US 20140294765A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0294765 A1 Cojocaru et al. (43) Pub. Date: Oct. 2, 2014

(54) LSRANTIBODIES, AND USES THEREOF FOR Publication Classification TREATMENT OF CANCER (51) Int. Cl. (71) Applicant: Compugen Ltd., Tel Aviv-Yafo (IL) C07K 6/28 (2006.01) (72) Inventors: Gad S. Cojocaru, Ramat HaSharon (IL); GOIN33/574 (2006.01) Liat Dassa, Yehud (IL); Galit Rotman, A63/675 (2006.01) Herzliyya (IL); Ofer Levi, Moshav A6II 45/06 (2006.01) Mesisraelat Zion (IL); Andrew Pow, San A 6LX39/395 (2006.01) Francisco, CA (US); Shirley Sameach-Greenwald, Kfar Saba (IL); (52) U.S. Cl. Zurit Levine, Herzliyya (IL) CPC ...... C07K 16/28 (2013.01); A61K 45/06 (2013.01); A61 K39/3955 (2013.01); A61 K (73) Assignee: COMPUGEN LTD., Tel Aviv-Yafo (IL) 31/675 (2013.01); G0IN33/57492 (2013.01) USPC ..... 424/85.2: 424/139.1; 424/85.7; 424/85.6; (21) Appl. No.: 14/361,571 424/85.5; 424/133.1; 424/136.1; 435/7.23 (22) PCT Fled: Jun. 19, 2013 (86) PCT NO.: PCT/IL2013/050527 (57) ABSTRACT S371 (c)(1), (2), (4) Date: May 29, 2014 This invention relates to antibodies and antigen binding frag Related U.S. Application Data ments and conjugates containing same, and/or alternative (60) Provisional application No. 61/662,470, filed on Jun. scaffolds, specific for LSR molecules, which are suitable 21, 2012. drugs for immunotherapy and treatment of specific cancer. Patent Application Publication Oct. 2, 2014 Sheet 1 of 30 US 2014/0294765 A1

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LSR ANTIBODIES, AND USES THEREOF FOR novel agents that are capable of modulating costimulatory TREATMENT OF CANCER signals, without compromising the immune systems ability to defend against pathogens, are highly advantageous for FIELD OF THE INVENTION treatment and prevention of such pathological conditions. 0001. This invention relates to LSR (lipolysis stimulated 0006 Costimulatory pathways play an important role in lipoprotein receptor)-specific antibodies, antibody frag tumor development. Interestingly, tumors have been shown to evade immune destruction by impeding T cell activation ments, conjugates and compositions comprising same, for through inhibition of co-stimmulatory factors in the treatment of cancer. B7-CD28 and TNF families, as well as by attracting regula tory T cells, which inhibit anti-tumor T cell responses (see BACKGROUND OF THE INVENTION Wang (2006) Immune Suppression by Tumor Specific CD4+ 0002 Naive T cells must receive two independent signals Regulatory T cells in Cancer. Semin Cancer. Biol. 16:73-79; from antigen-presenting cells (APC) in order to become pro Greenwald, et al. (2005) The B7 Family Revisited. Ann. Rev. ductively activated. The first, Signal 1, is antigen-specific and Immunol. 23:515-48: Watts (2005) TNF/TNFR Family occurs when T cell antigen receptors encounter the appropri Members in Co-stimulation of T Cell Responses Ann. Rev. ate antigen-MHC complex on the APC. The fate of the Immunol. 23:23-68; Sadum, et al. (2007) Immune Signatures immune response is determined by a second, antigen-inde of Murine and Human Cancers Reveal Unique Mechanisms pendent signal (Signal 2) which is delivered through a T cell of Tumor Escape and New Targets for Cancer Immuno costimulatory molecule that engages its APC-expressed therapy. Clin. Cane. Res. 13(13): 4016-4025). Such tumor ligand. This second signal could be either stimulatory (posi expressed co-stimulatory molecules have become attractive tive costimulation) or inhibitory (negative costimulation or cancer biomarkers and may serve as tumor-associated anti coinhibition). In the absence of a costimulatory signal, or in gens (TAAS). Furthermore, costimulatory pathways have the presence of a coinhibitory signal, T-cell activation is been identified as immunologic checkpoints that attenuate T impaired or aborted, which may lead to a state of antigen cell dependent immune responses, both at the level of initia specific unresponsiveness (known as T-cell anergy), or may tion and effector function within tumor metastases. As engi result in T-cell apoptotic death. neered cancer vaccines continue to improve, it is becoming 0003 Costimulatory molecule pairs usually consist of clear that such immunologic checkpoints are a major barrier ligands expressed on APCs and their cognate receptors to the vaccines’ ability to induce therapeutic anti-tumor expressed on T cells. The prototype ligand/receptor pairs of responses. In that regard, costimulatory molecules can serve costimulatory molecules are B7/CD28 and CD40/CD40L. as adjuvants for active (vaccination) and passive (antibody The B7 family consists of structurally related, cell-surface mediated) cancer immunotherapy, providing strategies to protein ligands, which may provide stimulatory or inhibitory thwart immune tolerance and stimulate the immune system. input to an immune response. Members of the B7 family are 0007. In addition, such agents could be of use in other structurally related, with the extracellular domain containing types of cancer immunotherapy, such as adoptive immuno at least one variable or constant immunoglobulin domain. therapy, in which tumor-specific T cell populations are 0004 Both positive and negative costimulatory signals expanded and directed to attack and kill tumor cells. Agents play critical roles in the regulation of cell-mediated immune capable of augmenting Such anti-tumor response have great responses, and molecules that mediate these signals have therapeutic potential and may be of value in the attempt to proven to be effective targets for immunomodulation. Based overcome the obstacles to tumor immunotherapy. Recently, on this knowledge, several therapeutic approaches that novel agents that modulate several costimulatory pathways involve targeting of costimulatory molecules have been were indeed introduced to the clinic as cancer immuno developed, and were shown to be useful for prevention and therapy. treatment of cancer by turning on, or preventing the turning 0008 Regulating costimulation using agonists and/or off of immune responses in cancer patients and for preven antagonists to various costimulatory proteins has been exten tion and treatment of autoimmune diseases and inflammatory sively studied as a strategy for treating autoimmune diseases, diseases, as well as rejection of allogenic transplantation, graft rejection, allergy and cancer. This field has been clini each by turning off uncontrolled immune responses, or by cally pioneered by CTLA4-Ig (Abatacept, OrenciaR) which induction of “off signal' by negative costimulation (or coin is approved for treatment of RA, mutated CTLA4-Ig (Belata hibition) in Subjects with these pathological conditions. cept, Nulojix(R) for prevention of acute kidney transplant 0005. Manipulation of the signals delivered by B7 ligands rejection and by the anti-CTLA4 antibody (Ipilimumab, Yer has shown potential in the treatment of autoimmunity, inflam voy CD), recently approved for the treatment of melanoma. matory diseases, and transplant rejection. Therapeutic strat Other costimulation regulators are currently in advanced egies include blocking of costimulation using monoclonal stages of clinical development including anti-PD-1 antibody antibodies to the ligand or to the receptor of a costimulatory (BMS-936558) which is in development for treatment of Non pair, or using soluble fusion proteins composed of the Small Cell Lung cancer and other cancers. Furthermore. Such costimulatory receptor that may bind and block its appropri agents are also inclinical development for viral infections, for ate ligand. Another approach is induction of co-inhibition example the anti PD-1 Ab, MDX-1 106, which is being tested using soluble fusion protein of an inhibitory ligand. These for treatment of hepatitis C, and the anti-CTLA-4 Ab CP-675, approaches rely, at least partially, on the eventual deletion of 206 (tremelimumab) which is in a clinical trial in hepatitis C auto- or allo-reactive T cells (which are responsible for the virus-infected patients with hepatocellular carcinoma. pathogenic processes in autoimmune diseases or transplanta 0009. Accumulations of inducible regulatory T cells tion, respectively), presumably because in the absence of (iTregs) are commonly seen in many tumors, and form the costimulation (which induces cell Survival genes) T cells major Subset of immune Suppressor cells in the tumor tissue. become highly susceptible to induction of apoptosis. Thus, Tregs create an immunosuppressive environment and regu US 2014/0294765 A1 Oct. 2, 2014

late anti-tumor immunity, and thus represent a major tumor NOs: 264, 265, 266, 267, 268, 269, 270, 271 or 272; or resistance mechanism from immune Surveillance. iTregs are alternatively at least one of SEQID NOs 274,275,276, 277, therefore viewed as important cellular targets for cancer or 282, and at least one of SEQID NOS: 264, 265,266, 267, therapy. 268,269,280,271, or 272; or alternatively at least one of SEQ 0010. In addition to their function in dampening effector T ID NOs303,304,305,306, or 307, and at least one of SEQID cell responses, multiple immune-checkpoint receptors, such NOs: 239, 240, 241, 252, 256, 287, 288, 284, or 285; or as CTLA4 and PD-1, and others like TIM3 and LAG3, are alternatively at least one of SEQID NOS 290, 291, 305,306, expressed at high levels on the Surface of iTregs and directly or 292, and at least one of SEQID NOs: 251,252,253,254, promote Treg cell-mediated Suppression of effector immune 256,287,288,258 or 259; or alternatively at least one of SEQ responses. Many of the immune-checkpoint antibodies in ID NOs303,304,305,306, or 307, and at least one of SEQID clinical testing most likely block the immunosuppressive NOs: 294, 295, 296,297,298,299, 300, 301 or 302. Option activity of iTregs as a mechanism of enhancing anti-tumor ally the immune molecule further comprises at least one of immunity. Indeed, two important factors in the mode of action SEQ ID NOs: 227-229, 239-243, 251-299, 264-272, 280, of CTLA4 blockade by ipilimumab are the enhancement of 284-285,287-288, 293-301 or 302 and at least one of SEQID effector T cell activity, and inhibition of Treg immunosup NOs 233-235, 245-249, 261-262, 274–278, 282, 290–292, pressive activity. 303-306 or 3.07. 0011. Several strategies, used alone or in combination 0017 Optionally the immune molecule is in the form of an with conventional treatments or immunotherapies, are in antibody fragment. development in order to disarm iTregs and restore antitumor 0018 Optionally the immune molecule comprises at least functions of effector T cells. two antigen-binding regions having amino acid sequences 0012 B cells play a critical role in recognition of foreign selected from the group consisting of SEQID NOs 239-243, antigens and they produce the antibodies necessary to provide 245-249, 251-259, 261-262, 264-272, 274–278, 280-281, protection against various type of infectious agents. T cell 282, 284-285, 287-288, 290–292, 294-307. help to B cells is a pivotal process of adaptive immune 0019. Optionally the immune molecule comprises at least responses. Follicular helper T (Tfh) cells are a subset of one antigen-binding region having an amino acid sequence CD4+ T cells specialized in B cell help (reviewed by Crotty, selected from the group consisting of SEQID NOS 227, 228, Annu. Rev. Immunol. 29: 621-663, 2011). Tfh cells express 229, 245, 246, 247, 248, 249, 261, 262, 274, 275,276, 277, the B cell homing chemokine receptor, CXCR5, which drives 278, 282,303, 304,305, 306, 307, 290, 291, and 292. Tfh cell migration into B cell follicles within lymph nodes in 0020 Optionally the immune molecule comprises at least a CXCL13-dependent manner. The requirement of Tfh cells one antigen-binding region having an amino acid sequence for B cell help and T cell-dependent antibody responses, selected from the group consisting of SEQID NOS 233,234, indicates that this cell type is of great importance for protec 235, 239, 240, 241, 287, 288, 242, 243, 251, 252,253, 254, tive immunity against various types of infectious agents, as 255, 256, 257, 258, 259,264, 265, 266, 267, 268, 269, 270, well as for rational vaccine design. 271, 272, 280, 294, 295, 296,297, 298, 299, 300, 301,302, 284, 285, 287, 288, 258, and 259. BRIEF SUMMARY OF THE INVENTION 0021 Optionally the immune molecule comprises a pro 0013 Despite recent progress in the understanding of can tein having the amino acid sequence of any of SEQID NOs: cer biology and cancer treatment, the Success rate for cancer 220, 244, 260, 273, 281, 289, 308, or a sequence 95% therapy remains low. Therefore, there is an unmet need for homologous thereto. new therapies which can Successfully treat cancer, Such as for 0022 Optionally the immune molecule comprises a pro example, specific blocking antibodies, which have a thera tein having the amino acid sequence of any of SEQID NOs: peutic application in stimulating the immune system against 218, 238, 250, 263, 279, 283, 286, 293, or a sequence 95% tumors. homologous thereto. 0014. By “blocking antibody' it is meant any antibody 0023 Optionally the immune molecule comprises a pro that binds to a particular protein or epitope on a protein, and tein having the amino acid sequence of SEQID NO: 220 and then optionally blocks interactions of that protein with one or of SEQID NO:218 or a sequence 95% homologous thereto; more other binding partners. 0024 or alternatively comprising a protein having the 0015. According to at least some embodiments there is amino acid sequence of SEQ ID NO: 244 and of SEQ ID provided an immune molecule, comprising an antigen-bind NO:238 or a sequence 95% homologous thereto; ing region having an amino acid sequence selected from the 0025 or alternatively comprising a protein having the group consisting of SEQ ID NOS 229, 235, 242-243, 245 amino acid sequence of SEQ ID NO: 260 and of SEQ ID 249, 258, 274–278, 264-272, 251-256, 280, 287, 284, 285, NO:259 or a sequence 95% homologous thereto; 290–292, 287,288, 259,295, 297-302, 306-307, wherein the 0026 or alternatively comprising a protein having the antigen-binding regions are adapted to specifically bind to a amino acid sequence of SEQ ID NO: 273 and of SEQ ID protein having the amino acid sequence of SEQID NO:10. NO:263 or a sequence 95% homologous thereto; 0016 Optionally the immune molecule comprises at least 0027 or alternatively comprising a protein having the one of SEQID NOs 227, 228 or 229 and at least one of SEQ amino acid sequence of SEQ ID NO: 281 and of SEQ ID ID NOs 233,234 or 235; or alternatively at least one of SEQ NO:279 or a sequence 95% homologous thereto; IDNOs 245,246,247,248, or 249, and at least one of SEQID 0028 or alternatively comprising a protein having the NOs: 239, 240, 241, 252, 256, 287, 288, 242 or 243; or amino acid sequence of SEQ ID NO:308 and of SEQ ID alternatively at least one of SEQID NOs303,304, 261,306, NO:283 or a sequence 95% homologous thereto; or 262, and at least one of SEQID NOs: 251,252,253,254, 0029 or alternatively comprising a protein having the 255,256,257,258 or 259; or alternatively at least one of SEQ amino acid sequence of SEQ ID NO: 289 and of SEQ ID IDNOs 274,275,276,277, or 278, and at least one of SEQID NO:286 or a sequence 95% homologous thereto; US 2014/0294765 A1 Oct. 2, 2014

