a longer nucleic acid sequence. In many em- [0122] Immunostimulatory agent: As used herein, the bodiments, a nucleic acid segment comprises at least 3, term "immunostimulatory agent" refers to an agent that 4, 5, 6, 7, 8, 9, 10, or more residues. In some embodi- modulates an immune response to an antigen but is not ments, a nucleic acid is or comprises natural nucleosides the antigen or derived from the antigen. "Modulate", as 45 (e.g., adenosine, thymidine, guanosine, cytidine, uridine, used herein, refers to inducing, enhancing, directing, or deoxyadenosine,deoxythymidine, deoxyguanosine, and redirecting an immune response. Such agents include deoxycytidine); nucleoside analogs (e.g., 2-aminoade- immunostimulatory agents that stimulate (or boost) an nosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3- immune response to an antigen but, as defined above, methyl adenosine, 5-methylcytidine, C-5 propynyl-cyti- is not the antigen or derived from the antigen. Immunos- 50 dine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bro- timulatory agents, therefore, include adjuvants. In some mouridine, C5-fluorouridine, C5-iodouridine, C5-propy- embodiments, the immunostimulatory agent is on the nyl-uridine, C5-propynylcytidine, C5-methylcytidine, 2- surface of the nanocarrier and is encapsulated within the aminoadenosine, 7-deazaadenosine, 7-deazaguanos- nanocarrier. In some embodiments, the immunostimula- ine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylgua- tory agent on the surface of the nanocarrier is different 55 nine, and 2-thiocytidine); chemically modified bases; bi- from the immunostimulatory agent encapsulated within ologically modified bases ( e.g., methylated bases); inter- the nanocarrier. In some embodiments, the nanocarrier calated bases; modified sugars (e.g., 2’-fluororibose, ri- comprises more than one type of immunostimulatory bose, 2’-deoxyribose, arabinose, and hexose); and/or

20 37 EP 2 630 966 B1 38 modified phosphate groups (e.g., phosphorothioates and desired immune response. "Sufficient", as used herein, 5’-N-phosphoramidite linkages). refers to the ability to elicit a detectable or protective im- [0124] Particle: As used herein, a "particle" refers to mune response when administered in a composition that any entity havinga diameter ofless than 10 microns ( mm). does not employ a nanocarrier described herein, e.g., as Typically, particles have a longest dimension ( e.g., diam- 5 free antigen mixed with adjuvant in the absence of a na- eter) of 1000 nm or less. In some embodiments, particles nocarrier. In some embodiments, the desired immune have a diameter of 300 nm or less. Particles include mi- response is to treat or prevent a disease or condition. In croparticles, nanoparticles, and picoparticles. In some certain embodiments, the desired immune response is embodiments, nanoparticles have a diameter of 200 nm to alleviate one or more symptoms of a disease or con- or less. In some embodiments, nanoparticles have a di- 10 dition. Poorly immunogenic antigens include, but are not ameter of 100 nm or less. In some embodiments, nano- limited to, self antigens, small molecules, and carbohy- particles have a diameter of 50 nm or less. In some em- drates. bodiments, nanoparticles have a diameter of 30 nm or [0126] Self antigen: As used herein, the term "self an- less. In some embodiments, nanoparticles have a diam- tigen" refers to a normal substance in the body of an eter of 20 nm or less. In some embodiments, nanoparti- 15 animal that when an immune response against the anti- cles have a diameter of 10 nm or less. In some embod- gen within the animal is triggered, autoimmunity (e.g., an iments, particles can be a matrix of polymers. In some autoimmune disease) can result. A self antigen can be embodiments, particles can be a non-polymeric particle a protein or peptide, lipoprotein, lipid, carbohydrate, or a (e.g., a metal particle, quantum dot, ceramic, inorganic nucleic acid. The nucleic acid can be a DNA or RNA. Self material, bone, etc.). Particles may also be liposomes 20 antigens include, but are not limited to enzymes, struc- and/or micelles. As used herein, the term "nanoparticle" tural proteins, secreted proteins, cell surface receptors, refers to any particle having a diameter of less than 1000 and cytokines. In some embodiments, the self antigen is nm. The nanocarriers have mean geometric diameter a cytokine, and the cytokine is TNF, IL-1, or IL-6. In some that is greater than 50 nm but less than 500 nm. In some embodiments, the self antigen is cholesteryl ester trans- embodiments, the mean geometric diameter of a popu- 25 fer protein (CETP), a serum protein responsible for cho- lation of nanocarriers is about 60 nm, 75 nm, 100 nm, lesterol transfer from high-density lipoprotein (HDL) to 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 low-density lipoprotein cholesterol (LDL), the Aβ protein nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, associated with Alzheimer’s, a proteolytic enzyme that 450 nm, or 475 nm. In some embodiments, the mean processes the pathological form of the Aβ protein, LDL geometric diameter isbetween 100-400 nm,100-300 nm, 30 associated with atherosclerosis, or a coreceptor for HIV- 100-250 nm, or 100-200 nm. In some embodiments, the 1. In some embodiments, the proteolytic enzyme that mean geometric diameter is between 60-400 nm, 60-350 processes the pathological form of the A β protein is beta- nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some em- secretase. In some embodiments, the LDL associated bodiments, the mean geometric diameter is between with atherosclerosis is oxidized or minimally modified. In 75-250 nm. In some embodiments, 30%, 40%, 50%,35 some embodiments, the coreceptor for HIV-1 is CCR5. 60%, 70%, 80%, 90%, or more of the nanocarriers of a [0127] Small molecule: In general, a "small molecule" population of nanocarriers have a diameter that is less is understood in the art to be an organic molecule that is than 500 nm. In some embodiments, 10%, 20%, 30%, less than about 2000 g/mol in size. In some embodi- 40%, 50%, 60%, 70%, 80%, 90%, or more of the nano- ments, the small molecule is less than about 1500 g/mol carriers of a population of nanocarriers have a diameter 40 or less than about 1000 g/mol. In some embodiments, that is greater than 50 nm but less than 500 nm. In some the small molecule is less than about 800 g/mol or less embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, than about 500 g/mol. In some embodiments, small mol- 80%, 90%, or more of the nanocarriers of a population ecules are non-polymeric and/or non-oligomeric. In some of nanocarriers have a diameter of about 60 nm, 75 nm, embodiments, small molecules are not proteins, pep- 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 45 tides, or amino acids. In some embodiments, small mol- nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, ecules are not nucleic acids or nucleotides. In some em- 425 nm, 450 nm, or 475 nm. In some embodiments, 10%, bodiments, small molecules are not saccharides or 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more polysaccharides. of the nanocarriers of a population of nanocarriers have [0128] Specific binding: As used herein, the term "spe- a diameter that is between 100-400 nm, 100-300 nm, 50 cific binding" refers to non-covalent physical association 100-250 nm, or 100-200 nm. In some embodiments, of a first and a second moiety wherein the association 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or between the first and second moieties is at least 2 times more of the nanocarriers of a population of nanocarriers as strong, at least 5 times as strong as, at least 10 times have a diameter that is between 60-400 nm, 60-350 nm, as strong as, at least 50 times as strong as, at least 100 60-300 nm, 60-250 nm, or 60-200 nm. 55 times as strong as, or stronger than the association of [0125] Poorly immunogenic antigen: As used herein, either moiety with most or all other moieties present in the term "poorly immunogenic antigen" refers to an an- the environment in which binding occurs. Binding of two tigen that does not trigger any or a sufficient level of a or more entities may be considered specific if the equi-

21 39 EP 2 630 966 B1 40

-3 -4 librium dissociation constant, Kd, is 10 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, 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 or is susceptible to tuberculosis, malaria, leishmaniasis, 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 5 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- 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- [0132] T cell antigen: As used herein, the term "T cell -3 10 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 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- 15 (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- 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 [0129] Subject: As used herein, the term "subject" or 20 response, a CD4+ T cell response, or both. The nano- "patient" refers to any organism to which a composition carriers, therefore, in some embodiments can effectively of this invention may be administered, e.g., for experi- stimulate both types of responses. mental, diagnostic, and/or therapeutic purposes. Typical [0133] Target: As used herein, the term "target" or subjects include animals (e.g., mammals such as mice, "marker" refers to any entity that is capable of specifically rats, rabbits, non-human primates, and humans) and/or 25 binding to a particular targeting moiety. In some embod- plants. iments, targets are specifically associated with one or [0130] Suffering from: An individual who is "suffering more particular tissue types. In some embodiments, tar- from" a disease, disorder, and/or condition has been di- gets are specifically associated with one or more partic- agnosed with, can be diagnosed with, or displays one or ular cell types. For example, a cell type specific marker more symptoms of the disease, disorder, and/or condi- 30 is typically expressed at levels at least 2 fold greater in tion. that cell type than in a reference population of cells. In [0131] Susceptible to: Anindividual who is "susceptible some embodiments, the cell type specific marker is to" a disease, disorder, and/or condition has not been present at levels at least 3 fold, at least 4 fold, at least 5 diagnosed with and/or may not exhibit symptoms of the fold, at least 6 fold, at least 7 fold, at least 8 fold, at least disease, disorder, and/or condition. In some embodi-35 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, ments, a disease, disorder, and/or condition is associat- or at least 1000 fold greater than its average expression ed with a microbial infection ( e.g., bacterial infection, viral in a reference population. Detection or measurement of infection, fungal infection, parasitic infection,etc. ). In a cell type specific marker may make it possible to dis- some embodiments, an individual who is susceptible to tinguish the cell type or types of interest from cells of a microbial infection may be exposed to a microbe (e.g., 40 many, most, or all other types. In some embodiments, a by ingestion, inhalation, physical contact, etc.). In some target can comprise a protein, a carbohydrate, a lipid, embodiments, an individual who is susceptible to a mi- and/or a nucleic acid, as described herein. crobial infection may be exposed to an individual who is [0134] Targeted: A substance is considered to be "tar- infected with the microbe. In some embodiments, an in- geted" for the purposes described herein if it specifically dividual who is susceptible to a microbial infection is one 45 binds to a target. In some embodiments, a targeting moi- who is in a location where the microbe is prevalent (e.g., ety specifically binds to a target under stringent condi- one who is traveling to a location where the microbe is tions. An inventive nanocarrier, such as a vaccine nano- prevalent). In some embodiments, an individual who is carrier, comprising a targeting moiety is considered to be susceptible to a disease, disorder, and/or condition will "targeted" if the targeting moiety specifically binds to a develop the disease, disorder, and/or condition. In some 50 target, thereby delivering the entire nanocarrier to a spe- embodiments, an individual who is susceptible to a dis- cific organ, tissue, cell, and/or subcellular locale. ease, disorder, and/or condition will not develop the dis- [0135] Targeting moiety: As used herein, the term "tar- ease, disorder, and/or condition. In some embodiments, geting moiety" refers to any moiety that binds to a com- the subject has or is susceptible to having cancer, an ponent of a cell. In some embodiments, the targeting moi- infectious disease, a non-autoimmune metabolic or de- 55 ety specifically binds to a component of a cell. Such a generative disease, or an addiction. In some embodi- component is referred to as a "target" or a "marker." A ments, the subject has or is susceptible to having a bac- targeting moiety may be a polypeptide, glycoprotein, nu- terial, fungal, protozoan, parisitic, or viral infection. The cleic acid, small molecule, carbohydrate, lipid,etc. In

22 41 EP 2 630 966 B1 42 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 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- 5 [0138] 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 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- 10 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 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 15 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 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- 20 subject. ment protein or a pre-existing antibody. In further em- [0139] Universal T cell antigen: As used herein, the bodiments, the targeting moiety is for delivery of the na- term "universal T cell antigen" refers to a T cell antigen nocarrier to antigen presenting cells, T cells, or B cells. that can promote T cell help and enhance an immune In some embodiments, the antigen presenting cells are response to a completely unrelated antigen. Universal T macrophages. In other embodiments, the macrophages 25 cell antigens include tetanus toxoid, as well as one or are subcapsular sinus macrophages. In still other em- more peptides derived from tetanus toxoid, Epstein-Barr bodiments, the antigen presenting cells are dendritic virus, or influenza virus. Universal T cell antigens also cells. In some embodiments, the antigen presenting cells include a component of influenza virus, such as hemag- are follicular dendritic cells. Specific non-limiting exam- glutinin, neuraminidase, or nuclear protein, or one or ples of targeting moieties include molecules that bind to 30 more peptides derived therefrom. CD11b, CD169, mannose receptor, DEC-205, CD11c, [0140] Vaccine Nanocarrier: As used herein, the term CD21/CD35, CX3CR1, or an Fc receptor. In some em- "vaccine nanocarrier" refers to an entity comprising at bodiments, the molecule that binds any of the foregoing least one immunomodulatory agent or immunostimula- is an antibody or antigen-binding fragment thereof (e.g., tory agent. In certain embodiments, a vaccine nanocar- ananti-CD169 antibody).In some embodiments,the mol- 35 rier includes at least two types of immunomodulatory ecule that binds an Fc receptor is one that comprises the agents. In some embodiments, the immunomodulatory Fc portion of an immunoglobulin (e.g., IgG). In other em- agents are antigens, and the vaccine nanocarrier com- bodiments, the Fc portion of an immunoglobulin is a hu- prises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens. The man Fc portion. In some embodiments, the molecule that different antigens can be or be derived from completely binds CX3CR1 is CX3CL1 (fractalkine). Targeting moi- 40 different antigenic molecules, or the different antigens eties that bind CD169 include anti-CD169 antibodies and can be different epitopes from the same antigenic mole- ligands of CD169, e.g., sialylated CD227, CD43, CD206, cule. In other embodiments, the vaccine nanocarrier or portions of these ligands that retain binding function, comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different e.g., soluble portions. epitopes from the same antigenic molecule. A vaccine [0136] Therapeutically effective amount: As used 45 nanocarrier, in some embodiments, is capable of stimu- herein, the term "therapeutically effective amount" lating an immune response in T cells and/or B cells. In means an amount of a therapeutic, prophylactic, and/or other embodiments, the vaccine nanocarrier is capable diagnostic agent e.g ( ., inventive vaccine nanocarrier) of enhancing, directing, or redirecting an immune re- that is sufficient, when administered to a subject suffering sponse. In some embodiments, any assay available in from or susceptible to a disease, disorder, and/or condi- 50 the art may be used to determine whether T cells and/or tion, to treat, alleviate, ameliorate, relieve, alleviate B cells have been stimulated. In some embodiments, T symptoms of, prevent, delay onset of, inhibit progression cell stimulation may be assayed by monitoring antigen- of, reduce severity of, and/or reduce incidence of the dis- induced production of cytokines, antigen-induced prolif- ease, disorder, and/or condition. The term is also intend- eration of T cells, and/or antigen-induced changes in pro- ed to refer to an amount of nanocarrier or composition 55 tein expression. In some embodiments, B cell stimulation thereof provided herein that modulates an immune re- may be assayed by monitoring antibody titers, antibody sponse in a subject. affinities, antibody performance in neutralization assays, [0137] Therapeutic agent: As used herein, the term class-switch recombination, affinity maturation of anti-

23 43 EP 2 630 966 B1 44 gen-specific antibodies, development of memory B cells, face carbohydrates or recombinant pathogen-derived development of long-lived plasma cells that can produce proteins that are sometimes fused to other molecules, large amounts of high-affinity antibodies for extended pe- particularly proteins that can confer adjuvant activity. riods of time, germinal center reactions, and/or antibody [0145] Vaccines used for intramuscular injections are performance in neutralization assays. In some embodi- 5 typically administered with an adjuvant carrier, most fre- ments, a vaccine nanocarrier further comprises at least quently alum (i.e., aluminum potassium sulphate), that one targeting moiety that can help deliver the vaccine is thought to establish a depot for prolonged release of nanocarrier to a particular target (e.g., organ, tissue, cell, antigenic material, but also exerts immunomodulatory and/or subcellular locale) within a subject. In some em- activities, such as skewing toward Th2 responses by bodiments, a vaccine nanocarrier further comprises at 10 mechanismsthat are incompletelyunderstood (Lindblad, least one immunostimulatory agent that can help stimu- 2004, Immunol. Cell. Biol., 82:497; and Jordan et al., late an immune response in T cells and/or B cells. In 2004, Science, 304:1808). some embodiments, vaccine nanocarriers comprise lip- [0146] Vaccines that utilize live attenuated or inactivat- ids, amphiphilic compounds, polymers, sugars, polymer- ed pathogens typically yield a vigorous immune re- ic matrices, and/or non-polymeric particles. 15 sponse, but their use has limitations. For example, live [0141] Water soluble, non-adhesive polymer : As used vaccine strains can sometimes cause infectious pathol- herein, the term "water soluble, non-adhesive polymer" ogies, especially when administered to immune-compro- refers to a polymer that is soluble in water and that can mised recipients. Moreover, many pathogens, particular- confer reduced biofouling properties. Water soluble, non- ly viruses, undergo continuous rapid mutations in their adhesive polymers include polyethylene glycol, polyeth- 20 genome, which allow them to escape immune responses ylene oxide, polyalkylene glycol, and polyalkylene oxide. to antigenically distinct vaccine strains. However, most or all pathogens are thought to possess certain antigenic Detailed Description of Certain Preferred Embodi- determinants that are not easily mutated because they ments of the Invention are associated with essential functions. Antibodies di- 25 rected against these conserved epitopes, rather than Vaccines more variable, non-essential epitopes can protect against highly mutable viruses (Baba et al., 2000, Nat. [0142] Vaccinations are typically either passive or ac- Med., 6:200). Vaccines based on live or killed intact path- tive in nature. In general, active vaccinations involve the ogens do not necessarily promote the recognition of exposure of a subject’s immune system to one or more 30 these critical epitopes, but may essentially "distract" the agents that are recognized as unwanted, undesired, immune system to focus its assault on highly variable and/or foreign and elicit an endogenous immune re- determinants. Thus, the present invention encompasses sponse resulting in the activation of antigen-specific na- the recognition that an engineered vaccine nanocarrier ive lymphocytes that then give rise to antibody-secreting that mimics the highly immunogenic particulate nature of B cells or antigen-specific effector and memory T cells 35 viral particles, but presents selectively essential, immu- or both. This approach can result in long-lived protective table epitopes, could yield much more potent and "es- immunity that may be boosted from time to time by re- cape-proof" neutralizing antibody and effector T cell re- newed exposure to the same antigenic material. The sponses than intact microorganisms. prospect of longevity of a successful immune response [0147] The precise mechanisms by which vaccines to active vaccination makes this strategy more desirable 40 stimulate antibody responses in draining lymph nodes in most clinical settings than passive vaccination where- (or fail to do so) are still incompletely understood. B and by a recipient is injected with preformed antibodies or T cells are initially sequestered in distinct anatomic re- with antigen-specific effector lymphocytes, which may gions, the superficially located B follicles and the sur- confer rapid ad hoc protection, but typically do not estab- rounding paracortex and deep cortex, respectively. Upon lish persistent immunity. 45 antigen challenge, antigen-specific B cells in follicles as [0143] Alarge variety of vaccine formulationsare being well as CD4 T cells in the T cell area become activated or have been employed in humans. The most common and then migrate toward the border zone between the route of administration in humans is by intramuscular two compartments. B cells that have phagocytosed (i.m.) injection, but vaccines may also be applied orally, lymph-borne antigens process the acquired material and intranasally, subcutaneously, inhalationly, or intrave-50 begin to present antigenic peptides in MHC class-II sur- nously. In most cases, vaccine-derived antigens are in- face molecules that are then recognized by the activated itially presented to naive lymphocytes in regional lymph CD4 T cells (the TFH cells). Antigen-recognition allows nodes. the TFH cells to provide help to B cells, which constitutes [0144] Some current vaccines against, e.g., microbial a potent survival signal and triggers the formation of ger- pathogens, consist of live attenuated or non-virulent var- 55 minal centers (GCs) within B follicles. The GC reaction iant strains of microorganisms, or killed or otherwise in- promotes class-switch recombination, affinity maturation activated organisms. Other vaccines utilize more or less of antigen-specific antibodies, and the formation of mem- purified components of pathogen lysates, such as sur- ory B cells and long-lived plasma cells that can produce

24 45 EP 2 630 966 B1 46 large amounts of high-affinity antibodies for extended pe- T Cells riods of time. Thus, the present invention encompasses the recognition that a vaccine nanocarrier may have com- [0151] The present invention provides vaccine nano- ponents that allow antigenic material to be efficiently rec- carriers for delivery of, for example, immunomodulatory ognized by both B and T cells and to induce vigorous GC 5 agents to the cells of the immune system. In some em- reactions (Figure 1). bodiments, vaccine nanocarriers comprise at least one [0148] The present invention describes systems for immunomodulatory agent which can be delivered to developing vaccine nanocarriers for vaccine delivery that APCs, which then process and deliver the immunomod- can overcome these aforementioned limitations of cur- ulatory agent(s) to T cells. rent vaccine technology. The present invention encom- 10 [0152] Professional APCs are very efficient at internal- passes the recognition that lymph-borne viral particles izing antigen, either by phagocytosis or by endocytosis, that measure tens to hundreds of nanometers in diameter and then display a fragment of the antigen, bound to ei- and induce potent cellular and antibody responses are ther a class II major histocompatibility complex (class II captured and retained on the surface of macrophages in MHC) molecule or a class I MHC molecule on the APC the subcapsular sinus of draining lymph nodes ( i.e., sub- 15 membrane. CD4 T cells recognize and interact with the capsular sinus macrophages, abbreviated SCS-Mph). antigen-class II MHC molecule complex on the APC These macrophages are involved in the efficient early membrane, whereas CD8 T cells recognize and interact presentation of intact viral particles to follicular B cells. with the antigen-class I MHC molecule complex. An ad- In some embodiments, inventive nanocarriers mimic viral ditional co-stimulatory signal as well as modulating cy- particles and target SCS-Mph. As shown in Example 1, 20 tokines are then produced by the APC, leading to T cell upon subcutaneous injection of Cy5 encapsulated poly activation. (lactic-coglycolic acid) (PLGA) nanoparticles (50 nm - 150 nm) that are surface stabilized with a monolayer of Immunomodulatory Agents lipid and polyethylene glycol, the injected nanoparticles readily enter lymphatics and are bound in the subcapsu- 25 [0153] The present invention provides vaccine nano- lar sinus of draining lymph nodes similar to lymph-borne carriers comprising one or more immunomodulatory viruses. Similar nanocarriers carrying immunomodulato- agents. In some embodiments, inventive nanocarriers ry agent(s) that stimulate B cells and/or T cells are par- comprising one or more immunomodulatory agents are ticularly useful in vaccinating a subject. used as vaccines. In some embodiments, an immu- [0149] Thus, the present invention encompasses the 30 nomodulatory agent may comprise isolated and/or re- recognition that nanocarriers, such as lymph-borne virus- combinant proteins or peptides, carbohydrates, glyco- sized nanocarriers carrying an immunomodulatory agent proteins, glycopeptides, proteoglycans, inactivated or- can be recognized in lymph nodes as if they were viruses ganisms and viruses, dead organisms and virus, genet- and may elicit a potent immune response, for example, ically altered organisms or viruses, and cell extracts. In when the particles include immunomodulatory agent(s) 35 some embodiments, an immunomodulatory agent may that are recognized by B cells and/or T cells. comprise nucleic acids, carbohydrates, lipids, and/or [0150] By carrying immunomodulatory agents on the small molecules. In some embodiments, an immu- surface and/or loading similar or distinct immunomodu- nomodulatory agent is one that elicits an immune re- latoryagents inside, nanocarrierscan simultaneously de- sponse. In other embodiments, an immunomodulatory liver these immunomodulatory agents to distinct cells of 40 agent is a polynucleotide that encodes a protein or pep- the immune system and stimulate them. Immunomodu- tide that when the protein or peptide is expressed an latory agents presented on nanocarrier surfaces stimu- immune response is elicited. In some embodiments, an late B cells, and immunomodulatory agents encapsulat- immunomodulatory agent is an antigen. In some embod- ed within the nanocarriers are processed by antigen-pre- iments, an immunomodulatory agent is a protein or pep- senting cells (APCs), such as dendritic cells (DCs), in 45 tide. In some embodiments, an immunomodulatory agent lymphoid tissues (and by B cells after activation) and pre- is used for vaccines. sented to T cells. In some embodiments, by modifying [0154] In some embodiments, an immunomodulatory the surface of nanocarriers with a targeting moiety (e.g., agent is any protein and/or other antigen derived from a antibody or fragment thereof, peptide or polypeptide, Af- pathogen. The pathogen may be a virus, bacterium, fun- fibody®, Nanobody™, AdNectin™, Avimer™, aptamer, 50 gus, protozoan, parasite, etc. In some embodiments, im- Spiegelmer®, small molecule, lipid, carbohydrate, etc.), munomodulatory agents may include antigens of bacte- nanocarriers can selectively deliver immunomodulatory rial organisms such as Borrelia species, Bacillus anthra- agents to specific antigen presenting cells, such as DCs, cis, Borrelia burgdorferi, Bordetella pertussis, Camphy- SCS-Mph, FDCs, T Cells, B cells, and/or combinations lobacter jejuni, Chlamydia species, Chlamydial psittaci, thereof. Vaccine nanocarriers are described in further 55 Chlamydial trachomatis, Clostridium species, Clostrid- detail in the section entitled " Vaccine Nanocarriers." ium tetani, Clostridium botulinum, Clostridium perfrin- gens, Corynebacterium diphtheriae, Coxiella species, an Enterococcus species, Erlichia species, Escherichia coli,

25 47 EP 2 630 966 B1 48

Francisella tularensis, Haemophilus species, Haemo- some embodiments, immunomodulatory agents may in- philus influenzae, Haemophilus parainfluenzae, Lacto- clude PfEMPl of Plasmodium falciparum or other para- bacillus species, a Legionella species, Legionella pneu- site-derived antigens expressed on plasmodium-infect- mophila, Leptospirosis interrogans, Listeria species, Lis- ed red blood cells or GRA7 of Toxoplasma gondi. teria monocytogenes, Mycobacterium species, Myco- 5 [0158] Any of the antigens described herein may be in bacterium tuberculosis, Mycobacterium leprae, Myco- the form of whole killed organisms, peptides, proteins, plasma species, Mycoplasma pneumoniae, Neisseria glycoproteins, glycopeptides, proteoglycans, nucleic ac- species, Neisseria meningitidis, Neisseria gonorrhoeae, ids thatencode aprotein or peptide, carbohydrates, small Pneumococcus species, Pseudomonas species, Pseu- molecules, or combinations thereof. domonas aeruginosa, Salmonella species, Salmonella 10 [0159] In some embodiments, an immunomodulatory typhi, Salmonella enterica, Rickettsia species, Rickettsia agent is derived from a microorganism for which at least ricketsii, Rickettsia typhi, Shigella species, Staphyloco- one vaccine already exists. In some embodiments, an ccus species, Staphylococcus aureus, Streptococcus immunomodulatory agent is derived from a microorgan- species, Streptococccus pnuemoniae, Streptococcus ism for which no vaccines have been developed. pyrogenes, Streptococcus mutans, Treponema species, 15 [0160] In some embodiments, a vaccine nanocarrier Treponema pallidum, a Vibrio species, Vibrio cholerae, comprises at least one type of immunomodulatory agent. Yersinia pestis, and the like. In some embodiments, all of the immunomodulatory [0155] In some embodiments, immunomodulatory agents of a vaccine nanocarrier are identical to one an- agents may include antigens of viral organisms such as other. In some embodiments, a vaccine nanocarrier com- pox viruses, smallpox (variola), ebola virus, hepadnavi- 20 prises a number of different immunomodulatory agents. rus, marburg virus, dengue fever virus, influenza A and In some embodiments, a vaccine nanocarrier comprises B, parainfluenza, respiratory syncytial virus, measles (ru- multiple individual immunomodulatory agents, all of beola virus), human immunodeficiency virus (HIV), hu- which are the same. In some embodiments, a vaccine man papillomavirus (HPV), varicellazoster, herpes sim- nanocarrier comprises exactly one type of immunomod- plex 1 and 2, cytomegalovirus, Epstein-Barr virus, JC 25 ulatory agent. In some embodiments, a vaccine nano- virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, or- carrier comprises exactly two distinct types of immu- thomyxovirus, papillomavirus, parvovirus, picornavirus, nomodulatory agents. In some embodiments, a vaccine poliovirus, mumps, rabies, reovirus, rubella, togavirus, nanocarrier comprises greater than two distinct types of retrovirus, coxsackieviruses, equine encephalitis, Japa- immunomodulatory agents. In some embodiments, a nese encephalitis,yellow fever, Rift Valleyfever, hepatitis 30 vaccine nanocarrier comprises 3, 4, 5, 6, 7, 8, 9, 10, or A, B, C, D, and E virus, and the like. Viral organisms more distinct types of immunomodulatory agents. include those that are dsDNA viruses, ssDNA viruses, [0161] In some embodiments, a vaccine nanocarrier dsRNA viruses, (+) ssRNA viruses (-) sRNA viruses, ss- comprises two types of immunomodulatory agents which RNA-RT viruses, and dsDNA-RT viruses. are both derived from a single genus of microorganism. [0156] In some embodiments, immunomodulatory35 In some embodiments, a vaccine nanocarrier comprises agents may include antigens of fungal, protozoan, and/or two types of immunomodulatory agents which are both parasitic organisms such as Aspergillus species, Cand- derived from a single genus and species of microorgan- ida species, Candida albicans, Candida tropicalis, Cryp- ism. In some embodiments, a vaccine nanocarrier com- tococcus species, Cryptococcus neoformans, Entamoe- prises two types of immunomodulatory agents which are bahistolytica, Histoplasma capsulatum, Leishmania spe- 40 both derived from a single genus, species, and strain of ceis, Nocardia asteroides, Plasmodium falciparum, Tox- microorganism. In some embodiments, a vaccine nano- oplasma gondii, Trichomonas vaginalis, Toxoplasma carrier comprises two types of immunomodulatory species, Trypanosoma brucei, Schistosoma mansoni, agents which are both derived from a single clone of a and the like. microorganism. [0157] In some embodiments, immunomodulatory45 [0162] In some embodiments, a vaccine nanocarrier agents may include E1 and/or E2 proteins of HCV. In comprises more than two types of immunomodulatory some embodiments, immunomodulatory agents may in- agents which are all derived from a single genus of mi- clude gpl20 of HIV. In some embodiments, immunomod- croorganism. In some embodiments, a vaccine nanocar- ulatory agents may include hemagglutinin and/or neu- rier comprises more than two types of immunomodula- raminidase of influenza virus. In some embodiments, im- 50 tory agents which are all derived from a single genus and munomodulatory agents may include pneumococcal species of microorganism. In some embodiments, a vac- polysaccharide or family 1 and/or family 2 PspA of Strep- cine nanocarrier comprises more than two types of im- tococcus pneumoniaeor capsular polysaccharides types munomodulatory agents which are all derived from a sin- 5 and 8 or microbial surface components recognizing ad- gle genus, species, and strain of microorganism. In some hesive matrix molecule ofStapylococcus aureus. In 55 embodiments, a vaccine nanocarrier comprises more some embodiments, immunomodulatory agents may in- than two types of immunomodulatory agents which are clude mannan of Candida albicans or cryptococcal cap- all derived from a single clone of a microorganism. sular polysaccharide of Cryptococcus neoformans. In [0163] In some embodiments, a vaccine nanocarrier

26 49 EP 2 630 966 B1 50 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 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- 5 one immunomodulatory agent is encapsulated within the cies of microorganism. In some embodiments, a vaccine lipid membrane of a vaccine nanocarrier. In some em- nanocarrier comprises two or more types of immunomod- bodiments, at least one type of immunomodulatory agent ulatory agent which are all derived from a single genus, may be located at multiple locations of a vaccine nano- species, and strain of microorganism. carrier. For example, a first type of immunomodulatory [0164] In some embodiments, a vaccine nanocarrier 10 agent may be embedded within a lipid membrane, and comprises two or more types of immunomodulatory a second type of immunomodulatory agent may be en- agents which are derived from different strains of a single capsulated within the lipid membrane of a vaccine nano- species of microorganism. In some embodiments, a vac- carrier. To give another example, a first type of immu- cine nanocarrier comprises two or more types of immu- nomodulatory agent may be associated with the exterior nomodulatory agents which are derived from different 15 surface of a lipid membrane, and a second type of im- species of the same genus of microorganism. In other munomodulatory agent may be associated with the inte- embodiments, a vaccine nanocarrier comprises two or rior surface of the lipid membrane of a vaccine nanocar- more types of immunomodulatory agents each derived rier. In some embodiments, a first type of immunomod- from different genera of microorganism. ulatory agent may be embedded within the lumen of a [0165] In some embodiments, a vaccine nanocarrier 20 lipid bilayer of a vaccine nanocarrier, and the lipid bilayer comprises a single type of immunomodulatory agent that may encapsulate a polymeric matrix throughout which a stimulates an immune response in both B cells and T second type of immunomodulatory agent is distributed. cells. In some embodiments, a vaccine nanocarrier com- In some embodiments, a first type of immunomodulatory prises two types of immunomodulatory agents, wherein agent and a second type of immunomodulatory agent the first immunomodulatory agent stimulates B cells, and 25 may be in the same locale of a vaccine nanocarrier ( e.g., the second type of immunomodulatory agent stimulates they may both be associated with the exterior surface of T cells. In certain embodiments, one or both agents may a vaccine nanocarrier; they may both be encapsulated stimulate T cells and B cells. In some embodiments, a within the vaccine nanocarrier; etc.). vaccine nanocarrier comprises greater than two types of [0168] The vaccine nanocarrier includes a polymer immunomodulatory agents, wherein one or more types 30 (e.g., a polymeric core). At least one type of immunomod- of immunomodulatory agents stimulate B cells, and one ulatory agent may be associated with the polymer. In or more types of immunomodulatory agents stimulate T some embodiments, at least one type of immunomodu- cells. latory agent is embedded within the polymer. In some [0166] In some embodiments, a vaccine nanocarrier embodiments, a vaccine nanocarrier comprises at least comprises at least one type of immunomodulatory agent 35 one type of immunomodulatory agent that is associated that is associated with the exterior surface of the vaccine with the interior surface of the polymer. In some embod- nanocarrier. In some embodiments, the association is iments, at least one type of immunomodulatory agent is covalent. In some embodiments, the covalent associa- encapsulated with the polymer of a vaccine nanocarrier. tion is mediated by one or more linkers. In some embod- In some embodiments, at least one type of immunomod- iments, the association is non-covalent. In some embod- 40 ulatory agent may be located at multiple locations of a iments, the non-covalent association is mediated by vaccine nanocarrier. For example, a first type of immu- charge interactions, affinity interactions, metal coordina- nomodulatory agent may be embedded within a polymer, tion, physical adsorption, host-guest interactions, hydro- and a second type of immunomodulatory agent may be phobic interactions, TT stacking interactions, hydrogen encapsulated within a lipid membrane surrounding the bonding interactions, van der Waals interactions, mag- 45 polymeric core of a vaccine nanocarrier. To give another netic interactions, electrostatic interactions, dipole-di- example, a first type of immunomodulatory agent may pole interactions, and/or combinations thereof. For a be associated with the exterior surface of a polymer, and more detailed description of how an immunomodulatory a second type of immunomodulatory agent may be em- agent may be associated with a vaccine nanocarrier, bedded within the polymer of a vaccine nanocarrier. please see the section below entitled "Production of Vac- 50 [0169] One of ordinary skill in the art will recognize that cine Nanocarriers." the preceding examples are only representative of the [0167] In some embodiments, a vaccine nanocarrier many different ways in which multiple immunomodulato- includes a lipid membrane (e.g., lipid bilayer, lipid mon- ry agents may be associated with different locales of vac- olayer, etc.). At least one immunomodulatory agent may cine nanocarriers. Multiple immunomodulatory agents be associated with the lipid membrane. In some embod- 55 may be located at any combination of locales of vaccine iments, at least one immunomodulatory agent is embed- nanocarriers. Additionally, the aforementioned examples ded within the lipid membrane. In some embodiments, can also apply to the other agents of a nanocarrier (e.g., at least one immunomodulatory agent is embedded with- an immunostimulatory agent).