0030 or alternatively comprising a protein having the 10, wherein said other portion of SEQID NO:10 comprises amino acid sequence of SEQ ID NO:308 and of SEQ ID amino acids 1-29 or amino acids 111 to 234 of SEQID NO: NO:293 or a sequence 95% homologous thereto; 10. 0031 or alternatively comprising a protein having the fol 0044 Optionally said antibody has an antigen-binding lowing amino acid sequences: one of SEQ ID NOs 245 or region that binds specifically to SEQID NO 215 or to SEQID 246; one of SEQID NOs 247 or 248: SEQID NO: 249; one NO 216 and that does not specifically bind to any other of SEQ ID NOs 239, 240, 241, or 252: one of SEQID NOs portion of SEQID NO 10, wherein said other portion of SEQ 256, 287, 288; and one of SEQID NOs 242 or 243: ID NO:10 comprises amino acids 1-80 or amino acids 99 to 0032 or alternatively comprising a protein having the fol 234 of SEQID NO: 10 for SEQID NO 215, or wherein said lowing amino acid sequences: one of SEQ ID NOS 303 or other portion of SEQID NO:10 comprises amino acids 1-117 or amino acids 136 to 234 of SEQID NO: 10 for SEQID NO 304; one of SEQID NOs 261 or 306: SEQID NO:262: one of 216. SEQID NOs 251, 252,253, or 254; one of SEQID NOs 255, 0045 Optionally the antibody is a fully human antibody, 256 or 257; and one of SEQID NOs 258 or 259; chimeric antibody, humanized or primatized antibody. 0033 or alternatively comprising a protein having the fol 0046) Optionally the antibody is selected from the group lowing amino acid sequences: one of SEQ ID NOs 274 or consisting of Fab, Fab'. F(ab')2, F(ab'), F(ab), Fv or schv 275; one of SEQID NOS:276 or 277; SEQID NO:278; one of fragment and minimal recognition unit. SEQID NOs:264,265,266, or 267: one of SEQID NOs:268, 0047 Optionally the antibody is coupled to a therapeutic 269, or 270; and one of SEQID NOS:271 or 272: agent selected from a drug, a radionuclide, a fluorophore, an 0034 or alternatively comprising a protein having the fol enzyme, a toxin, a therapeutic agent, or a chemotherapeutic lowing amino acid sequences: one of SEQ ID NOs 274 or agent; and wherein the detectable marker is a radioisotope, a 275; one of SEQID NOs:276 or 277; SEQID NO:282 one of metal chelator, an enzyme, a fluorescent compound, a biolu SEQID NOS:264, 265,266, 267: one of SEQ ID NOs:268, minescent compound or a chemiluminescent compound. 269, or 280; and one of SEQID NOS:271 or 272: 0048. According to at least some embodiments there is 0035 or alternatively comprising a protein having the fol provided a pharmaceutical composition comprising the lowing amino acid sequences: one of SEQ ID NOs: 303 or immune molecule, antibody or the antigen binding fragment 304; one of SEQID NOs: 305 or 306: SEQID NO:307; one as described herein. of SEQ ID NOs:239, 240, 241 or 252: one of SEQID NOs: 0049 According to at least some embodiments there is 256, 287 or 288; and one of SEQID NOS:284 or 285: provided use of the immune molecule, antibody, antibody 0036 or alternatively comprising a protein having the fol binding fragment or pharmaceutical composition as lowing amino acid sequences: one of SEQ ID NOS: 290 or described hereinfortreating a disease selected from the group 291; one of SEQID NOS:305 or 306: SEQID NO:292; one of consisting of cancer, immune condition or an infectious dis SEQID NOs:251,252,253, or 254; one of SEQID NOs:256, ease, in a subject in need thereof. 287, or 288; and one of SEQID NOS:258 or 259; 0050. Optionally said antibody or fragment modulates 0037 or alternatively comprising a protein having the fol immune cell activity, increases T cell activation, alleviates lowing amino acid sequences: one of SEQ ID NOs: 303 or T-cell Suppression, decreases immunosuppressive cytokine 304; one of SEQID NOS:305 or 306: SEQID NO:307; one of secretion, increases pro-inflammatory cytokine secretion, SEQID NOs:294,295, 296, or 297; one of SEQID NOs:298, increases IL-2 secretion; increases interferon-gamma pro 299 or 300; and one of SEQID NOS:301 or 302. duction by T-cells, promotes cancer epitope spreading, 0038 According to at least some embodiments there is increases T cell response in a mammal, decreases or elimi provided a polynucleotide encoding for the immune molecule nates M2 macrophages, reduces M2 macrophage pro-tumori as described herein. genic activity, enhances antigen-specific memory responses, 0039. Optionally the polynucleotide comprises a nucleic enhances apoptosis of cancer cells, enhances cytotoxic or cytostatic effect on cancer cells, enhances direct killing of acid sequence selected from the group consisting of SEQ cancer cells, induces complement dependent cytotoxicity NOs. 224, 225, 226, 230, 231, 232,217 and 219, and degen and/or antibody dependent cell-mediated cytotoxicity. erate variants thereof. 0051 Optionally said antibody or fragment increases 0040. According to at least some embodiments there is immune response against the cancer. provided a vector comprising the polynucleotide as described 0.052 Optionally the treatment is combined with another herein. therapeutic agent or therapy useful for treating cancer. 0041 According to at least some embodiments there is 0053) Optionally the therapy comprises one or more of provided a recombinant cell comprising the vector described radiotherapy, cryotherapy, antibody therapy, chemotherapy, herein, capable of expressing the immune molecule as photodynamic therapy, Surgery, hormonal deprivation or described herein. combination therapy with conventional drugs. 0042. According to at least some embodiments there is 0054 Optionally the therapeutic agent is selected from the provided a method for producing the immune molecule as group consisting of cytotoxic drugs, tumor vaccines, antibod described herein, comprising introducing the vector into a ies, peptides, pepti-bodies, Small molecules, chemotherapeu cell to form a recombinant cell; and producing the immune tic agents, cytotoxic and cytostatic agents, immunological molecule by the recombinant cell. modifiers, interferons, interleukins, immunostimulatory 0043. According to at least some embodiments there is growth hormones, cytokines, vitamins, minerals, aromatase provided an antibody or an antigenbinding fragment thereof, inhibitors, RNAi. Histone Deacetylase Inhibitors, and protea said antibody having an antigen-binding region that binds some inhibitors. specifically to amino acids 30-110 of SEQID NO 10 and that 0055 Optionally the immune molecule, antibody, anti does not specifically bind to any other portion of SEQID NO body binding fragment, composition as described herein is US 2014/0294765 A1 Oct. 2, 2014 administered to a Subject simultaneously or sequentially in 0061 Optionally the Therapeutic cancer vaccine is combination with one or more potentiating agents to obtain a selected from exogenous cancer vaccines including proteins therapeutic effect, wherein said one or more potentiating or peptides used to mount an immunogenic response to a agents is selected from the group consisting of radiotherapy, tumor antigen, recombinant virus and bacteria vectors encod conventional/classical anti-cancer therapy potentiating anti ing tumor antigens, DNA-based vaccines encoding tumor tumor immune responses, Targeted therapy potentiating anti antigens, proteins targeted to dendritic cells, dendritic cell tumor immune responses, Therapeutic agents targeting Tregs based vaccines, whole tumor cell vaccines, gene modified and/or MDSCs, Immunostimulatory antibodies, Cytokine tumor cells expressing GM-CSF, ICOS and/or Flt3-ligand, therapy. Therapeutic cancer vaccines, Adoptive cell transfer. oncolytic virus vaccines. 0056. Optionally the conventional/classical anti-cancer 0062 Optionally the Cytokine therapy is selected from agentis selected from platinum based compounds, antibiotics one or more of the following cytokines Such as IL-2, IL-7, with anti-cancer activity, Anthracyclines, Anthracenediones, IL-12, IL-15, IL-17, IL-18 and IL-21, IL23, IL-27, GM-CSF, alkylating agents, antimetabolites, Antimitotic agents, Tax IFNC (interferon alpha), IFNC-2b, IFNB, IFNY, and their anes, Taxoids, microtubule inhibitors, Vinca alkaloids, Folate different strategies for delivery. antagonists, Topoisomerase inhibitors, Antiestrogens, Anti 0063 Optionally the adoptive cell transfer therapy is car androgens, Aromatase inhibitors, GnRhanalogs, inhibitors of ried out following ex vivo treatment selected from expansion 5C-reductase, biphosphonates. of the patient autologous naturally occurring tumor specific T 0057 Optionally the Targeted therapy agent is selected cells or genetic modification of T cells to confer specificity for from the group consisting of histone deacetylase (HDAC) tumor antigens. inhibitors, proteasome inhibitors, mTOR pathway inhibitors, 0064. According to at least some embodiments there is JAK2 inhibitors, tyrosine kinase inhibitors (TKIs), PI3K provided a use of an antibody or immune molecule or a inhibitors, Protein kinase inhibitors, Inhibitors of serine/ pharmaceutical composition as described herein to perform threonine kinases, inhibitors of intracellular signaling, inhibi one or more of the following in a subject to treat a disease: (a) tors of Ras/Raf signaling, MEK inhibitors, AKT inhibitors, upregulating cytokines, (b) increases T-cell proliferation and/ inhibitors of Survival signaling proteins, cyclin dependent or expansion, (c) increases interferon-gamma production by kinase inhibitors, therapeutic monoclonal antibodies, TRAIL T-cells (d) increases IL-2 secretion (e) stimulates antibody pathway agonists, anti-angiogenic agents, metalloproteinase responses; (f) inhibits cancer cell growth, (g) promoting anti inhibitors, cathepsin inhibitors, inhibitors of urokinase plas genic specific T cell immunity, (g) promoting CD4+ and/or minogen activator receptor function, immunoconjugates. CD8+ T cell activation, (i) alleviating T-cell suppression, () antibody drug conjugates, antibody fragments, bispecific anti alleviating apoptosis or lysis of cancer cells, (k) cytotoxic or bodies, bispecific T cell engagers (BiTEs). cytostatic effect on cancer cells. 0065 According to at least some embodiments there is 0058 Optionally the antibody therapy is selected from provided a diagnostic method for diagnosing a disease in a cetuximab, panitumumab, nimotuZumab, trastuzumab, per Subject, wherein the disease is selected from the group con tuZumab, rituximab. ofatumumab, VeltuZumab, alemtu sisting of cancer or an autoimmune disease, wherein the Zumab, labetuZumab, adecatumumab, oregovomab, onartu diagnostic method is performed ex vivo, comprising contact Zumab, apomab, mapatumumab, lexatumumab, ing a tissue sample from the Subject with the immune mol conatumumab, tigatuZumab, catumaXomab, blinatumomab, ecule or antibody as described herein ex vivo and detecting ibritumomab triuxetan, to situmomab, brentuximab vedotin, specific binding thereto. gemtuzumab ozogamicin, clivatuZumab tetraxetan, pemtu 0066. According to at least some embodiments there is momab, trastuzumab emtansine, bevacizumab, etaraci provided a diagnostic method for diagnosing a disease in a Zumab, Volocliximab, ramucirumab, aflibercept. Subject, wherein the disease is selected from the group con 0059 Optionally the Therapeutic agent targeting immu sisting of cancer, autoimmune disease, or an infectious dis nosuppressive cells Tregs and/or MDSCs is selected from ease wherein the diagnostic method is performed in vivo, antimitotic drugs, cyclophosphamide, gemcitabine, mitox comprising administering the immune molecule or antibody antrone, fludarabine, thalidomide, thalidomide derivatives, as described herein to the subject and detecting specific bind COX-2 inhibitors, depleting or killing antibodies that directly ing of the immune molecule or antibody as described herein target Tregs through recognition of Treg cell Surface recep to a tissue of the subject. tors, anti-CD25 daclizumab, basiliximab, ligand-directed 0067. Optionally the diagnostic method is performed toxins, denileukin diftitox (Ontak)—a fusion protein of before administering the immune molecule or antibody or human IL-2 and diphtheria toxin, or LMB-2 a fusion pharmaceutical composition to the Subject. between an Schv against CD25 and the pseudomonas exo 0068. Optionally the use or method further comprises toxin, antibodies targeting Treg cell Surface receptors, TLR determining an LSR level in a tissue of the subject before modulators, agents that interfere with the adenosinergic path administering the immune molecule or antibody or pharma way, ectonucleotidase inhibitors, or inhibitors of the A2A ceutical composition to the Subject. adenosine receptor, TGF-B inhibitors, chemokine receptor 0069 Optionally said administering the immune molecule inhibitors, retinoic acid, all-trans retinoic acid (ATRA), Vita or antibody or pharmaceutical composition to the Subject min D3, phosphodiesterase 5 inhibitors, sildenafil. ROS only if said LSR level is sufficient. inhibitors and nitroaspirin. 0070 Optionally the use or method further comprises 0060 Optionally the Immunostimulatory antibody is determining said LSR level according to expression level of selected from antagonistic antibodies targeting one or more said LSR. of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, 0071 Optionally said determining said expression level B7-H3, VISTA, and/or Agonistic antibodies targeting one or comprises applying an IHC (immunohistochemistry) assay more of CD40, CD137, OX40, GITR, CD27, CD28 or ICOS. or a gene expression assay to a tissue of the Subject. US 2014/0294765 A1 Oct. 2, 2014

0072 Optionally said applying said IHC assay comprises Moderate to Poorly Differentiated Adenocarcinoma of the determining if a level of expression is at least 1 on a scale of cecum, Well to Poorly Differentiated Adenocarcinoma of the O to 3. colon, Grade 2 Tubular adenocarcinoma of the ascending 0073. Optionally said tissue comprises cancer cells or colon, colon adenocarcinoma Duke's stage C1, invasive immune infiltrate. adenocarcinoma of the colon, Adenocarcinoma of the rectum, 0074. Optionally said determining said LSR level in said Grade 3 Adenocarcinoma of the rectum, Moderately Differ tissue comprises contacting the tissue with the antibody or entiated Adenocarcinoma of the rectum, Moderately Differ immune molecule as described herein and detecting specific entiated Mucinous adenocarcinoma of the rectum, Well to binding thereto. Poorly differentiated Non-small cell carcinoma, Moderately 0075 According to at least some embodiments there is to poorly differentiated squamous carcinoma of the lung, provided an assay for diagnosing a disease in a tissue sample Moderately well differentiated keratinising squamous cell taken from a Subject, comprising the immune molecule or carcinoma of the lung, large cell adenocarcinoma of the lung, antibody as described herein and at least one reagent for prostate Adenocarcinoma Gleason Grade 7 to 9, prostate diagnosing a disease selected from the group consisting of Infiltrating adenocarcinoma, Moderately differentiated cancer, autoimmune disease, or infectious disease. adenocarcinoma of the prostate, serous papillary cystic car 0076 According to at least some embodiments there is cinoma of the ovary, Serous cystadenocarcinoma of the ovary, provided use as described herein for Screening for a disease, grade 4 Astrocytoma, Glioblastoma multiforme, Clear cell detecting a presence or a severity of a disease, providing renal cell carcinoma, Hepatocellular carcinoma, and Low prognosis of a disease, monitoring disease progression or Grade hepatocellular carcinoma; with the proviso that if the relapse, as well as assessment of treatment efficacy and/or cancer is ovarian cancer, it is not Granulosa cell tumor of the relapse of a disease, disorder or condition, as well as selecting ovary and with the proviso that if the cancer is brain cancer, it a therapy and/or a treatment for a disease, optimization of a is not Astrocytoma grade 2. given therapy for a disease, monitoring the treatment of a 0079. Optionally said breast cancer is selected from the disease, and/or predicting the Suitability of a therapy for spe group consisting of ductal-adenocarcinoma, infiltrating duc cific patients or subpopulations or determining the appropri tal carcinoma, lobular carcinoma, mucinous adenocarci ate dosing of a therapeutic product in patients or Subpopula noma, intra duct and invasive ductal carcinoma. tions. 0080 Optionally said breast cancer is Scirrhousadenocar 0077 Optionally said cancer, said immune cells infiltrat cinoma. ing the tumor or both express LSR at a sufficient level and I0081. Optionally said colon cancer is selected from the wherein said cancer is selected from the group consisting of group consisting of Moderate to Poorly Differentiated breast cancer, cervical cancer, ovary cancer, endometrial can Adenocarcinoma of the cecum, Well, Moderate and Poorly cer, melanoma, bladder cancer, lung cancer, pancreatic can Differentiated Adenocarcinoma of the colon, Tubular adeno cer, colon cancer, prostate cancer, leukemia, acute lympho carcinoma, preferably Grade 2 Tubular adenocarcinoma of cytic leukemia, chronic lymphocytic leukemia, B-cell the ascending colon, colonadenocarcinoma Duke's stage C1, lymphoma, Burkitt's lymphoma, multiple myeloma, Non invasive adenocarcinoma, Adenocarcinoma of the rectum, Hodgkin’s lymphoma, myeloid leukemia, acute myelog preferably Grade 3 Adenocarcinoma of the rectum, Moder enous leukemia (AML), chronic myelogenous leukemia, thy ately Differentiated Adenocarcinoma of the rectum, and roid cancer, thyroid follicular cancer, myelodysplastic Moderately Differentiated Mucinous adenocarcinoma of the syndrome (MDS), fibrosarcomas and rhabdomyosarcomas, rectum. melanoma, uveal melanoma, teratocarcinoma, neuroblas I0082 Optionally said lung cancer is selected from the toma, glioma, glioblastoma, benign tumor of the skin, kera group consisting of Well to Poorly differentiated Non-small toacanthomas, renal cancer, anaplastic large-cell lymphoma, cell carcinoma, Squamous Cell Carcinoma, preferably Mod esophageal squamous cells carcinoma, hepatocellular carci erately Differentiated Squamous Cell Carcinoma, Moder noma, follicular dendritic cell carcinoma, intestinal cancer, ately to poorly differentiated squamous carcinoma, Moder muscle-invasive cancer, seminal vesicle tumor, epidermal ately well differentiated keratinising squamous cell carcinoma, spleen cancer, bladder cancer, head and neck can carcinoma, large cell adenocarcinoma and Small cell lung cer, stomach cancer, liver cancer, bone cancer, brain cancer, CaCC. cancer of the retina, biliary cancer, Small bowel cancer, Sali I0083) Optionally said prostate cancer is selected from the vary gland cancer, cancer of uterus, cancer of testicles, cancer group consisting of Adenocarcinoma Gleason Grade 5 to 9, of connective tissue, prostatic hypertrophy, myelodysplasia, Infiltrating adenocarcinoma, High grade prostatic intraepi Waldenstrom's macroglobinaemia, nasopharyngeal, neu thelial neoplasia, and undifferentiated carcinoma. roendocrine cancer, myelodysplastic syndrome, mesothe I0084 Optionally said stomach cancer is moderately dif lioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesopha ferentiated gastric adenocarcinoma. gogastric, fallopian tube cancer, peritoneal cancer, papillary I0085 Optionally said ovarian cancer is selected from the serous mullerian cancer, malignant ascites, gastrointestinal group consisting of serous papillary cystic carcinoma, Serous stromal tumor (GIST), Li-Fraumeni syndrome and Von Hip cystadenocarcinoma and Invasive serous papillary carci pel-Lindau syndrome (VHL); with the proviso that if the Oa. cancer is ovarian cancer, it is not Granulosa cell tumor of the I0086 Optionally said brain cancer is selected from the ovary and with the proviso that if the cancer is brain cancer, it group consisting of Glioblastoma multiforme and Astrocy is not Astrocytoma grade 2. toma other than Astrocytoma grade 2. 0078. Optionally said cancer is selected from the group I0087 Optionally said astrocytoma is grade 4 Astrocy consisting of ductal-adenocarcinoma, infiltrating ductal car tOma. cinoma, Lobular carcinoma of breast, mucinous adenocarci I0088 Optionally said kidney cancer is Clear cell renal cell noma of the breast, Intra duct and invasive ductal carcinoma, carcinoma. US 2014/0294765 A1 Oct. 2, 2014

0089 Optionally liver cancer is Hepatocellular carci 0094 Optionally the treatment is combined with another Oa. moiety useful for treating immune related condition. 0090. Optionally said Hepatocellular carcinoma is Low 0.095 Optionally said disease is infectious disease and Grade hepatocellular carcinoma or Fibrolamellar Hepatocel wherein said infectious disease is selected from the disease lular Carcinoma. caused by bacterial infection, viral infection, fungal infection 0091. Optionally said hematological cancer is selected and/or other parasite infection. from the group consisting of large cell lymphoma, and High 0096 Optionally the infectious disease is selected from and low grade Non-Hodgkin’s Lymphoma. hepatitis B, hepatitis C, infectious mononucleosis, EBV, 0092 Optionally said disease is immune condition and cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria wherein said immune condition is selected from the group and Schistosomiasis. consisting of autoimmune disease, transplant rejection, and 0097. Optionally the treatment is combined with another graft versus host disease. moiety useful for treating infectious disease. 0093 Optionally said autoimmune disease is selected 0098. According to at least some embodiments there is from the group consisting of wherein the autoimmune disease provided use of an antibody or a fragment specifically binding is selected from a group consisting of multiple Sclerosis, to SEQID NO: 10 to treat or diagnose a subject suffering from psoriasis; rheumatoid arthritis; psoriatic arthritis, systemic a disease selected from the group consisting of ductal-adeno lupus erythematosus (SLE); ulcerative colitis; Crohn's dis carcinoma, infiltrating ductal carcinoma, lobular carcinoma, ease; benign lymphocytic angiitis, thrombocytopenic pur mucinous adenocarcinoma, intra duct and invasive ductal pura, idiopathic thrombocytopenia, idiopathic autoimmune carcinoma, Scirrhousadenocarcinoma, Moderate to Poorly hemolytic anemia, pure red cell aplasia, Sjogren's syndrome, Differentiated Adenocarcinoma of the cecum, Well, Moder rheumatic disease, connective tissue disease, inflammatory ate and Poorly Differentiated Adenocarcinoma of the colon, rheumatism, degenerative rheumatism, extra-articular rheu Tubular adenocarcinoma, preferably Grade 2 Tubular adeno matism, juvenile rheumatoid arthritis, arthritis uratica, mus carcinoma of the ascending colon, colon adenocarcinoma cular rheumatism, chronic polyarthritis, cryoglobulinemic Duke's stage C1, invasive adenocarcinoma, Adenocarcinoma vasculitis, ANCA-associated vasculitis, antiphospholipid of the rectum, preferably Grade 3 Adenocarcinoma of the syndrome, myasthenia gravis, autoimmune haemolytic rectum, Moderately Differentiated Adenocarcinoma of the anaemia, Guillian-Barre Syndrome, chronic immune poly rectum, Moderately Differentiated Mucinous adenocarci neuropathy, autoimmune thyroiditis, insulin dependent dia noma of the rectum, Well to Poorly differentiated Non-small betes mellitus, type I diabetes, Addison’s disease, membra cell carcinoma, Squamous Cell Carcinoma, preferably Mod nous glomerulonephropathy, Goodpasture's disease, erately Differentiated Squamous Cell Carcinoma, Moder autoimmune gastritis, autoimmune atrophic gastritis, perni ately to poorly differentiated squamous carcinoma, Moder cious anaemia, pemphigus, pemphigus Vulgarus, cirrhosis, ately well differentiated keratinising squamous cell primary biliary cirrhosis, dermatomyositis, polymyositis, carcinoma, large cell adenocarcinoma, Small cell lung can fibromyositis, myogelosis, celiac disease, immunoglobulin A cer, Adenocarcinoma Gleason Grade 5 to 9. Infiltrating nephropathy, Henoch-Schonlein purpura, Evans syndrome, adenocarcinoma, High grade prostatic intraepithelial neopla atopic dermatitis, psoriasis, psoriasis arthropathica, Graves sia, undifferentiated carcinoma, moderately differentiated disease, Graves ophthalmopathy, Scleroderma, systemic gastric adenocarcinoma, serous papillary cystic carcinoma, Scleroderma, progressive systemic Scleroderma, asthma, Serous cystadenocarcinoma, Invasive serous papillary carci allergy, primary biliary cirrhosis, Hashimoto's thyroiditis, noma, Glioblastoma multiforme, Astrocytoma, Astrocytoma primary myxedema, sympathetic ophthalmia, autoimmune grade 4, Clear cell renal cell carcinoma, Hepatocellular car uveitis, hepatitis, chronic action hepatitis, collagen diseases, cinoma, Low Grade hepatocellular carcinoma, Fibrolamellar ankylosing spondylitis, periarthritis humeroScapularis, Hepatocellular Carcinoma, large cell lymphoma, and High panarteritis nodosa, chondrocalcinosis, Wegener's granulo and low grade Non-Hodgkin’s Lymphoma; with the proviso matosis, microscopic polyangiitis, chronic urticaria, bullous that if the cancer is ovarian cancer, it is not Granulosa cell skin disorders, pemphigoid, atopic eczema, Devic's disease, tumor of the ovary and with the proviso that if the cancer is childhood autoimmune hemolytic anemia, Refractory or brain cancer, it is not Astrocytoma grade 2. chronic Autoimmune Cytopenias, Prevention of development 0099. According to at least some embodiments there is of Autoimmune Anti-Factor VIII Antibodies in Acquired provided a pharmaceutical composition comprising an anti Hemophilia A, Cold Agglutinin Disease, Neuromyelitis body or a fragment specifically binding to SEQID NO: 10 to Optica, Stiff Person Syndrome, gingivitis, periodontitis, pan treat a subject suffering from a disease selected from the creatitis, myocarditis, vasculitis, gastritis, gout, gouty arthri group consisting of ductal-adenocarcinoma, infiltrating duc tis, and inflammatory skin disorders, normocomplementemic tal carcinoma, lobular carcinoma, mucinous adenocarci urticarial vasculitis, pericarditis, myositis, anti-synthetase noma, intra duct and invasive ductal carcinoma, Scirrhous syndrome, Scleritis, macrophage activation syndrome, adenocarcinoma, Moderate to Poorly Differentiated Adeno Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, carcinoma of the cecum, Well, Moderate and Poorly Differ gout, adult and juvenile Still's disease, cryropyrinopathy, entiated Adenocarcinoma of the colon, Tubular adenocarci Muckle-Wells syndrome, familial cold-induced auto-inflam noma, preferably Grade 2 Tubular adenocarcinoma of the matory syndrome, neonatal onset multisystemic inflamma ascending colon, colon adenocarcinoma Duke's stage C1, tory disease, familial Mediterranean fever, chronic infantile invasive adenocarcinoma, Adenocarcinoma of the rectum, neurologic, cutaneous and articular syndrome, systemic juve preferably Grade 3 Adenocarcinoma of the rectum, Moder nile idiopathic arthritis, Hyper Igld syndrome, Schnitzler's ately Differentiated Adenocarcinoma of the rectum, Moder syndrome, autoimmune retinopathy, age-related macular ately Differentiated Mucinous adenocarcinoma of the rec degeneration, atherosclerosis, chronic prostatitis and TNF tum, Well to Poorly differentiated Non-small cell carcinoma, receptor-associated periodic syndrome (TRAPS). Squamous Cell Carcinoma, preferably Moderately Differen US 2014/0294765 A1 Oct. 2, 2014 tiated Squamous Cell Carcinoma, Moderately to poorly dif immune responses, Therapeutic agents targeting Tregs and/or ferentiated squamous carcinoma, Moderately well differen MDSCs, Immunostimulatory antibodies, Cytokine therapy, tiated keratinising squamous cell carcinoma, large cell Adoptive cell transfer. adenocarcinoma, Small cell lung cancer, Adenocarcinoma 0105 Optionally the conventional/classical anti-cancer Gleason Grade 5 to 9. Infiltrating adenocarcinoma, High agentis selected from platinum based compounds, antibiotics grade prostatic intraepithelial neoplasia, undifferentiated car with anti-cancer activity, Anthracyclines, Anthracenediones, cinoma, moderately differentiated gastric adenocarcinoma, alkylating agents, antimetabolites, Antimitotic agents, Tax serous papillary cystic carcinoma, Serous cystadenocarci anes, Taxoids, microtubule inhibitors, Vinca alkaloids, Folate noma, Invasive serous papillary carcinoma, Glioblastoma antagonists, Topoisomerase inhibitors, Antiestrogens, Anti multiforme, Astrocytoma, Astrocytoma grade 4. Clear cell androgens, Aromatase inhibitors, GnRhanalogs, inhibitors of renal cell carcinoma, Hepatocellular carcinoma, Low Grade 5C-reductase, biphosphonates. hepatocellular carcinoma, Fibrolamellar Hepatocellular Car cinoma, large cell lymphoma, and High and low grade Non 0106 Optionally the Targeted therapy agent is selected Hodgkin’s Lymphoma; with the proviso that if the cancer is from the group consisting of histone deacetylase (HDAC) ovarian cancer, it is not Granulosa cell tumor of the ovary and inhibitors, proteasome inhibitors, mTOR pathway inhibitors, with the proviso that if the cancer is brain cancer, it is not JAK2 inhibitors, tyrosine kinase inhibitors (TKIs), PI3K Astrocytoma grade 2. inhibitors, Protein kinase inhibitors, Inhibitors of serine/ threonine kinases, inhibitors of intracellular signaling, inhibi 0100 Optionally the cancer is selected from the group tors of Ras/Raf signaling, MEK inhibitors, AKT inhibitors, consisting of ductal-adenocarcinoma, infiltrating ductal car inhibitors of Survival signaling proteins, cyclin dependent cinoma, Lobular carcinoma of breast, mucinous adenocarci kinase inhibitors, therapeutic monoclonal antibodies, TRAIL noma of the breast, Intra duct and invasive ductal carcinoma, pathway agonists, anti-angiogenic agents, metalloproteinase Moderate to Poorly Differentiated Adenocarcinoma of the inhibitors, cathepsin inhibitors, inhibitors of urokinase plas cecum, Well to Poorly Differentiated Adenocarcinoma of the minogen activator receptor function, immunoconjugates, colon, Grade 2 Tubular adenocarcinoma of the ascending antibody drug conjugates, antibody fragments, bispecific anti colon, colon adenocarcinoma Duke's stage C1, invasive bodies, bispecific T cell engagers (BiTEs). adenocarcinoma, Adenocarcinoma of the rectum, Grade 3 Adenocarcinoma of the rectum, Moderately Differentiated 0107 Optionally the antibody is selected from cetuximab, Adenocarcinoma of the rectum, Moderately Differentiated panitumumab, nimotuZumab, trastuzumab, pertuzumab, rit Mucinous adenocarcinoma of the rectum, Well to Poorly uximab. ofatumumab, VeltuZumab, alemtuzumab, labetu differentiated Non-small cell carcinoma, Squamous Cell Car Zumab, adecatumumab, oregovomab, onartuzumab. apomab, cinoma: Moderately Differentiated, Moderately to poorly mapatumumab, lexatumumab, conatumumab, tigatuZumab, differentiated squamous carcinoma, Moderately well differ catumaXomab, blinatumomab, ibritumomab triuxetan, tosi entiated keratinising squamous cell carcinoma, large cell tumomab, brentuximab vedotin, gemtuzumab ozogamicin, adenocarcinoma, Adenocarcinoma Gleason Grade 7 to 9, clivatuZumab tetraxetan, pemtumomab, trastuzumab Infiltrating adenocarcinoma, Moderately differentiated emtansine, bevacizumab, etaracizumab, Volocliximab, ramu adenocarcinoma, serous papillary cystic carcinoma, Serous cirumab, aflibercept. cystadenocarcinoma, grade 4 Astrocytoma, Glioblastoma 0.108 Optionally the Therapeutic agent targeting immu multiforme, Clear cell renal cell carcinoma, Hepatocellular nosuppressive cells Tregs and/or MDSCs is selected from carcinoma, and Low Grade hepatocellular carcinoma; with antimitotic drugs, cyclophosphamide, gemcitabine, mitox the proviso that if the cancer is ovarian cancer, it is not antrone, fludarabine, thalidomide, thalidomide derivatives, Granulosa cell tumor of the ovary and with the proviso that if COX-2 inhibitors, depleting or killing antibodies that directly the cancer is brain cancer, it is not Astrocytoma grade 2. target Tregs through recognition of Treg cell Surface recep tors, anti-CD25 daclizumab, basiliximab, ligand-directed 0101. Optionally the treatment is combined with another toxins, denileukin diftitox (Ontak)—a fusion protein of therapeutic agent or therapy useful for treating cancer. human IL-2 and diphtheria toxin, or LMB-2 a fusion 0102 Optionally the therapy comprises one or more of between an Schv against CD25 and the pseudomonas exo radiotherapy, cryotherapy, antibody therapy, chemotherapy, toxin, antibodies targeting Treg cell Surface receptors, TLR photodynamic therapy, Surgery, hormonal deprivation or modulators, agents that interfere with the adenosinergic path combination therapy with conventional drugs. way, ectonucleotidase inhibitors, or inhibitors of the A2A 0103 Optionally the therapeutic agent is selected from the adenosine receptor, TGF-B inhibitors, chemokine receptor group consisting of cytotoxic drugs, tumor vaccines, antibod inhibitors, retinoic acid, all-trans retinoic acid (ATRA), Vita ies, peptides, pepti-bodies, Small molecules, chemotherapeu min D3, phosphodiesterase 5 inhibitors, sildenafil. ROS tic agents, cytotoxic and cytostatic agents, immunological inhibitors and nitroaspirin. modifiers, interferons, interleukins, immunostimulatory 0109 Optionally the Immunostimulatory antibody is growth hormones, cytokines, vitamins, minerals, aromatase selected from antagonistic antibodies targeting one or more inhibitors, RNAi. Histone Deacetylase Inhibitors, and protea of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, some inhibitors. B7-H3, VISTA, and/or Agonistic antibodies targeting one or 0104 Optionally the antibody or composition is adminis more of CD40, CD137, OX40, GITR, CD27, CD28 or ICOS. tered to a subject simultaneously or sequentially in combina 0110 Optionally the Therapeutic cancer vaccine is tion with one or more potentiating agents to obtain a thera selected from exogenous cancer vaccines including proteins peutic effect, wherein said one or more potentiating agents is or peptides used to mount an immunogenic response to a selected from the group consisting of radiotherapy, conven tumor antigen, recombinant virus and bacteria vectors encod tional/classical anti-cancer therapy potentiating anti-tumor ing tumor antigens, DNA-based vaccines encoding tumor immune responses, Targeted therapy potentiating anti-tumor antigens, proteins targeted to dendritic cell-based vaccines, US 2014/0294765 A1 Oct. 2, 2014

whole tumor cell vaccines, gene modified tumor cells I0123 FIG. 7 shows a specific knock down of the ectopic expressing GM-CSF, ICOS and/or Flt3-ligand, oncolytic expression of the Human WT LSR-flag protein demonstra virus Vaccines. eted by dramatic decrease in the signal intensity of the ~70 0111 Optionally the Cytokine therapy is selected from kDa band, corresponding to the HumanWTLSR-flag protein, one or more of the following cytokines Such as IL-2, IL-7, following transfection of the HEK293T cells stably trans IL-12, IL-15, IL-17, IL-18 and IL-21, IL23, IL-27, GM-CSF, fected with plRESpuro3 human WT LSR Flag, with the IFNC (interferon alpha), IFNC-2b, IFNB, IFNY, and their LSR specific siRNA (Thermo Scientific CathM-009672-00 different strategies for delivery. 0005) (lane 2), as compared to the scrambled siRNA (Thermo 0112 Optionally the adoptive cell transfer therapy is car Scientific CathD-001810-01-05) (lane 1). ried out following ex vivo treatment selected from expansion 0.124 FIG. 8 demonstrates the subcellular localization of of the patient autologous naturally occurring tumor specific T LSR P5a Flag m. LSR P5a Flag m (SEQ ID NO: 144) is cells or genetic modification of T cells to confer specificity for localized mainly to the cell cytoplasm, but can also be tumor antigens. detected on the cell surface as detected with anti Flag (Sigma 0113. According to at least some embodiments there is cati A9594) (FIG. 8A) and anti LSR antibodies as follows: provided a diagnostic method for determining whether to Abcam, cat ab59646 (FIG. 8B) Abnova, catiH00051599 perform the use or to administer the composition as described B01P (FIG. 8C) and Sigma cath HPAO07270 (FIG. 8D). herein, comprising performing the diagnostic method as 0.125 FIG. 9 presents confocal microscopy image demon described herein. strating the subcellular localization of human LSR WT pro 0114 Optionally the cancer is non-metastatic. tein (SEQ ID NOS:11) in human LSRWTHEK293T trans 0115 Optionally the cancer is invasive. fected cells. 0116. Optionally the cancer is metastatic. 0.126 FIG. 10 presents confocal microscopy image dem onstrating the subcellular localization of cyno LSR WT pro BRIEF DESCRIPTION OF THE FIGURES tein (SEQ ID NO: 211) in cyno LSR WT HEK293T trans 0117 FIG. 1 demonstrates Western Blot analysis of the fected cells. expression of LSR P5a Flag m protein (SEQ ID: 144) in I0127 FIG. 11 presents confocal microscopy image dem stably-transfected recombinant HEK293T cells, as detected onstrating the subcellular localization of mouse LSR WT with anti Flag (Sigma catiA8592) (FIG. 1A) and anti LSR protein (SEQ ID NO: 31) in mouse LSR WT transfected antibodies as follow: Abnova, catiH00051599-B01P (FIG. HEK293 cells. 1B) Abcam, cat ab59646 (FIG. 1C) and Sigma cath 0128 FIG. 12 presents confocal microscopy image dem HPAO07270 (FIG. 1D). Lane 1: HEK293T pIRESpuro3; onstrating the subcellular localization of cyno LSR skip4 lane 2: HEK293T pIRESpuro3 LSR P5a Flag. protein (SEQ ID NO:212) in HEK293T transfected cells. 0118 FIG.2 presents detection of human LSR WT with 3 I0129 FIG. 13 presents confocal microscopy image dem different commercial Abs against LSR: 2A refers to SIGMA onstrating the subcellular localization of mouse LSR WT Ab, whereas 2B to Abcam Ab and 2C to Abnova Ab (as protein (SEQ ID NOs: 31) in mouse LSR WT transfected detailed in Table 1). Lane #1 represents HEK293T pIRE CHO-K1 cells. Spuro3 empty vector transfected cells which were used as a I0130 FIGS. 9A, 10A, 11A, 12A and 13A present LSR negative control, whereas lane #2 represents HEK293T p protein localization with anti Flag antibody (Sigma cathi RESpuro3 human WT LSR Flag transfected cells. A9594). LSR protein localization with anti LSRantibodies is 0119 FIG. 3 presents detection of the human LSR skip4 presented in FIG.9C (Abcam, cat ab59646), and FIGS. 9B, protein using Abcam Ab. Lane #1 represents HEK293T 10B, 11B, 12B and 13B (Sigma cath HPAO07270). Figures pIRESpuro3 empty vector transfected cells which were used A-1, B-1 and C-1 in FIGS. 9-13 represent the results of empty as a negative control, whereas lane #2 represents HEK293T vector transfected cells which were used as a negative control. pIRESpuro3 human LSR skip4 Flag transfected cells. Arrows indicate membrane staining. Figures A-2, B-2 and 0120 FIG. 4 presents detection of the cyno LSR WT in C-2 in FIGS. 9-13 represent the results of the subcellular HEK293T pIRESpuro3 cyno WT LSR Flag transfected localization of human, cyno and mouse LSR WT protein in cells (lane 2) and LSR skip4 in HEK293T pIRESpuro3 recombinant cells expressing LSR proteins. cynoskip4 LSR Flag transfected cells (lane 3) proteins, both I0131 FIG. 14 presents the results of binding of different compared to the empty vector HEK293T pIRESpuro3 trans concentrations (1 lug/ml, 2.5ug/ml, 5ug/ml and 10 ug/ml) of fected cells lysate (lane 1). The detection was done using Anti 8C8 mAb to cells stably expressing human WT LSR protein LSR antibody (Abcam 1:4000). (SEQID NO:11), cyno WT LSR protein (SEQID NO:211), 0121 FIG. 5 presents detection by anti-flag Abs of mouse mouse WT LSR protein (SEQID NO:31), human Skip4 LSR LSR WT protein in CHO-K1 cells transfected with variant (SEQID NO:13) and cyno Skip4 LSR variant (SEQ pcDNA3.1 mouse WT LSR Flag (lane 2) and in HEK293 ID NO: 212). cells transfected with pcDNA3.1 mouse WTLSR Flag (lane I0132 FIG. 15 demonstrates endogenous expression of 4) compared to the corresponding empty vector transfected LSR proteins (corresponding to ~70kDa band) in various cell cells lysate (lanes 1 and 3, respectively). lines, as detailed below. Lanes 1 and 14 represent the positive 0122 FIG. 6 demonstrates the endogenous expression of control cells, corresponding to HEK293T cells stably trans LSR in various cell lines. A band at ~72 kDa corresponding to fected with plRESpuro3 human WT LSR Flag. Lane 2 cor LSR was detected with anti LSR antibody in cell extracts of responds to SKBR3; lane 3 corresponds to MCF7, lane 4 (1)Caov3, (2) ES2, (3) OV-90, (4) OVCAR, (5) SK-OV3, (6) corresponds to HepG2, lane 5 corresponds to Hela, lane 6 TOV112D, (7) CaCo2, (8) HeLa, (9) Hep G2, (10) MCF-7, corresponds to CaCO2, lane 7 corresponds to TOV112D, lane (11) SkBR and (12) 293T LSR P5a Flag (FIG. 3A). Anti 8 corresponds to SKOV3, lane 9 corresponds to OvCar3, lane GAPDH (Abcam cathi ab9484) served as a loading control 10 corresponds to OV-90, lane 11 corresponds to ES2, lane 12 (FIG. 3B). corresponds to CaOV3, lane 13 corresponds to HEK293T US 2014/0294765 A1 Oct. 2, 2014