27 51 EP 2 630 966 B1 52

[0170] 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 in- discussed in further detail below. tended. In this case, an initially small number of naive T [0175] A targeting moiety may be a nucleic acid, cells are stimulated to generate pathogen-specific effec- 5 polypeptide, glycoprotein, carbohydrate, lipid, small mol- tor and memory T cells. In some embodiments, the an- ecule, etc. For example, a targeting moiety can be a nu- tigen may be taken from an unrelated source, such as cleic acid targeting moiety (e.g. an aptamer, Spiegelm- an infectious agent to which wide-spread immunity al- er®, etc.) that binds to a cell type specific marker. In gen- ready exists (e.g., tetanus toxoid or a common compo- eral, an aptamer is an oligonucleotide (e.g., DNA, RNA, nent of influenza virus, such as hemagglutinin, neurami- 10 or an analog or derivative thereof) that binds to a partic- nidase, or nuclear protein). In the latter case, the vaccine ular target, such as a polypeptide. In some embodiments, exploits the presence of memory T cells that have arisen a targeting moiety may be a naturally occurring or syn- in response to prior infections or vaccinations. Memory thetic ligand for a cell surface receptor,e.g., a growth cells in general react more rapidly and vigorously to an- factor, hormone, LDL, transferrin, etc. A targeting moiety tigen rechallenge and, therefore, may provide a superior 15 can be an antibody, which term is intended to include source of help to B cells. antibody fragments, characteristic portions of antibodies, [0171] Other T cell antigens include, but are not limited single chain antibodies, etc. Synthetic binding proteins to,degenerative disease antigens,infectious disease an- such as Affibodies ®, Nanobodies™, AdNectins™, Avim- tigens, cancer antigens, allergens, alloantigens, atopic ers™, etc., can be used. Peptide targeting moieties can diseaseantigens, autoimmunedisease antigens, contact 20 be identified, e.g., using procedures such as phage dis- sensitizers, haptens,xenoantigens, or metabolic disease play. This widely used technique has been used to iden- enzymes or enzymatic products thereof. In some embod- tify cell specific ligands for a variety of different cell types. iments, the infectious disease antigen is a viral antigen, [0176] In accordance with the present invention, a tar- which includes any antigen derived from any of the vi- geting moiety recognizes one or more "targets" or "mark- ruses described herein. Examples of T cell antigens in- 25 ers"associated with a particularorgan, tissue, cell, and/or clude those provided elsewhere herein. subcellular locale. In some embodiments, a target may [0172] In some embodiments, T cell antigens are in- be a marker that is exclusively or primarily associated corporated into nanocarriers as intact proteins. In some with one or a few cell types, with one or a few diseases, embodiments, T cell antigens are incorporated into na- and/or with one or a few developmental stages. A cell nocarriers as modified proteins. In some embodiments, 30 type specific marker is typically expressed at levels at T cell antigens are incorporated into nanocarriers as mu- least 2 fold greater in that cell type than in a reference tated proteins. In some embodiments, T cell antigens are population of cells which may consist, for example, of a provided as a collection of overlapping peptides, which mixture containing an approximately equal amount of can boost antigen incorporation into MHC class II com- cells ( e.g., approximately equal numbers of cells, approx- plexes and, therefore, further promote a helper response. 35 imately equal volume of cells, approximately equal mass In some embodiments, T cell antigens are provided as a of cells, etc.). In some embodiments, the cell type specific collection of non-overlapping peptides, which can boost marker is present at levels at least 3 fold, at least 4 fold, antigen incorporation into MHC class II complexes and, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 therefore, further promote a helper response. In some fold, at least 9 fold, at least 10 fold, at least 50 fold, at embodiments, T cell antigens are provided as nucleic 40 least 100 fold, at least 500 fold, at least 1000 fold, at least acids that encode the antigens. 5000 fold, or at least 10,000 fold greater than its average [0173] In some embodiments, inventive nanocarriers, expression in a reference population. Detection or meas- such as vaccine nanocarriers, comprise less than less urement of a cell type specific marker may make it pos- than 90% by weight, less than 75% by weight, less than sible to distinguish the cell type or types of interest from 50% by weight, less than 40% by weight, less than 30% 45 cells of many, most, or all other types. by weight, less than 20% by weight, less than 15% by [0177] In some embodiments, a target can comprise a weight, less than 10% by weight, less than 5% by weight, protein, a carbohydrate, a lipid, and/or a nucleic acid. In less than 1% by weight, or less than 0.5% by weight of certain embodiments, a target can comprise a protein the immunomodulatory agent. and/or characteristic portion thereof, such as a tumor- 50 marker, integrin, cell surface receptor, transmembrane Targeting Moieties protein, intercellular protein, ion channel, membrane transporter protein, enzyme, antibody, chimeric protein, [0174] In some embodiments, inventive nanocarriers glycoprotein, etc. In certain embodiments, a target can comprise one or more targeting moieties. In certain em- comprise a carbohydrate and/or characteristic portion bodiments of the invention, nanocarriers are associated 55 thereof, such as a glycoprotein, sugar e.g( ., monosac- with one or more targeting moieties. A targeting moiety charide, disaccharide, polysaccharide), glycocalyx (i.e., is any moiety that binds to a component associated with the carbohydrate-rich peripheral zone on the outside sur- an organ, tissue, cell, extracellular matrix, and/or subcel- face of most eukaryotic cells)etc. In certain embodi-

28 53 EP 2 630 966 B1 54 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. 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 5 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, nucleic acid; RNA nucleic acid; modified DNA nucleic FDCs, and/or T cells. acid; modified RNA nucleic acid; nucleic acid that in- [0184] In some embodiments, expression of an APC, cludes any combination of DNA, RNA, modified DNA, DC, and/or T cell target in activated APCs, DCs, and/or and modified RNA; etc. 10 T cells is at least 2-fold, at least 3-fold, at least 4-fold, at [0178] In some embodiments, a targeting moiety could least 5-fold, at least 10-fold, at least 20-fold, at least 50- be the surface glycoprotein molecule from VSV. VSV fold, at least 100-fold, at least 500-fold, or at least 1000- comprises a single surface molecule, VSV-G, which is a fold overexpressed in activated APCs, DCs, and/or T toll-like receptor agonist. VSV is efficiently targeted to cells relative to a reference population. In some embod- cells of the immune system, so in some embodiments, 15 iments, a reference population may comprise non-acti- vaccine nanocarriers could comprise the VSV surface vated APCs, DCs, and/or T cells. molecule in order to target vaccine nanocarriers to cells [0185] In some embodiments, inventive nanocarriers, of the immune system. such as vaccine nanocarriers, comprise less than 50% [0179] In some embodiments, a target is a tumor mark- by weight, less than 40% by weight, less than 30% by er. In some embodiments, a tumor marker is an antigen 20 weight, less than 20% by weight, less than 15% by that is expressed in tumor cells but not in healthy and/or weight, less than 10% by weight, less than 5% by weight, normal cells. In some embodiments, a tumor marker is less than 1% by weight, or less than 0.5% by weight of an antigen that is more prevalent in tumor cells than in the targeting moiety. healthy and/or normal cells. Exemplary tumor markers [0186] In some embodiments, targeting moieties are include, but are not limited to, gp100; Melan-A; tyrosi- 25 covalently associated with a nanocarrier. In some em- nase; PSMA; HER-2/neu; MUC-1; topoisomerase II α; si- bodiments, covalent association is mediated by a linker. alyl-Tn; carcinoembryonic antigen; ErbB-3-binding pro- In some embodiments, targeting moieties are not cova- tein-1; alpha-fetoprotein; and the cancer-testis antigens lently associated with a nanocarrier. For example, tar- MAGE-A1, MAGE A4, and NY-ESO-1. geting moieties may be associated with the surface of, [0180] In some embodiments, a target is an APC mark- 30 encapsulated within, surrounded by, and/or distributed er. In some embodiments, an APC target is an antigen throughout the polymeric matrix of an inventive particle. that is expressed in APCs but not in non-APCs. In some For example, in some embodiments, a targeting moiety embodiments, an APC target is an antigen that is more can be encapsulated within, surrounded by, and/or dis- prevalent in APCs than in non-APCs. Exemplary APC persed throughout the liposomal membrane and/or pol- markers include, but are not limited to, CD11c, CD11b, 35 ymeric matrix of a nanocarrier. Alternatively or addition- CD14, CD40, CD45, CD163, CD169 (sialoadhesin), ally, a targeting moiety can be associated with a nano- DEC205 (CD205), MHC class II, DC-SIGN, CD21/CD35, carrier by charge interactions, affinity interactions, metal and Fc γ RI, PD-L2. In some embodiments, APC markers coordination, physical adsorption, host-guest interac- include any of DC and/or macrophage markers, exam- tions, hydrophobic interactions, TT stacking interactions, ples of which are described herein. 40 hydrogen bonding interactions, van der Waals interac- [0181] In certain embodiments, a target is a DC mark- tions, magnetic interactions, electrostatic interactions, di- er. In some embodiments, a DC target is an antigen that pole-dipole interactions, and/or combinations thereof. is expressed in DCs but not in non-DCs. In some em- Association of targeting moieties with vaccine nanocar- bodiments, a DC target is an antigen that is more prev- riers is described in further detail below, in the section alent in DCs than in non-DCs. Exemplary DC markers 45 entitled "Production of Vaccine Nanocarriers." are listed below in the section entitled "Dendritic Cells" and include those provided elsewhere herein. Dendritic Cells [0182] Incertain embodiments, a target isa T cell mark- er. In some embodiments, a T cell target is an antigen [0187] Dendritic Cells (DCs) are a type of myeloid leu- that is expressed in T cells but not in non-T cells. In some 50 kocytes; they are among the most potent antigen pre- embodiments, a T cell target is an antigen that is more senting cells for T lymphocytes. Resting DCs reside in prevalent in T cells than in non-T cells. Exemplary T cell many tissues, including lymph nodes, in an immature, markers are listed below in the section entitled T" Cell tolerogenic state, i.e., they present intermediate to high Targeting Moieties" and include those provided else- levels of peptide-MHC complexes, but with little or no where herein. 55 costimulatory molecules and without secreting cytokines [0183] In some embodiments, a target is preferentially that T cells need to differentiate into effector cells. T cells expressed in particular cell types. For example, expres- that are presented with a specific antigen by immature sion of an APC, DC, and/or T cell target in APCs, DCs, DCs begin to proliferate for a few days, but then they die

29 55 EP 2 630 966 B1 56 by apoptosis or become unresponsive to further activa- (LFA-3); CD64 (FcγRI); CD72 (Ly-19.2, Ly-32.2, Lyb-2); tion. The ensuing depletion of antigen-specific T cell re- CD73 (Ecto-5’nucloticlase); CD74 (Ii, invariant chain); sponses renders the host selectively tolerant to this an- CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15); tigen. By contrast, when DCs acquire antigens while they CD85a (ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); are exposed to maturation stimuli, the cells rapidly up- 5 CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); regulate MHC and costimulatory molecules and secrete CD86 (B7-2/B70); CD88 (C5aB); CD97 (BL-KDD/F12); several cytokines. The now mature DCs are potent in- CD101 (IGSF2, P126, V7); CD116 (GM-CSFR); α ducers of effector T cells and immunological memory. CD120a (TMFRI, p55); CD120b (TNFRII, p75, TNFR DC maturation can be induced by many signals, such as p80); CD123 (IL-3Rα); CD139; CD148 (HPTP-η, p260, certain inflammatory cytokines, ligation of DC-expressed 10 DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, CD40, agonists for TLRs, ( e.g., bacterial endotoxin), im- ADAM17, cSVP); CD157 (Mo5, BST-1); CD167a (DDR1, mune complexes, activated complement, necrotic cells, trkE, cak); CD168 (RHAMM, IHABP, HMMR); CD169 apoptotic cells, free urate, urate crystals, and/or HMGB- (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 1. (L1CAM, NILE); CD172 (SIRP-1α, MyD-1); CD172b [0188] DEC-205 (i.e., CD205) is a surface-expressed 15 (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD184 multi-functional lectin that is selectively expressed on (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); DCs and thymic epithelial cells in lymphoid tissues.In CD197 (CCR7 (ws CDw197)); CDw197 (CCR7, EBI1, vivo experiments with subcutaneously injected chimeric BLR2); CD200 (OX2); CD205 (DEC-205); CD206 α-DEC-205 monoclonal antibodies have shown that lig- (MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209 and binding to DEC-205 induces efficient internalization 20 (DC-SIGN); CDw218a (IL18Rα); CDw218b (IL8Rβ); and subsequent processing of the endocytosed material CD227 (MUC1, PUM, PEM, EMA); CD230 (Prion Protein for presentation in MHC molecules in both mice and hu- (PrP)); CD252 (OX40L, TNF (ligand) superfamily, mem- mans (Hawiger et al., 2001, J. Exp. Med. 194:769; Bon- ber 4); CD258 (LIGHT, TNF (ligand) superfamily, mem- ifaz et al., 2002, J. Exp. Med., 196:1627; and Bozzacco ber 14); CD265 (TRANCE-R, TNF-R superfamily, mem- et al., 2007, Proc. Natl. Acad. Sci., USA, 104:1289). Upon 25 ber 11a); CD271 (NGFR, p75, TNFR superfamily, mem- intra-cutaneous or subcutaneous injection, the chimeric ber 16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); antibody is transported via lymph vessels to the draining CD275 (B7H2, ICOSL); CD276 (B7H3); CD277 (BT3.1, lymph nodes where it binds specifically to resident DCs, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-like re- thus providing the means to target antigens to resting ceptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 DCs without causing their maturation. The targeted DCs 30 (LEPR); CD298 (ATP1B3, Na K ATPaseβ 3 submit); will then induce T cell tolerance to the presented antigen, CD300a (CMRF-35H); CD300c (CMRF-35A); CD301 rather than immunity. However, when DEC-205 is target- (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2); ed together with an immunostimulatory agent that induc- CD304 (BDCA4); CD312 (EMR2); CD317 (BST2); es DC maturation (e.g., α-CD40 or one or more ligands CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68 for DC-expressed TLRs; discussed in further detail below 35 (gyp110, Macrosialin); class II MHC; BDCA-1; Siglec-H; in the section entitled "Immunostimulatory Agents"), then wherein the names listed in parentheses represent alter- the vaccine acts as a potent immunostimulant promoting native names. preferentially cytotoxic and Thl-type effector T cell re- sponses. T Cell Targeting Moieties [0189] DC targeting can be accomplished by moieties 40 that bind DC-205, CD11c, class II MHC, CD80, CD86, [0191] In some embodiments, T cell targeting can be DC-SIGN, CD11b, BDCA-1, BDCA-2, BDCA-4, Siglec- accomplished by any targeting moiety that specifically H, CX3CR1, and/or Langerin. binds to any entity(e.g., protein, lipid, carbohydrate, [0190] In some embodiments, DC targeting can be ac- small molecule, etc.) that is prominently expressed complished by anytargeting moiety that specificallybinds 45 and/or present on T cells (i.e., a T cell marker). Exemplary to any entity ( e.g., protein, lipid, carbohydrate, small mol- T cell markers include, but are not limited to, CD2 (E- ecule, etc.) that is prominently expressed and/or present rosette R, T11, LFA-2); CD3 (T3); CD3 α; CD3 β; CD3 on DCs (i.e., a DC marker). Exemplary DC markers in- ε; CD4 (L3T4, W3/25, T4); CD5 (T1, Tp67, Leu-1, LY-1); clude, but are not limited to, CD1a (R4, T6, HTA-1); CD1b CD6 (T12); CD7 (gp40, Leu 9); CD8a (Leu2, T8, Lyt2,3); (R1); CD1c (M241, R7); CD1d (R3); CD1e (R2); CD11b 50 CD8b (CD8, Leu2, Lyt3); CD11a (LFA-1α, α Integrin (αM Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c (aX chain); CD11b α ( M Integrin chain, CR3, Mol, C3niR, Integrin, p150, 95, AXb2); CDw117 (Lactosylceramide, Mac-1); CD11c (aX Integrin, pl50, 95, AXb2); CD15s (Si- LacCer); CD19 (B4); CD33 (gp67); CD 35 (CR1,alyl Lewis X); CD15u (3’ sulpho Lewis X); CD15su (6 C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39 sulpho-sialylLewis X); CD16b (FcgR111b);CDw17 (Lac- (ATPdehydrogenase, NTPdehydrogenase-1); CD4055 tosylceramide, LacCer); CD18 (Integrin β2 CD11a, b, c (Bp50); CD45 (LCA, T200, B220, Ly5); CD45RA; β-subunit); CD26(DPP IV ectoeneyme, ADA bindingpro- CD45RB; CD45RC; CD45RO (UCHL-1); CD49d (VLA- tein); CD27 (T14, S152); CD28 (Tp44, T44); CD29 4α, α4 Integrin); CD49e (VLA-5α, α5 Integrin); CD58 (Platelet GPlla, β-1 integrin, GP); CD31 (PECAM-1, En-

30 57 EP 2 630 966 B1 58 docam); CD35 (CR1, C3b/C4b receptor); CD37R2, TNF-R superfamily, member 10b); CD263 (TRAIL- (gp52-40); CD38 (ADP-ribosyl/cyclase, T10); CD43 (Sia- R3, TNF-R superfamily, member 10c); CD264 (TRAIL- lophorin, Leukosialin); CD44 (ECMRII, H-CAM, Pgp-1); R4, TNF-R superfamily, member 10d); CD265 CD45 (LCA, T200, B220, Ly5); CD45RA (p561ck, (TRANCE-R, TNF-R superfamily, member 11a); CD268 p59fyn, Src kinases); CD45RB (p561ck, p59fyn, Src ki- 5 (BAFFR, TNF-R superfamily, member 13C); CD272 (BT- nases); CD45RC (p561ck, p59fyn, Src kinases); CD46 LA); CD275 (B7H2, ICOSL); CD277 (BT3.1, B7 family: (MCP); CD47 (gp42, IAP, OA3, Neurophillin); CD47R Butyrophilin 3); CD294 (CRTH2, PGRD2, G protein-cou- (MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); pled receptor 44); CD295 (LEPR); CD296 (ART1, ADP- CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, α4 In- ribosyltransferase 1); CD298 (ATP1B3, Na K ATPase β3 tegrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α, 10 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); α6 Integrin gplc); CD50 (ICAM-3); CD52 (CAMPATH-1, CD305 (LAIR1); CD314 (NKG2D); CD316 (EW12); HES); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321 CD56 (Leu-19, NKH-1, NCAM); CD57 (HNK1, Leu-7); (JAM1); CD322 (JAM2); CDw328 (Siglec7); and CD68 CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, (gp 110, Macrosialin); wherein the names listed in pa- MIRL, P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); 15 rentheses represent alternative names. CD60c (7-O acetyl GD3); CD62L (L-selectin, LAM-1, [0192] In some embodiments, T cell targeting can be LECAM-1, MEL-14, Leu8, TQ1); CD73 (Ecto-5’-nucloti- accomplished by any targeting moiety that binds, such dase); CD75 (sialo-masked Lactosamine); CD75S (α2, as specifically binds, to any entity (e.g., protein, lipid, car- 6 sialylated Lactosamine); CD81 (TAPA-1); CD82 (4F9, bohydrate, small molecule, etc.) that is prominently ex- C33, IA-4, KAI1, R2); CD84 (P75, GR6); CD85a (ILT5, 20 pressed and/or present on T cells upon activation (i.e., LIR3, HL9); CD85j (ILT2, LIR1, MIR7); CD87 (uPAR); activated T cell targets). Exemplary activated T cell tar- CDw92 (p70); CD94 (Kp43); CD95 (APO-1, FAS, geting moieties include, but are not limited to, CD1a (RA, TNFRSF6); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, T6, HTA-1); CD1b (R1); Cd1c (M241,R7); CD1d (R3); E2); CD99R (CD99 Mab restricted); CD100 (SEMA4D); CD9 (p24, DRAP-1, MRP-1); CD25 (Tac antigen, IL-2R α, CD102 (ICAM-2); CD108 (SEMA7A, JMH blood group 25 p55); CD30 (Ber-H2, Ki-1); CD39 (ATPdehydrogenase, antigen); CDw119 (IFNγR, IFNγRa); CD120a (TNFRI, NTPdehydrogenase-1); CD45RO (UCHL-1); CD49a p55); CD120b (TNFRII, p75, TNFR p80); CD121a (Type (VLA-1α, α1 Integrin); CD49b (VLA-2,α gpla, α2 In- 1IL-1R); CD121b (Type 2 IL-1R);CD122 (IL2R β); CD124 tegrin); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (IL-4Rα); CD126 (IL-6Rα); CD127 (p90, IL-7R, IL-7R α); (Ki-24, CD27 ligand); CD74 (Ii, invariant chain); CD80 CD128a (IL-8Ra, CXCR1, (Tentatively renamed as30 (B7, B7-1, BB1); CD86 (B7-2/B70); CD96 (TACTILE); CD181)); CD128b (IL-8Rb, CXCR2, (Tentatively re- CD97 (BL-KDD/F12); CD101 (IGSF2, P126, V7); CD103 named as CD182)); CD130 (gp130); CD132 (Common (HML-1, Integrin αE, ITGAE); CD107a (LAMP-1); γ chain, IL-2R γ); CD147 (Basigin, EMMPRIN, M6, OX47); CD107b (LAMP-2); CD109 (8A3, E123 7D1); CD134 CD148 (HPTP-η, p260, DEP-1); CD150 (SLAM, IPO-3); (OX40, TNFRSF4); CDwl37 (4-1BB, ILA); CD146 (Muc CD153 (CD3OL, TNSF8); CD156b (TACE, ADAM17, 35 18, S-endo, MCAM, Mel-CAM); CD152 (CTLA-4); cSVP); CD158a (KIR2DL1, p58.1); CD158b1 (KIR2DL2, CD154 (CD40L, gp39, TRAP-1, T-BAM); CD166 (AL- p58.2); CD158b2 (KIR2DL3, p58.3); CD158c (KIR2DS6, CAM, KG-CAM, SC-1, BEN, DM-GRASP); CD178 (Fas KIRX); CD158|e1/e2 (KIR3DLI/S1, p70); CD159F Ligand); CD227 (MUC1, PUM, PEM, EMA); CD253 (KIR2DL5); CD158g (KIR2DS5); CD158h (KIR2DS1, (TRAIL, TNF (ligand) superfamily, member 10); CD254 p50.1); CD158i (KIR2DS4, p50.3); CD158j (KIR2DS2, 40 (TRANCE, RANKL, TNF (ligand) superfamily, member p50.2); CCD158k (KIR3DL2, p140); CD159a (NKG2A); 11); CD258 (LIGHT, TMF (ligand) superfamily, member CD160 (BY55, NK1, NK28); CD161 (NKR, NKRP1A); 14); CD267 (TACI, TNF-R superfamily, member 13B); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD171 CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD278 (L1CAM, NILE); CD172g (SIRPg); CD181 (CXCR1, (For- (ICOS); CD279 (PD1); and CD312 (EMR2); wherein the merly known as CD128a)); CD182 (CXCR2, (Formerly 45 names listed in parentheses represent alternative known as CD128b)); CD183 (CXCR3, GPR9); CD184 names. (CXCR4, NPY3R); CD185 (CXCR5); CD186 (CXCR6); CD191 (CCR1); CD192 (CCR2); CD193 (CCR3); CD195 Molecular Characteristics of Targeting Moieties (CCR5); CD196 (CCR6); CD197 (CCR7 (was CDw197)); CDw197 (CCR7, EBI1, BLR2); CDw198 (CCR8);50 [0193] Nucleic Acid Targeting Moieties. As used here- CDw199 (CCR9); CD205 (DEC-205); CDw210 (CK); in, a "nucleic acid targeting moiety" is a nucleic acid that CDw217 (CK); CDw218a (IL18R α); CDw218b (IL18R β); binds selectively to a target. In some embodiments, a CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IG- nucleic acid targeting moiety is a nucleic acid aptamer. FII-R); CD223 (LAG-3); CD224 (GGT); CD225 (Leul3); An aptamer is typically a polynucleotide that binds to a CD226 (DNAM-1, PTA1); CD229 (Ly9); CD230 (Prion 55 specific target structure that is associated with a partic- Protein (PrP)); CD244 (2B4, P38, NAIL); CD245ular organ, tissue, cell, extracellular matrix component, (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL- and/or subcellular locale. In general, the targeting func- R1, TNF-R superfamily, member 10a); CD262 (TRAIL- tion of the aptamer is based on the three-dimensional

31 59 EP 2 630 966 B1 60 structure of the aptamer. In some embodiments, binding ring nucleic acid. U.S. Patents 6,403,779; 6,399,754; of an aptamer to a target is typically mediated by the 6,225,460; 6,127,533; 6,031,086; 6,005,087; 5,977,089; interaction between the two- and/or three-dimensional and references therein disclose a wide variety of specific structures of both the aptamer and the target. In some nucleotide analogs and modifications that may be used. embodiments, binding of an aptamer to a target is not 5 See Crooke, S. (ed.) Antisense Drug Technology: Prin- solely based on the primary sequence of the aptamer, ciples, Strategies, and Applications (1st ed), Marcel but depends on the three-dimensional structure(s) of the Dekker; ISBN: 0824705661, 1st edition (2001). For ex- aptamer and/or target. In some embodiments, aptamers ample, 2’-modifications include halo, alkoxy and allyloxy bind to their targets via complementary Watson-Crick groups. In some embodiments, the 2’-OH group is re- 10 base pairing which is interrupted by structures (e.g., hair- placed by a group selected from H, OR, R, halo, SH, SR 1, pin loops) that disrupt base pairing. NH2, NHR, NR2 or CN, wherein R is C 1-C6 alkyl, alkenyl, [0194] In some embodiments, a nucleic acid targeting or alkynyl, and halo is F, Cl, Br or I. Examples of modified moiety is a Spiegelmer®. In general, Spiegelmers® are linkages include phosphorothioate and 5’-N-phosphora- high-affinity L-enantiomeric oligonucleotide ligands that midite linkages. display high resistance to enzymatic degradation com- 15 [0199] Nucleic acids comprising a variety of different pared with D-oligonucleotides. In some embodiments, nucleotide analogs, modified backbones, or non-natural- Spiegelmers® can be designed and utilized just as an ly occurring internucleoside linkages can be utilized in aptamer would be designed and utilized. accordance with the present invention. Nucleic acids of [0195] One of ordinary skill in the art will recognize that the present invention may include natural nucleosides any nucleic acid that is capable of specifically binding to 20 (i.e., adenosine, thymidine, guanosine, cytidine, uridine, a target, as described herein, can be used in accordance deoxyadenosine,deoxythymidine, deoxyguanosine, and with the present invention. deoxycytidine) or modified nucleosides. Examples of [0196] Nucleic acids of the present invention (including modified nucleotides include base modified nucleosides nucleic acid targeting moieties and/or functional RNAs (e.g., aracytidine, inosine, isoguanosine, nebularine, to be delivered, e.g., RNAi agents, ribozymes, tRNAs, 25 pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thi- etc., described in further detail below) may be prepared othymidine, 3-deaza-5-azacytidine, 2’-deoxyuridine, 3- according to any available technique including, but not nitorpyrrole, 4-methylindole, 4-thiouridine, 4-thiothymi- limited to chemical synthesis, enzymatic synthesis, en- dine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, zymatic or chemical cleavage of a longer precursor, etc. 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6- Methods of synthesizing RNAs are known in the art (see, 30 chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8- e.g., Gait, M.J. (ed.) Oligonucleotide synthesis: a practi- azaadenosine, 8-azidoadenosine, benzimidazole, M1- calapproach, Oxford [Oxfordshire], Washington,DC: IRL methyladenosine, pyrrolo-pyrimidine, 2-amino-6-chloro- Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide purine, 3-methyl adenosine, 5-propynylcytidine, 5-propy- synthesis: methods and applications, Methods in molec- nyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcy- ular biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana 35 tidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoad- Press, 2005). enosine, 8-oxoguanosine, O(6)-methylguanine, and 2- [0197] The nucleic acid that forms the nucleic acid tar- thiocytidine), chemically or biologically modified bases geting moiety may comprise naturally occurring nucleo- (e.g., methylated bases), modified sugars (e.g., 2’-fluor- sides, modified nucleosides, naturally occurring nucleo- oribose, 2’-aminoribose, 2’-azidoribose, 2’-O-methylri- sides with hydrocarbon linkers (e.g., an alkylene) or a 40 bose, L-enantiomeric nucleosides arabinose, and hex- polyether linker ( e.g., a PEG linker) inserted between one ose), modified phosphate groups (e.g., phosphorothio- or more nucleosides, modified nucleosides with hydro- ates and 5’-N-phosphoramidite linkages), and combina- carbon or PEG linkers inserted between one or more tions thereof. Natural and modified nucleotide monomers nucleosides, or a combination of thereof. In some em- for the chemical synthesis of nucleic acids are readily bodiments, nucleotides or modified nucleotides of the nu- 45 available. In some cases, nucleic acids comprising such cleic acid targeting moiety can be replaced with a hydro- modifications display improved properties relative to nu- carbon linker or a polyether linker provided that the bind- cleic acids consisting only of naturally occurring nucle- ing affinity and selectivity of the nucleic acid targeting otides. In some embodiments, nucleic acid modifications moiety is not substantially reduced by the substitution described herein are utilized to reduce and/or prevent (e.g., the dissociation constant of the nucleic acid target- 50 digestion by nucleases (e.g. exonucleases, endonucle- ing moiety for the target should not be greater than about ases, etc.). For example, the structure of a nucleic acid 1 x 10-3 M). may be stabilized by including nucleotide analogs at the [0198] It will be appreciated by those of ordinary skill 3’ end of one or both strands order to reduce digestion. in the art that nucleic acids in accordance with the present [0200] Modified nucleic acids need not be uniformly invention may comprise nucleotides entirely of the types 55 modified along the entire length of the molecule. Different found in naturally occurring nucleic acids, or may instead nucleotide modifications and/or backbone structures include one or more nucleotide analogs or have a struc- may exist at various positions in the nucleic acid. One of ture that otherwise differs from that of a naturally occur- ordinary skill in the art will appreciate that the nucleotide