pIRESpuro3 negative control, lane 15 corresponds to HepG2. The mRNA levels of LSR were detected using mouse LSR lane 16 corresponds to HepG2C3A, lane 17 corresponds to Taqman probe (Applied Biosystems; cathi: Mm00660290 Hep3B, lane 18 corresponds to SNU182, lane 19 corresponds ml). to SkHep 1, lane 20 corresponds to PLC/PRF5, lane 21 cor 0.142 FIG. 25 shows that anti-LSR human IgG1 antibod responds to Chang Liver, lane 22 corresponds to HT29 cell ies demonstrate CDC activity against HEK293 expressing line. LSR. HEK293 cell lines expressing cyno LSR (Cyno LSR 0.133 FIG.16 presents a westen blot results demonstrating Hek) or GFP (GFPMT Hek) were incubated with antibodies a specific knockdown of endogenous LSR (SEQ ID NO:11) against LSR or isotype controls hIgG1 (ET901) or mIgG2a protein expression. (Mopc.173), in the presence of complement, and viability 0134 FIG. 17 demonstrates FACS analysis results of measured after 1 hr to assess % CDC activity. HT29 (FIG. 17A) and HepG2/C3A (FIG. 17B) cells follow 0.143 FIG. 26 shows FACS staining of anti-LSR antibod ing transient transfection with the LSR-specific siRNA ies on HEK293 cells ectopically expressing LSR. (Thermo Scientific CathM-009672-00-0005). 014.4 FIG. 26A represents binding of the anti-LSR mouse 0135 FIG. 18: shows the DNA sequence and the amino IgG2a monoclonal antibody 8C8, FIG. 26B, C, D, E, F, G, H acid sequence of the heavy (FIG. 18A) and light (FIG. 18B) represent binding of the anti-LSR human IgG1 monoclonal chains of the 8C8 antibody. The leader sequence is shown in antibodies S32-03.G11, S11-01.E02, S11-01.F08, S11-04. Italic font; the sequences of CDR1, CDR2, CDR3 are shown C11, S11-04.D11, S11-04.H07, S11-04.H09, respectively. in bold. The constant regions FRE FR2, FR3 and FR4 are The graphs show MFI values of the binding of the various shown in a regular font. antibodies to HEK293 cells expressing cyno LSR (“cyno”. 0.136 FIG. 19 presents sections of LSR positive cells fol circles) or HEK293 cells transfected with empty vector lowing pH9.0 antigen retrival (X60). Panel A shows the slides (“MT, squares). Binding of the Ig isotype control (hulgG1) incubated with LSR antibodies applied at 2 ug/ml. Panel B to both types of cells is shown in triangles. shows the adjacent no primary incubated sections. Positive (0145 FIG. 27 shows that LSR expressed on HEK-293T LSR immunoreactivity is seen in Panel A, demonstrating cells inhibits Jurkat cells activation. valid antibody working conditions. The adjacent no primary 0146 FIG.28 shows that HEK-293T cells expressing LSR section (Panel B) does not show apparent immunoreactivity. inhibit Jurkat cells activated with anti-CD3, as opposed to 0.137 FIG. 20 demonstrates binding assay results of HEK-293T cells expressing CD20. mouse LSRECD fused to mouse IgG2a Fc (SEQID NO:221) (at 2 g/5 ul/well) to anti CD3/CD28 or Con A activated DETAILED DESCRIPTION OF THE INVENTION murine T cells. SEQID NO: 223 corresponds to protein used 0147 The present invention, in at least some embodi as positive control. ments, relates to polyclonal and monoclonal antibodies and 0138 FIG. 21 shows binding of 2 ug. 3 ug, 4 ug or 5ug of fragments and/or conjugates thereof, and/or pharmaceutical biotin labeled mouse LSR-ECD fused to mouse IgG2a Fc composition comprising same, and/or diagnostic composi (SEQID NO:221) or of a 5ug biotin-labeled control mIgG2a tion comprising same, wherein these antibodies specifically to KARPAS-299 cells. FIG. 23A presents histograms show bind LSR proteins, and wherein said antibodies are adapted to ing Median Fluorescence Intensity (MFI). FIG. 23B presents be used as therapeutic and/or diagnostic agents, particularly the dose-dependent binding of LSR-ECD fused to mouse for treatment and/or diagnosis of specific cancer as described IgG2a Fc (SEQ ID NO:221) to Karpas-299 cells. The Y axis herein, particularly human, humanized or chimeric mono represents MFI ratio of mouse LSR-ECD fused to mouse clonal antibodies, including those that promote or inhibit IgG2a Fc (SEQ ID NO:221) vs. control-mIgG2a at various activities elicited by LSR. protein concentrations. 0.148. Without wishing to be limited by a closed list or by 0139 FIG.22 presents mRNA levels of INOS, which is a a single hypothesis, an antibody according to various embodi prototypic M1 marker, evaluated under the different stimula ments of the present invention may optionally have one or tion conditions in three independent experimental repetitions more of the following properties. Such neutralizing antibody (EXP1, EXP2 and EXP3). Axis Y represents the absolute may optionally promote Th2 to Th1 shift, thereby potentially value of mRNA expression. Axis X represents the different reverting the shift towards a Th2/M2 environment induced in stimulating conditions as detailed in Example 12. (IFNg, the tumor micro-environment that reducesthe immune LPS, IFNg+LPS, IL4, TGFb, PGE2, TSNT (tumor superna response towards the tumor. The antibody may therefore tant). NS stands for non-stimulated. optionally promote the immune system component which 0140 FIG. 23 presents mRNA levels of inducible acts against the tumor (Th1), while inhibiting the component ALOX15, which is a prototypic M2 marker, evaluated under which promotes the cancer (Th2). The antibody may promote the different stimulation conditions in three independent or inhibit activities elicited by LSR, including those relating experimental repetitions (EXP1, EXP2 and EXP3) Axis Y to modulation of immune costimulation, e.g. B7 related represents the absolute value of mRNA expression. Axis X costimulation, increases T cell activation and cytokine secre represents the different stimulating conditions as detailed in tion, or/and induce direct killing of cancer cells. Example 12 (IFNg, LPS, IFNg+LPS, IL4, TGFb, PGE2, 0149 According to at least some embodiments of the TSNT (tumor supernatant). NS stands for non-stimulated. present invention, such an antibody may optionally inhibit 0141 FIG. 24 presents mRNA levels of LSR evaluated iTregs accumulation and immunosuppressive function, and/ under the different stimulation conditions in three indepen or enhance effector T cell activity. dent experimental repetitions (EXP1, EXP2 and EXP3) Axis 0150. The term “cancer as used herein should be under Y represents the absolute value of mRNA expression. Axis X stood to encompass any neoplastic disease (whether invasive represents the different stimulating conditions as detailed in or metastatic) which is characterized by abnormal and uncon Example 12. (IFNg, LPS, IFNg+LPS, IL4, TGFb, PGE2, trolled cell division causing malignant growth or tumor, non TSNT (tumor supernatant). NS stands for non-stimulated. limiting examples of which are described herein. US 2014/0294765 A1 Oct. 2, 2014

0151. According to at least some embodiments of the the group consisting of any one of SEQ ID NOs: 95, 102, present invention, the antibodies are derived from particular and/or the amino acid sequences corresponding to the unique heavy and light chain germline sequences and/or comprise edges of SEQ ID NO: 18, and/or variants thereof, and/or particular structural features such as at least one CDR regions orthologs and/or fragments thereof, and/or nucleic acid comprising particular amino acid sequences. According to at sequences encoding for same, that are differentially least Some embodiments, the present invention provides iso expressed in cancer, on the cancer cells or in the immune cells lated antibodies, methods of making such antibodies, immu infiltrating the tumor. noconjugates and bispecific molecules comprising Such anti 0157 LSR protein is listed among many other proteins in bodies and pharmaceutical and diagnostic compositions WO2005019258; WO2005016962: WO2010105298, for the containing the antibodies, immunoconjugates, alternative diagnosis and treatment of immune related diseases. scaffolds or bispecific molecules according to at least some 0158 LSR protein is listed in US patent application No: embodiments of the present invention. US20070054268, among many other proteins proposed for 0152. According to at least some embodiments the present diagnosing ovarian cancer and/or a likelihood for Survival, or invention relates to in vitro and in vivo methods of using the recurrence of disease. antibodies and fragments thereof, to detect any one of LSR 0159 LSR protein is listed in US patent application No: proteins. US20070154889, among many other proteins for identifying 0153. According to at least some embodiments the present a melanoma, useful for distinguishing a malignant from invention further relates to methods of using the foregoing benign melanocyte. antibodies and fragments and/or conjugates thereof and/or (0160 LSR protein is listed in PCT application No: pharmaceutical and/or diagnostic composition comprising WO06133923, among many other proteins for diagnosis, same, to treat and/or to diagnose cancer, as described herein. prognosis, and prediction of breast cancer. 0154 LSR is a multimeric protein complex in the liver that (0161 LSR protein is listed in PCT application No: undergoes conformational changes upon binding of free fatty WO06138275, among many other proteins within stem cell acids, thereby revealing a binding site (s) that recognizes both gene signatures for use in the diagnosis and management of apoB and apoE. Complete inactivation of the LSR gene is CaCC. embryonic lethal in mice. Removal of a single LSR allele (0162 US patent Nos: U.S. Pat. No. 7,919,091, U.S. Pat. (LSR-/+) caused Statistically significant increases in both No. 6,635,431 and U.S. Pat. No. 7,291,709, and other related plasma triglyceride and cholesterol levels. The biology of patent family members disclose LSR proteins and LSR spe LSR has been thoroughly investigated in humans, rats and cific antibodies, particularly for treating obesity and other mice. LSR is mainly expressed in liver membranes and most metabolic disorders. elucidated function is to mediate clearance of chylomicrons (0163 US patent application No: 20120064100 disclose after activations of free fatty acids. However, other functions LSR protein among several other proteins for treating a con may exist, since LSR is also expressed in other tissues. LSR dition associated with regulatory T (Treg) cell-mediated Sup expression has been shown in tumor tissue like ovarian can pression of a immune system or for modulating an immune cer, and bladder cancer where LSR was one of thirty genes response. identified as a potential tumor marker. Upon LSR knockdown 0164. However, the above referenced patents and/or in epithelial cells, Tight Junction formation was affected and patent applications do not teach or Suggest or provide any the epithelial barrier function was diminished. (BMC Med. incentive that would direct a skilled artisan to use antibodies Genomics. 2008: 1: 31. Diabetes. 2009 May: 58(5): 1040 comprising one or more of the specific CDRs as described 1049, J. CellSci, 15 Feb. 2011 124, 548-555). herein, specifically binding to one or more polypeptides hav (O155 LSR protein is disclosed in PCT Application No: ing amino acid sequences selected from the group consisting PCT/IB2012/051868, owned in common with the present of SEQ ID NOS 215 and 216, and that does not specifically application, which is hereby incorporated by reference, as if bind to any other portion of SEQ ID NO 10, wherein said fully set forth herein. This application demonstrates that the other portion of SEQID NO:10 comprises amino acids 1-80 ECD sequence of mouse LSR molecule fused to mouse or amino acids 99 to 234 of SEQID NO: 10 for SEQID NO IgG2a inhibits mouse T-cell activation, induced by anti CD3 215, or wherein said other portion of SEQ ID NO:10 com and anti-CD28, cytokine secretion. The fusion protein ame prises amino acids 1-117 oramino acids 136 to 234 of SEQID liorates disease symptoms in mice model of multiple Sclerosis NO: 10 for SEQID NO 216, oramino acids 30-110 of SEQID (EAE model) demonstrating that LSR has an important role NO 10 and that does not specifically bind to any other portion in immune modulation. PCT/IB2012/051868 describes LSR of SEQ ID NO 10, wherein said other portion of SEQ ID antibodies that are potentially useful as therapeutic and/or NO:10 comprises amino acids 1-29 oramino acids 111 to 234 diagnostic agents (both in vitro and in vivo diagnostic meth of SEQID NO: 10. ods). Included in particular are antibodies and fragments that 0.165. Furthermore, the above referenced patents and/or are immune activating or immune Suppressing Such as anti patent applications do not teach or Suggest or provide any bodies or fragments that target cells via ADCC (antibody incentive that would direct a skilled artisan to use antibodies dependent cellular cytotoxicity) or CDC (complement depen specific to the LSR ECD for treatment and/or diagnosis of dent cytotoxicity) activities, particularly for treating condi cancer as described herein. Examples of the utility of such tions wherein the LSR antigen is expressed, including can antibodies for treatment and/or diagnosis of cancer are given cers, and/or in infectious disorders, and/or immune related below. disorders. 0166 Furthermore, the above referenced patents and/or 0156. As used herein, the term LSR refers to any one of the patent applications do not teach or Suggest or provide any proteins set forthin anyone of SEQID NOs: 10-18, 21, 22.31, incentive that would direct a skilled artisan to use antibodies 32, 47-50, 62-69, 143, 211-212, 237, and/or amino acid specific to the LSR ECD for treatment and/or diagnosis of sequences corresponding to LSRIGV domains selected from immune related disorders as described herein. Without wish US 2014/0294765 A1 Oct. 2, 2014

ing to be limited in any way, it is expected that these antibod 0.175. According to at least some embodiments, the ies have cytotoxic activity, including antibody-dependent or present invention provides blocking antibody that specifically complement dependent cytotoxic activity, on immune cells, binds any one of LSR proteins, selected from the group con resulting in their depletion, leading to amelioration of the sisting of any one of SEQ ID NOs: 10-18, 21, 22, 31, 32, immune disease. Furthermore, still without wishing to be 47-50, 62-69, 143, 211-212, and/or amino acid sequences limited in any way, it is expected that these antibodies may corresponding to extracellular domains thereof, selected enhance the inhibitory effect of LSR on T-cell activation, from the group consisting of any one of SEQID NOs: 12, 14, resulting in a dampening of immune cell response and ame 47-50, and/or fragments, and/or epitopes thereof, may lioration of the immune disease. optionally and preferably be specifically applied to cancer 0167 Furthermore, the above referenced patents and/or immunotherapy, alone or in combination with a potentiating patent applications do not teach or Suggest or provide any agent(s), which increase an endogenous anti-tumor incentive that would direct a skilled artisan to use antibodies responses. specific to the LSR ECD for treatment and/or diagnosis of 0176 Furthermore, surprisingly, it has been found that an infectious disease as described herein. Without wishing to be antibody that specifically binds any one of LSR proteins, limited in any way, it is expected that as an "infection' com selected from the group consisting of any one of SEQ ID prises a disorder, disease and/or condition caused by the NOs: 10-18, 21, 22, 31, 32, 47-50, 62-69, 95, 102, 143, persistence of foreign antigen, leading to diminished immune 211-212, and/or their corresponding extracellular domains, responses against the foreign antigen, the antibodies would be selected from the group consisting of any one of SEQ ID effective in activating the immune system to attack the infec NOs: 12, 14, 47-50, and/or fragments, and/or epitopes tious agent. Such diminished immune responses are charac thereof, may optionally and preferably be specifically applied terized by impaired functionality which can be manifested as to treatment of certain cancers, against which Such an anti T cell exhausting, reduced cell proliferation and cytokine body demonstrates particular efficacy. Pharmaceutical com production, and can be reversed by blocking inhibitory path positions comprising such an antibody, in conjunction with a ways using antibodies as described herein. pharmaceutically acceptable carrier, are also provided herein. 0168 As used herein, the term “antibody may optionally 0177. Furthermore, surprisingly, it has been found that refer to any of the following (and also optionally combina said antibody demonstrates particular efficacy in specific can tions of the following): monoclonal and/or polyclonal anti cers, including cancers in which LSR is expressed on malig bodies and antigenbinding fragments and/or alternative scaf nant cells, immune cells infiltrating into the tumor (such as folds and/or conjugates and/or immunoconjugates. T-cells, B-cell, macrophages, myeloid derive suppressor 0169. According to at least some embodiments, the cells, mast cells) and/or stromal tumor cells. LSR expression present invention provides antibodies and fragments as on any of the cells listed above could be either present prior to described herein, optionally and preferably wherein the anti treatment by standared of care agents or induced post treat body binds to human LSR with a KD of 1x10 Morless, and ment. wherein the antibody exhibits at least one of the following 0.178 Furthermore, surprisingly, it has been found that properties: modulates B7 related costimulation, increases T improved outcome can be achieved using the above LSR cell activation, alleviates T-cell Suppression, increases cytok antibodies for treatment of any one or more of: ine secretion, increases IL-2 secretion; increases interferon 0.179 Breast cancer, preferably any of ductal-adenocar gamma production by T-cells, increases Th1 response, cinoma, infiltrating ductal carcinoma, lobular carci decreases Th2 response, decreases or eliminates M2 mac noma, mucinous adenocarcinoma, intra duct and inva rophages, reduces M2 macrophage pro-tumorigenic activity, sive ductal carcinoma, preferably Scirrhous promotes cancer epitope spreading, reduces inhibition of T adenocarcinoma; cell activation, increases T cell response in a mammal, stimu lates antigen-specific memory responses, elicits apoptosis or 0180 Colorectal cancer, preferably any of Moderate to lysis of cancer cells, stimulates cytotoxic or cytostatic effect Poorly Differentiated Adenocarcinoma of the cecum, on cancer cells, induces direct killing of cancer cells, induces Well, Moderate and Poorly Differentiated Adenocarci complement dependent cytotoxicity and/or antibody depen noma of the colon, Tubular adenocarcinoma, preferably Grade 2 Tubular adenocarcinoma of the ascending dent cell-mediated cytotoxicity. colon, colon adenocarcinoma Duke's stage C1, invasive 0170 Optionally said antibody or fragment increases adenocarcinoma, Adenocarcinoma of the rectum, pref immune response against the cancer. erably Grade 3 Adenocarcinoma of the rectum, Moder 0171 Optionally said antibody or fragment reduces activ ately Differentiated Adenocarcinoma of the rectum, ity of regulatory T lymphocytes (T-regs). Moderately Differentiated Mucinous adenocarcinoma 0172 Optionally said antibody or fragment inhibits iTreg of the rectum; differentiation. 0181 Lung cancer, preferably any of Well to Poorly 0173 According to at least some embodiments, the differentiated Non-Small cell carcinoma, Squamous present invention provides the foregoing antibodies and frag Cell Carcinoma, preferably Moderately Differentiated ments thereof, wherein the antibody is a chimeric, human Squamous Cell Carcinoma, Moderately to poorly differ ized, fully human antibody and/or is an antibody or antibody entiated squamous carcinoma, Moderately well differ fragment having CDC or ADCC activities on target cells. entiated keratinising squamous cell carcinoma, large 0.174 Included in particular are antibodies and fragments cell adenocarcinoma, Small cell lung cancer, that are immune activating or immune Suppressing Such as 0182 Prostate cancer, preferably any of Adenocarci antibodies or fragments that target cells via ADCC (antibody noma Gleason Grade 5 to 9. Infiltrating adenocarci dependent cellular cytotoxicity) or CDC (complement depen noma, High grade prostatic intraepithelial neoplasia, dent cytotoxicity) activities. undifferentiated carcinoma; US 2014/0294765 A1 Oct. 2, 2014 12

0183 Stomach cancer, preferably moderately differen 0193 As used herein, when the term "epitopes thereof tiated gastric adenocarcinoma; appears, it may optionally and without limitation refer to 0.184 Ovary cancer, preferably any of serous papillary epitopes as embodied in SEQID NOs: 215, 216 and/or SEQ cystic carcinoma, Serous cystadenocarcinoma, Invasive ID NO 237. serous papillary carcinoma; 0194 Optionally the antibodies may be used for treatment 0185 Brain cancer, preferably any of Astrocytoma, of cancer as described herein. Optionally, said cancer, said preferably grade 4 Astrocytoma, Glioblastoma multi immune infiltrate or both express LSRata sufficient leveland forme; said cancer is as described herein. By immune infiltrate it is 0186 Kidney cancer, preferably Clear cell renal cell meant immune cells infiltrating to the tumor or to the area of the cancerous cells. By “expressing LSR at a sufficient level carcinoma, it is meant that Such cells express LSR protein at a high 0187 Liver cancer, preferably any of Hepatocellular enough level according to an assay. For example, if the assay carcinoma, preferably Low Grade hepatocellular carci is IHC (immunohistochemistry), and expression is measured noma, Fibrolamellar Hepatocellular Carcinoma; on a scale of 0 to 3 (0 no expression, 1—faint staining, 0188 Hematological cancer, preferably any of large 2—moderate and 3-strong expression), then a sufficient cell lymphoma, High and low grade Non-Hodgkin’s level of LSR expression would optionally be at least 1, pref Lymphoma. erably be at least 2 and more preferably be at least 3. Option 0189 It should be noted that surprisingly and contrary to ally the antibodies or immune molecules as described herein the art of record, the following cancer Subtypes, Hodgkin’s may be used for Such an assay. Lymphoma, Granulosa cell tumor of the ovary, and Astrocy 0.195 More preferably, the antibody binds to correspond toma grade 2, were found to frequently, if not typically, fail to ing human LSR antigen with a KD of 3x10-8 M or less, or express LSR at a sufficient level, and therefore patients suf with a KD of 1x10-9 M or less, or with a KD of 0.1x10-9 M fering from these subtypes are unlikely to benefit from treat or less, or with a KD Of 0.05x10-9 Morless or with a KD of ment with anti-LSR antibodies. between 1x10-9 and 1x10-11 M. 0190. Even more unexpectedly, it was found that ductal 0196. In addition, preferably these antibodies and/or con adenocarcinoma, infiltrating ductal carcinoma, Lobular car jugates thereof are effective in eliciting selective killing of cinoma of breast, mucinous adenocarcinoma of the breast, Such cancer cells and for modulating immune responses Intra duct and invasive ductal carcinoma, Moderate to Poorly involved in autoimmunity and cancer. Differentiated Adenocarcinoma of the cecum, Well to Poorly (0197) Standardassays to evaluate the binding ability of the Differentiated Adenocarcinoma of the colon, Grade 2 Tubular antibodies toward LSR are known in the art, including for adenocarcinoma of the ascending colon, colon adenocarci example, ELISAs, Western blots and RIAs. Suitable assays noma Duke's stage C1, invasive adenocarcinoma, Adenocar are described in detail in the Examples. The binding kinetics cinoma of the rectum, Grade 3 Adenocarcinoma of the rec (e.g., binding affinity) of the antibodies also can be assessed tum, Moderately Differentiated Adenocarcinoma of the by standard assays known in the art, Such as by Biacore rectum, Moderately Differentiated Mucinous adenocarci analysis. noma of the rectum, Well to Poorly differentiated Non-small 0198 Upon production of anti-LSR antibody sequences cell carcinoma, Squamous Cell Carcinoma: Moderately Dif from antibodies can bind to LSR the VH and VL sequences ferentiated, Moderately to poorly differentiated squamous can be "mixed and matched to create other anti-LSR, bind carcinoma, Moderately well differentiated keratinising squa ing molecules according to at least Some embodiments of the mous cell carcinoma, large cell adenocarcinoma, Adenocar invention. LSR binding of such “mixed and matched anti cinoma Gleason Grade 7 to 9. Infiltrating adenocarcinoma, bodies can be tested using the binding assays described Moderately differentiated adenocarcinoma, serous papillary above. e.g., ELISAs). Preferably, when VHandVL chains are cystic carcinoma, Serous cystadenocarcinoma, grade 4 Astro mixed and matched, a VH sequence from a particular VH/VL cytoma, Glioblastoma multiforme, Clear cell renal cell car pairing is replaced with a structurally similar VH sequence. cinoma, Hepatocellular carcinoma, and Low Grade hepato Likewise, preferably a VL sequence from a particular VH/VL cellular carcinoma, are especially susceptible to treatment pairing is replaced with a structurally similar VL sequence. with anti-LSR antibodies because of the significantly high For example, the VHand VL sequences of homologous anti levels of LSR expression found on these cancer cells. bodies are particularly amenable for mixing and matching. 0191). According to at least some embodiments, for any of 0199 Optionally, the antibody comprises CDR amino the above described cancers, optionally each of the above acid sequences selected from the group consisting of (a) described cancer type or Subtype may optionally form a sepa sequences as listed herein; (b) sequences that differ from rate embodiment and/or may optionally be combined as those CDRamino acid sequences specified in (a) by 1, 2, 3, 4, embodiments or Subembodiments. 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions 0.192 According to at least some embodiments, for any of except for the Serine residue in heavy chain CDR3 at position the above described cancers, methods of treatment and also 100A (Kabat numbering system); (c) amino acid sequences uses of the antibodies and pharmaceutical compositions having 90% or greater, 95% or greater, 98% or greater, or 99% described herein are provided wherein the cancer expresses or greater sequence identity to the sequences specified in (a) LSR polypeptides comprised in SEQID NOs: 10-18, 21, 22. or (b); (d) a polypeptide having an amino acid sequence 31, 32, 47-50, 62-69, 95, 102, 143, 211-212, and/or their encoded by a polynucleotide having a nucleic acid sequence corresponding extracellular domains, selected from the group encoding the amino acids as listed herein. consisting of any one of SEQID NOs: 12, 14, 47-50, and/or 0200 Optionally, for any antibody or fragment described fragments, such as for example SEQ ID NO:237, and/or herein, the antibody may be bispecific, meaning that one arm epitopes thereof, on the cancer cells or in the immune cells of the Ig molecule is specific for binding to the target protein infiltrating the tumor. or epitope as described herein, and the other arm of the Ig US 2014/0294765 A1 Oct. 2, 2014