32 61 EP 2 630 966 B1 62 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 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- 5 vided in the U.S. Code of Federal Regulations (C.F.R.). cifically bind to the aptamer target is not substantially [0205] One of ordinary skill in the art will appreciate affected. The modified region may be at the 5’-end and/or thatany small molecule thatspecifically binds toa desired the 3’-end of one or both strands. For example, modified target, as described herein, can be used in accordance aptamers in which approximately 1 to approximately 5 with the present invention. residues at the 5’ and/or 3’ end of either of both strands 10 [0206] Protein Targeting Moieties. In some embodi- are nucleotide analogs and/or have a backbone modifi- ments, a targeting moiety in accordance with the present cation can be employed. The modification may be a 5’ invention may be a protein or peptide. In certain embod- or 3’ terminal modification. A nucleic acid strand may iments, peptides range from about 5 to about 100, from comprise at least 50% unmodified nucleotides, at least about 5 to about 50, from about 10 to about 75, from 80% unmodified nucleotides, at least 90% unmodified 15 about 15 to about 50, or from about 20 to about 25 amino nucleotides, or 100% unmodified nucleotides. acids in size. In some embodiments, a peptide sequence [0201] Nucleic acids in accordance with the present can be based on the sequence of a protein. In some invention may, for example, comprise a modification to embodiments, a peptide sequence can be a random ar- a sugar, nucleoside, or internucleoside linkage such as rangement of amino acids. those described in U.S. Patent Publications20 [0207] The terms "polypeptide" and "peptide" are used 2003/0175950, 2004/0192626, 2004/0092470,interchangeably herein, with "peptide" typically referring 2005/0020525, and 2005/0032733. The present inven- to a polypeptide having a length of less than about 100 tion encompasses the use of any nucleic acid having any amino acids. Polypeptides may contain L-amino acids, one or more of the modification described therein. For D-amino acids, or both and may contain any of a variety example, a number of terminal conjugates,e.g., lipids 25 of amino acid modifications or analogs known in the art. such as cholesterol, lithocholic acid, aluric acid, or long Useful modifications include, e.g., terminal acetylation, alkyl branched chains have been reported to improve amidation, lipidation, phosphorylation, glycosylation, cellular uptake. Analogs and modifications may be tested acylation, farnesylation, sulfation, etc. using, e.g., using any appropriate assay known in the art, [0208] Exemplary proteins that may be used as target- for example, to select those that result in improved de- 30 ing moieties in accordance with the present invention in- livery of a therapeutic agent, improved specific binding clude, but are not limited to, antibodies, receptors, cy- of an aptamer to an aptamer target, etc. In some embod- tokines, peptide hormones, glycoproteins, glycopep- iments, nucleic acids in accordance with the present in- tides, proteoglycans, proteins derived from combinatorial vention may comprise one or more non-natural nucleo- libraries (e.g., Avimers™, Affibodies ®, etc.), and charac- side linkages. In some embodiments, one or more inter- 35 teristic portions thereof. Synthetic binding proteins such nal nucleotides at the 3’-end, 5’-end, or both 3’- and 5’- as Nanobodies™, AdNectins™, etc., can be used. In ends of the aptamer are inverted to yield a linkage such some embodiments, protein targeting moieties can be as a 3’ - 3’ linkage or a 5’ - 5’ linkage. peptides. [0202] In some embodiments, nucleic acids in accord- [0209] One of ordinary skill in the art will appreciate ance with the present invention are not synthetic, but are 40 that any protein and/or peptide that specifically binds to naturally-occurring entities that have been isolated from a desired target, as described herein, can be used in their natural environments. accordance with the present invention. [0203] Small Molecule Targeting Moieties. In some [0210] In some embodiments, a targeting moiety may embodiments, a targeting moiety in accordance with the be an antibody and/or characteristic portion thereof. The present invention may be a small molecule. In certain 45 term "antibody" refers to any immunoglobulin, whether embodiments, small molecules are less than about 2000 natural or wholly or partially synthetically produced and g/mol in size. In some embodiments, small molecules to derivatives thereof and characteristic portions thereof. are less than about 1500 g/mol or less than about 1000 An antibody may be monoclonal or polyclonal. An anti- g/mol. In some embodiments, small molecules are less body may be a member of any immunoglobulin class, than about 800 g/mol or less than about 500 g/mol. 50 including any of the human classes: IgG, IgM, IgA, IgD, [0204] In certain embodiments, a small molecule is ol- and IgE. igomeric. In certain embodiments, a small molecule is [0211] As used herein, an antibody fragment (i.e. char- non-oligomeric. In certain embodiments, a small mole- acteristic portion of an antibody) refers to any derivative cule is a natural product or a natural product-like com- of an antibody which is less than full-length. In some em- pound having a partial structure e.g.( , a substructure) 55 bodiments, an antibody fragment retains at least a sig- based on the full structure of a natural product. In certain nificant portion of the full-length antibody’s specific bind- embodiments, a small molecule is a synthetic product. ing ability. Examples of such antibody fragments include, In some embodiments, a small molecule may be from a but are not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv

33 63 EP 2 630 966 B1 64 diabody, and Fd fragments. Antibody fragments also in- present invention may comprise a carbohydrate. In some clude, but are not limited, to Fc fragments. embodiments, a carbohydrate may be a polysaccharide [0212] An antibody fragment may be produced by any comprising simple sugars (or their derivatives) connect- means. For example, an antibody fragment may be en- ed by glycosidic bonds, as known in the art. Such sugars zymatically or chemically produced by fragmentation of 5 may include, but are not limited to, glucose, fructose, an intact antibody and/or it may be recombinantly pro- galactose, ribose, lactose, sucrose, maltose, trehalose, duced from a gene encoding the partial antibody se- cellbiose, mannose, xylose, arabinose, glucoronic acid, quence. Alternatively or additionally, an antibody frag- galactoronic acid, mannuronic acid, glucosamine, gala- ment may be wholly or partially synthetically produced. tosamine, and neuramic acid. In some embodiments, a An antibody fragment may optionally comprise a single 10 carbohydrate may be one or more of pullulan, cellulose, chain antibody fragment. Alternatively or additionally, an microcrystalline cellulose, hydroxypropyl methylcellu- antibody fragment may comprise multiple chains which lose, hydroxycellulose, methylcellulose, dextran, cyclo- are linked together, for example, by disulfide linkages. dextran, glycogen, starch, hydroxyethylstarch, cara- An antibody fragment may optionally comprise a multi- geenan, glycon, amylose, chitosan, N,O-carboxylmeth- molecular complex. A functional antibody fragment will 15 ylchitosan, algin and alginic acid, starch, chitin, heparin, typicallycomprise atleast about 50amino acidsand more konjac, glucommannan, pustulan, heparin, hyaluronic typically will comprise at least about 200 amino acids. acid, curdlan, and xanthan. [0213] In some embodiments, antibodies may include [0221] In some embodiments, the carbohydrate may chimeric (e.g. "humanized") and single chain (recom- be aminated, carboxylated, and/or sulfated. In some em- binant) antibodies. In some embodiments, antibodies 20 bodiments, hydrophilic polysaccharides can be modified may have reduced effector functions and/or bispecific to become hydrophobic by introducing a large number molecules. In some embodiments, antibodies may in- of side-chain hydrophobic groups. In some embodi- clude fragments produced by a Fab expression library. ments, a hydrophobic carbohydrate may include cellu- [0214] Single-chain Fvs (scFvs) are recombinant anti- lose acetate, pullulan acetate, konjac acetate, amylose body fragments consisting of only the variable light chain 25 acetate, and dextran acetate. (VL) and variable heavy chain (VH) covalently connected [0222] One of ordinary skill in the art will appreciate to one another by a polypeptide linker. Either VL or VH that any carbohydrate that specifically binds to a desired may comprise the NH2-terminal domain. The polypep- target, as described herein, can be used in accordance tide linker may be of variable length and composition so with the present invention. long as the two variable domains are bridged without 30 [0223] Lipid Targeting Moieties. In some embodi- significant steric interference. Typically, linkers primarily ments, a targeting moiety in accordance with the present comprise stretches of glycine and serine residues with invention may comprise one or more fatty acid groups or some glutamic acid or lysine residues interspersed for salts thereof. In some embodiments, a fatty acid group solubility. may comprise digestible, long chain (e.g., C8-C50), sub- [0215] Diabodies are dimeric scFvs. Diabodies typical- 35 stituted or unsubstituted hydrocarbons. In some embod- ly have shorter peptide linkers than most scFvs, and they iments, a fatty acid group may be a C 10-C20 fatty acid or often show a preference for associating as dimers. salt thereof. In some embodiments, a fatty acid group [0216] An Fv fragment is an antibody fragment which may be a C15-C20 fatty acid or salt thereof. In some em- consists of one VH and one VL domain held together by bodiments, a fatty acid group may be a C 15-C25 fatty acid noncovalent interactions.The term "dsFv"as usedherein 40 or salt thereof. In some embodiments, a fatty acid group refers to an Fv with an engineered intermolecular di- may be unsaturated. In some embodiments, a fatty acid sulfide bond to stabilize the VH-VL pair. group may be monounsaturated. In some embodiments, [0217] An F(ab’)2 fragment is an antibody fragment es- a fatty acid group may be polyunsaturated. In some em- sentially equivalent to that obtained from immunoglobu- bodiments, a double bond of an unsaturated fatty acid lins by digestion with an enzyme pepsin at pH 4.0-4.5. 45 group may be in the cis conformation. In some embodi- The fragment may be recombinantly produced. ments, a double bond of an unsaturated fatty acid may [0218] A Fab’ fragment is an antibody fragment essen- be in the trans conformation. tially equivalent to that obtained by reduction of the di- [0224] In some embodiments, a fatty acid group may sulfide bridge or bridges joining the two heavy chain piec- be one or more of butyric, caproic, caprylic, capric, lauric, es in the F(ab’)2 fragment. The Fab’ fragment may be 50 myristic, palmitic, stearic, arachidic, behenic, or lignocer- recombinantly produced. ic acid. In some embodiments, a fatty acid group may be [0219] A Fab fragment is an antibody fragment essen- oneor more of palmitoleic,oleic, vaccenic, linoleic, alpha- tially equivalent to that obtained by digestion of immu- linoleic, gamma-linoleic, arachidonic, gadoleic, arachi- noglobulins with an enzyme ( e.g., papain). The Fab frag- donic, eicosapentaenoic,docosahexaenoic, or erucicac- ment may be recombinantly produced. The heavy chain 55 id. segment of the Fab fragment is the Fd piece. [0225] One of ordinary skill in the art will appreciate [0220] Carbohydrate Targeting Moieties . In some em- that any fatty acid group that specifically binds to a de- bodiments, a targeting moiety in accordance with the sired target, as described herein, can be used in accord-

34 65 EP 2 630 966 B1 66 ance with the present invention. embodiments, a vaccine nanocarrier comprises greater than two distinct types of immunostimulatory agents. Novel Targeting Moieties [0230] In some embodiments, a vaccine nanocarrier comprises a single type of immunostimulatory agent that [0226] Any novel targeting moiety can be utilized in the 5 stimulates both B cells and T cells. In some embodi- nanocarriers in accordance with the present invention. ments, a vaccine nanocarrier comprises two types of im- Any method known in the art can be used to design, iden- munostimulatory agents, wherein first type of immunos- tify, and/or isolate novel targeting moieties. For example, timulatory agent stimulates B cells, and the second type standard techniques utilizing libraries of molecules and of immunostimulatory agent stimulates T cells. In some in vitro binding assays can be utilized to identify novel 10 embodiments, a vaccine nanocarrier comprises greater targeting moieties. than two types of immunostimulatory agents, wherein [0227] Nucleic acid targeting moieties ( e.g. aptamers, oneor more typesof immunostimulatory agents stimulate Spiegelmers®) may be designed and/or identified using B cells, and one or more types of immunostimulatory any available method. In some embodiments, nucleic ac- agents stimulate T cells. id targeting moieties are designed and/or identified by 15 [0231] In some embodiments, a vaccine nanocarrier identifying nucleic acid targeting moieties from a candi- comprises at least one type of immunostimulatory agent date mixture of nucleic acids. Systemic Evolution of Lig- that is associated with the exterior surface of the vaccine ands by Exponential Enrichment (SELEX), or a variation nanocarrier. The association is covalent. In some em- thereof, is a commonly used method of identifying nucleic bodiments, the covalent association is mediated by one acid targeting moieties that bind to a target from a can- 20 or more linkers. Association of immunostimulatory didate mixture of nucleic acids (see, e.g., U.S. Patents agents with vaccine nanocarriers is described in further 6,482,594; 6,458,543; 6,458,539; 6,376,190; 6,344,318; detail below, in the section entitled "Production of Vac- 6,242,246; 6,184,364; 6,001,577; 5,958,691; 5,874,218; cine Nanocarriers." 5,853,984; 5,843,732; 5,843,653; 5,817,785; 5,789,163; [0232] In some embodiments, a vaccine nanocarrier 5,763,177; 5,696,249; 5,660,985; 5,595,877; 5,567,588; 25 comprises a lipid membrane ( e.g., lipid bilayer, lipid mon- and 5,270,163). Alternatively or additionally, Polyplex In olayer, etc.), wherein at least one type of immunostimu- Vivo Combinatorial Optimization (PICO) is a method that latory agent is associated with the lipid membrane. In can be used to identify nucleic acid targeting moieties some embodiments, at least one type of immunostimu- (e.g. aptamers) that bind to a target from a candidate latory agent is embedded within the lipid membrane. In mixture of nucleic acids in vivo and/or in vitro and is de- 30 some embodiments, at least one type of immunostimu- scribed in co-pending PCT Application US06/47975, en- latory agent is embedded within the lumen of a lipid bi- titled "System for Screening Particles," filed December layer. In some embodiments, a vaccine nanocarrier com- 15, 2006. prises at least one type of immunostimulatory agent that is associated with the interior surface of the lipid mem- Immunostimulatory Agents 35 brane. In some embodiments, at least one type of immu- nostimulatory agent may be located at multiple locations [0228] In some embodiments, nanocarriers may trans- of a vaccine nanocarrier. In some embodiments, a first port one or more immunostimulatory agents which can type of immunostimulatory agent and a second type of help stimulate immune responses. In some embodi- immunostimulatory agent may be in the same locale of ments, immunostimulatory agents boost immune re-40 a vaccine nanocarrier ( e.g., they may both be associated sponses by activating APCs to enhance their immunos- with the exterior surface of a vaccine nanocarrier). One timulatory capacity. In some embodiments, immunostim- of ordinary skill in the art will recognize that the preceding ulatory agents boost immune responses by amplifying examples are only representative of the many different lymphocyte responses to specific antigens. In some em- ways in which multiple immunostimulatory agents may bodiments, immunostimulatory agents boost immune re- 45 be associated with different locales of vaccine nanocar- sponses by inducing the local release of mediators, such riers. Multiple immunostimulatory agents may be located as cytokines from a variety of cell types. at any combination of locales of vaccine nanocarriers. [0229] In some embodiments, all of the immunostim- [0233] Immunostimulatory agents include interleukins, ulatory agents of a vaccine nanocarrier are identical to interferon, cytokines, etc. In specific embodiments, an one another. In some embodiments, a vaccine nanocar- 50 immunostimulatory agent may be a natural or synthetic rier comprises a number of different types of immunos- agonist for a Toll-like receptor (TLR). In specific embod- timulatory agents. In some embodiments, a vaccine na- iments, vaccine nanocarriers incorporate a ligand for toll- nocarrier comprises multiple individual immunostimula- like receptor (TLR)-7, such as CpGs, which induce type tory agents, all of which are identical to one another. In I interferon production. Immunostimulatory agents in- some embodiments, a vaccine nanocarrier comprises 55 clude an agonist for the DC surface molecule CD40. To exactly one type of immunostimulatory agent. In some stimulate immunity rather than tolerance, an immunos- embodiments, a vaccine nanocarrier comprises exactly timulatory agent may promote DC maturation (needed two distinct types of immunostimulatory agents. In some for priming of naive T cells) and the production of cy-

35 67 EP 2 630 966 B1 68 tokines, such as type I interferons, which promote anti- Assays for T Cell Activation body responses and anti-viral immunity. In some embod- iments, an immunostimulatory agent may be a TLR-4 [0237] In some embodiments, various assays can be agonist, such as bacterial lipopolysacharide (LPS), VSV- utilized in order to determine whether an immune re- G, and/or HMGB-1. Immunomodulatory agents include 5 sponse has been stimulated in a T cell or group of T cells cytokines, which are small proteins or biological factors (i.e., whether a T cell or group of T cells has become (in the range of 5 kD - 20 kD) that are released by cells "activated"). In some embodiments, stimulation of an im- and have specific effects on cell-cell interaction, commu- mune response in T cells can be determined by meas- nication and behavior of other cells. Immunostimulatory uring antigen-induced production of cytokines by T cells. agents include proinflammatory stimuli released from10 In some embodiments, stimulation of an immune re- necrotic cells (e.g., urate crystals). Immunostimulatory sponse in T cells can be determined by measuring anti- agents include activated components of the complement gen-induced production of IFNγ, IL-4, IL-2, IL-10, IL-17 cascade (e.g., CD21, CD35, etc.). Immunostimulatory and/or TNFα by T cells. In some embodiments, antigen- agents include activated components of immune com- produced production of cytokines by T cells can be meas- plexes. The immunostimulatory agents include TLR-1, 15 ured by intracellular cytokine staining followed by flow TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, cytometry. In some embodiments, antigen-induced pro- TLR-9, and TLR-10 agonists. Immunostimulatory agents duction of cytokines by T cells can be measured by sur- also include complement receptor agonists, such as a face capture staining followed by flow cytometry. In some molecule that binds to CD21 or CD35. A complement embodiments, antigen-induced production of cytokines receptor agonist induces endogenous complement op- 20 by T cells can be measured by determining cytokine con- sonization of the nanocarrier. Immunostimulatory agents centration in supernatants of activated T cell cultures. In also include cytokine receptor agonists, such as a cy- some embodiments, this can be measured by ELISA. tokine. Cytokine receptor agonists include a small mol- [0238] In some embodiments, antigen-produced pro- ecule, antibody, fusion protein, or aptamer. duction of cytokines by T cells can be measured by ELIS- [0234] In some embodiments, there are more than one 25 POT assay. In general, ELISPOT assays employ a tech- type of immunostimulatory agent. In some embodiments, nique very similar to the sandwich enzyme-linked immu- the different immunostimulatory agents each act on a nosorbent assay (ELISA) technique. An antibody e.g( . different pathway. The immunostimulatory agents, there- monoclonal antibody, polyclonal antibody, etc.) is coated fore, can be different Toll-like receptors, a Toll-like re- aseptically onto a PVDF (polyvinylidene fluoride) ceptor and CD40, a Toll-like receptor and a component 30 -backed microplate. Antibodies are chosen for their spe- of the inflammasome, etc. cificity for the cytokine in question. The plate is blocked [0235] In some embodiments, the present invention (e.g. with a serum protein that is non-reactive with any provides pharmaceutical compositions comprising vac- of the antibodies in the assay). Cells of interest are plated cine nanocarriers formulated with one or more adjuvants. out at varying densities, along with antigen or mitogen, 35 The term "adjuvant", as used herein, refers to an agent and then placed in a humidified 37°C CO2 incubator for that does not constitute a specific antigen, but boosts the a specified period of time. Cytokine secreted by activated immune response to the administered antigen. cells is captured locally by the coated antibody on the [0236] In some embodiments, vaccine nanocarriers high surface area PVDF membrane. After washing the are formulated with one or more adjuvants such as gel- wells to remove cells, debris, and media components, a type adjuvants e.g ( ., aluminum hydroxide, aluminum 40 secondary antibody (e.g., a biotinylated polyclonal anti- phosphate, calcium phosphate, etc.), microbial adju- body) specific for the cytokine is added to the wells. This vants (e.g., immunomodulatory DNA sequences that in- antibody is reactive with a distinct epitope of the target clude CpG motifs; endotoxins such as monophosphoryl cytokine and thus is employed to detect the captured lipid A; exotoxins such as cholera toxin, E. coli heat labile cytokine. Following a wash to remove any unbound bi- toxin, and pertussis toxin; muramyl dipeptide, etc.); oil- 45 otinylated antibody, the detected cytokine is then visual- emulsion and emulsifier-based adjuvants ( e.g., Freund’s ized using an avidin-HRP, and a precipitating substrate Adjuvant, MF59 [Novartis], SAF, etc.); particulate adju- (e.g., AEC, BCIP/NBT). The colored end product (a spot, vants (e.g., liposomes, biodegradable microspheres, sa- usually a blackish blue) typically represents an individual ponins, etc.); synthetic adjuvants e.g.,( nonionic block cytokine-producing cell. Spots can be counted manually copolymers, muramyl peptide analogues, polyphosp- 50 (e.g., with a dissecting microscope) or using an automat- hazene, synthetic polynucleotides, etc.), and/or combi- ed reader to capture the microwell images and to analyze nations thereof. Other exemplary adjuvants include spot number and size. In some embodiments, each spot some polymers (e.g., polyphosphazenes, described in correlates to a single cytokine-producing cell. U.S. Patent 5,500,161), QS21, squalene, tetrachlorode- [0239] In some embodiments, an immune response in caoxide, etc. 55 T cells is said to be stimulated if between about 1% and about 100% of antigen-specific T cells produce cy- tokines. In some embodiments, an immune response in T cells is said to be stimulated if at least about 1%, at

36 69 EP 2 630 966 B1 70 least about 5%, at least about 10%, at least about 25%, T cells is measured by assaying cytotoxicity by effector at least about 50%, at least about 75%, at least about CD8+ T cells against antigen-pulsed target cells. For ex- 90%, at least about 95%, at least about 99%, or about ample, a 51chromium (51Cr) release assay can be per- 100% of antigen-specific T cells produce cytokines. formed. In this assay, effector CD8 + T cells bind infected [0240] In some embodiments, an immune response in 5 cells presenting virus peptide on class I MHC and signal T cells is said to be stimulated if immunized subjects com- the infected cells to undergo apoptosis. If the cells are prise at least about 10-fold, at least about 50-fold, at least labeled with 51Cr before the effector CD8+ T cells are about 100-fold, at least about 500-fold, at least about added, the amount of 51Cr released into the supernatant 1000-fold, at least about 5000-fold, at least about 10,000- is proportional to the number of targets killed. fold, at least about 50,000-fold, at least about 100,000- 10 [0245] One of ordinary skill in the art will recognize that fold, or greater than at least about 100,000-fold more the assays described above are only exemplary methods cytokine-producing cells than do naive controls. which could be utilized in order to determine whether T [0241] In some embodiments, stimulation of an im- cell activation has occurred. Any assay known to one of mune response in T cells can be determined by meas- skill in the art which can be used to determine whether uring antigen-induced proliferation of T cells. In some 15 T cell activation has occurred falls within the scope of embodiments, antigen-induced proliferation may be this invention. The assays described herein as well as measured as uptake of H3-thymidine in dividing T cells additional assays that could be used to determine wheth- (sometimes referred to as "lymphocyte transformation er T cell activation has occurred are described in Current test, or "LTT"). In some embodiments, antigen-induced Protocols in Immunology (John Wiley & Sons, Hoboken, proliferation is said to have occurred if H 3-thymidine up- 20 NY, 2007). take (given as number of counts from a γ counter) is at least about 5-fold, at least about 10-fold, at least about B Cells 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least [0246] The present invention provides vaccine nano- about 5000-fold, at least about 10,000-fold, or greater 25 carriers for delivery of, for example, immunomodulatory than at least about 10,000-fold higher than a naive con- agents to the cells of the immune system. In some em- trol. bodiments, vaccine nanocarriers comprise at least one [0242] In some embodiments, antigen-induced prolif- immunomodulatory agent which can be presented to B eration may be measured by flow cytometry. In some cells (i.e., B cell antigens). embodiments, antigen-induced proliferation may be30 measured by a carboxyfluorescein succinimidyl ester Immunomodulatory Agents (CFSE) dilution assay. CFSE is a non-toxic, fluorescent, membrane-permeating dye that binds the amino groups [0247] B cells and T cells recognize antigen by different 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- 35 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 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 (CFSE low) and are distinguishable from other 40 bound-immunoglobulins. cells in culture (CFSEhigh). In some embodiments, anti- [0248] 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 CFSE low cells 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 45 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, [0243] In some embodiments, an immune response in the B cell antigen is a glycoprotein or glycopeptide asso- T cells is said to be stimulated if cellular markers of T cell ciated with an infectious agent. The infectious agent can activation are expressed at different levels (e.g. higher 50 be a bacterium, virus, fungus, protozoan, or parasite. In or lower levels) relative to unstimulated cells. In some some embodiments, the B cell antigen is a poorly immu- embodiments, CD11a CD27, CD25, CD40L, CD44, nogenic antigen. In some embodiments, the B cell anti- CD45RO, and/or CD69 are more highly expressed in ac- gen is an abused substance or a portion thereof. In some tivated T cells than in unstimulated T cells. In some em- embodiments, the B cell antigen is an addictive sub- bodiments, L-selectin (CD62L), CD45RA, and/or CCR7 55 stance or a portion thereof. Addictive substances include, are less highly expressed in activated T cells than in un- but are not limited to, nicotine, a narcotic, a cough sup- stimulated T cells. pressant, a tranquilizer, and a sedative. Examples of ad- [0244] In some embodiments, an immune response in dictive substances include those provided elsewhere

37 71 EP 2 630 966 B1 72 herein. immunomodulatory agents are used as vaccines. In [0249] In some embodiments, the B cell antigen is a some embodiments, antigen presentation to B cells can toxin, such as a toxin from a chemical weapon. In some be optimized by presenting structurally intact immu- embodiments, the toxin from a chemical weapon is bot- nomodulatory agents on the surface of nanocarriers. In ulinum toxin or phosphene. Toxins from a chemical5 some embodiments, structurally intact immunomodula- weapon include, but are not limited to, O-Alkyl (

38 73 EP 2 630 966 B1 74 that is expressed in FDCs but not in non-FDCs. In some geting SCS-Mph upon subcutaneous injection and pre- embodiments, an FDC target is an antigen that is more senting polyvalent conformationally intact antigens on prevalent in FDCs than in non-FDCs. Exemplary FDC their surface can stimulate a potent B cell response. markers are listed below in the section entitled "Follicular [0262] In some embodiments, SCS-Mph targeting is Dendritic Cells" and include those provided elsewhere 5 accomplished by moieties that bind CD169 ( i.e., siaload- herein. hesin), CD11b (i.e., CD11b/CD18, Mac-1, CR3 or αMβ2 [0258] In some embodiments, a target is preferentially integrin), and/or the mannose receptori.e., ( a multi- expressed in particular cell types. For example, expres- valent lectin), proteins which are all prominently ex- sion of an SCS-Mph, FDC, and/or B cell target in SCS- pressed on SCS-Mph. Examples of such moieties in- Mph, FDCs, and/or B cells is at least 2-fold, at least 3- 10 clude those provided elsewhere herein. fold, at least 4-fold, at least 5-fold, at least 10-fold, at least [0263] In some embodiments, SCS-Mph targeting can 20-fold, at least 50-fold, at least 100-fold, at least 500- be accomplished by any targeting moiety that specifically fold, or at least 1000-fold overexpressed in SCS-Mph, binds to any entity e.g ( ., protein, lipid, carbohydrate, FDCs, and/or B cells relative to a reference population. small molecule, etc.) that is prominently expressed In some embodiments, a reference population may com- 15 and/or present on macrophages (i.e., SCS-Mph mark- prise non-SCS-Mph, FDCs, and/or B cells. ers). Exemplary SCS-Mph markers include, but are not [0259] In some embodiments, expression of an SCS- limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, Mph, FDC, and/or B cell target in activated SCS-Mph, MRP-1); CD11a (LFA-1 α, αL Integrinchain); CD11b ( αM FDCs, and/or B cells is at least 2-fold, at least 3-fold, at Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c (aX In- least 4-fold, at least 5-fold, at least 10-fold, at least 20- 20 tegrin, p150, 95, AXb2); CDw12 (p90-120); CD13 (APN, fold, at least 50-fold, at least 100-fold, at least 500-fold, gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, or at least 1000-fold overexpressed in activated SCS- Lewis, X, SSEA-1, 3-FAL); CD15s (Sialyl Lewis X); Mph, FDCs, and/or B cells relative to a reference popu- CD15u (3’ sulpho Lewis X); CD15su (6 sulpho-sialyl lation. In some embodiments, a reference population Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb); CDw17 may comprise non-activated SCS-Mph, FDCs, and/or B 25 (Lactosylceramide, LacCer); CD18 (Integrinβ2, cells. CD11a,b,c β-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein); CD29 (Platelet GPIIa, β-1 integrin, GP); Subcapsular Sinus Macrophage Cells CD31 (PECAM-1, Endocam); CD32 (FCγRII); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, [0260] The present invention encompasses the recog- 30 GPIV, PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cy- nition that targeting of antigens to subcapsular sinus clase, T10); CD39 (ATPdehydrogenase, NTPdehydro- macrophages (SCS-Mph) is involved in efficient early genase-1); CD40 (Bp50); CD43 (Sialophorin, Leukosia- presentation of lymph-borne pathogens, such as viruses, lin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, to follicular B cells (Figure 2). As described in Example B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO 1, following subcutaneous injection of vesicular stomati- 35 (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neu- tis virus (VSV) or adenovirus (AdV) into the footpad of rophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, mice, viral particles were efficiently and selectively re- BCM-1, OX-45); CD49a (VLA-1α, α1 Integrin); CD49b tained by CD169+ SCS-Mph in the draining popliteal (VLA-2α, gpla, α2 Integrin); CD49c (VLA-3α, α3 In- lymph nodes. VSV-specific B cell receptor (BCR) trans- tegrin); CD49e (VLA-5 α, α5 Integrin); CD49f(VLA-6α , α6 genic B cells in these lymph nodes were rapidly activated 40 Integrin, gplc); CD50 (ICAM-3); CD51 (Integrin α, VNR- and generated extremely high antibody titers upon this α, Vitronectin-Rα); CD52 (CAMPATH-1, HE5); CD53 viral challenge. Depletion of SCS-Mph by injection of li- (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58 (LFA-3); posomes laden with clodronate (which is toxic for Mph) CD59 (1F5Ag, H19, Protectin, MACIF, MIRL, P-18); abolished early B cell activation, indicating that SCS-Mph CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP IIIa, are essential for the presentation of lymph-borne partic- 45 β3 Integrin); CD62L (L-selectin, LAM-1, LECAM-1, MEL- ulate antigens to B cells. 14, Leu8, TQ1); CD63 (LIMP, MLA1, gp55, NGA, LAMP- [0261] Bcells are morepotently activated by polyvalent 3, ME491); CD64 (FcγRI); CD65 (Ceramide, VIM-2); antigens that are presented to them on a fixed surface, CD65s (Sialylated-CD65, VIM2); CD72 (Ly-19.2, Ly- rather than in solution. While not wishing to be bound by 32.2, Lyb-2); CD74 (Ii, invariant chain); CD75 (sialo- any one theory, the present invention suggests a reason 50 masked Lactosamine); CD75S (α2,6 sialylated Lactos- why many enveloped viruses (such as VSV) elicit potent amine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 neutralizing antibody responses to their envelope glyco- (4F9, C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a protein: the antigenic protein is presented at a very high (ILT5, LIR2, HL9); CD85d (ILT4, LIR2, MIR10); CD85j density on the surface of the viral particles, and the viral (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86 particles are presented to B cells in a relatively immotile 55 (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc manner, i.e., bound to the plasma membrane of SCS- receptor, FcαR); CD91 (α2M-R, LRP); CDw92 (p70); Mph. The present invention encompasses the recogni- CDw93 (GR11); CD95 (APO-1, FAS, TNFRSF6); CD97 tion that vaccine carriers that mimic viral particles by tar- (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99

39 75 EP 2 630 966 B1 76

(MIC2, E2); CD99R (CD99 Mab restricted); CD100 tivated SCS-Mph marker). Exemplary activated SCS- (SEMA4D); CD101 (IGSF2, P126, V7); CD102 (ICAM- Mph markers include, but are not limited to, CD1a (R4, 2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD44R CD112(HveB, PRR2, PVRL2,Nectin2); CD114 (CSF3R, (CD44v, CD44v9); CD49d (VLA-4α, α4 Integrin); CD69 G-CSRF, HG-CSFR); CD115 (c-fms, CSF-1R, M-CS- 5 (AIM, EA 1, MLR3, gp34/28, VEA); CD105 (Endoglin); FR); CD116 (GM-CSFRα); CDw119 (IFNγR, IFNγRA); CD142 (Tissue factor, Thromboplastin, F3); CD143 CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFR (ACE, Peptidyl dipeptidase A, Kininase II); CD153 p80); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); CD123 (CD3OL, TNSF8); CD163 (M130, GHI/61, RM3/1); (IL-3Rα); CD124 (IL-4Rα); CD127 (p90, IL-7R, IL-7R α); CD166 (ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); CD128a (IL-8Ra, CXCR1, (Tentatively renamed as10 CD227 (MUC1, PUM, PEM, EMA); CD253 (TRAIL, TNF CD181)); CD128b (IL-8Rb, CSCR2, (Tentatively re- (ligand) superfamily, member 10); CD273 (B7DC, named as CD182)); CD130 (gp130); CD131 (Common PDL2); CD274 (B7H1,PDL1); CD275 (B7H2, ICOSL); β subunit); CD132 (Common γ chain, IL-2Rγ); CDw136 CD276 (B7H3); CD297 (ART4, ADP-ribosyltransferase (MSP-R, RON, p158-ron); CDw137 (4-1BB, ILA); 4; and Dombrock blood group glycoprotein; wherein the CD139; CD141 (Thrombomodulin, Fetomodulin); CD147 15 names listed in parentheses represent alternative (Basigin,EMMPRIN, M6, OX47); CD148 (HPTP- η, p260, names. Examples of such markers include those provid- DEP-1); CD155 (PVR); CD156a (CD156, ADAM8, MS2); ed elsewhere herein. CD156b (TACE, ADAM17, cSVP); CDw156C (ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); B Cell Targeting Moieties CD164 (MGC-24, MUC-24); CD165 (AD2, gp37); CD168 20 (RHAMM, IHABP, HMMR); CD169 (Sialoadhesin, Si- [0265] In some embodiments, B cell targeting can be glec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); accomplished by moieties that bind the complement re- CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 ceptors, CR1 (i.e., CD35) or CR2 (i.e., CD21), proteins (RP105, Bgp95, Ly64); CD181 (CXCR1, (Formerly which are expressed on B cells as well as FDCs. In some known as CD128a)); CD182 (CXCR2, (Formerly known 25 embodiments, B cell targeting can be accomplished by as CD128b)); CD184 (CXCR4, NPY3R); CD191 (CCR1); B cell markers such as CD19, CD20, and/or CD22. In CD192 (CCR2); CD195 (CCR5); CDw197 (CCR7 (was some embodiments, B cell targeting can be accom- CDw197)); CDw198 (CCR8); CD204 (MSR); CD205 plished by B cell markers such as CD40, CD52, CD80, (DEC-25); CD206 (MMR); CD207 (Langerin); CDw210 CXCR5, VLA-4, class II MHC, surface IgM or IgD, APRL, (CK); CD213a (CK); CDw217 (CK); CD220 (Insulin R); 30 and/or BAFF-R. The present invention encompasses the CD221 (IGF1 R); CD222 (M6P-R, IGFII-R); CD224 recognition that simultaneous targeting of B cells by moi- (GGT); CD226 (DNAM-1, PTA1); CD230 (Prion Protein eties specific for complement receptors or other APC- (PrP)); CD232 (VESP-R); CD244 (2B4, P38, NAIL); associated molecules boosts humoral responses. CD245 (p220/240); CD256 (APRIL, TALL2, TNF (ligand) [0266] In some embodiments, B cell targeting can be superfamily, member 13); CD257 (BLYS, TALL1, TNF 35 accomplished by any targeting moiety that specifically (ligand) superfamily, member 13b); CD261 (TRAIL-R1, binds to any entity(e.g., protein, lipid, carbohydrate, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, small molecule, etc.) that is prominently expressed TNF-R superfamily, member 10b); CD263 (TRAIL-R3, and/or present on B cells ( i.e., B cell marker). Exemplary TNBF-R superfamily, member 10c); CD264 (TRAIL-R4, B cell markers include, but are not limited to, CD1c TNF-R superfamily, member 10d); CD265 (TRANCE-R, 40 (M241, R7); CD1d (R3); CD2 (E-rosette R, T11, LFA-2); TNF-R superfamily, member 11a); CD277 (BT3.1, B7 CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9 (p24, family: Butyrophilin 3); CD280 (TEM22, ENDO180); DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD281 (TLR1, TOLL-like receptor 1); CD282 (TLR2, CD11b (αM Integrin chain, CR3, Mol, C3niR, Mac-1); TOLL-like receptor 2); CD284 (TLR4, TOLL-like receptor CD11c (aX Integrin, P150, 95, AXb2); CDw17 (Lactos- 4); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase, β3 45 ylceramide, LacCer); CD18 (Integrin β2, CD11a, b, c β- subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, CD300e (CMRF-35L1); CD302 (DCL1); CD305 (LAIR1); EBV-R, C3dR); CD22 (BL-CAM, Lyb8, Siglec-2); CD23 CD312 (EMR2); CD315 (CD9P1); CD317 (BST2); (FceRII, B6, BLAST-2, Leu-20); CD24 (BBA-1, HSA); CD321 (JAM1); CD322 (JAM2); CDw328 (Siglec7); CD25 (Tac antigen, IL-2Rα, p55); CD26 (DPP IV ec- CDw329 (Siglec9); CD68 (gp 110, Macrosialin); and/or 50 toeneyme, ADA binding protein); CD27 (T14, S152); mannose receptor; wherein the names listed in paren- CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM- theses represent alternative names. Examples of such 1, Endocam); CD32 (FCγRII); CD35 (CR1, C3b/C4b re- markers include those provided elsewhere herein. ceptor); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, [0264] In some embodiments, SCS-Mph targeting can T10); CD39 (ATPdehydrogenase, NTPdehydrogenase- be accomplished by any targeting moiety that specifically 55 1); CD40 (Bp50); CD44 (ECMRII, H-CAM, Pgp-1); CD45 binds to any entity e.g ( ., protein, lipid, carbohydrate, (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; small molecule, etc.) that is prominently expressed CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, and/or present on macrophages upon activation (i.e., ac- OA3, Neurophilin); CD47R (MEM-133); CD48 (Blast-1,

40 77 EP 2 630 966 B1 78

Hulym3, BCM-1, OX-45); CD49b (VLA-2α, gpla, α2 In- and/or present on B cells upon activation ( i.e., activated tegrin); CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, B cell marker). Exemplary activated B cell markers in- α4 Integrin); CD50 (ICAM-3); CD52 (CAMPATH-1, clude, but are not limited to, CD1a (R4, T6, HTA-1); CD1b HES); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); (R1); CD15s (Sialyl Lewis X); CD15u (3’ sulpho Lewis CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM- 5 X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki- 1, LECAM-1, MEL-14, Leu8, TQ1); CD72 (Ly-19.2, Ly- 1); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki- 32.2, Lyb-2); CD73 (Ecto-5’-nuciotidase); CD74 (Ii, in- 24, CD27 ligand); CD80 (B7, B7-1, BB1); CD86 variant chain); CD75 (sialo-masked Lactosamine); (B7-2/B70); CD97 (BL-KDD/F12); CD125 (IL-5Rα); CD75S (α2, 6 sialytated Lactosamine); CD77 (Pk anti- CD126 (IL-6Rα); CD138 (Syndecan-1, Heparan sulfate gen, BLA, CTH/Gb3); CD79a (Igα, MB1); CD79b (Igβ, 10 proteoglycan); CD152 (CTLA-4); CD252 (OX40L, B29); CD80; CD81 (TAPA-1); CD82 (4F9, C33, IA4, TNF(ligand) superfamily, member 4); CD253 (TRAIL, KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j TNF(ligand) superfamily, member 10); CD279 (PD1); (ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, CD289 (TLR9, TOLL-like receptor 9); and CD312 TNFRSF6); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, (EMR2); wherein the names listed in parentheses repre- E2); CD100 (SEMA4D); CD102 (ICAM-2); CD10815 sent alternative names. Examples of markers include (SEMA7A, JMH blood group antigen); CDw119 (IFNγR, those provided elsewhere herein. IFNγRa); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); Follicular Dendritic Cells CD124 (IL-4Rα); CD130 (gp130); CD132 (Commonγ chain, IL-2Rγ); CDw137 (4-1BB, ILA); CD139; CD147 20 [0268] B cells that initially detect a previously unknown (Basigin, EMMPRIN, M6, OX47); CD150 (SLAM, IPO- antigen typically express a B cell receptor (BCR, i.e., an 3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); antibody with a transmembrane domain) with suboptimal CD166 (ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); binding affinity for that antigen. However, B cells can in- CD167a (DDR1, trkE, cak); CD171 (L1CMA, NILE); crease by several orders of magnitude the affinity of the CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95,25 antibodies they make when they enter into a germinal Ly64); CD184 (CXCR4, NPY3R); CD185 (CXCR5); center (GC) reaction. This event, which typically lasts CD192 (CCR2); CD196 (CCR6); CD197 (CCR7 (was several weeks, depends on FDC that accumulate, retain CDw197));CDw197 (CCR7, EBI1,BLR2); CD200 (OX2); and present antigenic material to the activated B cells. B CD205 (DEC-205); CDw210 (CK); CD213a (CK); cells, while proliferating vigorously, repeatedly mutate CDw217 (CK); CDw218a (IL18R α); CDw218b (IL18R β); 30 the genomic sequences that encode the antigen binding CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IG- site of their antibody and undergo class-switch recombi- FII-R); CD224 (GGT); CD225 (Leu13); CD226 (DNAM- nation to form secreted high-affinity antibodies, mostly 1, PTA1); CD227 (MUC1, PUM, PEM, EMA); CD229 of the IgG isotype. GC reactions also stimulate the gen- (Ly9); CD230 (Prion Protein (Prp)); CD232 (VESP-R); eration of long-lived memory B cells and plasma cells CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 35 that maintain high protective antibody titers, often for (TRAIL-R1, TNF-R superfamily, member 10a); CD262 many years. Vaccine carriers that target FDC upon sub- (TRAIL-R2, TNF-R superfamily, member 10b); CD263 cutaneous injection and that are retained on the FDC (TRAIL-R3, TNF-R superfamily, member 10c); CD264 surface for long periods of time are predicted to boost (TRAIL-R4, TNF-R superfamily, member 10d); CD265 GC reactions in response to vaccination and improve the (TRANCE-R, TNF-R superfamily, member 11a); CD267 40 affinity and longevity of desired humoral immune re- (TACI, TNF-R superfamily, member 13B); CD268 (BAF- sponses. FR, TNF-R superfamily, member 13C); CD269 (BCMA, [0269] In some embodiments, FDC targeting can be TNF-R superfamily, member 16); CD275 (B7H2, accomplished by moieties that bind the complement re- ICOSL); CD277 (BT3.1.B7 family: Butyrophilin 3); ceptors, CR1 (i.e., CD35) or CR2 (i.e., CD21), proteins CD295 (LEPR); CD298 (ATP1B3 Na K ATPase β3 sub- 45 which are expressed on FDCs as well as B cells. Exam- unit); CD300a (CMRF-35H); CD300c (CMRF-35A); ples of moieties include those provided elsewhere herein. CD305 (LAIR1); CD307 (IRTA2); CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319 (CRACC, Vaccine Nanocarriers Comprising Multiple Targeting SLAMF7); CD321 (JAM1); CD322 (JAM2); CDw327 Moieties (Siglec6, CD33L); CD68 (gp 100, Macrosialin); CXCR5; 50 VLA-4; class II MHC; surface IgM; surface IgD; APRL; [0270] GC reactions and B cell survival not only require and/or BAFF-R; wherein the names listed in parentheses FDC, but also are dependent on help provided by acti- represent alternative names. Examples of markers in- vated CD4 T cells. Help is most efficiently provided when clude those provided elsewhere herein. a CD4 T cell is first stimulated by a DC that presents a [0267] In some embodiments, B cell targeting can be 55 cognate peptide in MHC class II (pMHC) to achieve a accomplished by any targeting moiety that specifically follicular helper (TFH) phenotype. The newly generated binds to any entity e.g., ( protein, lipid, carbohydrate, TFH cell then migrates toward the B follicle and provides small molecule, etc.) that is prominently expressed help to those B cells that present them with the same

41 79 EP 2 630 966 B1 80 pMHC complex. For this, B cells first acquire antigenic comprises at least one targeting moiety that is associated material (e.g., virus or virus-like vaccine), internalize and with the exterior surface of the vaccine nanocarrier. In process it (i.e., extract peptide that is loaded into MHC some embodiments, the association is covalent. In some class II), and then present the pMHC to a T FH cell. embodiments, the covalent association is mediated by [0271] Thus, the present invention encompasses the 5 one or more linkers. In some embodiments, the associ- recognition that a vaccine that stimulates optimal humor- ation is non-covalent. In some embodiments, the non- al immunity can combine several features and compo- covalent association is mediated by charge interactions, nents (Figure 1): (a) antigenic material for CD4 T cells affinity interactions, metal coordination, physical adsorp- that is targeted to and presented by DCs; (b) high density tion, host-guest interactions, hydrophobic interactions, surface antigens that can be presented in their native 10 TT stacking interactions, hydrogen bonding interactions, form by SCS-Mph to antigen-specific follicular B cells; van der Waals interactions, magnetic interactions, elec- (c) the capacity to be acquired and processed by follicular trostatic interactions, dipole-dipole interactions, and/or

B cells for presentation to T FH cells (the present invention combinations thereof. encompasses the recognition that B cells readily acquire [0275] In some embodiments, a vaccine nanocarrier and internalize particulate matter from SCS-Mph); (d) the 15 comprises a lipid membrane ( e.g., lipid bilayer, lipid mon- ability to reach FDC and be retained on FDC in intact olayer, etc.), wherein at least one targeting moiety is as- form and for long periods of time; and (e) adjuvant activity sociated with the lipid membrane. In some embodiments, to render APC fully immunogenic and to avoid or over- at least one targeting moiety is embedded within the lipid come tolerance. membrane. In some embodiments, at least one targeting [0272] In some embodiments, a vaccine nanocarrier 20 moiety is embedded within the lumen of a lipid bilayer. comprises at least one targeting moiety. In some embod- In some embodiments, at least one targeting moiety may iments, all of the targeting moieties of a vaccine nano- be located at multiple locations of a vaccine nanocarrier. carrier are identical to one another. In some embodi- For example, a first targeting moiety may be embedded ments, a vaccine nanocarrier a number of different types within a lipid membrane, and a second immunostimula- of targeting moieties. In some embodiments, a vaccine 25 tory agent may be associated with the exterior surface nanocarrier comprises multiple individual targeting moi- of a vaccine nanocarrier. To give another example, a first eties, all of which are identical to one another. In some targeting moiety and a second targeting moiety may both embodiments, a vaccine nanocarrier comprises exactly be associated with the exterior surface of a vaccine na- one type of targeting moiety. In some embodiments, a nocarrier. vaccine nanocarrier comprises exactly two distinct types 30 of targeting moieties. In some embodiments, a vaccine Immunostimulatory Agents nanocarrier comprises greater than two distinct types of targeting moieties. [0276] As described above, the vaccine nanocarriers [0273] In some embodiments, a vaccine nanocarrier may transport one or more immunostimulatory agents comprises a single type of targeting moiety that directs 35 which can help stimulate immune responses. In some delivery of the vaccine nanocarrier to a single cell type embodiments, a vaccine nanocarrier comprises a single (e.g., delivery to SCS-Mph only). In some embodiments, type of immunostimulatory agent that stimulates both B a vaccine nanocarrier comprises a single type of target- cells and T cells. In some embodiments, a vaccine na- ing moiety that directs delivery of the vaccine nanocarrier nocarrier comprises two types of immunostimulatory to multiple cell types ( e.g., delivery to both SCS-Mph and 40 agents, wherein first type of immunostimulatory agent FDCs). In some embodiments, a vaccine nanocarrier stimulates B cells, and the second type of immunostim- comprises two types of targeting moieties, wherein the ulatory agent stimulates T cells. In some embodiments, first type oftargeting moiety directsdelivery ofthe vaccine a vaccine nanocarrier comprises greater than two types nanocarrier to one cell type, and the second type of tar- of immunostimulatory agents, wherein one or more types geting moiety directs delivery of the vaccine nanocarrier 45 of immunostimulatory agents stimulate B cells, and one to a second cell type. In some embodiments, a vaccine or more types of immunostimulatory agents stimulate T nanocarrier comprises greater than two types of targeting cells. See the section above for a more detailed descrip- moieties, wherein one or more types of targeting moieties tion of immunostimulatory agents that can be used in direct delivery of the vaccine nanocarrier to one cell type, accordance with the present invention. and one or more types of targeting moieties direct deliv- 50 ery of the vaccine nanocarrier to a second cell type. To Assays for B Cell Activation give but one example, a vaccine nanocarrier may com- prise two types of targeting moieties, wherein the first [0277] In some embodiments, various assays can be type of targeting moiety directs delivery of the vaccine utilized in order to determine whether an immune re- nanocarrier to DCs, and the second type of targeting moi- 55 sponse has been stimulated in a B cell or group of B cells ety directs delivery of the vaccine nanocarrier to SCS- (i.e., whether a B cell or group of B cells has become Mph. "activated"). In some embodiments, stimulation of an im- [0274] In some embodiments, a vaccine nanocarrier mune response in B cells can be determined by meas-

42 81 EP 2 630 966 B1 82 uring antibody titers. In general, "antibody titer" refers to [0282] In some embodiments, an immune response in the ability of antibodies to bind and neutralize antigens B cells is said to be stimulated if a vigorous germinal at particular dilutions. For example, a high antibody titer centerreaction occurs. In some embodiments, a vigorous refers to the ability of antibodies to bind and neutralize germinal center reaction can be assessed visually by per- antigens even at high dilutions. In some embodiments, 5 forming histology experiments. In some embodiments, an immune response in B cells is said to be stimulated vigorous germinal center reaction can be assayed by per- if antibody titers are measured to be positive at dilutions forming immunohistochemistry of antigen-containing at least about 5-fold greater, at least about 10-fold great- lymphoid tissues (e.g., vaccine-draining lymph nodes, er, at least about 20-fold greater, at least about 50-fold spleen, etc.). In some embodiments, immunohistochem- greater, at least about 100-fold greater, at least about 10 istry is followed by flow cytometry. 500-fold greater, at least about 1000 fold greater, or more [0283] In some embodiments, stimulation of an im- than about 1000-fold greater than in non-immunized in- mune response in B cells can be determined by identi- dividuals or pre-immune serum. fying antibody isotypes (e.g., IgG, IgA, IgE, IgM). In cer- [0278] In some embodiments, stimulation of an im- tain embodiments, production of IgG isotype antibodies mune response in B cells can be determined by meas- 15 by B cells is a desirable immune response in a B cell. uring antibody affinity. In particular, an immune response [0284] In some embodiments, an immune response in in B cells is said to be stimulated if an antibody has an B cells is determined by analyzing antibody function in -7 equilibrium dissociation constant (Kd) less than 10 M, neutralization assays. In particular, the ability of a micro- less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less organism (e.g., virus, bacterium, fungus, protozoan, par- than 10-11 M, less than 10-12 M, or less. 20 asite, etc.) to infect a susceptible cell line in vitro in the [0279] In some embodiments, a T cell-dependent im- absence of serum is compared to conditions when dif- mune response in B cells is said to be stimulated if class- ferent dilutions of immune and non-immune serum are switch recombinationhas occurred. In particular, aswitch added to the culture medium in which the cells are grown. from IgM to an IgG isotype or to IgA or to a mixture of In certain embodiments, an immune response in a B cell these isotypes is indicative of a T cell dependent immune 25 is said to be stimulated if infection of a microorganism is response in B cells. neutralized at a dilution of about 1:5, about 1:10, about [0280] In some embodiments, an immune response in 1:50, about 1:100, about 1:500, about 1:1000, about B cells is determined by measuring affinity maturation of 1:5000, about 1:10,000, or less. antigen-specific antibodies. Affinity maturation occurs [0285] In some embodiments, the efficacy of vaccines during the germinal center reaction whereby activated B 30 in animal models may be determined by infecting groups cells repeatedly mutate a region of the immunoglobulin of immunized and non-immunized mice (e.g., 3 or more gene that encodes the antigen-binding region. B cells weeks after vaccination) with a dose of a microorganism producing mutated antibodies which have a higher affin- that is typically lethal. The magnitude and duration of ity for antigen are preferentially allowed to survive and survival of both group is monitored and typically graphed proliferate. Thus, over time, the antibodies made by B 35 a Kaplan-Meier curves. To assess whether enhanced cells in GCs acquire incrementally higher affinities. In survival is due to B cell responses, serum from immune some embodiments, the readout of this process is the mice can be transferred as a "passive vaccine" to assess 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 [0286] One of ordinary skill in the art will recognize that dilutions). 40 the assays described above are only exemplary methods [0281] 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 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 45 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 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). cells and/or long-lived plasma cells that can produce50 large amounts of high-affinity antibodies for extended pe- Vaccine Nanocarriers riods of time. In some embodiments, memory B cells and/or long-lived plasma cells that can produce large [0287] 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- 55 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. In some embodi- rapid and result in higher titers after a later booster vac- ments, nanocarriers are biodegradable and biocompat- cination than during the initial sensitization). ible. In general, a biocompatible substance is not toxic

43 83 EP 2 630 966 B1 84 to cells. In some embodiments, a substance is consid- composition. ered to be biocompatible if its addition to cells results in [0291] A variety of different nanocarriers can be used less than a certain threshold of cell death ( e.g. less than in accordance with the present invention. In some em- 50%, 20%, 10%, 5%, or less cell death). In some em- bodiments, nanocarriers are spheres or spheroids. In bodiments, a substance is considered to be biocompat- 5 some embodiments, nanocarriers are spheres or sphe- ible if its addition to cells does not induce adverse effects. roids. In some embodiments, nanocarriers are flat or In general, a biodegradable substance is one that under- plate-shaped. In some embodiments, nanocarriers are goes breakdown under physiological conditions over the cubes or cuboids. In some embodiments, nanocarriers course of a therapeutically relevant time periode.g ( ., are ovals or ellipses. In some embodiments, nanocarriers weeks, months, or years). In some embodiments, a bio- 10 are cylinders, cones, or pyramids. degradable substance is a substance that can be broken [0292] Nanocarriers can be hollow and can comprise down by cellular machinery. In some embodiments, a one or more layers. In some embodiments, each layer biodegradable substance is a substance that can be bro- has a unique composition and unique properties relative ken downby chemical processes.In someembodiments, to the other layer(s). To give but one example, nanocar- a nanocarrier is a substance that is both biocompatible 15 riers may have a core/shell structure, wherein the core and biodegradable. In some embodiments, a nanocarrier is one layer e.g.( a polymeric core) and the shell is a is a substance that is biocompatible, but not biodegrad- second layer (e.g. a lipid bilayer or monolayer). Nano- able. In some embodiments, a nanocarrier is a substance carriers may comprise a plurality of different layers. In that is biodegradable, but not biocompatible. some embodiments, one layer may be substantially [0288] In some embodiments, nanocarriers have a di- 20 cross-linked, a second layer is not substantially cross- ameter of approximately 500 nm. In some embodiments, linked, and so forth. In some embodiments, one, a few, nanocarriers have a diameter of approximately 450 nm. or all of the different layers may comprise one or more In some embodiments, nanocarriers have a diameter of immunomodulatory agents, targeting moieties, immu- approximately 400 nm. In some embodiments, nanocar- nostimulatory agents, and/or combinations thereof. In riers have a diameter of approximately 350 nm. In some 25 some embodiments, one layer comprises an immu- embodiments, nanocarriers have a diameter of approx- nomodulatory agent, targeting moiety, and/or immunos- imately 300 nm. In some embodiments, nanocarriers timulatory agent, a second layer does not comprise an have a diameter of approximately 275 nm. In some em- immunomodulatory agent, targeting moiety, and/or im- bodiments, nanocarriers have a diameter of approxi- munostimulatory agent, and so forth. In some embodi- mately 250 nm. In some embodiments, nanocarriers30 ments, each individual layer comprises a different immu- have a diameter of approximately 225 nm. In some em- nomodulatory agent, targeting moiety, immunostimula- bodiments, nanocarriers have a diameter of approxi- tory agent, and/or combination thereof. mately 200 nm. In some embodiments, nanocarriers have a diameter of approximately 175 nm. In some em- Lipid Vaccine Nanocarriers bodiments, nanocarriers have a diameter of approxi-35 mately 150 nm. In some embodiments, nanocarriers [0293] In some embodiments, nanocarriers may op- have a diameter of approximately 125 nm. In some em- tionally comprise one or more lipids. In some embodi- bodiments, nanocarriers have a diameter of approxi- ments, a nanocarrier may comprise a lipid bilayer. In mately 100 nm. In some embodiments, nanocarriers some embodiments, a nanocarrier may comprise a lipid have a diameter of approximately 75 nm. In some em- 40 monolayer. In some embodiments, a nanocarrier may bodiments, nanocarriers have a diameter of approxi- comprise a core comprising a polymeric matrix surround- mately 50 nm. ed by a lipid layer ( e.g., lipid bilayer, lipid monolayer, etc.). [0289] In certain embodiments, nanocarriers are small [0294] In some embodiments, nanocarriers may com- enough to avoid clearance of nanocarriers from the prise a lipid bilayer oriented such that the interior and bloodstream by the liver ( e.g., nanocarriers having diam- 45 exterior of the nanocarrier are hydrophilic, and the lumen eters of less than 1000 nm). In general, physiochemical of the lipid bilayer is hydrophobic. Comparative examples features of nanocarriers should allow a nanocarrier to of vaccine nanocarriers comprising lipid bilayers are de- circulate longer in plasma by decreasing renal excretion scribed in Example 2 and shown in Figures 3-8. In some and liver clearance. embodiments, hydrophobic immunomodulatory agents, [0290] It is often desirable to use a population of na- 50 targeting moieties, and/or immunostimulatory agents nocarriers that is relatively uniform in terms of size, may be associated with (e.g., embedded within) the lu- shape, and/or composition so that each nanocarrier has men of the lipid bilayer. In some embodiments, hy- similar properties. For example, at least 80%, at least drophilic immunomodulatory agents, targeting moieties, 90%, or at least 95% of the nanocarriers may have a and/or immunostimulatory agents may be associated diameter or greatest dimension that falls within 5%, 10%, 55 with (e.g., covalently or non-covalently associated with, or 20% of the average diameter or greatest dimension. encapsulated within, etc.) the interior and/or exterior of In some embodiments, a population of nanocarriers may the nanocarrier. In some embodiments, hydrophilic im- be heterogeneous with respect to size, shape, and/or munomodulatory agents, targeting moieties, and/or im-

44 85 EP 2 630 966 B1 86 munostimulatory agents may be associated with e.g.,( bond of an unsaturated fatty acid group may be in the cis covalently or non-covalently associated with, encapsu- conformation. In some embodiments, a double bond of lated within, etc.) the interior and/or exterior surface of an unsaturated fatty acid may be in the trans conforma- the lipid bilayer. In some embodiments, the interior, hy- tion. drophilic surface of the lipid bilayer is associated with an 5 [0298] In some embodiments, a fatty acid group may amphiphilic entity. In some embodiments, the am- be one or more of butyric, caproic, caprylic, capric, lauric, phiphilic entity is oriented such that the hydrophilic end myristic, palmitic, stearic, arachidic, behenic, or lignocer- of the amphiphilic entity is associated with the interior ic acid. In some embodiments, a fatty acid group may be surface of the lipid bilayer, and the hydrophobic end of oneor more of palmitoleic,oleic, vaccenic, linoleic, alpha- the amphiphilic entity is oriented toward the interior of 10 linolenic, gamma-linoleic, arachidonic, gadoleic, arachi- the nanocarrier, producing a hydrophobic environment donic, eicosapentaenoic,docosahexaenoic, or erucicac- within the nanocarrier interior. id. [0295] In some embodiments, nanocarriers may com- [0299] In some embodiments, the oil is a liquid triglyc- prise a lipid monolayer oriented such that the interior of eride. the nanocarrier is hydrophobic, and the exterior of the 15 [0300] Suitable oils for use with the present invention nanocarrier is hydrophilic. Comparative examples of vac- include, but are not limited to, almond, apricot kernel, cine nanocarriers comprising lipid monolayers are de- avocado, babassu, bergamot, black current seed, bor- scribed in Example 2 and shown in Figures 9 and 10. In age, cade, camomile, canola, caraway, carnauba, cas- some embodiments, hydrophobic immunomodulatory tor, cinnamon, cocoa butter, coconut, cod liver, coffee, agents, targeting moieties, and/or immunostimulatory 20 corn, cotton seed, emu, eucalyptus, evening primrose, agents may be associated with (e.g., covalently or non- fish, flaxseed, geraniol, gourd, grape seed, hazel nut, covalently associated with, encapsulated within, etc.) the hyssop, jojoba, kukui nut, lavandin, lavender, lemon, lits- interior of the nanocarrier and/or the interior surface of ea cubeba, macademia nut, mallow, mango seed, mead- the lipid monolayer. In some embodiments, hydrophilic owfoam seed, mink, nutmeg, olive, orange, orange immunomodulatory agents, targeting moieties, and/or 25 roughy, palm, palm kernel, peach kernel, peanut, poppy immunostimulatory agents may be associated with ( e.g., seed, pumpkin seed, rapeseed, rice bran, rosemary, saf- covalently or non-covalently associated with, encapsu- flower, sandalwood, sasquana, savoury, sea buckthorn, lated within, etc.) the exterior of the nanocarrier and/or sesame, shea butter, silicone, soybean, sunflower, tea the exterior surface of the lipid monolayer. In some em- tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils, bodiments, the interior, hydrophobic surface of the lipid 30 and combinations thereof. Suitable oils for use with the bilayer is associated with an amphiphilic entity. In some present invention include, but are not limited to, butyl embodiments, the amphiphilic entity is oriented such that stearate, caprylic triglyceride, capric triglyceride, cyclom- the hydrophobic end of the amphiphilic entity is associ- ethicone, diethyl sebacate, dimethicone 360, isopropyl ated with the interior surface of the lipid bilayer, and the myristate, mineral oil, octyldodecanol, oleyl alcohol, sili- hydrophilic end of the amphiphilic entity is oriented to- 35 cone oil, and combinations thereof. ward the interior of the nanocarrier, producing a hy- In some embodiments, a lipid is a hormone (e.g. estro- drophilic environment within the nanocarrier interior. gen, testosterone), steroid (e.g., cholesterol, bile acid), [0296] In some embodiments, a nanocarrier may com- vitamin (e.g. vitamin E), phospholipid (e.g. phosphatidyl prise one or more nanoparticles associated with the ex- choline), sphingolipid e.g. ( ceramides), or lipoprotein terior surface of the nanocarrier. Comparative examples 40 (e.g. apolipoprotein). of vaccine nanocarriers comprising nanoparticles asso- ciated with the exterior surface of the nanocarrier are Nanocarriers Comprising a Polymeric Matrix described in Example 2 and shown in Figures 4, 6, and 8. [0297] In some embodiments, lipids are oils. In gener- [0301] The nanocarriers comprise one or more poly- al, any oil known in the art can be included in nanocarri- 45 mers. In some embodiments, a polymeric matrix can be ers. In some embodiments, an oil may comprise one or surrounded by a coating layer ( e.g., liposome, lipid mon- more fatty acid groups or salts thereof. In some embod- olayer, micelle, etc.). In some embodiments, an immu- iments, a fatty acid group may comprise digestible, long nomodulatory agent, targeting moiety, and/or immunos- chain (e.g., C8-C50), substituted or unsubstituted hydro- timulatory agent can be associated with the polymeric carbons. In some embodiments, a fatty acid group may 50 matrix. In such embodiments, the immunomodulatory be a C10-C20 fatty acid or salt thereof. In some embodi- agent, targeting moiety, and/or immunostimulatory agent ments, a fatty acid group may be a C 15-C20 fatty acid or is effectively encapsulated within the nanocarrier. salt thereof. In some embodiments, a fatty acid group [0302] In some embodiments, an immunomodulatory may be a C15-C25 fatty acid or salt thereof. In some em- agent, targeting moiety, and/or immunostimulatory agent bodiments, a fatty acid group may be unsaturated. In 55 can be covalently associated with a polymeric matrix. In some embodiments, a fatty acid group may be monoun- some embodiments, covalent association is mediated by saturated. In some embodiments, a fatty acid group may a linker. In some embodiments, an immunomodulatory be polyunsaturated. In some embodiments, a double agent and/or targeting moiety can be non-covalently as-

45 87 EP 2 630 966 B1 88 sociated with a polymeric matrix. For example, in some are described in further detail below. In some embodi- embodiments, an immunomodulatory agent and/or tar- ments, a fatty acid group may be one or more of butyric, geting moiety can be encapsulated within, surrounded caproic, caprylic, capric, lauric, myristic, palmitic, stearic, by, and/or dispersed throughout a polymeric matrix. Al- arachidic, behenic, or lignoceric acid. In some embodi- ternatively or additionally, an immunomodulatory agent 5 ments, a fatty acid group may be one or more of palmi- and/or targeting moiety can be associated with a poly- toleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-li- meric matrix by hydrophobic interactions, charge inter- noleic, arachidonic, gadoleic, arachidonic, eicosapen- actions, van der Waals forces, etc. taenoic, docosahexaenoic, or erucic acid. [0303] A wide variety of polymers and methods for [0310] Polymers include polyesters, including copoly- forming polymeric matrices therefrom are known in the 10 mers comprising lactic acid and glycolic acid units, such art of drug delivery. In general, a polymeric matrix com- as poly(lactic acid-co-glycolic acid) and poly(lactide-co- prises one or more polymers. Polymers may be natural glycolide), collectively referred to herein as "PLGA"; and or unnatural (synthetic) polymers. Polymers may be homopolymers comprising glycolic acid units, referred to homopolymers or copolymers comprising two or more herein as "PGA," and lactic acid units, such as poly-L- monomers. In terms of sequence, copolymers may be 15 lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly- random, block, or comprise a combination of random and L-lactide, poly-D-lactide, and poly-D,L-lactide, collective- block sequences. Typically, polymers in accordance with ly referred to herein as "PLA." the present invention are organic polymers. [0311] In some embodiments, a polymer may be [0304] Examples of polymers include polyethylenes, PLGA. PLGA is a biocompatible and biodegradable co- polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhy- 20 polymer of lactic acid and glycolic acid, and various forms drides (e.g. poly(sebacic anhydride)), polyhydroxyacids of PLGA are characterized by the ratio of lactic acid:gly- (e.g. poly(β-hydroxyalkanoate)), polypropylfumerates, colic acid. Lactic acid can be L-lactic acid, D-lactic acid, polycaprolactones, polyamides (e.g. polycaprolactam), or D,L-lactic acid. The degradation rate of PLGA can be polyacetals, polyethers, polyesters ( e.g., polylactide, pol- adjusted by altering the lactic acid:glycolic acid ratio. In yglycolide), poly(orthoesters), polycyanoacrylates, poly- 25 some embodiments, PLGA to be used in accordance with vinyl alcohols, polyurethanes, polyphosphazenes, poly- the present invention is characterized by a lactic acid:gly- acrylates, polymethacrylates, polyureas, polystyrenes, colic acid ratio of approximately 85:15, approximately and polyamines. 75:25, approximately 60:40, approximately 50:50, ap- [0305] Polymers include polymers which have been proximately 40:60, approximately 25:75, or approximate- approved for use in humans by the U.S. Food and Drug 30 ly 15:85. Administration (FDA) under 21 C.F.R. § 177.2600, in- cluding polyesters (e.g., polylactic acid, poly(lactic-co- Nanocarriers Comprising Amphiphilic Entities glycolic acid), polycaprolactone, polyvalerolactone, po- ly(1,3-dioxan-2one)); polyanhydrides ( e.g., poly(sebacic [0312] In some embodiments, nanocarriers may op- anhydride)); polyethers (e.g., polyethylene glycol); poly- 35 tionally comprise one or more amphiphilic entities. In urethanes; polymethacrylates; polyacrylates; and poly- some embodiments, an amphiphilic entity can promote cyanoacrylates. the production of nanocarriers with increased stability, [0306] Polymers can be hydrophilic. For example, pol- improved uniformity, or increased viscosity. In some em- ymers may comprise anionic groups e.g., ( phosphate bodiments, amphiphilic entities can be associated with group, sulphate group, carboxylate group); cationic40 the interior surface of a lipid membrane ( e.g., lipid bilayer, groups (e.g., quaternary amine group); or polar groups lipid monolayer, etc.). For example, if the interior surface (e.g., hydroxyl group, thiol group, amine group). A nano- of a lipid membrane is hydrophilic, the space encapsu- carrier comprising a hydrophilic polymeric matrix gener- lated within the lipid nanocarrier is hydrophilic. However, ates a hydrophilic environment within the nanocarrier. if an amphiphilic entity is associated with the interior sur- [0307] Polymers can be hydrophobic. In some embod- 45 face of the hydrophilic lipid membrane such that the hy- iments,a nanocarriercomprising ahydrophobic polymer- drophilic end of the amphiphilic entity is associated with ic matrix generates a hydrophobic environment within the interior surface of the hydrophilic lipid membrane and the nanocarrier. the hydrophobic end of the amphiphilic entity is associ- [0308] In some embodiments, polymers may be mod- ated with the interior of the nanocarrier, the space en- ified with one or more moieties and/or functional groups. 50 capsulated within the nanocarrier is hydrophobic. Any moiety or functional group can be used in accord- [0313] Any amphiphilic entity known in the art is suit- ance with the present invention. In some embodiments, able for use in making nanocarriers in accordance with polymers may be modified with polyethylene glycol the present invention. Such amphiphilic entities include, (PEG), with a carbohydrate, and/or with acyclic polya- but are not limited to, phosphoglycerides; phosphatidyl- cetals derived from polysaccharides (Papisov, 2001,55 cholines; dipalmitoyl phosphatidylcholine (DPPC); dio- ACS Symposium Series, 786:301). leylphosphatidyl ethanolamine (DOPE); dioleyloxypro- [0309] In some embodiments, polymers may be mod- pyltriethylammonium (DOTMA); dioleoylphosphatidyl- ified with a lipid or fatty acid group, properties of which choline; cholesterol; cholesterol ester; diacylglycerol; di-