molecule has a different specificity that can enhance or redi their receptors on T cells during antigen-specific T cell rect the biological activity of the antibody or fragment. In this responses. Without wishing to be limited by a single hypoth regard, a multi-specific antibody is also considered to be at esis, the antigen-specific T cell response is believed to be least bispecific. The antibody or fragment also can be multi mediated by two signals: 1) engagement of the T cell Recep specific in the sense of being multi-valent. tor (TCR) with antigenic peptide presented in the context of 0201 According to at least some embodiments the inven MHC (signal 1), and 2) a second antigen-independent signal tion relates to protein scaffolds with specificities and affinities delivered by contact between different costimulatory recep in a range similar to specific antibodies. According to at least tor/ligand pairs (signal 2). Without wishing to be limited by a Some embodiments the present invention relates to an anti single hypothesis, this “second signal' is critical in determin gen-binding construct comprising a protein scaffold which is ing the type of T cell response (activation VS inhibition) as linked to one or more epitope-binding domains. Such engi well as the strength and duration of that response, and is neered protein scaffolds are usually obtained by designing a regulated by both positive and negative signals from costimu random library with mutagenesis focused at a loop region or latory molecules, such as the B7 family of proteins. at an otherwise permissible Surface area and by selection of 0211. As used herein, the term “B7 polypeptide means a variants against a given target via phage display or related member of the B7 family of proteins that costimulate T cells techniques. According to at least some embodiments the including, but not limited to B7-1, B7-2, B7-DC, B7-H5, invention relates to alternative scaffolds including, but not B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3 and biologi limited to, anticalins, DARPins, Armadillo repeat proteins, cally active fragments and/or variants thereof. Representative protein A, lipocalins, fibronectin domain, ankyrin consensus biologically active fragments include the extracellular repeat domain, thioredoxin, chemically constrained peptides domain or fragments of the extracellular domain that and the like. According to at least Some embodiments the costimulate T cells. invention relates to alternative scaffolds that are used as thera 0212. As used herein, a “variant' polypeptide contains at peutic agents for treatment of cancer as recited herein, as well least one amino acid sequence alteration as compared to the as for in vivo diagnostics. amino acid sequence of the corresponding wild-type 0202 According to at least some embodiments, there is polypeptide. provided a method of performing one or more of the follow 0213. As used herein, "conservative” amino acid substitu ing in a Subject: tions are Substitutions wherein the Substituted amino acid has 0203 (a) upregulating cytokines, (b) increases T-cell pro similar structural or chemical properties. As used herein, the liferation and/or expansion, (c) increases interferon-gamma term "host cell” refers to prokaryotic and eukaryotic cells into production by T-cells (d) increases IL-2 secretion (e) stimu which a recombinant vector can be introduced. lates antibody responses, (f) inhibits cancer cell growth, (g) 0214. As used herein, the term “an edgeportion” or “a new promoting antigenic specific T cell immunity, (g) promoting junction” refers to a connection between two portions of a CD4+ and/or CD8+ T cell activation, (i) alleviating T-cell splice variant according to the present invention that were not Suppression, () alleviating apoptosis or lysis of cancer cells, joined in the wildtype or known protein. An edge may option (k) cytotoxic or cytostatic effect on cancer cells, ally arise due to a join between the above “known protein’ 0204 comprising administering an antibody or immune portion of a variant and the tail, for example, and/or may molecule as described herein or a pharmaceutical composi occur if an internal portion of the wild type sequence is no tion as described herein to the subject. longerpresent, such that two portions of the sequence are now 0205. In order that the present invention in various contiguous in the splice variant that were not contiguous in embodiments may be more readily understood, certain terms the known protein. A "bridge' may optionally be an edge are first defined. Additional definitions are set forth through portion as described above, but may also include a join out the detailed description. between ahead and a “known protein’ portion of a variant, or 0206. As used herein the term "isolated’ refers to a com a join between a tail and a “known protein’ portion of a pound of interest (for example a polynucleotide or a polypep variant, or a join between an insertion and a "known protein' tide) that is in an environment different from that in which the portion of a variant. compound naturally occurs e.g. separated from its natural 0215. In some embodiments, a bridge between a tail or a milieu Such as by concentrating a peptide to a concentration at head or a unique insertion, and a “known protein’ portion of which it is not found in nature. “Isolated includes com a variant, comprises at least about 10amino acids, or in some pounds that are within Samples that are Substantially enriched embodiments at least about 20 amino acids, or in some for the compound of interest and/or in which the compound of embodiments at least about 30 amino acids, or in some interest is partially or substantially purified. embodiments at least about 40 amino acids, in which at least 0207. An “immune cell” refers to any cell from the one amino acid is from the tail/head/insertion and at least one hemopoietic origin including but not limited to T cells, B amino acid is from the “known protein’ portion of a variant. cells, monocytes, dendritic cells, and macrophages. In some embodiments, the bridge may comprise any number 0208. As used herein, the term “polypeptide' refers to a of amino acids from about 10 to about 40 amino acids (for chain of amino acids of any length, regardless of modification example, 10, 11, 12, 13 . . . 37, 38, 39, 40 amino acids in (e.g., phosphorylation or glycosylation). length, or any number in between). 0209. As used herein, a “costimulatory polypeptide' or 0216. It should be noted that a bridge cannot be extended "costimulatory molecule' is a polypeptide that, upon interac beyond the length of the sequence in either direction, and it tion with a cell-surface molecule on T cells, modulates T cell should be assumed that every bridge description is to be read responses. in Such manner that the bridge length does not extend beyond 0210. As used herein, "costimulatory signaling is the sig the sequence itself. naling activity resulting from the interaction between 0217. Furthermore, bridges are described with regard to a costimulatory polypeptides on antigen presenting cells and sliding window in certain contexts below. For example, cer US 2014/0294765 A1 Oct. 2, 2014 tain descriptions of the bridges feature the following format: chains thereof. An “antibody' refers to a glycoprotein com a bridge between two edges (in which a portion of the known prising at least two heavy (H) chains and two light (L) chains protein is not present in the variant) may optionally be inter-connected by disulfide bonds, or an antigen binding described as follows: a bridge portion of the protein, com portion thereof. Each heavy chain is comprised of at least one prising a polypeptide having a length “n”, whereinn is at least heavy chain variable region (abbreviated herein as VH) and a about 10 amino acids in length, optionally at least about 20 heavy chain constant region. The heavy chain constant region amino acids, at least about 30 amino acids, at least about 40 is comprised of three domains, CH1, CH2 and CH3. Each amino acids, or at least about 50 amino acids in length, light chain is comprised of at least one light chain variable wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), region (abbreviated herein as VL) and a light chain constant having a structure as follows (numbering according to the region. The light chain constant region is comprised of one sequence of the protein): a sequence starting from any of domain, CL. The VH and VL regions can be further subdi amino acid numbers 49-X to 49 (for example); and ending at vided into regions of hyperVariability, termed complementa any of amino acid numbers 50+(n-2)-X) (for example), in rity determining regions (CDR), interspersed with regions which X varies from 0 to n-2. In this example, it should also that are more conserved, termed framework regions (FR). be read as including bridges in which n is any number of Each VH and VL is composed of three CDRs and four FRS, amino acids between 10-50 amino acids in length. Further arranged from amino-terminus to carboxy-terminus in the more, the bridge polypeptide cannot extend beyond the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. sequence, so it should be read Such that 49-X (for example) is The variable regions of the heavy and light chains contain a not less than 1, nor 50+(n-2)-X) (for example) greater than binding domain that interacts with an antigen. The constant the total sequence length. regions of the antibodies may mediate the binding of the 0218. As used herein, the term “vaccine” refers to a bio immunoglobulin to host tissues or factors, including various logical preparation that improves immunity to a particular cells of the immune system (e.g., effector cells) and the first disease, wherein the vaccine includes cancer antigen, against component (Cld) of the classical complement system. which immune responses are elicited. A vaccine typically includes an adjuvant as immune potentiator to stimulate the 0223) The term “antigen-binding portion of an antibody immune system. As used herein, the term “therapeutic vac (or simply “antibody portion'), as used herein, refers to one or cine” and/or “therapeutic vaccination” refers to a vaccine more fragments of an antibody that retain the ability to spe used to treat cancer. cifically bind to an antigen (e.g., LSR molecules, and/or a 0219. As used herein, the term “adjuvant” refers to an fragment thereof). It has been shown that the antigen-binding agent used to stimulate the immune system and increase the function of an antibody can be performed by fragments of a response to a vaccine, without having any specific antigenic full-length antibody. Examples of binding fragments encom effect in itself. passed within the term “antigen-binding portion of an anti 0220. As used herein, the terms “immunologic”, “immu body include (i) a Fab fragment, a monovalent fragment nological' or “immune' response is the development of a consisting of the V Light, V Heavy, Constant light (CL) and beneficial humoral (antibody mediated) and/or a cellular (me CH1 domains; (ii) a F(ab').2 fragment, a bivalent fragment diated by antigen-specific T cells or their secretion products) comprising two Fab fragments linked by a disulfide bridge at response directed againstapeptide in a recipient patient. Such the hinge region; (iii) a Fd fragment consisting of the VHand a response can be an active response induced by administra CH1 domains; (iv) a Fv fragment consisting of the VL and VH tion of immunogen or a passive response induced by admin domains of a single arm of an antibody, (v) a dAb fragment istration of antibody or primed T-cells. Without wishing to be (Wardet al., (1989) Nature 341:544-546), which consists of a limited by a single hypothesis, a cellular immune response is VH domain; and (vi) an isolated complementarity determin elicited by the presentation of polypeptide epitopes in asso ing region (CDR). Furthermore, although the two domains of ciation with Class II or Class I MHC molecules to activate the Fv fragment, VL and VH, are coded for by separate genes, antigen-specific CD4+T helper cells and/or CD8+ cytotoxic they can be joined, using recombinant methods, by a synthetic T cells, respectively. The response may also involve activa linker that enables them to be made as a single protein chain tion of monocytes, macrophages, NK cells, basophils, den in which the VL and VH regions pair to form monovalent dritic cells, astrocytes, microglia cells, eosinophils, activation molecules (known as single chain FV (ScFV); see e.g., Bird et or recruitment of neutrophils or other components of innate al. (1988) Science 242:423-426; and Huston et al. (1988) immunity. The presence of a cell-mediated immunological Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain response can be determined by proliferation assays (CD4+T antibodies are also intended to be encompassed within the cells) or CTL (cytotoxic T lymphocyte) assays. The relative term “antigen-binding portion of an antibody. These anti contributions of humoral and cellular responses to the pro body fragments are obtained using conventional techniques tective or therapeutic effect of an immunogen can be distin known to those with skill in the art, and the fragments are guished by separately isolating antibodies and T-cells from an screened for utility in the same manner as are intact antibod immunized Syngeneic animal and measuring protective or 1CS therapeutic effect in a second Subject. 0224. An "isolated antibody', as used herein, is intended 0221. An "immunogenic agent' or “immunogen' is to refer to an antibody that is substantially free of other capable of inducing an immunological response against itself antibodies having different antigenic specificities (e.g., an on administration to a mammal, optionally in conjunction isolated antibody that specifically binds LSR proteins and/or with an adjuvant. fragments thereof, and is substantially free of antibodies that 0222. The term “antibody” as referred to herein includes specifically bind antigens other than LSR, respectively. An whole polyclonal and monoclonal antibodies and any antigen isolated antibody that specifically binds LSR proteins may, binding fragment (i.e., “antigen-binding portion') or single however, have cross-reactivity to other antigens, such as LSR US 2014/0294765 A1 Oct. 2, 2014 molecules from other species, respectively. Moreover, an iso 0231. As used herein, an antibody that “specifically binds lated antibody may be substantially free of other cellular to human LSR proteins” is intended to refer to an antibody material and/or chemicals. that binds to LSR proteins, preferably one with a KD of 0225. The terms “monoclonal antibody” or “monoclonal 5x10 M or less, more preferably 3x10 M or less, even antibody composition' as used herein refer to a preparation of more preferably 1x10 M or less, even more preferably antibody molecules of single molecular composition. A 1x10' M, even more preferably 1x10' M and even more monoclonal antibody composition displays a single binding preferably 1x10'’M or less. specificity and affinity for a particular epitope. 0232. The term “K-assoc' or “Ka', as used herein, is 0226. The term “human antibody', as used herein, is intended to refer to the association rate of a particular anti intended to include antibodies having variable regions in body-antigen interaction, whereas the term “Kdiss’ or “Kd.” which both the framework and CDR regions are derived from as used herein, is intended to refer to the dissociation rate of human germline immunoglobulin sequences. Furthermore, if a particular antibody-antigen interaction. The term “KD’, as the antibody contains a constant region, the constant region used herein, is intended to refer to the dissociation constant, also is derived from human germline immunoglobulin which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and sequences. The human antibodies according to at least some is expressed as a molar concentration (M). KD values for embodiments of the present invention may include amino antibodies can be determined using methods well established acid residues not encoded by human germline immunoglo in the art. A preferred method for determining the KD of an bulin sequences (e.g., mutations introduced by random or antibody is by using Surface Plasmon resonance, preferably site-specific mutagenesis in vitro or by Somatic mutation in using a biosensor system such as a Biacore R system. vivo). However, the term “human antibody, as used herein, is 0233. As used herein, the term “high affinity’ for an IgG not intended to include antibodies in which CDR sequences antibody refers to an antibody having a KD of 10 Morless, derived from the germline of another mammalian species, more preferably 10 M or less and even more preferably Such as a mouse, have been grafted onto human framework 10' Morless for a target antigen. However, “high affinity” Sequences. binding can vary for other antibody isotypes. For example, 0227. The term “human monoclonal antibody” refers to “high affinity’ binding for an IgM isotype refers to an anti antibodies displaying a single binding specificity which have body having a KD of 107M or less, more preferably 10 M variable regions in which both the framework and CDR or less. regions are derived from human germline immunoglobulin 0234. As used herein, the term “subject' or “patient’ sequences. In one embodiment, the human monoclonal anti includes any human or nonhuman animal. The term “nonhu bodies are produced by a hybridoma which includes a B cell man animal' includes all vertebrates, e.g., mammals and obtained from a transgenic nonhuman animal, e.g., a trans non-mammals, such as nonhuman primates, sheep, dogs, genic mouse, having a genome comprising a human heavy cats, horses, cows, chickens, amphibians, reptiles, etc. chain transgene and a light chain transgene fused to an 0235 Antibodies Having Particular Germline Sequences immortalized cell. 0236. In certain embodiments, an antibody of the inven 0228. The term “recombinant human antibody', as used tion comprises a heavy chain variable region from a particular herein, includes all human antibodies that are prepared, germline heavy chain immunoglobulin gene and/or a light expressed, created or isolated by recombinant means, such as chain variable region from a particular germline light chain (a) antibodies isolated from an animal (e.g., a mouse) that is immunoglobulin gene. transgenic or transchromosomal for human immunoglobulin 0237 As used herein, a human antibody comprises heavy genes or a hybridoma prepared therefrom (described further or light chain variable regions that is “the product of or below), (b) antibodies isolated from a host cell transformed to "derived from a particular germline sequence if the variable express the human antibody, e.g., from a transfectoma, (c) regions of the antibody are obtained from a system that uses antibodies isolated from a recombinant, combinatorial human germline immunoglobulin genes. Such systems human antibody library, and (d) antibodies prepared, include immunizing a transgenic mouse carrying human expressed, created or isolated by any other means that involve immunoglobulin genes with the antigen of interest or screen splicing of human immunoglobulin gene sequences to other ing a human immunoglobulin gene library displayed on DNA sequences. Such recombinant human antibodies have phage with the antigen of interest. A human antibody that is variable regions in which the framework and CDR regions are “the product of or "derived from a human germline immu derived from human germline immunoglobulin sequences. In noglobulin sequence can be identified as Such by comparing certain embodiments, however, such recombinant human the amino acid sequence of the human antibody to the amino antibodies can be subjected to in vitro mutagenesis (or, when acid sequences of human germline immunoglobulins and an animal transgenic for human Ig sequences is used, in vivo selecting the human germline immunoglobulin sequence that Somatic mutagenesis) and thus the amino acid sequences of is closest in sequence (i.e., greatest '% identity) to the the VH and VL regions of the recombinant antibodies are sequence of the human antibody. sequences that, while derived from and related to human 0238 A human antibody that is “the product of or germline VH and VL sequences, may not naturally exist "derived from a particular human germline immunoglobulin within the human antibody germline repertoire in vivo. sequence may contain amino acid differences as compared to 0229. As used herein, “isotype” refers to the antibody the germline sequence, due to, for example, naturally-occur class (e.g., IgM or IgG1) that is encoded by the heavy chain ring somatic mutations or intentional introduction of site constant region genes. directed mutation. However, a selected human antibody typi 0230. The phrases “an antibody recognizing an antigen” cally is at least 90% identical in amino acids sequence to an and “an antibody specific for an antigen” are used inter amino acid sequence encoded by a human germline immu changeably herein with the term “an antibody which binds noglobulin gene and contains amino acid residues that iden specifically to an antigen.” tify the human antibody as being human when compared to US 2014/0294765 A1 Oct. 2, 2014

the germline immunoglobulin amino acid sequences of other CDR1, CDR2 and CDR3 sequences and a light chain variable species (e.g., murine germline sequences). In certain cases, a region comprising CDR1, CDR2 and CDR3 sequences, human antibody may beat least 95, 96.97,98 or 99%, or even wherein one or more of these CDR sequences comprise speci at least 96%, 97%, 98%, or 99% identical in amino acid fied amino acid sequences based on preferred anti-anti-LSR sequence to the amino acid sequence encoded by the germline antibodies isolated and produced using methods herein, or immunoglobulin gene. Typically, a human antibody derived conservative modifications thereof, and wherein the antibod from a particular human germline sequence will display no ies retain the desired functional properties of anti-LSR anti more than 10 amino acid differences from the amino acid bodies according to at least Some embodiments of the inven sequence encoded by the human germline immunoglobulin tion, respectively. gene. In certain cases, the human antibody may display no 0246. In various embodiments, the anti-LSRantibody can more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the be, for example, human antibodies, humanized antibodies or germline immunoglobulin gene. chimeric antibodies. 0239 Homologous Antibodies 0247. As used herein, the term “conservative sequence 0240. In yet another embodiment, an antibody of the modifications” is intended to refer to amino acid modifica invention comprises heavy and light chain variable regions tions that do not significantly affect or alter the binding char comprising amino acid sequences that are homologous to acteristics of the antibody containing the amino acid isolated anti-LSR amino acid sequences of preferred anti sequence. Such conservative modifications include amino LSR antibodies, respectively, wherein the antibodies retain acid Substitutions, additions and deletions. Modifications can the desired functional properties of the parent anti-LSR anti be introduced into an antibody according to at least some bodies. embodiments of the invention by standard techniques known 0241. As used herein, the percent homology between two in the art, Such as site-directed mutagenesis and PCR-medi amino acid sequences is equivalent to the percent identity ated mutagenesis. Conservative amino acid Substitutions are between the two sequences. The percent identity between the ones in which the amino acid residue is replaced with an two sequences is a function of the number of identical posi amino acid residue having a similar side chain. Families of tions shared by the sequences (i.e., 96 homology it of iden amino acid residues having similar side chains have been tical positions/total # of positionsX100), taking into account defined in the art. These families include amino acids with the number of gaps, and the length of each gap, which need to basic side chains (e.g., lysine, arginine, histidine), acidic side be introduced for optimal alignment of the two sequences. chains (e.g., aspartic acid, glutamic acid), uncharged polar The comparison of sequences and determination of percent side chains (e.g., glycine, asparagine, glutamine, serine, identity between two sequences can be accomplished using a threonine, tyrosine, cysteine, tryptophan), nonpolar side mathematical algorithm, as described in the non-limiting chains (e.g., alanine, Valine, leucine, isoleucine, proline, phe examples below. nylalanine, methionine), beta-branched side chains (e.g., 0242. The percent identity between two amino acid threonine, Valine, isoleucine) and aromatic side chains (e.g., sequences can be determined using the algorithm of E. Mey tyrosine, phenylalanine, tryptophan, histidine). Thus, one or ers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) more amino acid residues within the CDR regions of an which has been incorporated into the ALIGN program (ver antibody according to at least some embodiments of the sion 2.0), using a PAM120 weight residue table, a gap length invention can be replaced with other amino acid residues penalty of 12 and a gap penalty of 4. In addition, the percent from the same side chain family and the altered antibody can identity between two amino acid sequences can be deter be tested for retained function (i.e., the functions set forth in mined using the Needleman and Wunsch (J. Mol. Biol. (c) through () above) using the functional assays described 48:444-453 (1970)) algorithm which has been incorporated herein. into the GAP program in the GCG software package (avail 0248 Antibodies that Bind to the Same Epitope as Anti able commercially), using either a Blossum 62 matrix or a LSR According to at Least Some Embodiments of the Inven PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 tion. and a length weight of 1, 2, 3, 4, 5, or 6. 0249. In another embodiment, the invention provides anti 0243 Additionally or alternatively, the protein sequences bodies that bind to preferred epitopes on human LSR which of the present invention can further be used as a “query possess desired functional properties such as modulation of sequence' to perform a search against public databases to, for co-stimulation and related functions. Other antibodies with example, identify related sequences. Such searches can be desired epitope specificity may be selected and will have the performed using the XBLAST program (version 2.0) of Alts ability to cross-compete for binding to LSR antigen with the chul, et al. (1990).J. Mol. Biol. 215:403-10. BLAST protein desired antibodies. searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences (0250 Engineered and Modified Antibodies homologous to the antibody molecules according to at least 0251 An antibody according to at least some embodi Some embodiments of the invention. To obtain gapped align ments of the invention further can be prepared using an anti ments for comparison purposes, Gapped BLAST can be uti body having one or more of the VH and/or VL sequences lized as described in Altschuletal. (1997) Nucleic Acids Res. derived from an anti-LSR antibody starting material to engi 25(17):3389-3402. When utilizing BLAST and Gapped neer a modified antibody, which modified antibody may have BLAST programs, the default parameters of the respective altered properties from the starting antibody. An antibody can programs (e.g., XBLAST and NBLAST) can be used. be engineered by modifying one or more residues within one 0244 Antibodies with Conservative Modifications or both variable regions (i.e., VH and/or VL), for example 0245. In certain embodiments, an antibody of the inven within one or more CDR regions and/or within one or more tion comprises a heavy chain variable region comprising framework regions. Additionally or alternatively, an antibody US 2014/0294765 A1 Oct. 2, 2014 can be engineered by modifying residues within the constant can be identified by comparing the antibody framework regions, for example to alter the effector functions of the sequences to the germline sequences from which the antibody antibody. is derived. 0252 One type of variable region engineering that can be 0256 In addition or alternative to modifications made within the framework or CDR regions, antibodies according performed is CDR grafting. Antibodies interact with target to at least Some embodiments of the invention may be engi antigens predominantly through amino acid residues that are neered to include modifications within the Fc region, typi located in the six heavy and light chain complementarity cally to alter one or more functional properties of the anti determining regions (CDRS). For this reason, the amino acid body, such as serum half-life, complement fixation, Fc sequences within CDRs are more diverse between individual receptor binding, and/or antigen-dependent cellular cytotox antibodies than sequences outside of CDRs. Because CDR icity. Furthermore, an antibody according to at least some sequences are responsible for most antibody-antigen interac embodiments of the invention may be chemically modified tions, it is possible to express recombinant antibodies that (e.g., one or more chemical moieties can be attached to the mimic the properties of specific naturally occurring antibod antibody) or be modified to alter its glycosylation, again to ies by constructing expression vectors that include CDR alter one or more functional properties of the antibody. Such sequences from the specific naturally occurring antibody embodiments are described further below. The numbering of grafted onto framework sequences from a different antibody residues in the Fc region is that of the EU index of Kabat. with different properties (see, e.g., Riechmann, L. et al. 0257. In one embodiment, the hinge region of CH1 is (1998) Nature 332:323-327: Jones, P. et al. (1986) Nature modified such that the number of cysteine residues in the 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. hinge region is altered, e.g., increased or decreased. This U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, approach is described further in U.S. Pat. No. 5,677,425 by and U.S. Pat. Nos. 5,530,101: 5,585,089: 5,693,762 and Bodmer et al. The number of cysteine residues in the hinge 6,180,370 to Queen et al.) region of CH1 is altered to, for example, facilitate assembly 0253) Suitable framework sequences can be obtained from of the light and heavy chains or to increase or decrease the public DNA databases or published references that include stability of the antibody. germline antibody gene sequences. For example, germline 0258. In another embodiment, the Fc hinge region of an DNA sequences for human heavy and light chain variable antibody is mutated to decrease the biological half life of the region genes can be found in the “VBase human germline antibody. More specifically, one or more amino acid muta sequence database (available on the Internet), as well as in tions are introduced into the CH2-CH3 domain interface Kabat, E. A., et al. (1991) Sequences of Proteins of Immuno region of the Fc-hinge fragment such that the antibody has logical Interest, Fifth Edition, U.S. Department of Health and impaired Staphylococcyl protein A (SpA) binding relative to Human Services, NIH Publication No. 91-3242; Tomlinson, native Fc-hinge domain SpA binding. This approach is I. M., et al. (1992) “The Repertoire of Human Germline VH described in further detail in U.S. Pat. No. 6,165,745 by Ward Sequences Reveals about Fifty Groups of VHSegments with et al. Different Hypervariable Loops' J. Mol. Biol. 227:776-798: 0259. In another embodiment, the antibody is modified to and Cox, J. P. L. et al. (1994) “A Directory of Human Germ increase its biological half life. Various approaches are pos line VHSegments Reveals a Strong Bias in their Usage' Eur. sible. For example, one or more of the following mutations J. Immunol. 24:827-836; the contents of each of which are can be introduced: T252L. T254S, T256F, as described in expressly incorporated herein by reference. U.S. Pat. No. 6,277.375 to Ward. Alternatively, to increase the 0254 Another type of variable region modification is to biological half life, the antibody can be altered within the mutate amino acid residues within the VHand/or VLCDR1, CH1 or CL region to contain a salvage receptor binding CDR2 and/or CDR3 regions to thereby improve one or more epitope taken from two loops of a CH2 domain of an Fc region binding properties (e.g., affinity) of the antibody of interest. of an IgG, as described in U.S. Pat. Nos. 5,869,046 and Site-directed mutagenesis or PCR-mediated mutagenesis can 6,121,022 by Presta et al. be performed to introduce the mutations and the effect on 0260. In yet other embodiments, the Fc region is altered by antibody binding, or other functional property of interest, can replacing at least one amino acid residue with a different be evaluated in appropriate in vitro or in vivo assays. Prefer amino acid residue to alter the effector functions of the anti ably conservative modifications (as discussed above) are body. For example, one or more amino acids selected from introduced. The mutations may be amino acid Substitutions, amino acid residues 234, 235, 236, 237, 297, 318, 320 and additions or deletions, but are preferably substitutions. More 322 can be replaced with a different amino acid residue such over, typically no more than one, two, three, four or five that the antibody has an altered affinity for an effector ligand residues within a CDR region are altered. but retains the antigen-binding ability of the parent antibody. 0255 Engineered antibodies according to at least some The effector ligand to which affinity is altered can be, for embodiments of the invention include those in which modi example, an Fc receptor or the C1 component of complement. fications have been made to framework residues within VH This approach is described in further detail in U.S. Pat. Nos. and/or VL, e.g. to improve the properties of the antibody. 5,624,821 and 5,648,260, both by Winter et al. Typically such framework modifications are made to 0261. In another example, one or more amino acids decrease the immunogenicity of the antibody. For example, selected from amino acid residues 329, 331 and 322 can be one approach is to “backmutate' one or more framework replaced with a different amino acid residue such that the residues to the corresponding germline sequence. More spe antibody has altered Cld binding and/or reduced or abolished cifically, an antibody that has undergone Somatic mutation complement dependent cytotoxicity (CDC). This approach is may contain framework residues that differ from the germline described in further detail in U.S. Pat. No. 6,194.551 by sequence from which the antibody is derived. Such residues Idusogie et al. US 2014/0294765 A1 Oct. 2, 2014