46 89 EP 2 630 966 B1 90 acylglycerolsuccinate; diphosphatidyl glycerol (DPPG); iments, the carbohydrate is a sugar alcohol, including but hexanedecanol; fatty alcohols such as polyethylene gly- not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, col (PEG); polyoxyethylene-9-lauryl ether; a surface ac- and lactitol. tive fatty acid, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; 5 Particles Associated with Vaccine Nanocarriers fatty acid amides; sorbitan trioleate (Span ®85) glycocho- late; sorbitan monolaurate (Span®20); polysorbate 20 [0317] In some embodiments, vaccine nanocarriers in (Tween®20); polysorbate 60 (Tween®60); polysorbate accordance with the present invention may comprise one 65 (Tween®65); polysorbate 80 (Tween®80); polysorb- or more particles. In some embodiments, one or more ate 85 (Tween ®85); polyoxyethylene monostearate; sur- 10 particles are associated with a vaccine nanocarrier. In factin; a poloxomer; a sorbitan fatty acid ester such as some embodiments, vaccine nanocarriers comprise one sorbitan trioleate; lecithin; lysolecithin; phosphatidylser- or more particles associated with the outside surface of ine; phosphatidylinositol; sphingomyelin; phosphati- the nanocarrier. In some embodiments, particles may be dylethanolamine (cephalin); cardiolipin; phosphatidic ac- associated with vaccine nanocarriers via covalent link- id; cerebrosides; dicetylphosphate; dipalmitoylphos-15 age. In some embodiments, particles may be associated phatidylglycerol; stearylamine; dodecylamine; hexade- with vaccine nanocarriers via non-covalent interactions cyl-amine; acetyl palmitate; glycerol ricinoleate; hexade- (e.g., charge interactions, affinity interactions, metal co- cyl sterate; isopropyl myristate; tyloxapol; poly(ethylene ordination, physical adsorption, host-guest interactions, glycol)5000-phosphatidylethanolamine; poly(ethylene hydrophobic interactions, TT stacking interactions, hy- glycol)400-monostearate; phospholipids; synthetic20 drogen bonding interactions, van der Waals interactions, and/or natural detergents having high surfactant proper- magnetic interactions, electrostatic interactions, dipole- ties; deoxycholates; cyclodextrins; chaotropic salts; ion dipole interactions, and/or combinations thereof). In pairing agents; and combinations thereof. An amphiphilic some embodiments, vaccine nanocarriers comprise one entity component may be a mixture of different am- or more particles encapsulated within the nanocarrier. In phiphilic entities. These amphiphilic entities may be ex- 25 some embodiments, vaccine nanocarriers comprise one tracted and purified from a natural source or may be pre- or more particles embedded within the surface of the na- pared synthetically in a laboratory. In certain specific em- nocarrier (e.g., embedded within a lipid bilayer). In some bodiments, amphiphilic entities are commercially availa- embodiments, particles associated with a nanocarrier al- ble. low for tunable membrane rigidity and controllable lipo- [0314] Those skilled in the art will recognize that this 30 some stability. is an exemplary, not comprehensive, list of substances [0318] In some embodiments, particles to be associ- with surfactant activity. Any amphiphilic entity may be ated with a vaccine nanocarrier may comprise a poly- used in the production of nanocarriers to be used in ac- meric matrix, as described above. In some embodiments, cordance with the present invention. particles to be associated with a vaccine nanocarrier may 35 comprise non-polymeric components (e.g., metal parti- Vaccine Nanocarriers Comprising Carbohydrates cles, quantum dots, ceramic particles, bone particles, vi- ral particles, etc.), as described above. [0315] In some embodiments, nanocarriers may op- [0319] In some embodiments, a particle to be associ- tionally comprise one or more carbohydrates. ated with a vaccine nanocarrier may have a negative [0316] Carbohydrates may be natural or synthetic. A 40 charge. In some embodiments, a particle to be associat- carbohydrate may be a derivatized natural carbohydrate. ed with a vaccine nanocarrier may have a positive In certain embodiments, a carbohydrate is a monosac- charge. In some embodiments, a particle to be associat- charide, including but not limited to glucose, fructose, ed with a vaccine nanocarrier may be electrically neutral. galactose, ribose, lactose, sucrose, maltose, trehalose, [0320] In some embodiments, the particle has one or cellbiose, mannose, xylose, arabinose, glucoronic acid, 45 more amine moieties on its surface. The amine moieties galactoronic acid, mannuronic acid, glucosamine, gala- can be, for example, aliphatic amine moieties. In certain tosamine, and neuramic acid. In certain embodiments, a embodiments, the amine is a primary, secondary, terti- carbohydrate is a disaccharide, including but not limited ary, or quaternary amine. In certain embodiments, the to lactose, sucrose, maltose, trehalose, and cellobiose. particle comprises an amine-containing polymer. In cer- In certain embodiments, a carbohydrate is a polysaccha- 50 tain embodiments, the particle comprises an amine-con- ride, including but not limited to pullulan, cellulose, mi- taining lipid. In certain embodiments, the particles com- crocrystalline cellulose, hydroxypropyl methylcellulose prises a protein or a peptide that is positively charged at (HPMC), hydroxycellulose (HC), methylcellulose (MC), neutrol pH. In some embodiments, the particle with the dextran, cyclodextran, glycogen, starch, hydroxyethyl- one or more amine moieties on its surface has a net pos- starch, carageenan, glycon, amylose, chitosan, N,O-car- 55 itive charge at neutral pH. Other chemical moieties that boxylmethylchitosan, algin and alginic acid, starch, chi- provide a positive charge at neutrol pH may also be used tin, heparin, konjac, glucommannan, pustulan, heparin, in the inventive particles. hyaluronic acid, curdlan, and xanthan. In certain embod- [0321] Zeta potential is a measurement of surface po-

47 91 EP 2 630 966 B1 92 tential of a particle. In some embodiments, the particle [0325] Methods for making microparticles for delivery has a positive zeta potential. In some embodiments, par- of encapsulated agents are described in the literature ticles have a zeta potential ranging between -50 mV and (see, e.g., Doubrow, Ed., "Microcapsules and Nanopar- +50 mV. In some embodiments, particles have a zeta ticles in Medicine and Pharmacy," CRC Press, Boca Ra- potential ranging between -25 mV and +25 mV. In some 5 ton, 1992; Mathiowitz et al., 1987, J. Control. Release, embodiments, particles have a zeta potential ranging be- 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; tween -10 mV and +10 mV. In some embodiments, par- and Mathiowitz et al., 1988, J. Appl. Polymer Sci., ticles have a zeta potential ranging between -5 mV and 35:755). +5 mV. In some embodiments, particles have a zeta po- [0326] If particles prepared by any of the above meth- tential ranging between 0 mV and +50 mV. In some em- 10 ods have a size range outside of the desired range, par- bodiments, particles have a zeta potential ranging be- ticles can be sized, for example, using a sieve. tween0 mVand +25 mV. Insome embodiments, particles have a zeta potential ranging between 0 mV and +10 Production of Vaccine Nanocarriers mV. In some embodiments, particles have a zeta poten- tial ranging between 0 mV and +5 mV. In some embod- 15 [0327] Vaccine nanocarriers may be prepared using iments, particles have a zeta potential ranging between any method known in the art. For example, particulate -50 mV and 0 mV. In some embodiments, particles have nanocarrier formulations can be formed by methods as a zeta potential ranging between -25 mV and 0 mV. In nanoprecipitation, flow focusing using fluidic channels, some embodiments, particles have a zeta potential rang- spray drying, single and double emulsion solvent evap- ing between -10 mV and 0 mV. In some embodiments, 20 oration, solvent extraction, phase separation, milling, mi- particles have a zeta potential ranging between -5 mV croemulsion procedures, microfabrication, nanofabrica- and 0 mV. In some embodiments, particles have a sub- tion, sacrificial layers, simple and complex coacervation, stantially neutral zeta potential ( i.e. approximately 0 mV). and other methods well known to those of ordinary skill [0322] A variety of different particles can be used in in the art. Alternatively or additionally, aqueous and or- accordance with the present invention. In some embod- 25 ganic solvent syntheses for monodisperse semiconduc- iments, particles are spheres or spheroids. In some em- tor, conductive, magnetic, organic, and other nanoparti- bodiments, particles are spheres or spheroids. In some cles have been described (Pellegrino et al., 2005, Small, embodiments, particles are flat or plate-shaped. In some 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; embodiments, particles are cubes or cuboids. In some and Trindade et al., 2001, Chem. Mat., 13:3843). embodiments, particles are ovals or ellipses. In some 30 [0328] In certain embodiments, vaccine nanocarriers embodiments, particles are cylinders, cones, or pyra- are prepared by the nanoprecipitation process or spray mids. drying. Conditions used in preparing nanocarriers may [0323] Particles (e.g., nanoparticles, microparticles) be altered to yield particles of a desired size or property may be prepared using any method known in the art. For (e.g., hydrophobicity, hydrophilicity, external morpholo- example, particulate formulations can be formed by35 gy, "stickiness," shape, etc.). The method of preparing methods as nanoprecipitation, flow focusing using fluidic the nanocarrier and the conditions ( e.g., solvent, temper- channels, spray drying, single and double emulsion sol- ature, concentration, air flow rate, etc.) used may depend vent evaporation, solvent extraction, phase separation, on the composition and/or resulting architecture of the milling, microemulsion procedures, microfabrication, na- vaccine nanocarrier. nofabrication, sacrificial layers, simple and complex40 [0329] Methods for making microparticles for delivery coacervation, and other methods well known to those of of encapsulated agents are described in the literature ordinary skill in the art. Alternatively or additionally, aque- (see, e.g., Doubrow, Ed., "Microcapsules and Nanopar- ous and organic solvent syntheses for monodisperse ticles in Medicine and Pharmacy," CRC Press, Boca Ra- semiconductor, conductive, magnetic, organic, and other ton, 1992; Mathiowitz et al., 1987, J. Control. Release, nanoparticles have been described (Pellegrino et al.,45 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. and Mathiowitz et al., 1988, J. Appl. Polymer Sci., Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 35:755). 13:3843). [0330] Inventive vaccine nanocarriers may be manu- [0324] In certain embodiments, particles are prepared factured using any available method. It is desirable to by the nanoprecipitation process or spray drying. Condi- 50 associate immunomodulatory agents, targeting moie- tions used in preparing particles may be altered to yield ties, and/or immunostimulatory agents to vaccine nano- particles of a desired size or property ( e.g., hydrophobic- carriers without adversely affecting the 3-dimensional ity, hydrophilicity, external morphology, "stickiness," characteristic and conformation of the immunomodula- shape, etc.). The method of preparing the particle and tory agents, targeting moieties, and/or immunostimula- the conditions ( e.g., solvent, temperature, concentration, 55 tory agents. It is desirable that the vaccine nanocarrier air flow rate, etc.) used may depend on the therapeutic should be able to avoid uptake by the mononuclear agent to be delivered and/or the composition of the pol- phagocytic system after systemic administration so that ymer matrix. it is able to reach specific cells in the body.

48 93 EP 2 630 966 B1 94

[0331] Immunomodulatory agents are released by dif- glycosidase sensitive carbohydrate linkers, pH sensitive fusion, degradation of the vaccine nanocarrier, and/or linkers, hypoxia sensitive linkers, photo-cleavable link- combination thereof. In some embodiments, polymers ers, heat-labile linkers, enzyme cleavable linkers e.g.( degrade by bulk erosion. In some embodiments, poly- esterase cleavable linker), ultrasound-sensitive linkers, mers degrade by surface erosion. 5 x-ray cleavable linkers, etc. In some embodiments, the [0332] Release and delivery of the immunomodulatory linker is not a cleavable linker. agent to a target site occurs by disrupting the association. [0337] Any of a variety of methods can be used to as- For example, if an immunomodulatory agent is associat- sociate a linker with a vaccine nanocarrier. General strat- ed with a nanocarrier by a cleavable linker, the immu- egies include passive adsorption (e.g., via electrostatic nomodulatory agent is released and delivered to the tar- 10 interactions), multivalent chelation, high affinity non-cov- get site upon cleavage of the linker. alent binding between members of a specific binding pair, [0333] Physical association can be achieved in a vari- covalent bond formation, etc. (Gao et al., 2005, Curr. Op. ety of different ways. Physical association may be cov- Biotechnol., 16:63). In some embodiments, click chem- alent or non-covalent. The vaccine nanocarrier, immu- istry can be used to associate a linker with a particle ( e.g. nomodulatory agent, targeting moiety, immunostimula- 15 Diels-Alder reaction, Huigsen 1,3-dipolar cycloaddition, tory agent, and/or nanoparticle may be directly associat- nucleophilic substitution, carbonyl chemistry, epoxida- ed with one another, e.g., by one or more covalent bonds, tion, dihydroxylation, etc.). or may be associated by means of one or more linkers. [0338] A bifunctional cross-linking reagent can be em- In one embodiment, a linker forms one or more covalent ployed. Such reagents contain two reactive groups, or non-covalent bonds with the immunomodulatory20 thereby providing a means of covalently associating two agent, targeting moiety, immunostimulatory agent, target groups. The reactive groups in a chemical cross- and/or nanoparticle and one or more covalent or non- linking reagent typically belong to various classes of func- covalent bonds with the immunomodulatory agent, tar- tional groups such as succinimidyl esters, maleimides, geting moiety, immunostimulatory agent, and/or nano- and pyridyldisulfides. Exemplary cross-linking agents in- particle, thereby attaching them to one another. In some 25 clude, e.g., carbodiimides, N-hydroxysuccinimidyl-4-azi- embodiments, a first linker forms a covalent or non-cov- dosalicylic acid (NHS-ASA), dimethyl pimelimidate dihy- alent bond with the vaccine nanocarrier and a second drochloride (DMP), dimethylsuberimidate (DMS), 3,3’- linker forms a covalent or non-covalent bond with the dithiobispropionimidate (DTBP), N-Succinimidyl 3-[2-py- immunomodulatory agent, targeting moiety, immunos- ridyldithio]-propionamido (SPDP), succimidyl α-methyl- timulatory agent, and/or nanoparticle. The two linkers 30 butanoate, biotinamidohexanoyl-6-amino-hexanoic acid form one or more covalent or non-covalent bond(s) with N-hydroxysuccinimide ester (SMCC), succinimidyl-[(N- each other. maleimidopropionamido)-dodecaethyleneglycol] ester [0334] Any suitable linker can be used in accordance (NHS-PEO12), etc. For example, carbodiimide-mediat- with the present invention. Linkers may be used to form ed amide formation and active ester maleimide-mediated amide linkages, ester linkages, disulfide linkages,etc. 35 amine and sulfhydryl coupling are widely used approach- Linkers may contain carbon atoms or heteroatoms ( e.g., es. nitrogen, oxygen, sulfur, etc.). Typically, linkers are 1 to [0339] In some embodiments, a vaccine nanocarrier 50 atoms long, 1 to 40 atoms long, 1 to 25 atoms long, can be formed by coupling an amine group on one mol- 1 to 20 atoms long, 1 to 15 atoms long, 1 to 10 atoms ecule to a thiol group on a second molecule, sometimes long, or 1 to 10 atoms long. Linkers may be substituted 40 by a two- or three-step reaction sequence. A thiol-con- with various substituents including, but not limited to, hy- taining molecule may be reacted with an amine-contain- drogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, ing molecule using a heterobifunctional cross-linking re- dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, agent, e.g., a reagent containing both a succinimidyl es- aryl, heterocyclic, aromatic heterocyclic, cyano, amide, ter and either a maleimide, a pyridyldisulfide, or an io- carbamoyl, carboxylic acid, ester, thioether, alkylth-45 doacetamide. Amine-carboxylic acid and thiol-carboxylic ioether, thiol, and ureido groups. As would be appreciat- acid cross-linking, maleimide-sulfhydryl coupling chem- ed by one of skill in this art, each of these groups may in istries (e.g., the maleimidobenzoyl-N-hydroxysuccinim- turn be substituted. ide ester (MBS) method), etc., may be used. Polypep- [0335] In some embodiments, a linker is an aliphatic tides can conveniently be attached to particles via amine or heteroaliphatic linker. In some embodiments, the linker 50 or thiol groups in lysine or cysteine side chains respec- is a polyalkyl linker. In certain embodiments, the linker is tively, or by an N-terminal amino group. Nucleic acids a polyether linker. In certain embodiments, the linker is such as RNAs can be synthesized with a terminal amino a polyethylene linker. In certain specific embodiments, group. A variety of coupling reagents (e.g., succinimidyl the linker is a polyethylene glycol (PEG) linker. 3-(2-pyridyldithio)propionate (SPDP) and sulfosuccinim- [0336] In some embodiments, the linker is a cleavable 55 idyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker. To give but a few examples, cleavable linkers in- (sulfo-SMCC) may be used to associate the various com- clude protease cleavable peptide linkers, nuclease sen- ponents of vaccine nanocarriers. Vaccine nanocarriers sitive nucleic acid linkers, lipase sensitive lipid linkers, can be prepared with functional groups, e.g., amine or

49 95 EP 2 630 966 B1 96 carboxyl groups, available at the surface to facilitate as- targeting moiety, and/or nanoparticle via affinity interac- sociation with a biomolecule. tions. For example, biotin may be attached to the surface [0340] Non-covalent specific binding interactions can of the vaccine nanocarrier and streptavidin may be at- be employed. For example, either a particle or a biomol- tached to the immunomodulatory agent, targeting moie- ecule can be functionalized with biotin with the other be- 5 ty, and/or nanoparticle; or conversely, biotin may be at- ing functionalized with streptavidin. These two moieties tached to the immunomodulatory agent, targeting moie- specifically bind to each other non-covalently and with a ty, and/or nanoparticle and the streptavidin may be at- high affinity, thereby associating the particle and the bi- tached to the surface of the vaccine nanocarrier. The omolecule. Other specific binding pairs could be similarly biotin group and streptavidin may be attached to the vac- used. Alternately, histidine-tagged biomolecules can be 10 cine nanocarrier or to the immunomodulatory agent, tar- associated with particles conjugated to nickel-nitrolot- geting moiety, and/or nanoparticle via a linker, such as riaceteic acid (Ni-NTA). an alkylene linker or a polyether linker. Biotin and strepta- [0341] Any biomolecule to be attached to a particle, vidin bind via affinity interactions, thereby binding the targeting moiety, and/or therapeutic agent. The spacer vaccine nanocarrier to the immunomodulatory agent, tar- can be, for example, a short peptide chain, e.g., between 15 geting moiety, and/or nanoparticle. 1 and 10 amino acids in length, e.g., 1, 2, 3, 4, or 5 amino [0346] In some embodiments, a vaccine nanocarrier acids in length, a nucleic acid, an alkyl chain, etc. may be associated with an immunomodulatory agent, [0342] For additional general information on associa- targeting moiety, and/or nanoparticle via metal coordina- tion and/or conjugation methods and cross-linkers, see tion. For example, a polyhistidine may be attached to one the journal Bioconjugate Chemistry, published by the 20 end of the immunomodulatory agent, targeting moiety, American Chemical Society, Columbus OH, PO Box and/or nanoparticle, and a nitrilotriacetic acid can be at- 3337, Columbus, OH, 43210; "Cross-Linking," Pierce tached to the surface of the vaccine nanocarrier. A metal, Chemical Technical Library, available at the Pierce web such as Ni 2+, will chelate the polyhistidine and the nitrilo- site and originally published in the 1994-95 Pierce Cat- triacetic acid, thereby binding the immunomodulatory alog, and references cited therein; Wong SS, Chemistry 25 agent, targeting moiety, and/or nanoparticle to the vac- of Protein Conjugation and Cross-linking, CRC Press cine nanocarrier. Publishers, Boca Raton, 1991; and Hermanson, G. T., [0347] In some embodiments, a vaccine nanocarrier Bioconjugate Techniques, Academic Press, Inc., San Di- may be associated with an immunomodulatory agent, ego, 1996. targeting moiety, and/or nanoparticle via physical ad- [0343] Alternatively or additionally, vaccine nanocarri- 30 sorption. For example, a hydrophobic tail, such as ers can be attached to immunomodulatory agents, tar- polymethacrylate or an alkyl group having at least about geting moieties, and/or nanoparticles directly or indirectly 10 carbons, may be attached to one end of the immu- via non-covalent interactions. Non-covalent interactions nomodulatory agent, targeting moiety, and/or nanoparti- include but are not limited to charge interactions, affinity cle. The hydrophobic tail will adsorb onto the surface of interactions, metal coordination, physical adsorption,35 a hydrophobic vaccine nanocarrier, thereby binding the host-guest interactions, hydrophobic interactions, TT immunomodulatory agent, targeting moiety, and/or nan- stacking interactions, hydrogen bonding interactions, oparticle to the vaccine nanocarrier. van der Waals interactions, magnetic interactions, elec- [0348] In some embodiments, a vaccine nanocarrier trostatic interactions, dipole-dipole interactions, and/or may be associated with an immunomodulatory agent, combinations thereof. 40 targeting moiety, and/or nanoparticle via host-guest in- [0344] In some embodiments, a vaccine nanocarrier teractions. For example, a macrocyclic host, such as cu- may be associated with an immunomodulatory agent, curbituril or cyclodextrin, may be attached to the surface targeting moiety, and/or nanoparticle via charge interac- of the vaccine nanocarrier and a guest group, such as tions. For example, a vaccine nanocarrier may have a an alkyl group, a polyethylene glycol, or a diaminoalkyl cationic surface or may be reacted with a cationic poly- 45 group, may be attached to the immunomodulatory agent, mer, such as poly(lysine) or poly(ethylene imine), to pro- targeting moiety, and/or nanoparticle; or conversely, the vide a cationic surface. The vaccine nanocarrier surface host group may be attached to the immunomodulatory can then bind via charge interactions with a negatively agent, targeting moiety, and/or nanoparticle and the charged immunomodulatory agent, targeting moiety, guest group may be attached to the surface of the vaccine and/or nanoparticle. One end of the immunomodulatory 50 nanocarrier. In some embodiments, the host and/or the agent, targeting moiety, and/or nanoparticle is, typically, guest molecule may be attached to the immunomodula- attached to a negatively charged polymer e.g ( ., a po- tory agent, targeting moiety, and/or nanoparticle or the ly(carboxylic acid)) or an additional oligonucleotide se- vaccine nanocarrier via a linker, such as an alkylene link- quence that can interact with the cationic polymer surface er or a polyether linker. without disrupting the function of the immunomodulatory 55 [0349] In some embodiments, a vaccine nanocarrier agent, targeting moiety, and/or nanoparticle. may be associated with an immunomodulatory agent, [0345] In some embodiments, a vaccine nanocarrier targeting moiety, and/or nanoparticle via hydrogen bond- may be associated with an immunomodulatory agent, ing interactions. For example, an oligonucleotide having

50 97 EP 2 630 966 B1 98 a particular sequence may be attached to the surface of both of which enable tuning of membrane rigidity. This the vaccine nanocarrier, and an essentially complemen- aspect is significant for vaccine delivery using liposomes tary sequence may be attached to one or both ends of to mimic viruses whose stiffness depends on the com- the immunomodulatory agent, targeting moiety, and/or position of other biological components within virus mem- nanoparticle such that it does not disrupt the function of 5 brane. Moreover, adsorbed nanoparticles form a the immunomodulatory agent, targeting moiety, and/or charged shell which protects liposomes against fusion, nanoparticle. The immunomodulatory agent, targeting thereby enhancing liposome stability. Small nanoparti- moiety, and/or nanoparticle then binds to the vaccine na- cles are mixed with liposomes under gentle vortex, and nocarrier via complementary base pairing with the oligo- the nanoparticles stick to liposome surface spontaneous- nucleotide attached to the vaccine nanocarrier. Two oli- 10 ly. Small nanoparticles can be, but are not limited to, pol- gonucleotides are essentially complimentary if about ymeric nanoparticles, metallic nanoparticles, inorganic 80% of the nucleic acid bases on one oligonucleotide or organic nanoparticles, hybrids thereof, and/or combi- form hydrogen bonds via an oligonucleotide base pairing nations thereof. system, such as Watson-Crick base pairing, reverse [0352] In a comparative example, liposome-polymer Watson-Crick base pairing, Hoogsten base pairing, etc., 15 nanocarriers are used to deliver one or a plurality of im- with a base on the second oligonucleotide. Typically, it munomodulatory agents to cells of the immune system is desirable for an oligonucleotide sequence attached to (Figure 5). Instead of keeping the liposome interior hol- the vaccine nanocarrier to form at least about 6 comple- low, hydrophilic immunomodulatory agents can be en- mentary base pairs with a complementary oligonucle- capsulated. Figure 3 shows liposomes that are loaded otide attached to the immunomodulatory agent, targeting 20 with di-block copolymer nanoparticles to form liposome- moiety, and/or nanoparticle. coated polymeric nanocarriers, which have the merits of [0350] In some embodiments, vaccine nanocarriers both liposomes and polymeric nanoparticles, while ex- are made by self-assembly. For a detailed example of cluding some of their limitations. The liposome shell can self-assembly of vaccine nanocarriers, see Examples 1 be used to carry lipophilic or conjugate hydrophilic im- and 2. In a comparative example, small liposomes (10 25 munomodulatory agents, and the polymeric core can be nm - 1000 nm) are manufactured and employed to deliver used to deliver hydrophobic immunomodulatory agents. one or multiple immunomodulatory agents to cells of the [0353] Pre-formulated polymeric nanoparticles are immune system (Figure 3). In general, liposomes are ar- mixed with small liposomes (20 nm - 100 nm) under gen- tificially-constructed spherical lipid vesicles, whose con- tle vortex to induce liposome fusion onto polymeric nan- trollable diameter from tens to thousands of nm signifies 30 oparticle surface. that individual liposomes comprise biocompatible com- [0354] Nanoparticle-stabilized liposome-polymer na- partments with volume from zeptoliters (10-21 L) to fem- nocarriers are used to deliver one or a plurality of immu- toliters (10-15 L) that can be used to encapsulate and nomodulatory agents (Figure 6). By adsorbing small na- store various cargoes such as proteins, enzymes, DNA noparticles (1 nm - 30 nm) to the liposome-polymer na- and drug molecules. Liposomes may comprise a lipid 35 nocarrier surface, the nanocarrier has not only the merit bilayer which has an amphiphilic property: both interior of both aforementioned nanoparticle-stabilized lipo- and exterior surfaces of the bilayer are hydrophilic, and somes (Figure 4) and aforementioned liposome-polymer the bilayer lumen is hydrophobic. Lipophilic molecules nanoparticles (Figure 5), but also tunable membrane ri- can spontaneously embed themselves into liposome gidity and controllable liposome stability. membrane and retain their hydrophilic domains outside, 40 [0355] Liposome-polymer nanocarriers containing re- and hydrophilic molecules can be chemically conjugated verse micelles are used to deliver one or a plurality of to the outer surface of liposome taking advantage of immunomodulatory agents (Figure 7). Since the afore- membrane biofunctionality. mentioned liposome-polymer nanocarriers (Figures 5 [0351] In a comparative example, nanoparticle-stabi- and 6) are limited to carry hydrophobic immunomodula- lized liposomes are used to deliver one or a plurality of 45 tory agents within polymeric nanoparticles, here small immunomodulatory agents to cells of the immune system reverse micelles (1 nm - 20 nm) are formulated to encap- (Figure 4). By allowing small charged nanoparticles (1 sulate hydrophilic immunomodulatory agents and then nm - 30 nm) to adsorb on liposome surface, liposome- mixed with the di-block copolymers to formulate polymer- nanoparticle complexes have not only the merits of afore- ic core of liposomes. mentioned bare liposomes (Figure 3), but also tunable 50 [0356] A hydrophilic immunomodulatory agent to be membrane rigidity and controllable liposome stability. encapsulated is first incorporated into reverse micelles When small charged nanoparticles approach the surface by mixing with naturally derived and non-toxic am- of liposomes carrying either opposite charge or no net phiphilic entities in a volatile, water-miscible organic sol- charge, electrostatic or charge-dipole interaction be- vent. The amphiphilic entity can be, but is not limited to, tween nanoparticles and membrane attracts the nano- 55 one or a plurality of the following: phosphatidylcholine, particles to stay on the membrane surface, being partially lipid A, cholesterol, dolichol, shingosine, sphingomyelin, wrapped by lipid membrane. This induces local mem- ceramide, glycosylceramide, cerebroside, sulfatide, brane bending and globule surface tension of liposomes, phytosphingosine, phosphatidylethanolamine, phos-

51 99 EP 2 630 966 B1 100 phatidylglycerol, phosphatidylinositol, phosphatidylser- or isopropyl alcohol. The partially water miscible organic ine, cardiolipin, phosphatidic acid, and lysophophatides. solvent can be: acetonitrile, tetrahydrofuran, ethyl ace- The volatile, water-miscible organic solvent can be, but tate, isopropyl alcohol, isopropyl acetate, or dimethylfor- is not limited to: tetrahydrofuran, acetone, acetonitrile, or mamide. The resulting polymer solution is added to the dimethylformamide. A biodegradable polymer can be 5 aqueous solution of conjugated and unconjugated lipid added to this mixture after reverse micelle formation is to yield nanoparticles by the rapid diffusion of the organic complete. The resulting biodegradable polymer-reverse solvent into the water and evaporation of the organic sol- micelle mixture is combined with a polymer-insoluble hy- vent. drophilic non-solvent to form nanoparticles by the rapid [0360] Lipid monolayer stabilized polymeric nanopar- diffusion of the solvent into the non-solvent and evapo- 10 ticles comprising reverse micelles can be used to deliver ration of the organic solvent. The polymer-insoluble hy- one or a plurality of immunomodulatory agents (Figure drophilic non-solvent can be one or a plurality of the fol- 10). Since the aforementioned lipid-stabilized polymeric lowing: water, ethanol, methanol, and mixtures thereof. nanocarriers (Figure 9) are limited to carry hydrophobic Reverse micelle contained polymeric nanoparticles are immunomodulatory agents, here, small reverse micelles mixed with lipid molecules to form the aforementioned 15 (1 nm - 20 nm) are formulated to encapsulate hydrophilic liposome-polymer complex structure (Figure 5). immunomodulatory agents and mixed with biodegrada- [0357] Nanoparticle-stabilized liposome-polymer na- ble polymers to form polymeric nanocarrier core. nocarriers containing reverse micelles can be used to [0361] It is to be understood that the compositions of deliver one or a plurality of immunomodulatory agents the invention can be made in any suitable manner. Se- (Figure 8). By adsorbing small nanoparticles (1 nm - 30 20 lection of an appropriate method may require attention nm) to a liposome-polymer nanocarrier surface, the na- to the properties of the particular moieties being associ- nocarrier has not only the merit of both aforementioned ated. nanoparticle-stabilized liposomes (Figure 4) and afore- [0362] If desired, various methods may be used to sep- mentioned reverse micelle contained liposome-polymer arate vaccine nanocarriers with an attached immu- nanoparticles (Figure 7), but also tunable membrane ri- 25 nomodulatory agent, targeting moiety, immunostimula- gidity and controllable liposome stability. tory agent, and/or nanoparticle from vaccine nanocarri- [0358] Lipid monolayer stabilized polymeric nanocar- ers to which the immunomodulatory agent, targeting moi- riers can be used to deliver one or a plurality of immu- ety, immunostimulatory agent, and/or nanoparticle has nomodulatory agents (Figure 9). As compared to afore- not become attached, or to separate vaccine nanocarri- mentioned liposome-polymer nanocarrier (Figures 5-8), 30 ers having different numbers of immunomodulatory this system has the merit of simplicity in terms to both agents, targeting moieties, immunostimulatory agents, agents and manufacturing. A hydrophobic homopolymer and/or nanoparticles attached thereto. For example, size can form the polymeric core in contrast to the di-block exclusion chromatography, agarose gel electrophoresis, copolymer used in Figures 5-8, which has both hydro- or filtration can be used to separate populations of vac- phobic and hydrophilic segments. Lipid-stabilized poly- 35 cine nanocarriers having different numbers of entities at- meric nanocarriers can be formed within one single step tached thereto and/or to separate vaccine nanocarriers instead of formulating polymeric nanoparticle and lipo- from other entities. Some methods include size-exclu- some separately followed by fusing them together. sion or anionexchange chromatography. [0359] A hydrophilic immunomodulatory molecule can [0363] In some embodiments, inventive vaccine nano- be first chemically conjugated to lipid headgroup. The 40 carriers are manufactured under sterile conditions. This conjugate is mixed with a certain ratio of unconjugated can ensure that resulting vaccines are sterile and non- lipid molecules in an aqueous solution containing one or infectious, thus improving safety when compared to live more water-miscible solvents. The amphiphilic entity can vaccines. This provides a valuable safety measure, es- be one or a plurality of the following: phosphatidylcholine, pecially when subjects receiving vaccine have immune lipid A, cholesterol, dolichol, shingosine, sphingomyelin, 45 defects, are suffering from infection, and/or are suscep- ceramide, cerebroside, sulfatide, phytosphingosine, tible to infection. phosphatidylethanolamine, glycosylceramide, phos- [0364] In some embodiments, inventive vaccine nano- phatidylglycerol, phosphatidylinositol, phosphatidylser- carriers may be lyophilized and stored in suspension or ine, cardiolipin, phosphatidic acid, and lysophosphati- as lyophilized powder depending on the formulation strat- des. The water miscible solvent can be: acetone, ethanol, 50 egy for extended periods without losing activity. methanol, and isopropyl alcohol. A biodegradable poly- meric material is mixed with the hydrophobic immu- Applications nomodulatory agents to be encapsulated in a water mis- cible or partially water miscible organic solvent. The bi- [0365] Thecompositions described herein can be used odegradable polymer can be one or a plurality of the fol- 55 to induce, enhance, direct, or redirect an immune re- lowing: poly(D,L-lactic acid), poly(D,L-glycolic acid), or sponse. The compositions described herein can be used their copolymers at various molar ratios. The water mis- for the prophylaxis and/or treatment of any cancer, infec- cible organic solvent can be: acetone, ethanol, methanol, tious disease, metabolic disease, degenerative disease,

52 101 EP 2 630 966 B1 102 autoimmune disease, allergic disease, inflammatory dis- of microbial infection, exposure to the toxin, abused sub- ease, immunological disease, or other disorder and/or stance, addictive substance, etc. and/or onset of symp- condition. The compositions described herein can also toms related thereto; at risk individuals ( e.g., patients ex- be used for the treatment of an addiction, such as an posed to individuals suffering from microbial infection, addiction to any of the addictive substances described 5 traveling to locations where microbes/toxins are preva- herein. The compositions described herein can also be lent; etc.) can be treated substantially contemporaneous- used for the prophylaxis and/or treatment of a condition ly with (e.g., within 48 hours, within 24 hours, or within resulting from the exposure to a toxin, hazardous sub- 12hours of) the onset ofsymptoms of and/or exposure/in- stance, environmental toxin, or other harmful agent. Sub- gestion. Of course individuals known to have microbial jects include, but are not limited to, humans and/or other 10 infection, have been exposed to a toxin, or ingested an primates; mammals, including commercially relevant abused or additive substance may receive treatment at mammals such as cattle, pigs, horses, sheep, cats, any time. and/or dogs; and/or birds, including commercially rele- [0369] Prophylactic and/or therapeutic protocols in- vant birds such as chickens, ducks, geese, and/or tur- volve administering a therapeutically effective amount of keys. 15 one or more inventive vaccine nanocarriers to a subject [0366] In some embodiments, vaccine nanocarriers in such that an immune response is stimulated in both T accordance with the present invention may be used to cells and B cells. treat, alleviate, ameliorate, relieve, delay onset of, inhibit [0370] By combining selected immunomodulatory progression of, reduce severity of, and/or reduce inci- agents with targeting moieties and immunostimulatory dence of one or more symptoms or features of a disease, 20 agents for different APCs, immune responses e.g( . ef- disorder, and/or condition. In some embodiments, inven- fector responses) can be tailored to preferentially elicit tive vaccine nanocarriers may be used to treat, alleviate, the most desirable type of immune response for a given ameliorate, relieve, delay onset of, inhibit progression of, indication, e.g., humoral response, type 1 T cell re- reduce severity of, and/or reduce incidence of one or sponse,type 2 Tcell response, cytotoxicT cell, response, more symptoms or features of microbial infection e.g( . 25 and/ora combination ofthese responses. Thus,the same bacterial infection, fungal infection, viral infection, para- platform may be used for a broad range of different clin- sitic infection, etc.). ical applications, including prophylactic vaccines to a [0367] In one aspect of the invention, a nanocarrier for host of pathogens as well as immunotherapy of existing the prophylaxis and/or treatment of a disease, disorder, diseases, such as infections, allergies, autoimmune dis- or condition (e.g., a microbial infection) is provided. In 30 eases, and/or cancer. some embodiments, the prophylaxis and/or treatment of [0371] Cancers include but are not limited to biliary the disease, disorder, or condition comprises adminis- tract cancer; braincancer; breast cancer; cervical cancer; tering a therapeutically effective amount of inventive vac- choriocarcinoma; colon cancer; endometrial cancer; es- cine nanocarriers to a subject in need thereof, in such ophageal cancer; gastric cancer; intraepithelial neo- amounts and for such time as is necessary to achieve 35 plasms;lymphomas; liver cancer; lung cancer (e.g., small the desired result. In certain embodiments of the present cell and non-small cell); melanoma; neuroblastomas; invention a "therapeutically effective amount" of an in- oral cancer; ovarian cancer; pancreatic cancer; prostate ventive vaccine nanocarrier is that amount effective for cancer; rectal cancer; sarcomas; skin cancer; testicular treating, alleviating, ameliorating, relieving, delaying on- cancer; thyroid cancer; and renal cancer, as well as other set of, inhibiting progression of, reducing severity of,40 carcinomas and sarcomas. and/or reducing incidence of one or more symptoms or [0372] Autoimmune diseases include, but are not lim- features of microbial infection. In some embodiments, a ited to, rheumatoid arthritis, rheumatic fever, ulcerative "therapeutically effective amount" is an amount effective colitis, celiac disease, Crohn’s disease, inflammatory to modulate the immune system. Such an amount may bowel disease, insulin-dependent diabetes mellitus, di- be an immunogenic amount, i.e., an amount sufficient to 45 abetes mellitus, juvenile diabetes, spontaneous autoim- elicit a detectable immune response in a subject, e.g., a mune diabetes, gastritis, autoimmune atrophic gastritis, detectable antibody response and/or detectable T cell autoimmune hepatitis, thyroiditis, Hashimoto’s thyroidi- response. tis, autoimmune thyroiditis, insulitis, oophoritis, orchitis, [0368] Inventive prophylactic and/or therapeutic proto- uveitis, phacogenic uveitis, multiple sclerosis, myasthe- cols involve administering a therapeutically effective50 nia gravis, primary myxoedema, thyrotoxicosis, perni- amount of one or more inventive vaccine nanocarriers to cious anemia, autoimmune haemolytic anemia, Addi- a healthy subject (e.g., a subject who does not display son’s disease, scleroderma, Goodpasture’s syndrome, any symptoms of microbial infection and/or who has not Guillain-Barre syndrome, Graves’ disease, glomerulone- been diagnosed with microbial infection; a subject who phritis, psoriasis, pemphigus vulgaris, pemphigoid, sym- has not yet been exposed to a toxin, a subject who has 55 pathetic opthalmia, idiopathic thrombocylopenic purpu- not yet ingested an abused or addictive substance, etc.). ra, idiopathic feucopenia, Siogren’s syndrome, Wegen- For example, healthy individuals may be vaccinated us- er’s granulomatosis, poly/dermatomyositis or systemic ing inventive vaccine nanocarrier(s) prior to development lupus erythematosus.

53 103 EP 2 630 966 B1 104

[0373] Allergic diseases include, but are not limited to, mercially relevant mammals such as cattle, pigs, horses, eczema, allergic rhinitis or coryza, hay fever, conjuncti- sheep, cats, and/or dogs; and/or birds, including com- vitis, asthma, urticaria (hives), topic allergic reactions, mercially relevant birds such as chickens, ducks, geese, food allergies, anaphylaxis, atopic dermatitis, hypersen- and/or turkeys. sitivity reactions, and other allergic conditions. The aller- 5 [0377] The formulations of the pharmaceutical compo- gic reaction may be the result of an immune reaction to sitions described herein may be prepared by any method any allergen including but not limited to, common dust, known or hereafter developed in the art of pharmaceu- pollen, plants, animal dander, drugs, food allergens, in- tics. In general, such preparatory methods include the sect venom, viruses, or bacteria. step of bringing the active ingredient into association with [0374] Inflammatory disease/disorders include, for ex- 10 one or more excipients and/or one or more other acces- ample, cardiovascular disease, chronic obstructive pul- sory ingredients, and then, if necessary and/or desirable, monary disease (COPD), bronchiectasis, chronic chole- shaping and/or packaging the product into a desired sin- cystitis,tuberculosis, Hashimoto’sthyroiditis, sepsis, sar- gle- or multi-dose unit. coidosis, silicosis and other pneumoconioses, and an im- [0378] A pharmaceutical composition of the invention planted foreign body in a wound, but are not so limited. 15 may be prepared, packaged, and/or sold in bulk, as a As used herein, the term "sepsis" refers to a well-recog- single unit dose, and/or as a plurality of single unit doses. nized clinical syndrome associated with a host’s systemic As used herein, a "unit dose" is discrete amount of the inflammatory response to microbial invasion. The term pharmaceutical composition comprising a predeter- "sepsis" as used herein refers to a condition that is typi- mined amount of the active ingredient. The amount of cally signaled by fever or hypothermia, tachycardia, and 20 the active ingredient is generally equal to the dosage of tachypnea, and in severe instances can progress to hy- the active ingredient which would be administered to a potension, organ dysfunction, and even death. subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dos- Pharmaceutical Compositions age. 25 [0379] The relative amounts of the active ingredient, [0375] The present invention provides novel composi- the pharmaceutically acceptable excipient(s), and/or any tions comprising a therapeutically effective amount of additional ingredients in a pharmaceutical composition one or more vaccine nanocarriers and one or more phar- of the invention will vary, depending upon the identity, maceutically acceptable excipients. In some embodi- size, and/or condition of the subject treated and further ments, the present invention provides for pharmaceutical 30 depending upon the route by which the composition is to compositions comprising inventive vaccine nanocarriers be administered. By way of example, the composition and/or any of the compositions thereof described herein. may comprise between 0.1% and 100% (w/w) active in- Such pharmaceutical compositions may optionally com- gredient. prise one or more additional therapeutically-active sub- [0380] Pharmaceutical formulations of the present in- stances. In accordance with some embodiments, admin- 35 vention may additionally comprise a pharmaceutically istering a pharmaceutical composition comprising inven- acceptableexcipient, which, as usedherein, includes any tive compositions to a subject in need thereof is provided. and all solvents, dispersion media, diluents, or other liq- In some embodiments, inventive compositions are ad- uid vehicles, dispersion or suspension aids, surface ac- ministered to humans. For the purposes of the present tive agents, isotonic agents, thickening or emulsifying invention, the phrase "active ingredient" generally refers 40 agents, preservatives, solid binders, lubricants and the to an inventive vaccine nanocarrier comprising at least like, as suited to the particular dosage form desired. Rem- one immunomodulatory agent and optionally comprising ington’sThe Science andPractice of Pharmacy,21st Edi- one or more targeting moieties, and/or nanoparticles. tion, A. R. Gennaro, (Lippincott, Williams & Wilkins, Bal- [0376] Although the descriptions of pharmaceutical timore, MD, 2006) discloses various excipients used in compositions provided herein are principally directed to 45 formulating pharmaceutical compositions and known pharmaceutical compositions which are suitable for ad- techniques for the preparation thereof. Except insofar as ministration to humans, it will be understood by the skilled any conventional excipient is incompatible with a sub- artisan that such compositions are generally suitable for stance or its derivatives, such as by producing any un- administration toanimals ofall sorts. Modification of phar- desirable biological effect or otherwise interacting in a maceutical compositions suitable for administration to 50 deleterious manner with any other component(s) of the humans in order to render the compositions suitable for pharmaceutical composition, its use is contemplated to administration to various animals is well understood, and be within the scope of this invention. the ordinarily skilled veterinary pharmacologist can de- [0381] In some embodiments, the pharmaceutically sign and/or perform such modification with merely ordi- acceptable excipient is at least 95%, 96%, 97%, 98%, nary, if any, experimentation. Subjects to which admin- 55 99%, or 100% pure. In some embodiments, the excipient istration of the pharmaceutical compositions of the inven- is approved for use in humans and for veterinary use. In tion is contemplated include, but are not limited to, hu- some embodiments, the excipient is approved by United mans and/or other primates; mammals, including com- States Food and Drug Administration. In some embodi-

54 105 EP 2 630 966 B1 106 ments, the excipient is pharmaceutical grade. In some 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. 5 ylene esters e.g (. polyoxyethylene monostearate [0382] Pharmaceutically acceptableexcipients used in [Myrj®45], polyoxyethylene hydrogenated castor oil, pol- 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- 10 ethers, (e.g. polyoxyethylene lauryl ether [Brij®30]), po- servatives, buffering agents, lubricating agents, and/or ly(vinyl-pyrrolidone), diethylene glycol monolaurate, tri- oils. Such excipients may optionally be included in the ethanolamine oleate, sodium oleate, potassium oleate, inventive formulations. Excipients such as cocoa butter ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sul- and suppository waxes, coloring agents, coating agents, fate, Pluronic F 68, Poloxamer 188, cetrimonium bro- sweetening, flavoring, and perfuming agents can be15 mide, cetylpyridinium chloride, benzalkonium chloride, present in the composition, according to the judgment of docusate sodium, etc. and/or combinations thereof. the formulator. [0386] Exemplary binding agents include, but are not [0383] 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- 20 molasses, lactose, lactitol, mannitol,); natural and syn- drogen phosphate, sodium phosphate lactose, sucrose, 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- [0384] Exemplary granulating and/or dispersing25 ypropyl methylcellulose, microcrystalline cellulose, cel- agents include, but are not limited to, potato starch, corn lulose acetate, poly(vinyl-pyrrolidone), magnesium alu- starch, tapioca starch, sodium starch glycolate, clays, minum silicate (Veegum), and larch arabogalactan); al- alginic acid, guar gum, citrus pulp, agar, bentonite, cel- ginates; polyethylene oxide; polyethylene glycol; inor- lulose and wood products, natural sponge, cation-ex- ganic calcium salts; silicic acid; polymethacrylates; wax- change resins, calcium carbonate, silicates, sodium car- 30 es; water; alcohol; etc.; and combinations thereof. bonate, cross-linked poly(vinyl-pyrrolidone) (crospovi- [0387] Exemplary preservatives may include antioxi- done), sodium carboxymethyl starch (sodium starch gly- dants, chelating agents, antimicrobial preservatives, an- colate), carboxymethyl cellulose, cross-linked sodium tifungal preservatives, alcohol preservatives, acidic pre- carboxymethyl cellulose (croscarmellose), methylcellu- servatives, and other preservatives. Exemplary antioxi- lose, pregelatinized starch (starch 1500), microcrystal- 35 dants include, but are not limited to, alpha tocopherol, line starch, water insoluble starch, calcium carboxyme- ascorbic acid, acorbyl palmitate, butylated hydroxy- thyl cellulose, magnesium aluminum silicate (Veegum), anisole, butylated hydroxytoluene, monothioglycerol, po- sodium lauryl sulfate, quaternary ammonium com- tassium metabisulfite, propionic acid, propyl gallate, so- pounds, etc., and combinations thereof. dium ascorbate, sodium bisulfite, sodium metabisulfite, [0385] Exemplary surface active agents and/or emul- 40 and sodium sulfite. Exemplary chelating agents include sifiers include, but are not limited to, natural emulsifiers ethylenediaminetetraacetic acid (EDTA), citric acid (e.g. acacia, agar, alginic acid, sodium alginate, traga- monohydrate, disodium edetate, dipotassium edetate, canth, chondrux, cholesterol, xanthan, pectin, gelatin, edetic acid, fumaric acid, malic acid, phosphoric acid, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sodium edetate, tartaric acid, and trisodium edetate. Ex- colloidal clays e.g ( . bentonite [aluminum silicate] and 45 emplary antimicrobial preservatives include, but are not Veegum [magnesium aluminum silicate]), long chain limited to, benzalkonium chloride, benzethonium chlo- amino acid derivatives, high molecular weight alcohols ride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin chloride, chlorhexidine, chlorobutanol, chlorocresol, monostearate, ethylene glycol distearate, glyceryl mon- chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, ostearate, and propylene glycol monostearate, polyvinyl 50 imidurea, phenol, phenoxyethanol, phenylethyl alcohol, alcohol), carbomers (e.g. carboxy polymethylene, poly- phenylmercuric nitrate, propylene glycol, and thimerosal. acrylic acid, acrylic acid polymer, and carboxyvinyl pol- Exemplary antifungal preservatives include, but are not ymer), carrageenan, cellulosic derivativese.g (. car- limited to, butyl paraben, methyl paraben, ethyl paraben, boxymethylcellulose sodium, powdered cellulose, hy- propyl paraben, benzoic acid, hydroxybenzoic acid, po- droxymethyl cellulose, hydroxypropyl cellulose, hydrox- 55 tassium benzoate, potassium sorbate, sodium benzoate, ypropyl methylcellulose, methylcellulose), sorbitan fatty sodium propionate, and sorbic acid. Exemplary alcohol acid esters (e.g. polyoxyethylene sorbitan monolaurate preservatives include, but are not limited to, ethanol, pol- [Tween®20], polyoxyethylene sorbitan [Tween®60], yethylene glycol, phenol, phenolic compounds, bisphe-

55 107 EP 2 630 966 B1 108 nol, chlorobutanol, hydroxybenzoate, and phenylethyl al- sebacate, dimethicone 360, isopropyl myristate, mineral cohol. Exemplary acidic preservatives include, but are oil, octyldodecanol, oleyl alcohol, silicone oil, and com- not limited to, vitamin A, vitamin C, vitamin E, beta-car- binations thereof. otene, citric acid, acetic acid, dehydroacetic acid, ascor- [0391] Liquid dosage forms for oral and parenteral ad- bic acid, sorbic acid, and phytic acid. Other preservatives 5 ministration include, but are not limited to, pharmaceuti- include, but are not limited to, tocopherol, tocopherol ac- cally acceptable emulsions, microemulsions, solutions, etate, deteroxime mesylate, cetrimide, butylated hy- suspensions, syrups and elixirs. In addition to the active droxyanisol (BHA), butylated hydroxytoluened (BHT), ingredients, the liquid dosage forms may comprise inert ethylenediamine, sodium lauryl sulfate (SLS), sodium diluents commonly used in the art such as, for example, lauryl ether sulfate (SLES), sodium bisulfite, sodium me- 10 water or other solvents, solubilizing agents and emulsi- tabisulfite, potassium sulfite, potassium metabisulfite, fiers such as ethyl alcohol, isopropyl alcohol, ethyl car- Glydant Plus ®, Phenonip ®, methylparaben, Germall 115, bonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Germaben II, Neolone™, Kathon™, and Euxyl®. In cer- propylene glycol, 1,3-butylene glycol, dimethylforma- tain embodiments, the preservative is an anti-oxidant. In mide, oils (in particular, cottonseed, groundnut, corn, other embodiments,the preservative isa chelating agent. 15 germ, olive, castor, and sesame oils), glycerol, tetrahy- [0388] 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, 20 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- cium phosphate, calcium hydroxide phosphate, potassi- fied oils, glycols, polysorbates, cyclodextrins, polymers, um acetate, potassium chloride, potassium gluconate, 25 and combinations thereof. potassium mixtures, dibasic potassium phosphate, [0392] Injectable formulations, for example, sterile in- monobasic potassium phosphate, potassium phosphate jectable aqueous or oleaginous suspensions may be for- mixtures, sodium acetate, sodium bicarbonate, sodium mulated according to the known art using suitable dis- chloride, sodium citrate, sodium lactate, dibasic sodium persing or wetting agents and suspending agents. A ster- phosphate, monobasic sodium phosphate, sodium phos- 30 ile injectable preparation may be a sterile injectable so- phate mixtures, tromethamine, magnesium hydroxide, lution, suspension or emulsion in a nontoxic parenterally aluminum hydroxide, alginic acid, pyrogen-free water, acceptable diluent or solvent, for example, as a solution isotonic saline, Ringer’s solution, ethyl alcohol, etc., and in 1,3-butanediol. Among the acceptable vehicles and combinations thereof. solvents that may be employed are water, Ringer’s so- [0389] Exemplary lubricating agents include, but are 35 lution, U.S.P. and isotonic sodium chloride solution. In not limited to, magnesium stearate, calcium stearate, addition, sterile, fixed oils are conventionally employed stearic acid, silica, talc, malt, glyceryl behanate, hydro- as a solvent or suspending medium. For this purpose genated vegetable oils, polyethylene glycol, sodium ben- any bland fixed oil can be employed including synthetic zoate, sodium acetate, sodium chloride, leucine, mag- mono- or diglycerides. In addition, fatty acids such as nesium lauryl sulfate, sodium lauryl sulfate,etc., and 40 oleic acid are used in the preparation of injectables. combinations thereof. [0393] Injectable formulations can be sterilized, for ex- [0390] Exemplary oils include, but are not limited to, ample, by filtration through a bacterial-retaining filter, or almond, apricot kernel, avocado, babassu, bergamot, by incorporating sterilizing agents in the form of sterile black current seed, borage, cade, camomile, canola, car- solid compositions which can be dissolved or dispersed away, carnauba, castor, cinnamon, cocoa butter, coco- 45 in sterile water or other sterile injectable medium prior to nut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, use. evening primrose, fish, flaxseed, geraniol, gourd, grape [0394] In order to prolong the effect of a drug, it is often seed, hazel nut, hyssop, isopropyl myristate, jojoba, desirable to slow the absorption of the drug from subcu- kukui nut, lavandin, lavender, lemon, litsea cubeba, taneous or intramuscular injection. This may be accom- macademia nut, mallow, mango seed, meadowfoam50 plished by the use of a liquid suspension of crystalline or seed, mink, nutmeg, olive, orange, orange roughy, palm, amorphous material with poor water solubility. The rate palm kernel, peach kernel, peanut, poppy seed, pumpkin of absorption of the drug then depends upon its rate of seed, rapeseed, rice bran, rosemary, safflower, sandal- dissolution which, in turn, may depend upon crystal size wood, sasquana, savoury, sea buckthorn, sesame, shea and crystalline form. Alternatively, delayed absorption of butter, silicone, soybean, sunflower, tea tree, thistle,55 a parenterally administered drug form may be accom- tsubaki, vetiver, walnut, and wheat germ oils. Exemplary plished by dissolving or suspending the drug in an oil oils include, but are not limited to, butyl stearate, caprylic vehicle. triglyceride, capric triglyceride, cyclomethicone, diethyl [0395] Compositions for rectal or vaginal administra-

56 109 EP 2 630 966 B1 110 tion are typically suppositories which can be prepared by rystalline cellulose. In the case of capsules, tablets and mixing vaccine nanocarriersof thisinvention with suitable pills, dosage forms may comprise buffering agents. They non-irritating excipients such as cocoa butter, polyethyl- may optionally comprise opacifying agents and can be ene glycol or a suppository wax which are solid at ambient of a composition that they release the active ingredient(s) temperature but liquid at body temperature and therefore 5 only, or preferentially, in a certain part of the intestinal melt in the rectum or vaginal cavity and release active tract, optionally, in a delayed manner. Examples of em- ingredient. bedding compositions which can be used include poly- [0396] Solid dosage forms for oral administration in- meric substances and waxes. clude capsules, tablets, pills, powders, and granules. In [0399] Dosage forms for topical and/or transdermal ad- such solid dosage forms, the active ingredient is mixed 10 ministration of vaccine nanocarriers in accordance with with at least one inert, pharmaceutically acceptable ex- the invention may include ointments, pastes, creams, lo- cipient such as sodium citrate or dicalcium phosphate tions, gels, powders, solutions, sprays, inhalants and/or and/or a) fillers or extenders such as starches, lactose, patches. Generally, active ingredient is admixed under sucrose, glucose, mannitol, and silicic acid, b) binders sterile conditions with a pharmaceutically acceptable ex- such as, for example, carboxymethylcellulose, alginates, 15 cipient and/or any needed preservatives and/or buffers gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) as may be required. Additionally, the present invention humectants such as glycerol, d) disintegrating agents contemplates the use of transdermal patches, which of- such as agar, calcium carbonate, potato or tapioca ten have the added advantage of providing controlled starch, alginic acid, certain silicates, and sodium carbon- delivery of an active ingredient to the body. Such dosage ate, e) solution retarding agents such as paraffin, f) ab- 20 forms may be prepared, for example, by dissolving and/or sorption accelerators such as quaternary ammonium dispensing the active ingredient in the proper medium. compounds, g) wetting agents such as, for example, cetyl Alternatively or additionally, the rate may be controlled alcohol and glycerol monostearate, h) absorbents such by either providing a rate controlling membrane and/or as kaolin and bentonite clay, and i) lubricants such as by dispersing the active ingredient in a polymer matrix talc, calcium stearate, magnesium stearate, solid poly- 25 and/or gel. ethylene glycols, sodium lauryl sulfate, and mixtures [0400] Suitable devices for use in delivering intrader- thereof. In the case of capsules, tablets and pills, the mal pharmaceutical compositions described herein in- dosage form may comprise buffering agents. clude short needle devices such as those described in [0397] Solid compositions of a similar type may be em- U.S. Patents 4,886,499; 5,190,521; 5,328,483; ployed as fillers in soft and hard-filled gelatin capsules 30 5,527,288; 4,270,537; 5,015,235; 5,141,496; and using such excipients as lactose or milk sugar as well as 5,417,662. Intradermal compositions may be adminis- high molecular weight polyethylene glycols and the like. tered by devices which limit the effective penetration Solid dosage forms of tablets, dragees, capsules, pills, length of a needle into the skin, such as those described and granules can be prepared with coatings and shells in PCT publication WO 99/34850 and functional equiva- such as enteric coatings and other coatings well known 35 lents thereof. Jet injection devices which deliver liquid in the pharmaceutical formulating art. They may option- vaccines to the dermis via a liquid jet injector and/or via ally comprise opacifying agents and can be of a compo- a needle which pierces the stratum corneum and produc- sition that they release the active ingredient(s) only, or es a jet which reaches the dermis are suitable. Jet injec- preferentially, in a certain part of the intestinal tract, op- tion devices are described, for example, in U.S. Patents tionally, in a delayed manner. Examples of embedding 40 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; compositions which can be used include polymeric sub- 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; stances and waxes. Solid compositions of a similar type 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; may be employed as fillers in soft and hard-filled gelatin 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT capsules using such excipients as lactose or milk sugar publications WO 97/37705 and WO 97/13537. Ballistic as well as high molecular weight polethylene glycols and 45 powder/particle delivery devices which use compressed the like. gas to accelerate vaccine in powder form through the [0398] Active ingredients can be in micro-encapsulat- outer layers of the skin to the dermis are suitable. Alter- ed form with one or more excipients as noted above. natively or additionally, conventional syringes may be Solid dosage forms of tablets, dragees, capsules, pills, used in the classical mantoux method of intradermal ad- and granules can be prepared with coatings and shells 50 ministration. such as enteric coatings, release controlling coatings and [0401] Formulations suitable for topical administration other coatings well known in the pharmaceutical formu- include, but are not limited to, liquid and/or semi liquid lating art. In such solid dosage forms, active ingredient preparations such as liniments, lotions, oil in water and/or may be admixed with at least one inert diluent such as water in oil emulsions such as creams, ointments and/or sucrose, lactose or starch. Such dosage forms may com- 55 pastes, and/or solutions and/or suspensions. Topically- prise, as is normal practice, additional substances other administrable formulations may, for example, comprise than inert diluents, e.g., tableting lubricants and other from about 1% to about 10% (w/w) active ingredient, al- tableting aids such a magnesium stearate and microc- though the concentration of the active ingredient may be

57 111 EP 2 630 966 B1 112 as high as the solubility limit of the active ingredient in 500 mm. Such a formulation is administered in the man- the solvent. Formulations for topical administration may ner in which snuff is taken, i.e. by rapid inhalation through further comprise one or more of the additional ingredients the nasal passage from a container of the powder held described herein. close to the nares. [0402] A pharmaceutical composition of the invention 5 [0406] 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 10 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 usinga device comprising adry powder reservoir to which tablets and/or lozenges made using conventional meth- a stream of propellant may be directed to disperse the ods, and may, for example, 0.1% to 20% (w/w) active powder and/or using a self propelling solvent/powder dis- 15 ingredient, the balance comprising an orally dissolvable pensing container such as a device comprising the active and/or degradable composition and, optionally, one or ingredient dissolved and/or suspended in a low-boiling more of the additional ingredients described herein. Al- propellant in a sealed container. Such powders comprise ternately, formulations suitable for buccal administration particles wherein at least 98% of the particles by weight may comprise a powder and/or an aerosolized and/or have a diameter greater than 0.5 mm and at least 95% 20 atomized solution and/or suspension comprising the ac- of the particles by number have a diameter less than 7 tive ingredient. Such powdered, aerosolized, and/or aer- mm. Alternatively, at least 95% of the particles by weight osolized formulations, when dispersed, may have an av- have a diameter greater than 1 mm and at least 90% of erage particle and/or droplet size in the range from about the particles by number have a diameter less than 6 mm. 0.1 mm to about 200 mm, and may further comprise one Dry powder compositions may include a solid fine powder 25 or more of the additional ingredients described herein. diluent such as sugar and are conveniently provided in [0407] A pharmaceutical composition of the invention a unit dose form. may be prepared, packaged, and/or sold in a formulation [0403] Low boiling propellants generally include liquid suitable for ophthalmic administration. Such formulations propellants having a boiling point of below 65°F at at- may, for example, be in the form of eye drops including, mospheric pressure. Generally the propellant may con- 30 for example, a 0.1%/1.0% (w/w) solution and/or suspen- stitute 50% to 99.9% (w/w) of the composition, and the sion of the active ingredient in an aqueous or oily liquid active ingredient may constitute 0.1% to 20% (w/w) of excipient. Such drops may further comprise buffering the composition. The propellant may further comprise agents, salts, and/or one or more other of the additional additional ingredients such as a liquid nonionic and/or ingredients described herein. Other opthalmically-ad- solid anionic surfactant and/or a solid diluent (which may 35 ministrable formulations which are useful include those have a particle size of the same order as particles com- which comprise the active ingredient in microcrystalline prising the active ingredient). form and/or in a liposomal preparation. Ear drops and/or [0404] Pharmaceutical compositions of the invention eye drops are contemplated as being within the scope formulated for pulmonary delivery may provide the active of this invention. ingredient in the form of droplets of a solution and/or sus- 40 [0408] General considerations in the formulation pension. Such formulations may be prepared, packaged, and/or manufacture of pharmaceutical agents may be and/or sold as aqueous and/or dilute alcoholic solutions found,for example,in Remington:The Science and Prac- and/or suspensions, optionally sterile, comprising the ac- tice of Pharmacy 21st ed., Lippincott Williams & Wilkins, tive ingredient, and may conveniently be administered 2005. using any nebulization and/or atomization device. Such 45 formulations may further compriseone or more additional Administration ingredients including, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, abuffering agent, [0409] In some embodiments, a therapeutically effec- a surface active agent, and/or a preservative such as tive amount of an inventive vaccine nanocarrier compo- methylhydroxybenzoate. The droplets provided by this 50 sition is delivered to a patient and/or animal prior to, si- route of administration may have an average diameter multaneously with, and/or after diagnosis with a disease, in the range from about 0.1 mm to about 200 mm. disorder, and/or condition. In some embodiments, a ther- [0405] The formulations described herein as being apeutic amount of an inventive composition is delivered useful for pulmonary delivery are useful for intranasal to a patient and/or animal prior to, simultaneously with, deliveryof a pharmaceutical composition of the invention. 55 and/or after onset of symptoms of a disease, disorder, Another formulation suitable for intranasal administration and/or condition. In some embodiments, the amount of is a coarse powder comprising the active ingredient and a vaccine nanocarrier is sufficient to treat, alleviate, amel- having an average particle from about 0.2 mm to about iorate, relieve, delay onset of, inhibit progression of, re-