0262. In another example, one or more amino acid resi cation No. 200401 10704 by Yamane et al. and Yamane dues within amino acid positions 231 and 239 are altered to Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As thereby alter the ability of the antibody to fix complement. another example, EP1,176,195 by Hanaietal. describes a cell This approach is described further in PCT Publication WO line with a functionally disrupted FUT8 gene, which encodes 94/29351 by Bodmer et al. a fucosyltransferase, such that antibodies expressed in Such a 0263. In yet another example, the Fc region is modified to cell line exhibit hypofucosylation by reducing or eliminating increase the ability of the antibody to mediate antibody the alpha 1.6 bond-related enzyme. Hanai et al. also describe dependent cellular cytotoxicity (ADCC) and/or to increase cell lines which have a low enzyme activity for adding fucose the affinity of the antibody for an Fcy receptor by modifying to the N-acetylglucosamine that binds to the Fc region of the one or more amino acids at the following positions: 238,239, antibody or does not have the enzyme activity, for example 248, 249, 252,254, 255, 256, 258, 265, 267,268, 269, 270, the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, Publication WO 03/035835 by Presta describes a variant 295, 296,298,301,303, 305,307, 309, 312,315, 320, 322, CHO cell line, Lec13 cells, with reduced ability to attach 324, 326,327, 329, 330, 331,333,334, 335, 337,338, 340, fucose to ASn(297)-linked carbohydrates, also resulting in 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, hypofucosylation of antibodies expressed in that host cell 434, 435, 437, 438 or 439. This approach is described further (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277: in PCT Publication WO 00/42072 by Presta. Moreover, the 26733-26740). PCT Publication WO99/54342 by Umana et binding sites on human IgG1 for Fc gamma RI. Fc gamma al. describes cell lines engineered to express glycoprotein RII, Fc gammaRIII and FcRn have been mapped and variants modifying glycosyl transferases (e.g., beta(1,4)-N-acetyl with improved binding have been described (see Shields, R. glucosaminyltransferase III (GnTIII)) such that antibodies L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific expressed in the engineered cell lines exhibit increased mutations at positions 256, 290, 298,333,334 and 339 are bisecting GlcNac structures which results in increased ADCC shown to improve binding to FcyRIII. Additionally, the fol activity of the antibodies (see also Umana et al. (1999) Nat. lowing combination mutants are shown to improve Fcgam Biotech. 17:176-180). Alternatively, the fucose residues of ma.RIII binding: T256A/S298A, S298A/E333A, S298A/ the antibody may be cleaved off using a fucosidase enzyme. K224A and S298A/E333A/K334A. Furthermore, mutations For example, the fucosidase alpha-L-fucosidase removes such as M252Y/S254T/T256E or M428L/N434S improve fucosyl residues from antibodies (Tarentino, A. L. et al. binding to FcRn and increase antibody circulation half-life (1975) Biochem. 14:5516-23). (see Chan CA and Carter PJ (2010) Nature Rev Immunol 0266. Another modification of the antibodies herein that is 10:301-316). contemplated by the invention is pegylation. An antibody can 0264. In still another embodiment, the glycosylation of an be pegylated to, for example, increase the biological (e.g., antibody is modified. For example, an aglycoslated antibody serum) half life of the antibody. To pegylate an antibody, the can be made (i.e., the antibody lacks glycosylation). Glyco antibody, or fragment thereof, typically is reacted with poly Sylation can be altered to, for example, increase the affinity of ethylene glycol (PEG), such as a reactive ester or aldehyde the antibody for antigen. Such carbohydrate modifications derivative of PEG, under conditions in which one or more can be accomplished by, for example, altering one or more PEG groups become attached to the antibody or antibody sites of glycosylation within the antibody sequence. For fragment. Preferably, the pegylation is carried out via an example, one or more amino acid Substitutions can be made acylation reaction or an alkylation reaction with a reactive that result in elimination of one or more variable region PEG molecule (or an analogous reactive water-soluble poly framework glycosylation sites to thereby eliminate glycosy mer). As used herein, the term “polyethylene glycol is lation at that site. Such aglycosylation may increase the affin intended to encompass any of the forms of PEG that have ity of the antibody for antigen. Such an approach is described been used to derivatize other proteins, such as mono (C1 in further detail in U.S. Pat. Nos. 5,714,350 and 6.350,861 by C10) alkoxy- or aryloxy-polyethylene glycolor polyethylene Co et al. glycol-maleimide. In certain embodiments, the antibody to be 0265 Additionally or alternatively, an antibody can be pegylated is an aglycosylated antibody. Methods for pegylat made that has an altered type of glycosylation, Such as a ing proteins are known in the art and can be applied to the hypoflucosylated antibody having reduced amounts of fuco antibodies according to at least Some embodiments of the Syl residues or an antibody having increased bisecting invention. See for example, EPO 154316 by Nishimura et al. GlcNac structures. Such altered glycosylation patterns have and EP 0401 384 by Ishikawa et al. been demonstrated to increase the ADCC ability of antibod 0267 Methods of Engineering Antibodies ies. Such carbohydrate modifications can be accomplished 0268 As discussed above, anti-LSRantibodies having VH by, for example, expressing the antibody in a host cell with and VK sequences disclosed herein can be used to create new altered glycosylation machinery. Cells with altered glycosy anti-LSR antibodies, respectively, by modifying the VHand/ lation machinery have been described in the art and can be or VL sequences, or the constant regions attached thereto. used as host cells in which to express recombinant antibodies Thus, in another aspect according to at least some embodi according to at least Some embodiments of the invention to ments of the invention, the structural features of an anti-LSR thereby produce an antibody with altered glycosylation. For antibody according to at least some embodiments of the example, the cell lines Ms704, Ms705, and Ms709 lack the invention, are used to create structurally related anti-LSR fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltrans antibodies that retain at least one functional property of the ferase), such that antibodies expressed in the Ms704, Ms705, antibodies according to at least Some embodiments of the and Ms709 cell lines lack fucose on their carbohydrates. The invention, Such as binding to human LSR, respectively. For Ms704, Ms705, and Ms709 FUT8.-/- cell lines are created example, one or more CDR regions of one LSR antibody or by the targeted disruption of the FUT8 gene in CHO/DG44 mutations thereof, can be combined recombinantly with cells using two replacement vectors (see U.S. Patent Publi known framework regions and/or other CDRs to create addi US 2014/0294765 A1 Oct. 2, 2014 tional, recombinantly-engineered, anti-LSR antibodies carrying human immunoglobulin genes as described further according to at least Some embodiments of the invention, as below), cDNAs encoding the light and heavy chains of the discussed above. Other types of modifications include those antibody made by the hybridoma can be obtained by standard described in the previous section. The starting material for the PCR amplification or cDNA cloning techniques. Forantibod engineering method is one or more of the VH and/or VK ies obtained from an immunoglobulin gene library (e.g., sequences provided herein, or one or more CDR regions using phage display techniques), nucleic acid encoding the thereof. To create the engineered antibody, it is not necessary antibody can be recovered from the library. to actually prepare (i.e., express as a protein) an antibody 0277 Once DNA fragments encoding VH and VL seg having one or more of the VHand/or VK sequences provided ments are obtained, these DNA fragments can be further herein, or one or more CDR regions thereof. Rather, the manipulated by standard recombinant DNA techniques, for information contained in the sequences is used as the starting example to convert the variable region genes to full-length material to create a 'second generation’ sequences derived antibody chain genes, to Fab fragment genes or to a schv from the original sequences and then the 'second generation” gene. In these manipulations, a VL- or VH-encoding DNA sequences is prepared and expressed as a protein. fragment is operatively linked to another DNA fragment 0269 Standard molecular biology techniques can be used encoding another protein, Such as an antibody constant region to prepare and express altered antibody sequence. or a flexible linker. (0270 Preferably, the antibody encoded by the altered anti 0278. The term “operatively linked’, as used in this con body sequences is one that retains one, Some or all of the text, is intended to mean that the two DNA fragments are functional properties of the anti-LSR antibodies, respec joined Such that the amino acid sequences encoded by the two tively, produced by methods and with sequences provided DNA fragments remain in-frame. herein, which functional properties include binding to LSR (0279. The isolated DNA encoding the VH region can be antigen with a specific KD level or less and/or modulating B7 converted to a full-length heavy chain gene by operatively costimulation and/or selectively binding to desired target linking the VH-encoding DNA to another DNA molecule cells such as for example, that express LSR antigen. encoding heavy chain constant regions (CH1, CH2 and CH3). 0271 The functional properties of the altered antibodies The sequences of human heavy chain constant region genes can be assessed using standard assays available in the art are known in the art (see e.g., Kabat, E. A., et al. (1991) and/or described herein. Sequences of Proteins of Immunological Interest, Fifth Edi 0272. In certain embodiments of the methods of engineer tion, U.S. Department of Health and Human Services, NIH ing antibodies according to at least some embodiments of the Publication No. 91-3242) and DNA fragments encompassing invention, mutations can be introduced randomly or selec these regions can be obtained by standard PCR amplification. tively along all or part of an anti-LSR antibody coding The heavy chain constant region can be an IgG1, IgG2, IgG3. sequence and the resulting modified anti-LSR antibodies can IgG4, IgA, IgE, IgM or Ig|D constant region, but most pref be screened for binding activity and/or other desired func erably is an IgG1 or IgG4 constant region. For a Fab fragment tional properties. heavy chain gene, the VH-encoding DNA can be operatively 0273 Mutational methods have been described in the art. linked to another DNA molecule encoding only the heavy For example, PCT Publication WO 02/092780 by Short chain CH1 constant region. describes methods for creating and Screening antibody muta 0280. The isolated DNA encoding the VL region can be tions using Saturation mutagenesis, synthetic ligation assem converted to a full-length light chain gene (as well as a Fab bly, or a combination thereof. Alternatively, PCT Publication light chain gene) by operatively linking the VL-encoding WO 03/074679 by Lazaret al. describes methods of using DNA to another DNA molecule encoding the light chain computational screening methods to optimize physiochemi constant region, CL. The sequences of human light chain cal properties of antibodies. constant region genes are known in the art (see e.g., Kabat, E. 0274 Nucleic Acid Molecules Encoding Antibodies A., et al. (1991) Sequences of Proteins of Immunological 0275 Another aspect of the invention pertains to nucleic Interest, Fifth Edition, U.S. Department of Health and Human acid molecules that encode the antibodies according to at Services, NIH Publication No. 91-3242) and DNA fragments least some embodiments of the invention. The nucleic acids encompassing these regions can be obtained by standard PCR may be present in whole cells, in a cell lysate, or in a partially amplification. The light chain constant region can be a kappa purified or substantially pure form. A nucleic acid is “iso or lambda constant region, but most preferably is a kappa lated' or “rendered substantially pure' when purified away constant region. from other cellular components or other contaminants, e.g., 0281 To create a schv gene, the VH and VL-encoding other cellular nucleic acids or proteins, by Standard tech DNA fragments are operatively linked to another fragment niques, including alkaline/SDS treatment, CsCl banding, col encoding a flexible linker, e.g., encoding the amino acid umn chromatography, agarose gel electrophoresis and others sequence (Gly-4-Ser)3, such that the VH and VL sequences well known in the art. See, F. Ausubel, et al., ed. (1987) can be expressed as a contiguous single-chain protein, with Current Protocols in Molecular Biology, Greene Publishing the VL and VH regions joined by the flexible linker (see e.g., and Wiley Interscience, New York. A nucleic acid according Bird et al. (1988) Science 242:423-426; Huston et al. (1988) to at least some embodiments of the invention can be, for Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., example, DNA or RNA and may or may not contain intronic (1990) Nature 348:552-554). sequences. In a preferred embodiment, the nucleic acid is a 0282 Production of Anti-LSR Monoclonal Antibodies cDNA molecule. 0283 Monoclonal antibodies (mAbs) of the present 0276 Nucleic acids according to at least some embodi invention can be produced by a variety of techniques, includ ments of the invention can be obtained using standard ing conventional monoclonal antibody methodology e.g., the molecular biology techniques. For antibodies expressed by standard somatic cell hybridization technique of Kohler and hybridomas (e.g., hybridomas prepared from transgenic mice Milstein (1975) Nature 256:495. Although somatic cell US 2014/0294765 A1 Oct. 2, 2014 20 hybridization procedures are preferred, in principle, other No. 5,545,807 to Surani et al., PCT Publication Nos. WO techniques for producing monoclonal antibody can be 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO employed e.g., viral or oncogenic transformation of B lym 98/24884 and WO 99/45962, all to Lonberg and Kay; and phocytes. PCT Publication No. WO 01/14424 to Korman et al. 0284. A preferred animal system for preparing hybrido 0287. In another embodiment, human antibodies accord mas is the murine system. Hybridoma production in the ing to at least some embodiments of the invention can be mouse is a very well-established procedure Immunization raised using a mouse that carries human immunoglobulin protocols and techniques for isolation of immunized spleno sequences on transgenes and transchomosomes, such as a cytes for fusion are known in the art. Fusion partners (e.g., mouse that carries a human heavy chain transgene and a murine myeloma cells) and fusion procedures are also human light chain transchromosome. Such mice, referred to known. herein as “KM mice TM.”, are described in detail in PCT 0285 Chimeric or humanized antibodies of the present Publication WOO2/43478 to Ishida et al. invention can be prepared based on the sequence of a murine 0288 Still further, alternative transgenic animal systems monoclonal antibody prepared as described above. DNA expressing human immunoglobulin genes are available in the encoding the heavy and light chain immunoglobulins can be art and can be used to raise anti-LSR antibodies according to obtained from the murine hybridoma of interest and engi at least some embodiments of the invention. For example, an neered to contain non-murine (e.g., human) immunoglobulin alternative transgenic system referred to as the Xenomouse sequences using standard molecular biology techniques. For (Abgenix, Inc.) can be used; Such mice are described in, for example, to create a chimeric antibody, the murine variable example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598: regions can be linked to human constant regions using meth 6,150,584 and 6,162.963 to Kucherlapati et al. ods known in the art (see e.g., U.S. Pat. No. 4,816,567 to 0289 Moreover, alternative transchromosomic animal Cabilly et al.). To create a humanized antibody, the murine systems expressing human immunoglobulin genes are avail CDR regions can be inserted into a human framework using able in the art and can be used to raise anti-LSR antibodies methods known in the art (see e.g., U.S. Pat. No. 5.225,539 to according to at least Some embodiments of the invention. For Winter, and U.S. Pat. Nos. 5,530,101: 5,585,089: 5,693,762 example, mice carrying both a human heavy chain transchro and 6,180.370 to Queen et al.). mosome and a human light chain transchromosome, referred 0286 According to at least some embodiments of the to as “TC mice’ can be used; such mice are described in invention, the antibodies are human monoclonal antibodies. Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722 Such human monoclonal antibodies directed against LSR can 727. Furthermore, cows carrying human heavy and light be generated using transgenic or transchromosomic mice car chain transchromosomes have been described in the art rying parts of the human immune system rather than the (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) mouse system. These transgenic and transchromosomic mice and can be used to raise anti-LSR antibodies according to at include mice referred to herein as the HuMAb Mouse RTM least Some embodiments of the invention. and KM Mouse RTM, respectively, and are collectively 0290 Human monoclonal antibodies according to at least referred to herein as “human Ig mice.” The HuMAb Mouse Some embodiments of the invention can also be prepared TM. (Medarex. Inc.) contains human immunoglobulin gene using phage display methods for screening libraries of human miniloci that encode unrearranged human heavy (mu.. and. immunoglobulin genes. Such phage display methods for iso gamma.) and kappa. light chain immunoglobulin sequences, lating human antibodies are established in the art. See for together with targeted mutations that inactivate the endog example: U.S. Pat. Nos. 5.223,409:5,403.484; and 5,571,698 enous.mu.. and kappa. chain loci (see e.g., Lonberg, et al. to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to (1994) Nature 368(6474): 856-859). Accordingly, the mice Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to exhibit reduced expression of mouse IgM or kappa., and in McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; response to immunization, the introduced human heavy and 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et light chain transgenes undergo class Switching and Somatic al. mutation to generate high affinity human IgGkappa. mono 0291 Human monoclonal antibodies according to at least clonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, Some embodiments of the invention can also be prepared N. (1994) Handbook of Experimental Pharmacology 1 13:49 using SCID mice into which human immune cells have been 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immu reconstituted Such that a human antibody response can be nol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann generated upon immunization. Such mice are described in, N.Y. Acad. Sci. 764:536-546). The preparation and use of the for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wil HuMab Mouse RTM., and the genomic modifications carried son et al. by such mice, is further described in Taylor, L. et al. (1992) 0292 Immunization of Human Ig Mice Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) 0293 When human Ig mice are used to raise human anti International Immunology 5:647-656: Tuaillon et al. (1993) bodies according to at least Some embodiments of the inven Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) tion, Such mice can be immunized with a purified or enriched Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. preparation of LSR antigen and/or recombinant LSR, or LSR 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912 fusion protein, as described by Lonberg, N. et al. (1994) 2920; Taylor, L. et al. (1994) International Immunology Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnol Biotechnology 14: 845-851; and PCT Publication WO ogy 14: 845-851, the contents of all of which are hereby 98/24884 and WO 01/14424. Preferably, the mice will be specifically incorporated by reference in their entirety. See 6-16 weeks of age upon the first infusion. For example, a further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; purified or recombinant preparation (5-50.mu.g) of LSR anti 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; gen can be used to immunize the human Ig mice intraperito 5,874.299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. neally. US 2014/0294765 A1 Oct. 2, 2014