58 113 EP 2 630 966 B1 114 duce severity of, and/or reduce incidence of one or more [0412] In general the most appropriate route of admin- symptoms or features of the disease, disorder, and/or istration will depend upon a variety of factors including condition. In some embodiments, the amount of a vac- the nature of the vaccine nanocarrier ( e.g., its stability in cine nanocarrier is sufficient to elicit a detectable immune the environment of the gastrointestinal tract), the condi- response in a subject. In some embodiments, the amount 5 tion of the subject (e.g., whether the subject is able to of a vaccine nanocarrier is sufficient to elicit a detectable tolerate oral administration), etc. The invention encom- antibody response in a subject. In some embodiments, passes the delivery of the inventive pharmaceutical com- the amount of a vaccine nanocarrier is sufficient to elicit position by any appropriate route taking into considera- a detectable T cell response in a subject. In some em- tion likely advances in the sciences of drug delivery. bodiments, the amount of a vaccine nanocarrier is suffi- 10 [0413] In certain embodiments, the vaccine nanocar- cient to elicit a detectable antibody and T cell response riers of the invention may be administered in amounts in a subject. In some embodiments, an advantage of the ranging from about 0.001 mg/kg to about 100 mg/kg, from nanocarriers provided is that the nanocarriers can elicit about 0.01 mg/kg to about 50 mg/kg, from about 0.1 potent responses with a much lower concentration of an- mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about tigen than required with a conventional vaccine. 15 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from [0410] The compositions may be administered using about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg any amount and any route of administration effective for to about 25 mg/kg, of subject body weight per day, one treatment. The exact amount required will vary from sub- or more times a day, to obtain the desired therapeutic ject to subject, depending on the species, age, and gen- effect. The desired dosage may be delivered three times eral condition of the subject, the severity of the infection, 20 a day, two times a day, once a day, every other day, the particular composition, its mode of administration, its every third day, every week, every two weeks, every three mode of activity, and the like. The compositions of the weeks, or every four weeks. In certain embodiments, the invention are typically formulated in dosage unit form for desired dosage may be delivered using multiple admin- ease of administration and uniformity of dosage. It will istrations (e.g., two, three, four, five, six, seven, eight, be understood, however, that the total daily usage of the 25 nine, ten, eleven, twelve, thirteen, fourteen, or more ad- compositions of the present invention will be decided by ministrations). theattending physician within thescope of sound medical [0414] In some embodiments, the present invention judgment. The specific therapeutically effective dose lev- encompasses "therapeutic cocktails" comprising popu- el for any particular subject or organism will depend upon lations of inventive vaccine nanocarriers. In some em- a variety of factors including the disorder being treated 30 bodiments, all of the vaccine nanocarriers within a pop- and the severity of the disorder; the activity of the specific ulation of vaccine nanocarriers comprise a single species active ingredient employed; the specific composition em- of targeting moiety which can bind to multiple targets ( e.g. ployed; the age, body weight, general health, sex and can bind to both SCS-Mph and FDCs). In some embod- diet of the subject; the time of administration, route of iments, different vaccine nanocarriers within a population administration, and rate of excretion of the specific active 35 of vaccine nanocarriers comprise different targeting moi- ingredient employed; the duration of the treatment; drugs eties, and all of the different targeting moieties can bind used in combination or coincidental with the specific ac- to the same target. In some embodiments, different vac- tive ingredient employed; and like factors well known in cine nanocarriers comprise different targeting moieties, the medical arts. and all of the different targeting moieties can bind to dif- [0411] The pharmaceutical compositions of 40 the ferent targets. In some embodiments, such different tar- present invention may be administered by any route. In gets may be associated with the same cell type. In some some embodiments, the pharmaceutical compositions of embodiments, such different targets may be associated the present invention are administered by a variety of with different cell types. routes, including oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, subcutaneous, intra- 45 Combination Therapies ventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, [0415] It will be appreciated that vaccine nanocarriers creams, and/or drops), transdermal, mucosal, nasal, and pharmaceutical compositions of the present inven- buccal, enteral, sublingual; by intratracheal instillation, tion can be employed in combination therapies. The par- bronchial instillation, and/or inhalation; and/or as an oral 50 ticular combination of therapies (therapeutics or proce- spray, nasal spray, and/or aerosol. Specifically contem- dures) to employ in a combination regimen will take into plated routes are oral administration, intravenous injec- account compatibility of the desired therapeutics and/or tion, intramuscular injection, and/or subcutaneous injec- procedures and the desired therapeutic effect to be tion. In some embodiments, inventive vaccine nanocar- achieved. It will be appreciated that the therapies em- riers are administered parenterally. In some embodi-55 ployedmay achieve a desired effectfor the same purpose ments, inventive vaccine nanocarriers are administered (for example, an inventive vaccine nanocarrier useful for intravenously. In some embodiments, inventive vaccine vaccinating against a particular type of microbial infection nanocarriers are administered orally. may be administered concurrently with another agent

59 115 EP 2 630 966 B1 116 useful for treating the same microbial infection), or they with one or more additional therapeutic agents which may achieve different effects (e.g., control of any adverse treat the symptoms of microbial infection. To give but one effects attributed to the vaccine nanocarrier). example, upon exposure to rabies virus, nanocarriers [0416] In some embodiments, pharmaceutical compo- comprising immunomodulatory agents useful for vacci- sitions of the present invention may be administered ei- 5 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 matic of rabies virus infection). these methods of delivery are within the scope of the10 [0422] 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 therapeuticsor medicalprocedures. In gen- less than 30% by weight, less than 20% by weight, less eral, each agent will be administered at a dose and/or on than 15% by weight, less than 10% by weight, less than a time schedule determined for that agent. Additionally, 15 5% by weight, less than 1% by weight, or less than 0.5% the invention encompasses the delivery of the inventive by weight of an agent to be delivered. pharmaceutical compositions in combination with agents [0423] In some embodiments, vaccine nanocarriers thatmay improve their bioavailability, reduce and/ormod- are administered in combination with one or more small ify their metabolism, inhibit their excretion, and/or modify molecules and/or organic compounds with pharmaceu- their distribution within the body. 20 tical activity. In some embodiments, the agent is a clini- [0417] 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 anticancer agent, antibiotic, anti-viral agent, anti-HIV regimen will take into account compatibility of the desired agent, anti-parasite agent, anti-protozoal agent, anes- therapeutics and/or procedures and/or the desired ther- thetic, anticoagulant, inhibitor of an enzyme, steroidal apeutic effect to be achieved. It will be appreciated that 25 agent, steroidal or non-steroidal anti-inflammatory agent, the therapies employed may achieve a desired effect for antihistamine, immunosuppressant agent, anti-neoplas- the same disorder (for example, an inventive vaccine na- tic agent, antigen, vaccine, antibody, decongestant, sed- nocarrier may be administered concurrently with another ative, opioid, analgesic, anti-pyretic, birth control agent, therapeutic agent used to treat the same disorder), hormone, prostaglandin, progestational agent, anti-glau- and/or they may achieve different effects ( e.g., control of 30 coma agent, ophthalmic agent, anti-cholinergic, analge- any adverse effects attributed to the vaccine nanocarri- sic, anti-depressant, anti-psychotic, neurotoxin, hypnot- er). In some embodiments, vaccine nanocarriers of the ic, tranquilizer, anti-convulsant, muscle relaxant, anti- invention are administered with a second therapeutic Parkinson agent, anti-spasmodic, muscle contractant, agent that is approved by the U.S. Food and Drug Ad- channel blocker, miotic agent, anti-secretory agent, anti- ministration. 35 thrombotic agent, anticoagulant, anti-cholinergic,β- [0418] In will further be appreciated that therapeutically adrenergic blockingagent, diuretic, cardiovascular active active agents utilized in combination may be adminis- agent, vasoactive agent, vasodilating agent, anti-hyper- tered together in a single composition or administered tensive agent, angiogenic agent, modulators of cell-ex- separately in different compositions. tracellular matrix interactions (e.g. cell growth inhibitors [0419] In general, it is expected that agents utilized in 40 and anti-adhesion molecules), inhibitors of DNA, RNA, combination with be utilized at levels that do not exceed or protein synthesis, etc. the levels at which they are utilized individually. In some [0424] In certain embodiments, a small molecule agent embodiments, the levels utilized in combination will be can be any drug. In some embodiments, the drug is one lower than those utilized individually. that has already been deemed safe and effective for use [0420] In some embodiments, inventive vaccine nano- 45 in humans or animals by the appropriate governmental carriers may be administered in combination with an agency or regulatory body. For example, drugs approved agent, including, for example, therapeutic, diagnostic, for human use are listed by the FDA under 21 C.F.R. §§ and/or prophylactic agents. Exemplary agents to be de- 330.5, 331 through 361, and 440 through 460; drugs for livered in accordance with the present invention include, veterinary use are listed by the FDA under 21 C.F.R. §§ but are not limited to, small molecules, organometallic 50 500 through 589. All listed drugs are considered accept- compounds, nucleic acids, proteins (including multimeric able for use in accordance with the present invention. proteins, protein complexes, etc.), peptides, lipids, car- [0425] A more complete listing of classes and specific bohydrates,hormones, metals, radioactive elementsand drugs suitable for use in the present invention may be compounds, drugs, vaccines, immunological agents, found in Pharmaceutical Drugs: Syntheses, Patents, Ap- etc., and/or combinations thereof. 55 plications by Axel Kleemann and Jurgen Engel, Thieme [0421] In certain embodiments, vaccine nanocarriers Medical Publishing, 1999 and the Merck Index: An En- which delay the onset and/or progression of a particular cyclopedia of Chemicals, Drugs and Biologicals, Ed. by microbial infection may be administered in combination Budavari et al., CRC Press, 1996.

60 117 EP 2 630 966 B1 118

[0426] In some embodiments, vaccine nanocarriers 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 5 are used to delay the onset and/or progression of a par- Kits ticular microbial infection may be administered in com- bination with RNAi agents which reduce expression of [0431] 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 10 example, the invention provides a kit comprising an in- Targeting Moieties." ventive vaccine nanocarrier and instructions for use. A [0427] 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. delivered may be a peptide, hormone, erythropoietin, in- 15 [0432] Kits typically include instructions for use of in- sulin, cytokine, antigen for vaccination, etc. In some em- ventive vaccine nanocarriers. Instructions may, for ex- bodiments, the agent to be delivered may be an antibody ample, comprise protocols and/or describe conditions for and/or characteristic portion thereof. Molecular proper- production of vaccine nanocarriers, administration of ties of which are described in the section above entitled vaccine nanocarriers to a subject in need thereof,etc. "Protein Targeting Moieties." 20 Kits generally include one or more vessels or containers [0428] In some embodiments, vaccine nanocarriers so that some or all of the individual components and re- are administered in combination with one or more car- agents may be separately housed. Kits may also include bohydrates, such as a carbohydrate that is associated a means for enclosing individual containers in relatively with a protein (e.g. glycoprotein, proteogycan,etc. ). A close confinement for commercial sale,e.g., a plastic carbohydrate may be natural or synthetic. A carbohy- 25 box, in which instructions, packaging materials such as drate may also be a derivatized natural carbohydrate. In styrofoam, etc., may be enclosed. An identifier, e.g., a certain embodiments, a carbohydrate may be a simple bar code, radio frequency identification (ID) tag, etc., may or complex sugar. In certain embodiments, a carbohy- be present in or on the kit or in or one or more of the drate is a monosaccharide, including but not limited to vessels or containers included in the kit. An identifier can glucose, fructose, galactose, and ribose. In certain em- 30 be used, e.g., to uniquely identify the kit for purposes of bodiments, a carbohydrate is a disaccharide, including quality control, inventory control, tracking, movement be- but not limited to lactose, sucrose, maltose, trehalose, tween workstations, etc. and cellobiose. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to cellulose, Exemplification microcrystalline cellulose, hydroxypropyl methylcellu- 35 lose (HPMC), methylcellulose (MC), dextrose, dextran, Example 1: Subcapsular sinus macrophages in lymph glycogen, xanthan gum, gellan gum, starch, and pullulan. nodes clear lymph-borne viruses and present them to In certain embodiments, a carbohydrate is a sugar alco- antiviral B cells hol, including but not limited to mannitol, sorbitol, xylitol, erythritol, malitol, and lactitol. Molecular properties of car- 40 Materials and Methods bohydrates are described in the section above entitled "Vaccine Nanocarriers Comprising Carbohydrates." Method Summary [0429] In some embodiments, vaccine nanocarriers are administered in combination with one or more lipids, [0433] VSV-IND and VSV-NJ virions were purified such as a lipid that is associated with a protein (e.g. li- 45 from culture supernatants of infected BSRT7 cells and poprotein). Exemplary lipids that may be used in accord- used either unmodified or fluorescently labeled with Al- ance with the present invention include, but are not lim- exa-568 (red) or Alexa-488 (green). Fluorescent viruses ited to, oils, fatty acids, saturated fatty acid, unsaturated used for tissue imaging were UV-irradiated to prevent fatty acids, essential fatty acids, cis fatty acids, trans fatty generation of non-fluorescent progeny. Fluorescent la- acids, glycerides, monoglycerides, diglycerides, triglyc- 50 beling or UV-irradiation of VSV-IND particles did not af- erides, hormones, steroids ( e.g., cholesterol, bile acids), fect their antigenicity or their ability to elicit a calcium flux vitamins (e.g. vitamin E), phospholipids, sphingolipids, in VI10YEN cells (not shown). Following fluorescent virus and lipoproteins. Molecular properties of lipids are de- injection into footpads, draining popliteal LNs were har- scribed in the section above entitled "Lipid Vaccine Na- vested for analysis by electron microscopy or to generate nocarriers." 55 frozen sections for immunostaining and confocal micro- [0430] Those skilled in the art will recognize that this scopy. To image adoptively transferred B cells in LNs, is an exemplary, not comprehensive, list of therapeutic, VI10YEN and wildtype B cells were fluorescently labeled diagnostic, and/or prophylactic agents that can be deliv- and co-transferred by i.v. injection into wildtype or mutant

61 119 EP 2 630 966 B1 120 recipient mice. 18 hours later, when B cells had homed et al., 2003, Proc. Natl. Acad. Sci., USA, 100:12883) was to B cell follicles, mice were injected with labeled or un- produced from hybridoma supernatants according to labeledVSV inthe rightfootpad. At different timeintervals standard methods. thereafter,the drainingpopliteal LN was observed by MP- IVM or harvested for confocal microscopy or for flow cy- 5 Flow Cytometry tometry to analyze the activation state of virus-specific and control B cells. In some experiments, macrophages [0438] Flow cytometric analysis of blood samples was in the popliteal LN were depleted by sc injections of CLL, performed afterretro-orbital phlebotomy ofmice andlysis and animals were used for experiments 7-10 days later. of erythrocytes with ACK buffer (0.15 M NH4Cl, 1 mM 10 MP-IVM, electron microscopy, immunohistochemistry KHCO3, 0.1 mM EDTA (disodium salt), pH 7.2). Single- and flow cytometry for various markers was performed cell suspensions of LNs and spleens for flow cytometry on LNs with and without prior CLL treatment. VSV prop- were generated by careful mincing of tissues and sub- agation from the footpad injection site to the blood and sequent digestion at 37°C for 40 minutes in DMEM (In- other organs was assessed by injecting a defined amount vitrogen-Gibco) in the presence of 250 mg/ml liberase CI of live VSV into footpads followed by tissue harvest at 15 (Roche)plus 50 mg/mlDNase-I (Roche). After 20 minutes two hours or six hours after VSV injection. To measure of digestion, samples were vigorously passed through viral titers, tissues were homogenized and used in plaque an 18G needle to ensure complete organ dissociation. assays. Some viral propagation experiments were per- All flow cytometric analyses were performed in FACS formed after cannulation of the thoracic duct. buffer containing PBS with 2 mM EDTA and 2% FBS 20 (Invitrogen-GIBCO) on a FACScalibur (BD Pharmingen), Mice and Antibodies and analyzed by FlowJo software (Treestar Inc., Ash- land, OR). For calcium flux, cells were labeled with 4 mM [0434] C57BL/6 and BALB/c mice were purchased Fluo-LOJO (Teflabs) in DMEM containing 10% FCS for from Taconic Farms (Germantown, NY). VI10YEN 90 minutes at 37°C. Cells were spun through FCS and (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA, 25 used immediately. 100:12883), C3 -/- (Wessels et al., 1995, Proc. Natl. Acad. Sci., USA, 92:11490), MHCII-EGFP (Boes et al., 2002, Viruses and VSV Plaque Assay Nature, 418: 983), Act-EGFP (Wright et al., 2001, Blood, 97:2278), and DH-LMP2A mice (Casola et al., 2004, Nat. [0439] VSV serotypes Indiana (VSV-IND, Mudd-Sum- Immunol., 5:317) were bred in barrier animal facilities at 30 mers derived clone, in vitro rescued (Whelan et al., 1995, Harvard Medical School and the Immune Disease Insti- Proc. Natl. Acad. Sci., USA, 92:8388) and plaque puri- tute (IDI). Radiation chimeras were generated by irradi- fied) or New Jersey (VSV-NJ, Pringle Isolate, plaque pu- ation of Act(EGFP) mice with two doses of 650 rad and rified) were propagated at a MOI of 0.01 on BSRT7 cells. reconstitution with C57BL/6 bone marrow, and were al- Supernatants of infected cells were cleared from cell de- lowed to reconstitute for 8 weeks prior to use. In some 35 bris by centrifugation at 2000 x g, filtered through 0.45 experiments, SCS macrophages were depleted by foot- mm sterile filters and subjected to ultracentrifugation at pad injections of 30 ml clodronate liposomes (CLL), 7-10 40,000 x g for 90 minutes. Pellets were resuspended in days prior to the experiment. PBS and purified by ultracentrifugation (157,000 x g, 60 [0435] Clodronate was a gift of Roche Diagnostics Gm- minutes) through a cushion of 10% sucrose in NTE (0.5 bH, Mannheim, Germany. Other reagents for preparation 40 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8). of liposomes were: Phosphatidylcholine (LIPOID E PC, After resuspension in PBS overnight, virus protein was Lipoid GmbH, Ludwigshafen, Germany) and cholesterol quantified by BCA assay (Pierce), and infectivity was (Sigma-Aldrich). quantified by plaque assay. Some batches were labeled [0436] Mice were housed under specific pathogen-free with carboxylic acid succinimidyl esters of AlexaFluor- andanti-viral antibody-free conditionsin accordance with 45 488 or AlexaFluor-568 (Invitrogen-Molecular Probes) at National Institutes of Health guidelines. All experimental a 104-105-fold molar excess of Alexa dye over virus par- animal procedures were approved by the Institutional An- ticles. Unconjugated dye was removed by ultracentrifu- imal Committees of Harvard Medical School and the IDI. gation through 10% sucrose in NTE, pellets resuspended [0437] Antibodies were purchased from BD Bioscienc- in PBS and stored frozen. Infectivity of VSV preparations es (San Jose, CA), except anti-B220-Alexa647 (Invitro- 50 was quantified by plaque assay on green monkey kidney gen-Caltag), anti-LYVE-1 (Millipore-Upstate), goat-anti- cells (Vero). VSV titers from organs of infected mice were rabbit-APC (Invitrogen), goat-anti-GFP-FITC (Rock- determined similarly, after homogenization of the organs land), anti-FITC-Alexa488 (Invitrogen), and Fab anti- with a Potter-Elvejhem homogenizer. When necessary, IgM-FITC (Jackson Immunoresearch). The following an- during viral preparation, the approximately 4 ml super- tibodies were purchased from AbD-Serotec: anti-CD68- 55 natants from the 157,000 x g ultracentrifugation were col- Alexa647, anti-CD11b-Alexa647, F4/80-Alexa647, anti- lected and concentrated with a 10,000 MWCO Amicon CD169-FITC (3D6). The anti-idiotypic antibody 35.61 for Ultra (Millipore). In order to account for residual infectivity detection of the VI10 BCR in VI10YEN mice (Hangartner in concentrated supernatants, VSV stocks were diluted

62 121 EP 2 630 966 B1 122 to levels of infectivity equal to that of the concentrated utes. For other experiments, mice were transfused with supernatants and calcium flux in VI10YEN B cells was 1 x 107 negatively selected naive B cells from VI10YEN compared over further 100 fold dilutions of VSV and su- x MHCII-EGFP mice one day prior to the experiment. At pernatant. UV-inactivated, AlexaFluor-568 labeled Ade- predetermined time points, popliteal LNs were fixed in novirus 5 (AdV5) was generated following standard pro- 5 situ by footpad injections of phosphate buffered L-lysine cedures (Leopold et al., 1998, Human Gene Therapy, with 1% paraformaldehyde/periodate (PLP). After re- 9:367). All infectious work was performed in designated moval of popliteal LNs and 3-5 hours incubation in PLP BL2+ workspaces, in accordance with institutional guide- at 4°C, popliteal LNs were washed in 0.1 M PBS, pH 7.2 lines, and approved by the Harvard Committee on Micro- and cryoprotected by an ascending series of 10%, 20%, biological Safety. 10 and 30% sucrose in PBS. Samples were snap-frozen in TBS tissue freezing liquid (Triangle Biomedical Scienc- VSV Neutralization Assay es, Durham NC) and stored at -80°C. Sections of 40 mm thickness were mounted on Superfrost Plus slides (Fish- [0440] Serum of immunized mice was prediluted 40- erbrand) and stained with fluorescent antibodies in a hu- fold in MEM containing 2% FCS. Serial two-fold dilutions 15 midified chamber after Fc receptor blockade with 1 mg/ml were mixed with equal volumes of VSV (500 pfu/ml) and antibody 2.4G2 (BD Pharmingen). Samples were mount- incubated for 90 minutes at 37°C in 5% CO2. 100 ml of ed in FluorSave reagent solution (EMD-Calbiochem) and serum-virus mixture was transferred onto Vero cell mon- stored at 4°C until analysis. Images were collected with olayers in 96-well plates and incubated for 1 hour at 37°C. a BioRad confocal microscopy system using an Olympus The monolayers were overlaid with 100 ml DMEM con- 20 BX50WImicroscope and 10x/0.4 or60x/1.2W objectives. taining 1% methylcellulose and incubated for 24 hours Images were analyzed using LaserSharp2000 software at 37°C. Subsequently, the overlay was discarded, and (BioRad Cell Science, Hemel Hempstead, Great Britain) the monolayer was fixed and stained with 0.5% crystal and Photoshop CS (Adobe). Quantification of T/B border violet. The highest dilution of serum that reduced the localized B cells was done by counting cells that were number of plaques by 50% was taken as titer. To deter- 25 within 50 mm of the T/B border, as denoted by B220 coun- mine IgG titers, undiluted serum was pretreated with an terstain, any cells localized in more central regions were equal volume of 0.1 mM β-mercaptoethanol in saline. considered follicular.

Adhesion Assays Electron Microscopy 30 [0441] 96-well plates (Corning) were coated overnight [0443] Popliteal LNs were fixed in situ by footpad in- with dilutions of recombinant murine VCAM-1-Fc or jection of 2% formaldehyde and 2.5 % glutaraldehyde in 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 35 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 water) for 1 hour at room temperature in the dark. After washed. Naive 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, 40 stained for 2 hours in 1% uranyl acetate in maleate buffer Germany) and added to the plates at 3 x 10 5/wellin HBSS and washed three times in water. Samples were dehy- + + with 1% BSA, 1mM Ca 2 and 1 mM Mg 2 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 45 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. 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 50 3.1.2 software, Molecular Devices Corporation) after 30 Intravital Multiphoton Microscopy (MP-IVM) of the Pop- minutes. liteal LN

Confocal Microscopy [0444] Naive B cells were negatively selected by mag- 55 netic isolation using CD43 beads (Miltenyi). VI10YEN B [0442] For some analyses, C57BL/6 mice were inject- cells were labeled for 20 minutes at 37°C with 10m M ed into both hind footpads with 20 mg AlexaFluor-568 or 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

63 123 EP 2 630 966 B1 124 labeled for 25 minutes at 37°C with 10m M 7-amino-4- North Chicago, IL) containing 1 U/ml heparin (American chloromethylcoumarin (CMAC; Invitrogen). In some ex- Pharmaceutical partners, Los Angeles, CA). Using a dis- periments, labels were swapped between wildtype and secting microscope, the TD was exposed through a left VI10YEN B cells to exclude unspecific dye effects. 5-6 x subcostal incision. Silastic® silicon tubing (0.012" I.D., 106 B cells of each population were mixed and adoptively 5 Dow Corning, Midland, USA) was flushed with transferred by tail vein injection into C57BL/6 recipient heparinised (50 U/ml) phosphate-buffered saline (DPBS, mice one day before analysis. In some experiments, re- Mediatech, Herndon, VA), inserted into the cisterna chyli cipient C57BL/6 mice had received an injection of 30 ml through an approximately 0.3 mm incision and fixed with CLL into the hind footpad 7-10 days before the experi- isobutyl cyanoacrylate monomer (Nexaband®, Abbott ment to eliminate SCS macrophages (Delemarre et al., 10 Laboratories). The remaining part of the tubing was ex- 1990, J. Leukoc. Biol., 47:251). Eighteen hours following teriorized through the posterior abdominal wall. Subse- adoptive B cell transfer, recipient mice were anaesthe- quently, the abdominal incision was closed using a 6-0 tized by intraperitoneal injection on of ketamine (50 nonabsorbable running suture (Sofsilk, Tyco Healthcare mg/kg) and xylazine (10 mg/kg). The right popliteal LN Group, Norwalk, CO). Following a 30 minute equ ilibration was prepared microsurgically for MP-IVM and positioned 15 of lymph flow, animals were footpad injected with 108 on a custom-built microscope stage as described (Mem- pfu of VSV-IND and lymph samples were collected on pel et al., 2004, Nature, 427:154). Care was taken to ice for 6 hours. Lymph and organs were taken after 6 spare blood vessels and afferent lymph vessels. The ex- hours of thoracic duct lymph collection and plaqued as posed LN was submerged in normal saline and covered described above. Lymph and organs were plaqued as with a glass coverslip. A thermocouple was placed next 20 described above. In some experiments the draining pop- to the LN to monitor local temperature, which was main- liteal and paraaortic lymph nodes were surgically excised tained at 36-38°C. MP-IVM was performed on a BioRad and the surrounding lymph vessels cauterized to prevent 2100MP system at an excitation wavelength of 800 nm, lymph borne viral access to the blood. from a tunable MaiTai Ti:sapphire laser (Spectra-Phys- ics). Fluorescently labeled VSV (20 mg in 20 ml) was in- 25 Results and Discussion jected through a 31G needle into the right hind footpad of recipient mice concomitant to observation. For four- [0446] Lymph nodes (LNs) prevent systemic dissemi- dimensional off-line analysis of cell migration, stacks of nation of pathogens, such as viruses that enter the body’s 11 optical x-y sections with 4 mm z spacing were acquired surfaces, from peripheral sites of infection. They are also every 15 seconds with electronic zooming to 1.8x-3x30 the staging ground of adaptive immune responses to through a 20x/0.95 water immersion objective (Olym- pathogen-derived antigens (von Andrian and Mempel, pus). Emitted fluorescence and second harmonic signals 2003, Nat. Rev. Immunol., 3:867; and Karrer et al., 1997, were detected through 400/40 nm, 450/80 nm, 525/50 J. Exp. Med., 185:2157). It is unclear how virus particles nm, and 630/120 nm band-pass filters with non-des- arecleared from afferentlymph and presented to cognate canned detectors to generate three-color images. Se- 35 B cells to induce antibody responses. Here, we identify quences of image stacks were transformed into volume- a population of CD1 1b+CD169+MHCII+ macrophages rendered, four-dimensional time-lapse movies using Vo- on the floor of the subcapsular sinus (SCS) and in the locity software (Improvision). 3D instantaneous velocities medulla of LNs that capture viral particles within minutes were determined by semi-automated cell tracking with after subcutaneous (s.c.) injection. SCS macrophages Volocity and computational analysis by Matlab (Math- 40 translocated surface-bound viral particles across the works). Accumulation of cells at the SCS was determined SCS floor and presented them to migrating B cells in the by manual movie analysis performed by blinded observ- underlying follicles. Selective depletion of these macro- ers. Every 2 minutes, the VI10YEN B cells and polyclonal phages compromised local viral retention, exacerbated B cells were counted at the SCS, in the superficial follicle viremia of the host, and impaired local B cell activation. (<50 mm distance from the SCS) and the deep follicle 45 These findings indicate that CD169 + macrophages have (>50 mm distance from the SCS), and ratios ofa dual physiological function. They act as innate "flypa- VI10YEN/polyclonal B cells was expressed for each com- per" by preventing the systemic spread of lymph-borne partment in the entire 30 minute movie. pathogens and as critical gatekeepers at the lymph-tis- sueinterface that facilitate B cell recognition ofparticulate Thoracic Duct Cannulation 50 antigens and initiate humoral immune responses. [0447] We have investigated how virus particles that [0445] For thoracic duct cannulation, mice received enter peripheral tissues are handled within draining LNs. 200 ml olive oil p.o. 30 minutes prior to cannulation to Hind footpads of mice were injected with fluorescently facilitate visualization of the lymph vessels. Animals were labeled UV-inactivated vesicular stomatitis virus (VSV), then anesthetized with xylazine (10 mg/kg) and ketamine 55 a cytopathic rhabdovirus that is transmittable by insect HCl (50 mg/kg). A polyethylene catheter (PE-10) was bites (Mead et al., 2000, Ann. N.Y. Acad. Sci., 916:437) inserted into the right jugular vein for continuous infusion and elicits T-independent neutralizing B cell responses (2 ml/hour) of Ringer’s lactate (Abbott Laboratories, (Bachmann et al., 1995, Eur. J. Immunol., 25:3445). Us-

64 125 EP 2 630 966 B1 126 ing multiphoton intravital microscopy (MP-IVM) in pop- popliteal LNs (Figure 11F). Therefore, VSV fixation in liteal LNs (Mempel et al., 2004, Nature, 427:154) draining LNs occurs via a mechanism distinct from that used by the injected footpad, we observed that VSV accumulated splenic marginal zone macrophages, which require C3 in discrete patches on the SCS floor within minutes after and natural antibodies to capture blood-borne VSV (Och- sc injection, while the parenchyma and roof of the SCS 5 senbein et al., 1999, J. Exp. Med., 190:1165; and Och- remained free of virus (Figure 11A). The viral deposits senbein et al., 1999, Science, 286:2156). Conceivably, became progressively denser forming conspicuous irreg- the VSV surface glycoprotein (VSV-G) may be recog- ular reticular patterns, which remained fixed in place for nized in LNs by macrophage-expressed carbohydrate- hours. binding scavenger receptors (Taylor et al., 2005, Ann. [0448] To characterize the predilection sites for VSV 10 Rev. Immunol., 23:901), but the precise mechanism will binding in LNs, we reconstituted irradiated Act(EGFP) require further investigation. mice with wildtype bone marrow. The resulting[0451] What are the consequences of viral capture by B6→Act(EGFP) chimeras expressed EGFP in non-he- macrophages for virus dissemination and anti-viral im- matopoietic cells, presumably lymphatic endothelial munity? To address this question, we depleted LN-resi- cells, on the SCS floor and roof. Upon footpad injection 15 dent macrophages by footpad injection of clodronate li- of fluorescent VSV into C57BL/6→ Act(EGFP) chimeras, posomes (CLL; Delemarre et al., 1990, J. Leukoc. Biol., viral particles flooded the SCS. Three hours later, un- 47:251). At the dose used, sc injected CLL selectively bound lumenal VSV had disappeared, but the SCS floor eliminated macrophages in LNs draining the injection displayed prominent patches of VSV that did not colocal- site, including the popliteal, inguinal and paraortic LNs ize with EGFP + cells, suggesting that VSV was captured 20 (Delemarre et al., 1990, J. Leukoc. Biol., 47:251), while by hematopoietic cells (Figure 11B). To characterize the macrophages in distal LNs and spleen were spared (Fig- putative VSV-capturing leukocytes, we performed elec- ures 13A, B). Among the different LN-resident tron microscopy on popliteal LNs that were harvested 5 CD11b+MHCII+ phagocytes, CLL preferentially removed min afterVSV injection (Figure 11C). Bullet-shaped,elec- the CD169+ subset, whereas LYVE-1 + cells and conven- tron-dense VSV particles were selectively bound to dis- 25 tional DCs remained unchanged. CLL-treated popliteal crete regions on the surface of scattered large cells that LNs had increased B cell numbers and enlarged follicles resided within the SCS or just below the SCS floor. VSV- 7 days after treatment, but other morphological param- binding cells that were located beneath the SCS floor eters, e.g. demarcation of the T/B border and SCS ul- were typically in contact with the lymph compartment via trastructure remained unaltered (Figures 13 C-E). protrusions that extended into the SCS lumen. 30 [0452] Compared to untreated LNs, we recovered ap- [0449] Ultrastructural studies of LNs have shown that proximately10-fold lower viral titers fromthe drainingLNs the SCS contains many macrophages (Clark, 1962, Am. of CLL-treated mice (Figure 11G), suggesting that mac- J. Anat., 110:217; and Farr et al., 1980, Am. J. Anat., rophage depletion rendered lymph filtration inefficient. 157:265), so we hypothesized that the VSV-retaining Indeed, VSV titers were dramatically increased in blood, cells belonged to this population. Indeed, confocal mi- 35 spleen, and non-draining LNs of CLL-treated mice. Viral croscopy of frozen LN sections obtained thirty minutes dissemination from the injection site to the blood depend- after footpad injection showed that VSV co-localized in ed strictly on lymph drainage, because circulating VSV the SCS with a macrophage marker, CD169/sialoadhes- was undetectable when virus was injected into footpads in (Figure 11D). Using flow cytometry, we detected of mice that carried an occluding catheter in the thoracic CD169 on approximately 1%-2% of mononuclear cells 40 duct (TD), even in CLL-treated mice. Viral titers were low, (MNCs) in LNs, which uniformly co-expressed CD11b but detectable in TD lymph fluid of untreated mice, but and MHC-II, indicating that the VSV-binding cells are in- increased significantly in CLL-treated animals (Figure deed macrophages (Figure 12). Most CD169 + cells also 11H). This indicates that the principal conduit for early expressed other macrophage markers, including CD68 viral dissemination from peripheral tissues is the lymph, and F4/80, while few expressed the granulocyte/mono- 45 which is monitored by LN-resident, CLL-sensitive mac- cyte marker Gr-1. CD 169 + cells also expressed CD 11c, rophages that prevent the systemic spread of lymph- but at lower levels than CD 11c high conventional dendritic borne VSV. cells (DCs). We conclude that intact virions enter the [0453] This capture mechanism was not unique to lymph within minutes after transcutaneous deposition VSV; CD169+ SCS macrophages also retained adeno- and accumulate rapidly and selectively on macrophages 50 virus (AdV; Figures 14 A-C) and vaccinia virus (W, Figure in the medulla and SCS of draining LNs. 14D), indicating that macrophages act as guardians [0450] To explore mechanisms for virus fixation, live against many structurally distinct pathogens. In contrast, VSV (20 mg containing 2 x 10 8 pfu) was injected into hind virus-sized latex beads (200 nm) were poorly retained in footpads and viral titers in draining LNs were assessed the SCS after footpad injection (Figure 14E). Thus, SCS 2 hours later. There was no defect in VSV retention in 55 macrophages discriminate between lymph-borne virus- draining LNs of complement C3-deficient mice (Figure es and other particles of similar size. Fluorescent VSV, 11E). DH-LMP2a mice, which lack secreted immu- AdV and W also accumulated in the medulla of draining noglobulins, had reduced virus titers in spleen, but not in LNs, where they were not only bound by CD169 low cells

65 127 EP 2 630 966 B1 128

(Figure 11D) but also by CD169-LYVE-1+ lymphatic en- was not retained in the SCS and VI10YEN B cells failed dothelial cells (Figures 14C, D). This was corroborated to congregate in that region, indicating that SCS macro- in CLL-treated LNs, where VSV accumulated exclusively phages are essential for both events (Figure 17B). on medullary LYVE-1+ cells (Figure 15). [0456] To rigorously quantify VI10YEN B cell distribu- [0454] Next, we examined how captured VSV is rec- 5 tion, LNs were harvested 30 minutes after VSV challenge ognized by B cells. Popliteal LNs contain rare B cells in and analyzed by confocal microscopy. While the entire the SCS lumen (Figure 16A), but we found no evidence follicular VI10YEN population retained its overall distri- for virus-binding lymphocytes within the SCS on electron bution (Figure 17D), the subset of cells residing ≤50 mm micrographs. Instead, viral particles were presented to below the SCS shifted toward the SCS in VSV-IND, but B cells within superficial follicles by macrophages that 10 not VSV-NJ containing LNs (Figure 17E). It seems un- extended across the SCS floor. Following injection of ei- likely that VI10YEN B cells redistributed to the SCS be- ther VSV (Figure 17A) or AdV (Figures 16B-E), virions cause of chemoattractant signals, since unresponsive were readily detectable at B cell-macrophage interfaces polyclonal B cells express the same chemoattractant re- for at least 4 hours. This suggested that SCS macrophag- ceptors. More likely, the random contacts of motile es shuttle viral particles across the SCS floor for presen- 15 VI10YEN cells with macrophage-bound VSV-IND trig- tation to B cells. Transcytosis seemed unlikely, because gered a BCR-dependent "stop signal" (Okada et al., the few vesicles containing VSV in SCS macrophages 2005, PLoS Biol., 3:e150): Short-term exposure to VSV- showed evidence of viral degradation. In addition, we did IND activates LFA-1 and/or α4 integrins (Dang and Rock, not detect substantial motility of virus-binding macro- 1991, J. Immunol., 146:3273) on VI10YEN B cells, re- phages by MP-IVM, at least during the first 6 hours after 20 sulting in adhesion to the respective ligands, ICAM-1 and challenge. Therefore, viral particles most likely reached VCAM-1, which are both expressed in the SCS (Figure the LN parenchyma by moving along the macrophage 19). Additionally, VSV-IND bound to SCS macrophages surface. Of note, VSV and other antigens are also pre- may provide a substrate for VI10YEN B cell adhesion sented to B cells by DCs immigrating from peripheral directly via the BCR. locations (Ludewig et al., 2000, Eur. J. Immunol., 30:185; 25 [0457] To investigate how captured virions are proc- and Qi et al., 2006, Science, 312:1672), but footpad-de- essed upon detection by B cells, we tested B cells from rived DCs are not likely to play a role during these very VI10YENxMHCII-EGFP mice, which allowed us to visu- early events, because their migration into popliteal LNs alize endocytosed VSV co-localizing with endosomal takes much longer. We conclude that the SCS floor is MHC-II as an indicator of B cell priming (Vascotto et al., not unsurmountable for lymph-borne viruses; CD169+ 30 2007, Curr., Opin., Immunol., 19:93). Within 30 minutes macrophages appear to act as gatekeepers and facilita- after injection, VI10YENxMHCII-EGFP B cells in the su- tors of viral translocation and presentation to B cells. perficial follicle had extensively internalized VSV-IND, [0455] Next, we explored how naive B cells respond but not VSV-NJ particles (Figures 20A, B). Virus-carrying to viral encounter using two VSV serotypes, Indiana VSV-specific B cells were infrequent, but detectable in (VSV-IND) and New Jersey (VSV-NJ) (Figure 18; Roost 35 deep follicles. These cells may have acquired virions et al., 1996, J. Immunol. Methods, 189:233). We com- from rare polyclonal B cells that carried VSV on their sur- pared wildtype B cells to B cells from VI10YEN mice, face, or may correspond to VI10YEN cells which failed which express a VSV-IND-specific B cell receptor that to arrest at the SCS after acquiring VSV-IND. does not bind VSV-NJ (Hangartner et al., 2003, Proc. [0458] While our histological findings demonstrate that Natl. Acad. Sci., USA, 100:12883). By contrast, a small 40 intact virions are preferentially detected and acquired by fraction (2%-5%) of wildtype B cells bound both sero- B cells in the SCS and superficial follicle, MP-IVM meas- types without being activated. This might reflect low-af- urements of B cell motility revealed broader antigen dis- finity reactivity with VSV-G or indirect interactions,e.g . semination. After VSV-IND injection, VI10YEN cells ex- via complement (Rossbacher and Shlomchik, 2003, J. hibited a rapid drop in velocity throughout the entire B Exp. Med., 198:591). To assess in vivo responses, dif- 45 follicle, (Figure 21). This was equally observed in CLL- ferentially labeled wildtype and VI10YEN B cells were treated and control LNs, indicating that viral antigen adoptively transferred and allowed to home to LN folli- reached B cells independent of macrophages. This an- cles. Fluorescent UV-inactivated virus was then injected tigenic material was most likely composed of free viral into footpads and popliteal LNs were recorded by MP- protein, an inevitable by-product of natural infections. In- IVM about 5-35 minutes later. In virus-free LNs or after 50 deed, purified supernatant of our VSV stocks induced a injection of VSV-NJ, VI10YEN and control B cells dis- potent calcium flux in VI10YEN B cells (Figure 18E). played the same distribution (Figures 17B-C). In contrast, Small lymph-borne proteins are known to diffuse rapidly upon VSV-IND injection VI10YEN cells rapidly accumu- into follicles and activate cognate B cells (Pape et al., lated below and at the SCS floor. There was no difference 2007, Immunity, 26:491). Accordingly, injection of viral in baseline B cell motility and distribution between CLL- 55 supernatant suppressed themotility of follicular VI10YEN treated and untreated LNs, suggesting that VSV-specific B cells without inducing their accumulation at the SCS, B cells are equally likely to probe the SCS in both con- indicatingthat free VSV-G wascontained and active with- ditions. However, in CLL-treated LNs, fluorescent virus in the viral inoculum. This can explain the macrophage-

66 129 EP 2 630 966 B1 130 independent pan-follicular effect of VSV-IND injection. comprise a lipid bilayer which has an amphiphilic prop- [0459] To determine the kinetics of VI10YEN B cell ac- erty: both interior and exterior surfaces of the bilayer are tivation upon viral encounter, we measured common ac- hydrophilic, and the bilayer lumen is hydrophobic. Li- tivation markers (Figure 22). The costimulatory molecule pophilic molecules can spontaneously embed them- CD86 was first up-regulated 6 hours after VSV-IND chal- 5 selves into liposome membrane and retain their hy- lenge. CD69 was induced more rapidly, but also on pol- drophilicdomains outside, and hydrophilic molecules can yclonal B cells, presumably by pleiotropic IFN- α signaling be chemically conjugatedto the outer surface of liposome (Barchet et al., 2002, J. Exp. Med., 195:507; and Shiow taking advantage of membrane biofunctionality. et al., 2006, Nature, 440:540). Surface IgM (Figures 20C, [0463] Lipids are mixed with a lipophilic immunomod- D) was down-regulated as early as 30 minutes after chal- 10 ulatory agent, and then formed into thin films on a solid lenge reaching a maximum within 2h when >70% of surface. A hydrophilic immunomodulatory agent is dis- VI10YEN cells were BCRlow/neg. Therefore, BCR inter- solved in an aqueous solution, which is added to the lipid nalization provided the earliest specific readout for virus- films to hydrolyze lipids under vortex. Liposomes with specific B cell activation. Remarkably, VI10YEN B cells lipophilic immunomodulatory agents incorporated into in CLL-treated LNs failed to downregulate their BCR dur- 15 the bilayer wall and hydrophilic immunomodulatory ing the first 2 hours after subcutaneous injection of 20 agents inside the liposome lumen are spontaneously as- mg VSV-IND (Figure 20E), indicating that SCS macro- sembled. phages are necessary for efficient early presentation of captured virions to B cells. Nanoparticle-Stabilized Liposome Nanocarriers [0460] Primed B cells eventually solicit help from CD4 + 20 T cells (Vascotto et al., 2007, Curr., Opin., Immunol., [0464] This is a comparative example. Nanoparticle- 19:93) for class switch recombination and germinal cent- stabilized liposomes are used to deliver one or a plurality er formation. To contact T cells, newly activated B cells of immunomodulatory agents to cells of the immune sys- migrate toward the T/B border (Okada et al., 2005, PLoS tem (Figure 4). When small charged nanoparticles ap- Biol., 3:e150; and Reif et al., Nature, 416:94). This mech- 25 proach the surface of liposomes carrying either opposite anism operated efficiently in macrophage-sufficient charge or no net charge, electrostatic or charge-dipole mice; most VI10YEN B cells redistributed to the T/B bor- interaction between nanoparticles and membrane at- der within 6h after footpad injection of as little as 40 ng tracts the nanoparticles to stay on the membrane surface, VSV-IND (Figures 20F, H and 23). By contrast, a 100- being partially wrapped by lipid membrane. This induces fold higher viral dose was needed to elicit full redistribu- 30 local membrane bending and globule surface tension of tion of VI10YEN B cells in CLL-treated mice (Figures liposomes, both of which enable tuning of membrane ri- 20G, H). By 12 hours after injection, most VSV-specific gidity. This aspect is significant for vaccine delivery using cells reached the T-B border, irrespective of the injected liposomes to mimic viruses whose stiffness depends on dose. Thus, even without SCS macrophages follicular B the composition of other biological components within cells are eventually activated by VSV-derived antigen, 35 virus membrane. Moreover, adsorbed nanoparticles albeit less efficiently. form a charged shell which protects liposomes against [0461] In conclusion, we demonstrate a dual role for fusion, thereby enhancing liposome stability. Small nan- CD169+ macrophages in LNs: they capture lymph-borne oparticles are mixed with liposomes under gentle vortex, viruses preventing their systemic dissemination and they and the nanoparticles stick to liposome surface sponta- guide captured virions across the SCS floor for efficient 40 neously. presentation and activation of follicular B cells. Liposome-Polymer Nanocarrier Example 2: Exemplary Lipid-Based Vaccine Nanotech- nology Architectures [0465] This is a comparative example. Liposome-pol- 45 ymer nanocarriers are used to deliver one or a plurality Liposome Nanocarriers of immunomodulatory agents to cells of the immune sys- tem (Figure 5). Instead of keeping the liposome interior [0462] This is a comparative example. Small lipo- hollow, hydrophilic immunomodulatory agents may be somes (10 nm- 1000 nm) are manufactured and em- encapsulated. Figure 3 shows liposomes that are loaded ployed to deliver one or multiple immunomodulatory50 with di-block copolymer nanoparticles to form liposome- agents to cells of the immune system (Figure 3). In gen- coated polymeric nanocarriers, which have the merits of eral, liposomes are artificially-constructed spherical lipid both liposomes and polymeric nanoparticles, while ex- vesicles, whose controllable diameter from tens to thou- cluding some of their limitations. The liposome shell can sands of nm signifies that individual liposomes comprise be used to carry lipophilic or conjugate hydrophilic im- biocompatible compartments with volume from zeptolit- 55 munomodulatory agents, and the polymeric core can be ers (10-21 L) to femtoliters (10-15 L) that can be used to used to deliver hydrophobic immunomodulatory agents. encapsulate and store various cargoes such as proteins, Pre-formulated polymeric nanoparticles (40 nm - 1000 enzymes, DNA and drug molecules. Liposomes may nm) are mixed with small liposomes (20 nm - 100 nm)

67 131 EP 2 630 966 B1 132 under gentle vortex to induce liposome fusion onto pol- Lipid Monolayer-Stabilized Polymeric Nanocarrier ymeric nanoparticle surface. [0470] This is a comparative example. Lipid monolayer Nanoparticle-Stabilized Liposome-Polymer Nanocarri- stabilized polymeric nanocarriers are used to deliver one ers 5 or a plurality of immunomodulatory agents (Figure 9). As compared to aforementioned liposome-polymer nano- [0466] This is a comparative example. Nanoparticle- carrier (Figures 5-8), this system has the merit of sim- stabilized liposome-polymer nanocarriers are used to de- plicity in terms to both agents and manufacturing. A hy- liver one or a plurality of immunomodulatory agents (Fig- drophobic homopolymer can form the polymeric core in ure 6). By adsorbing small nanoparticles (1 nm - 30 nm) 10 contrast to the di-block copolymer used in Figures 5-8, to the liposome-polymer nanocarrier surface, the nano- which has both hydrophobic and hydrophilic segments. carrier has not only the merit of both aforementioned na- Lipid-stabilized polymeric nanocarriers can be formed noparticle-stabilized liposomes (Figure 4) and aforemen- within one single step instead of formulating polymeric tioned liposome-polymer nanoparticles (Figure 5), but al- nanoparticle and liposome separately followed by fusing so tunable membrane rigidity and controllable liposome 15 them together. stability. [0471] A hydrophilic immunomodulatory molecule is first chemically conjugated to a lipid headgroup. The con- Liposome-Polymer Nanocarriers Comprising Reverse jugate is mixed with a certain ratio of unconjugated lipid Micelles molecules in an aqueous solution containing one or more 20 water-miscible solvents. A biodegradable polymeric ma- [0467] This is a comparative example. Liposome-pol- terial is mixed with the hydrophobic immunomodulatory ymer nanocarriers containing reverse micelles are used agents to be encapsulated in a water miscible or partially to deliver one or a plurality of immunomodulatory agents water miscible organic solvent. The resulting polymer so- (Figure 7). Since the aforementioned liposome-polymer lution is added to the aqueous solution of conjugated and nanocarriers (Figures 5 and 6) are limited to carry hydro- 25 unconjugated lipid to yield nanoparticles by the rapid dif- phobic immunomodulatory agents within polymeric nan- fusion of the organic solvent into the water and evapo- oparticles, here small reverse micelles (1 nm - 20 nm) ration of the organic solvent. are formulated to encapsulate hydrophilic immunomod- ulatory agents and then mixed with the di-block copoly- Lipid Monolayer-Stabilized Polymeric Nanocarrier Com- mers to formulate polymeric core of liposomes. 30 prising Reverse Micelles [0468] A hydrophilic immunomodulatory agent to be encapsulated is first incorporated into reverse micelles [0472] This is a comparative example. Lipid monolayer by mixing with naturally derived and non-toxic am- stabilized polymeric nanoparticles comprising reverse phiphilic entities in a volatile, water-miscible organic sol- micelles are used to deliver one or a plurality of immu- vent. The resulting biodegradable polymer-reverse mi- 35 nomodulatory agents (Figure 10). Since the aforemen- celle mixture is combined with a polymer-insoluble hy- tioned lipid-stabilized polymeric nanocarriers (Figure 9) drophilic non-solvent to form nanoparticles by the rapid are limited to carry hydrophobic immunomodulatory diffusion of the solvent into the non-solvent and evapo- agents, here, small reverse micelles (1 nm - 20 nm) are ration of the organic solvent. Reverse micelle contained formulated to encapsulate hydrophilic immunomodulato- polymeric nanoparticles are mixed with lipid molecules 40 ry agents and mixed with biodegradable polymers to form to form the aforementioned liposome-polymer complex polymeric nanocarrier core. structure (Figure 5). Example 3: In vivo targeting of SCS-Mph using Fc frag- Nanoparticle-Stabilized Liposome-Polymer Nanocarri- ments from human IgG ers Comprising Reverse Micelles 45 [0473] This is a comparative example. Fluorescent un- [0469] This is a comparative example. Nanoparticle- modified control nanoparticles (top panel, Figure 24A) or stabilized liposome-polymer nanocarriers containing re- Fc surface-conjugated targeted nanoparticles (middle verse micelles are used to deliver one or a plurality of and lower panel, Figure 24A) were injected into footpads immunomodulatory agents (Figure 8). By adsorbing50 of anesthetized mice, and the draining popliteal lymph small nanoparticles (1 nm - 30 nm) to a liposome-polymer node was excised 1 hour later and single-cell suspen- nanocarrier surface, the nanocarrier has not only the sions were prepared for flow cytometry. Targeted nano- merit of both aforementioned nanoparticle-stabilized li- particles were also injected into mice one week after posomes (Figure 4) and aforementioned reverse micelle lymph node macrophageshad been depleted by injection contained liposome-polymer nanoparticles (Figure 7), 55 of clodronate-laden liposomes (lower panel, Figure 24A). but also tunable membrane rigidity and controllable lipo- The cell populations in gates were identified as nanopar- some stability. ticle-associated macrophages based on high expression of CD11b. These results indicate that (i) nanoparticle

68 133 EP 2 630 966 B1 134 binding depends on the presence of clodronate-sensitive nificantly higher neutralizing anti-VSV titers than any oth- macrophages and (ii) targeted nanoparticles are bound er group (the two animals with the highest titers in that to - twice as many macrophages as control nanoparticles. group completely neutralized plaque formation at the [0474] The Panels on the right of Figure 24 show flu- highest dilution tested, so actual titers may have been orescent micrographs of frozen lymph node sections af- 5 even higher). ter injection of blue fluorescent control (top panel, Figure [0477] The induced immune response elicited by na- 24A) or targeted (middle and lower panels, Figure 24A) noparticle (NP) vaccines confers potent protection from nanoparticles. Sections were counter-stained with anti- a lethal dose of VSV. While all vaccinated groups showed CD169 and a marker that identifies either the medulla (in some protection, only the group that received VSV-G top and bottom panel, Figure 24A) or B cells (in middle 10 conjugates to Fc-targeted NPs plus alum showed 100% panel, Figure 24A). At one hour after nanoparticle injec- protection from lethal infection. Recipients of free VSV- tion most control particles are found in the medulla (top, G (VSV-G + alum) received ∼10-fold more antigen than Figure 24A), while targeted nanoparticles colocalise with animals that were given VSV-G conjugated to nanopar- CD169+ SCS-Mph adjacent to B cell follicles (middle, ticles. As a negative control, one group of mice received Figure 24A). At 24 hours after injection, discrete cell- 15 Fc-targeted nanoparticles (NP-Fc) without VSV-G, which sized accumulations of targeted nanoparticles are seen did not confer protection. in the cortical region between the SCS and the medulla, suggesting uptake and transport by migratory dendritic Example5: In vivo T cell activation by immunomodulatory cells. nanoparticles [0475] Mice were injected i.v. with red fluorescent B 20 cells and in a footpad with a 1:1 mixture of control and [0478] C57BL6J mice were injected i.v. with CFSE-la- Fc targeted nanoparticles. 24 hours later, when some of beled CD4 T cells from OT-II donor mice, which express the transferred B cells had migrated into B cell follicles, a transgenic TCR specific for chicken ovalbumin (OVA) the draining popliteal lymph node was excised and sec- presented in MHC class II. Subsequently, immunization tioned for confocal microscopy and quantitative image 25 experiments were performed by injecting one footpad analysis of green:blue fluorescent ratios. The subcapsu- with free OVA or with nanoparticles composed of either lar sinus (SCS) region contained similar levels of blue PLA or PLGA that encapsulated an equivalent amount and green nanoparticles (cells encircled on the right, Fig- of OVA as a model antigen. All antigenic mixtures also ure 24B), while green fluorescence associated with Fc- contained CpG (a TLR9 agonist) as an adjuvant. The targeted nanoparticles was about twice higher in the30 animals were injected, sacrificed three days after immu- SCS. There were also prominent accumulations of green nization, and OT-II T cell activation was assessed by flow nanoparticles within B follicles delineated by scattered cytometry in single-cell suspensions from different tis- red B cells. These regions have the characteristic size, sues. shape, and distribution of follicular dendritic cells (FDC), [0479] Unstimulated 5,6-carboxy-succinimidyl-fluo- which like macrophages and dendritic cells are known to 35 rescein-ester (CFSE)-labeled T cells do not divide and, express abundant Fc receptors. therefore, carry a uniformly high concentration of CFSE resulting in a single narrow peak of brightly fluorescent Example 4: Antigen-bearing targeted nanoparticles are cells. By contrast, activated T cells divide and in the proc- highly immunogenic and induce high antibody titers ess split the fluorescent dye evenly between the two 40 daughter cells resulting in an incremental decrease in [0476] This is a comparative example. Groups of mice fluorescence intensity upon each successive division. (5/group) were immunized with: UV-inactivated vesicular Thus, the greater the left shift in CFSE, fluorescence the stomatits virus (VSV, serotype Indiana) or with the puri- stronger T cells were activated. The results indicate that: fied immunogenic envelope glycoprotein (VSV-G) of (i) nanoparticle-encapsulated antigen generated a more VSV. VSV-G was either given in soluble form mixed with 45 potent CD4 T cell response than free antigen in the drain- alum or conjugated to non-targeted or targeted (with sur- ing popliteal lymph node (popLN, top row); (ii) only nan- face immobilized human Fc) PLGA nanoparticles with or oparticles, but not free OVA induced local T cell prolifer- without alum as an adjuvant. The dose of free VSV-G ation in distal lymphoid tissues, including the brachial was estimated to be ∼10-fold higher than the dose of lymph node (middle row) and the spleen (bottom row). VSV-G delivered with nanoparticles. Mice received a50 In recipients of free OVA, the brachial LN and spleen booster injection at day 55 after the primary immuniza- contained only undivided cells or cells with very low CFSE tion, and serum was obtained after 10 weeks and tested content. The latter population does not indicate local T for neutralization of VSV-mediated plaque formation on cell activation but migration of T cells that were activated Vero cells. Results show titers as the highest serum di- elsewhere. lution that blocked plaque formation by at least 50%.55 [0480] C57BL6J mice were injected i.v. with CFSE-la- Each symbol reflects the neutralizing anti-VSV titer in beled CD8 T cells from OT-I donor mice, which express one mouse. The group of mice immunized with VSV-G a transgenic T cell receptor (TCR) specific for chicken presented on Fc-targeted nanoparticles generated sig- ovalbumin (OVA) presented in MHC class I. The exper-

69 135 EP 2 630 966 B1 136 imental protocol was otherwise identical as immediately 5. The nanocarrier of claim 4, wherein the targeting described above. agent targets the nanocarriers to antigen presenting [0481] C57BL6J mice were injected i.v. with CFSE-la- cells, T cells or B cells. beled CD8 T cells from OT-I donor mice as above. How- ever, in this experiment CL097, an imidazoquinoline5 6. The nanocarrier of claim 5, wherein the antigen pre- compound that activates TLR-7 and TLR-8, was used as senting cell is selected from: a dendritic cell, for ex- adjuvant and different methods of adjuvant delivery were ample a follicular dendritic cell; and a macrophage, tested. T cell activation in this case was assessed by for example a subcapsular sinus macrophage. counting the total number of OT-I T cells in the draining popliteal lymph node three days after footpad injection 10 7. The nanocarrier of any preceding claim, wherein the of either free OVA (1 mg or 100 ng) mixed with free ad- toll-like receptor agonist is selected from the group juvant (160 ng). All animals that received nanoparticles consisting of: a TLR-1 agonist, a TLR-2 agonist, a were given 100 ng OVA with or without 160 ng CL097. TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a Material that was encapsulated within nanoparticles, but TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a not covalently attached to the PLA polymer is shown in 15 TLR-9 agonist, and a TLR-10 agonist. []. Covalent linkage of CL097 to PLA is identified by hy- phenation. Materials that were mixed in free form within 8. The nanocarrier of claim 7, wherein the toll-like re- the same compartment are separated by "+". These re- ceptor agonist is selected from the group consisting sults revealed a marked increase in CD8 T cell prolifer- of: a TLR-3 agonist, a TLR-7 agonist, a TLR-8 ago- ation in animals that received encapsulated OVA in na- 20 nist, and a TLR-9 agonist. noparticles in which the adjuvant was covalently linked to the excipient. 9. The nanocarrier of any of claims 1-6, wherein the toll-like receptor agonist is imiquimod, R848 or CL097. Claims 25 10. A nanocarrier of any preceding claim, wherein the 1. A nanocarrier comprising: nanocarrier nanoparticle is formed by self-assembly.

an immunostimulatoryagent, whereinthe immu- 11. A method of preparing a nanocarrier of any preced- nostimulatory agent is a toll-like receptor ago- 30 ing claim by nanoprecipitation, flow focusing using nist; fluidic channels, spray drying, single or double emul- a T cell antigen; and, sion solvent evaporation, or solvent extraction. a polymer selected from the group consisting of: polylactic acid (PLA), poly(glycolic acid) (PGA), 12. A nanocarrier of any of Claims 1-10, for use in med- and poly(lactic acid/glycolic acid) (PLGA); 35 icine. wherein the nanocarrier has a mean geometric diameter of between 50 nm and 500 nm; wherein the immunostimulatory agent is covalently Patentansprüche bound to the nanocarrier or the immunostimu- latory agent is covalently bound to a polymer 40 1. Nanoträger, Folgendes umfassend: from which the nanocarrier is formed, and the T cell antigen is encapsulated within the nanocar- ein Immunstimmulans, wobei das Immunstim- rier or the T cell antigen is covalently bound to mulans ein Toll-like-Rezeptor-Agonist ist; the nanocarrier. ein T-Zell-Antigen; und 45 ein Polymer, ausgewählt aus der Gruppe beste- 2. The nanocarrier of claim 1, wherein the PLGA has hend aus: Polymilchsäure (PLA), Poly(glycol- a lactic acid:glycolic acid ratio of 85:15, approximate- säure) (PGA) und Poly(milchsäure/glycolsäure) ly 75:25, approximately 60:40, approximately 50:50, (PLGA); approximately 40:60, approximately 25:75, or ap- wobei der Nanoträger einen mittleren geomet- proximately 15:85. 50 rischen Durchmesser zwischen 50 nm und 500 nm aufweist; wobei das Immunstimmulans ko- 3. The nanocarrier of claim 1 or 2, wherein the T cell valent an den Nanoträger gebunden ist oder das antigen is a protein or peptide, an infectious disease Immunstimmulans kovalent an ein Polymer, aus antigen, or a cancer antigen. dem der Nanoträger ausgebildet ist, gebunden 55 ist und das T-Zell-Antigen in dem Nanoträger 4. The nanocarrier of any preceding claim, wherein the eingekapselt ist oder das T-Zell-Antigen kova- nanocarrier further comprises a targeting agent. lent an den Nanoträger gebunden ist.

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2. Nanoträger nach Anspruch 1, wobei das PLGA ein Revendications Milchsäure:Glycolsäure-Verhältnis von 85:15, etwa 75:25, etwa 60:40, etwa 50:50, etwa 40:60, etwa 1. Nanovecteur comprenant : 25:75 oder etwa 15:85 aufweist. 5 un agent immunostimulateur, lequel agent im- 3. Nanoträger nach Anspruch 1 oder 2, wobei das T- munostimulateur est un agoniste du récepteur Zell-Antigen ein Protein oder ein Peptid, ein Infekti- de type toll ; onskrankheit-Antigen oder ein Krebs-Antigen ist. un antigène T ; et un polymère choisi dans le groupe constitué 4. Nanoträger nach einem der vorhergehenden An- 10 par : l’acide polylactique (PLA), l’acide polygly- sprüche, wobei der Nanoträger ferner ein Targeting- colique (PGA) et l’acide polylactique-co-glycoli- mittel umfasst. que (PLGA) ;

5. Nanoträger nach Anspruch 4, wobei das Targeting- lequel nanovecteur a un diamètre géométrique mittel die Nanoträger auf antigenpräsentierende Zel- 15 moyen compris entre 50 nm et 500 nm ; dans lequel len, T-Zellen oder B-Zellen richtet. l’agent immunostimulateur est lié de façon covalente au nanovecteur ou l’agent immunostimulateur est lié 6. Nanoträger nach Anspruch 5, wobei die antigenprä- de façon covalente à un polymère à partir duquel le sentierende Zelle ausgewählt ist aus: einer dendri- nanovecteur est formé, et l’antigène T est encapsulé tischen Zelle, beispielsweise einer follikulären den- 20 dans le nanovecteur ou l’antigène T est lié de façon dritischen Zelle; und einem Makrophagen, beispiels- covalente au nanovecteur. weise einem Makrophagen eines subkapsulären Si- nus. 2. Nanovecteur selon la revendication 1, dans lequel le PLGA a un rapport acide lactique/acide glycolique 7. Nanoträger nach einem der vorhergehenden An- 25 de 85/15, d’environ 75/25, d’environ 60/40, d’environ sprüche, wobei der Toll-like-Rezeptor-Agonist aus- 50/50, d’environ 40/60, d’environ 25/75 ou d’environ gewählt ist aus der Gruppe bestehend aus: einem 15/85. TLR-1-Agonisten, einem TLR-2-Agonisten, einem TLR-3-Agonisten, einem TLR-4-Agonisten, einem 3. Nanovecteur selon la revendication 1 ou 2, dans le- TLR-5-Agonisten, einem TLR-6-Agonisten, einem 30 quel l’antigène T est une protéine ou un peptide, un TLR-7-Agonisten, einem TLR-8-Agonisten, einem antigène de maladie infectieuse ou un antigène du TLR-9-Agonisten und einem TLR-10-Agonisten. cancer.

8. Nanoträger nach Anspruch 7, wobei der Toll-like- 4. Nanovecteur selon l’une quelconque des revendica- Rezeptor-Agonist ausgewählt ist aus der Gruppe be- 35 tions précédentes, lequel nanovecteur comprend en stehend aus: einem TLR-3-Agonisten, einem TLR- outre un agent de ciblage. 7-Agonisten, einem TLR-8-Agonisten und einem TLR-9-Agonisten. 5. Nanovecteur selon la revendication 4, dans lequel l’agent de ciblage cible les nanovecteurs sur les cel- 9. Nanoträger nach einem der Ansprüche 1-6, wobei 40 lules présentatrices d’antigène, les lymphocytes T der Toll-like-Rezeptor-Agonist ein Imiquimod, R848 ou les lymphocytes B. oder CL097 ist. 6. Nanovecteur selon la revendication 5, dans lequel 10. Nanoträger nach einem der vorhergehenden An- la cellule présentatrice d’antigène est choisie parmi : sprüche, wobei das Nanoträgernanopartikel durch 45 une cellule dendritique, par exemple une cellule den- Selbstorganisation ausgebildet wird. dritique folliculaire ; et un macrophage, par exemple un macrophage du sinus sous-capsulaire. 11. Verfahren zum Herstellen eines Nanoträgers nach einem der vorhergehenden Ansprüche durch Nano- 7. Nanovecteur selon l’une quelconque des revendica- präzipitation, Flussfokussierung unter Verwendung 50 tions précédentes, dans lequel l’agoniste du récep- von Fluidkanälen, Sprühtrocknen, Einfach- oder teur de type toll est choisi dans le groupe constitué Doppelemulsionslösungsmittelverdampfung oder par : un agoniste TLR-1, un agoniste TLR-2, un ago- Lösungsmittelextraktion. niste TLR-3, un agoniste TLR-4, un agoniste TLR- 5, un agoniste TLR-6, un agoniste TLR-7, un ago- 12. Nanoträger nach einem der Ansprüche 1-10 zur Ver- 55 niste TLR-8, un agoniste TLR-9 et un agoniste TLR- wendung in der Medizin. 10.

8. Nanovecteur selon la revendication 7, dans lequel

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l’agoniste du récepteur de type toll est choisi dans le groupe constitué par : un agoniste TLR-3, un ago- niste TLR-7, un agoniste TLR-8 et un agoniste TLR- 9. 5 9. Nanovecteur selon l’une quelconque des revendica- tions 1 à 6, dans lequel l’agoniste du récepteur de type toll est l’imiquimod, R848 ou CL097.

10. Nanovecteur selon l’une quelconque des revendica- 10 tions précédentes, lequel nanovecteur se forme par auto-assemblage.

11. Procédé de préparation d’un nanovecteur selon l’une quelconque des revendications précédentes 15 par nanoprécipitation, focalisation de flux à l’aide de canaux fluidiques, séchage par atomisation, évapo- ration de solvant simple ou double émulsion ou ex- traction au solvant. 20 12. Nanovecteur selon l’une quelconque des revendica- tions 1 à 10, pour une utilisation en médecine.

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