0294 Prior experience with various antigens by others has 0298 Generation of Transfectomas Producing Mono shown that the transgenic mice respond when initially immu clonal Antibodies nized intraperitoneally (IP) with antigen in complete Fre 0299 Antibodies according to at least some embodiments und's adjuvant, followed by every other week IP immuniza according to at least Some embodiments of the invention also tions (up to a total of 6) with antigen in incomplete Freund's can be produced in a host cell transfectoma using, for adjuvant. However, adjuvants other than Freunds are also example, a combination of recombinant DNA techniques and found to be effective. In addition, whole cells in the absence gene transfection methods as is well known in the art (e.g., of adjuvant are found to be highly immunogenic. The immune Morrison, S. (1985) Science 229:1202). response can be monitored over the course of the immuniza 0300 For example, to express the antibodies, or antibody tion protocol with plasma samples being obtained by retro fragments thereof. DNAS encoding partial or full-length light orbital bleeds. The plasma can be screened by ELISA (as and heavy chains, can be obtained by standard molecular described below), and mice with sufficient titers of anti-LSR biology techniques (e.g., PCR amplification or cDNA cloning human immunoglobulin can be used for fusions. Mice can be using a hybridoma that expresses the antibody of interest) and boosted intravenously with antigen 3 days before sacrifice the DNAs can be inserted into expression vectors such that the and removal of the spleen. It is expected that 2-3 fusions for genes are operatively linked to transcriptional and transla each immunization may need to be performed. Between 6 and tional control sequences. In this context, the term “opera 24 mice are typically immunized for each antigen. Usually tively linked' is intended to mean that an antibody gene is both HCo7 and HCo12 strains are used. In addition, both ligated into a vector Such that transcriptional and translational HCo7 and HCo12 transgene can be bred together into a single control sequences within the vector serve their intended func mouse having two different human heavy chain transgenes tion of regulating the transcription and translation of the (HCo7/HCo 12). Alternatively or additionally, the KM antibody gene. The expression vector and expression control Mouse. RTM. Strain can be used. sequences are chosen to be compatible with the expression 0295 Generation of Hybridomas Producing Human host cell used. The antibody light chain gene and the antibody Monoclonal Antibodies heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression 0296 To generate hybridomas producing human mono vector. The antibody genes are inserted into the expression clonal antibodies according to at least some embodiments of vector by standard methods (e.g., ligation of complementary the invention, splenocytes and/or lymph node cells from restriction sites on the antibody gene fragment and vector, or immunized mice can be isolated and fused to an appropriate blunt end ligation if no restriction sites are present). The light immortalized cell line. Such as a mouse myeloma cell line. and heavy chain variable regions of the antibodies described The resulting hybridomas can be screened for the production herein can be used to create full-length antibody genes of any of antigen-specific antibodies. For example, single cell Sus antibody isotype by inserting them into expression vectors pensions of Splenic lymphocytes from immunized mice can already encoding heavy chain constant and light chain con be fused to one-sixth the number of P3X63-Ag8.653 nonse stant regions of the desired isotype such that the VH segment creting mouse myeloma cells (ATCC, CRL 1580) with 50% is operatively linked to the CH segments within the vector and PEG. Cells are plated at approximately 2x10-5 in flat bottom the VK segment is operatively linked to the CL segment microtiter plate, followed by a two week incubation in selec within the vector. Additionally or alternatively, the recombi tive medium containing 20% fetal Clone Serum, 18% “653” nant expression vector can encode a signal peptide that facili conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 tates secretion of the antibody chain from a host cell. The mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercap antibody chain gene can be cloned into the vector Such that toethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 the signal peptide is linked in-frame to the amino terminus of mg/ml gentamycin and 1xEHAT (Sigma; the HAT is added 24 the antibody chain gene. The signal peptide can be an immu hours after the fusion). After approximately two weeks, cells noglobulin signal peptide or a heterologous signal peptide can be cultured in medium in which the HAT is replaced with (i.e., a signal peptide from a non-immunoglobulin protein). HT. Individual wells can then be screened by ELISA for 0301 In addition to the antibody chain genes, the recom human monoclonal IgM and IgG antibodies. Once extensive binant expression vectors according to at least Some embodi hybridoma growth occurs, medium can be observed usually ments of the invention carry regulatory sequences that control after 10-14 days. The antibody secreting hybridomas can be the expression of the antibody chain genes in a host cell. The replated, screened again, and if still positive for human IgG, term “regulatory sequence' is intended to include promoters, the monoclonal antibodies can be subcloned at least twice by enhancers and other expression control elements (e.g., poly limiting dilution. The stable subclones can then be cultured in adenylation signals) that control the transcription or transla vitro to generate Small amounts of antibody in tissue culture tion of the antibody chain genes. Such regulatory sequences medium for characterization. are described, for example, in Goeddel (Gene Expression 0297 To purify human monoclonal antibodies, selected Technology. Methods in Enzymology 185, Academic Press, hybridomas can be grown in two-liter spinner-flasks for San Diego, Calif. (1990)). It will be appreciated by those monoclonal antibody purification. Supernatants can be fil skilled in the art that the design of the expression vector, tered and concentrated before affinity chromatography with including the selection of regulatory sequences, may depend protein A-Sepharose (Pharmacia, Piscataway, N.J.). Eluted on such factors as the choice of the host cell to be transformed, IgG can be checked by gel electrophoresis and high perfor the level of expression of protein desired, etc. Preferred regu mance liquid chromatography to ensure purity. The buffer latory sequences for mammalian host cell expression include solution can be exchanged into PBS, and the concentration viral elements that direct high levels of protein expression in can be determined by OD280 using 1.43 extinction coeffi mammalian cells, such as promoters and/or enhancers cient. The monoclonal antibodies can be aliquoted and stored derived from cytomegalovirus (CMV), Simian Virus 40 at -80 degrees C. (SV40), adenovirus, (e.g., the adenovirus major late promoter US 2014/0294765 A1 Oct. 2, 2014 22

(AdMLP) and polyoma. Alternatively, nonviral regulatory example, standard ELISA. Briefly, microtiter plates are sequences may be used, such as the ubiquitin promoter coated with purified LLSR at 0.25.mu.g/ml in PBS, and then or.beta-globin promoter. Still further, regulatory elements blocked with 5% bovine serum albumin in PBS. Dilutions of composed of sequences from different sources, such as the antibody (e.g., dilutions of plasma from -immunized mice) SRalpha. promoter system, which contains sequences from are added to each well and incubated for 1-2 hours at 37 the SV40 early promoter and the long terminal repeat of degrees C. The plates are washed with PBS/Tween and then human T cell leukemia virus type 1 (Takebe. Y. et al. (1988) incubated with secondary reagent (e.g., for human antibodies, Mol. Cell. Biol. 8:466-472). a goat-anti-human IgG Fc-specific polyclonal reagent) con 0302) In addition to the antibody chain genes and regula jugated to alkaline phosphatase for 1 hour at 37 degrees C. tory sequences, the recombinant expression vectors accord After washing, the plates are developed with pNPP substrate ing to at least Some embodiments of the invention may carry (1 mg/ml), and analyzed at OD of 405-650. Preferably, mice additional sequences, such as sequences that regulate repli which develop the highest titers will be used for fusions. cation of the vector in host cells (e.g., origins of replication) 0307 An ELISA assay as described above can also be and selectable marker genes. The selectable marker gene used to screen for hybridomas that show positive reactivity facilitates selection of host cells into which the vector has with LSR immunogen. Hybridomas that bind with high avid been introduced (see, e.g., U.S. Pat. Nos. 4.399.216, 4,634. ity to LSRare subcloned and further characterized. One clone 665 and 5,179,017, all by Axel et al.). For example, typically from each hybridoma, which retains the reactivity of the the selectable marker gene confers resistance to drugs, such parent cells (by ELISA), can be chosen for making a 5-10 vial as G418, hygromycin or methotrexate, on a host cell into cell bank stored at -140 degrees C., and for antibody purifi which the vector has been introduced. Preferred selectable cation. marker genes include the dihydrofolate reductase (DHFR) 0308 To purify anti-LSR antibodies, selected hybridomas gene (for use in dhfr-host cells with methotrexate selection/ can be grown in two-liter spinner-flasks for monoclonal anti amplification) and the neogene (for G418 selection). body purification. Supernatants can be filtered and concen 0303 For expression of the light and heavy chains, the trated before affinity chromatography with protein expression vectors encoding the heavy and light chains is A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can transfected into a host cell by standard techniques. The vari be checked by gel electrophoresis and high performance liq ous forms of the term “transfection' are intended to encom uid chromatography to ensure purity. The buffer Solution can pass a wide variety of techniques commonly used for the be exchanged into PBS, and the concentration can be deter introduction of exogenous DNA into a prokaryotic or eukary mined by OD280 using 1.43 extinction coefficient. The otic host cell, e.g., electroporation, calcium-phosphate pre monoclonal antibodies can be aliquoted and stored at -80 cipitation, DEAE-dextran transfection and the like. Although degrees C. it is theoretically possible to express the antibodies according 0309 To determine if the selected anti-LSR monoclonal to at least some embodiments of the invention in either antibodies bind to unique epitopes, each antibody can be prokaryotic or eukaryotic host cells, expression of antibodies biotinylated using commercially available reagents (Pierce, in eukaryotic cells, and most preferably mammalian host Rockford, Ill.). Competition studies using unlabeled mono cells, is the most preferred because Such eukaryotic cells, and clonal antibodies and biotinylated monoclonal antibodies can in particular mammalian cells, are more likely than prokary be performed using LSR coated-ELISA plates as described otic cells to assemble and secrete a properly folded and immu above. Biotinylated mAb binding can be detected with a nologically active antibody. Prokaryotic expression of anti strep-avidin-alkaline phosphatase probe. body genes has been reported to be ineffective for production 0310. To determine the isotype of purified antibodies, iso of highyields of active antibody (Boss, M.A. and Wood, C. R. type ELISAS can be performed using reagents specific for (1985) Immunology Today 6:12-13). antibodies of a particular isotype. For example, to determine 0304 Preferred mammalian host cells for expressing the the isotype of a human monoclonal antibody, wells of micro recombinant antibodies according to at least some embodi titer plates can be coated with 1...mu.g/ml of anti-human ments of the invention include Chinese Hamster Ovary (CHO immunoglobulin overnight at 4 degrees C. After blocking cells) (including dhfr-CHO cells, described in Urlaub and with 1% BSA, the plates are reacted with 1 mug/ml or less of ChasM, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, test monoclonal antibodies or purified isotype controls, at used with a DHFR selectable marker, e.g., as described in R. ambient temperature for one to two hours. The wells can then J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), be reacted with either human IgG1 or human IgM-specific NSO myeloma cells, COS cells and SP2 cells. In particular, alkaline phosphatase-conjugated probes. Plates are devel for use with NSO myeloma cells, another preferred expres oped and analyzed as described above. sion system is the GS gene expression system disclosed in 0311 Anti-LSR human IgGs can be further tested for WO 87/04462, WO 89/01036 and EP338,841. When recom reactivity with LSR antigen, respectively, by Western blot binant expression vectors encoding antibody genes are intro ting. Briefly, LSR antigen can be prepared and Subjected to duced into mammalian host cells, the antibodies are produced Sodium dodecyl Sulfate polyacrylamide gel electrophoresis. by culturing the host cells for a period of time sufficient to After electrophoresis, the separated antigens are transferred allow for expression of the antibody in the host cells or, more to nitrocellulose membranes, blocked with 10% fetal calf preferably, secretion of the antibody into the culture medium serum, and probed with the monoclonal antibodies to be in which the host cells are grown. Antibodies can be recov tested. Human IgG binding can be detected using anti-human ered from the culture medium using standard protein purifi IgG alkaline phosphatase and developed with BCIP/NBT cation methods. substrate tablets (Sigma Chem. Co., St. Louis, Mo.). 0305 Characterization of Antibody Binding to Antigen 0312. Alternative Scaffolds 0306 Antibodies according to at least some embodiments 0313 According to at least some embodiments the inven of the invention can be tested for binding to LSR by, for tion relates to protein scaffolds with specificities and affinities US 2014/0294765 A1 Oct. 2, 2014 in a range similar to specific antibodies. According to at least immunoglobulin single variable domain may be a human Some embodiments the present invention relates to an anti antibody variable domain, but also includes single antibody gen-binding construct comprising a protein scaffold which is variable domains from other species such as rodent (for linked to one or more epitope-binding domains. Such engi example, as disclosed in WO 00/29004), nurse shark and neered protein scaffolds are usually obtained by designing a Camelid V-HH dAbs. Camelid V-HH are immunoglobulin random library with mutagenesis focused at a loop region or single variable domain polypeptides that are derived from at an otherwise permissible Surface area and by selection of species including camel, llama, alpaca, dromedary, and gua variants against a given target via phage display or related naco, which produce heavy chain antibodies naturally devoid techniques. According to at least some embodiments the of light chains. Such V-HH domains may be humanised invention relates to alternative scaffolds including, but not according to standard techniques available in the art, and Such limited to, anticalins, DARPins, Armadillo repeat proteins, domains are still considered to be “domain antibodies' protein A, lipocalins, fibronectin domain, ankyrin consensus according to the invention. As used herein “VH includes repeat domain, thioredoxin, chemically constrained peptides camelid V-HH domains. NARV are another type of immuno and the like. According to at least Some embodiments the globulin single variable domain which were identified in car invention relates to alternative scaffolds that are used as thera tilaginous fish including the nurse shark. These domains are peutic agents for treatment of cancer, autoimmune and infec also known as Novel Antigen Receptor variable region (com tious diseases as well as for in Vivo diagnostics. monly abbreviated to V(NAR) or NARV). For further details 0314. According to at least some embodiments the inven see MoI. Immunol. 44, 656-665 (2006) and tion further provides a pharmaceutical composition compris US2005OO43519A. ing an antigen binding construct as described herein a phar 0318. The term “epitope-binding domain refers to a maceutically acceptable carrier. domain that specifically binds an antigen or epitope indepen 0315. The term Protein Scaffold as used herein includes dently of a differentV region or domain, this may be a domain but is not limited to an immunoglobulin (Ig) scaffold, for antibody (dAb), for example a human, camelid or shark example an IgG scaffold, which may be a four chain or two immunoglobulin single variable domain or it may be a chain antibody, or which may comprise only the Fc region of domain which is a derivative of a scaffold selected from the an antibody, or which may comprise one or more constant group consisting of CTLA-4 (Evibody); lipocalin: Protein A regions from an antibody, which constant regions may be of derived molecules such as Z-domain of Protein A (Affibody, human or primate origin, or which may be an artificial chi SpA), A-domain (Avimer/Maxibody); Heat shock proteins mera of human and primate constant regions. Such protein such as GroEI and GroES; transferrin (trans-body); ankyrin scaffolds may comprise antigen-binding sites in addition to repeat protein (DARPin); peptide aptamer; C-type lectin the one or more constant regions, for example where the domain (Tetranectin); human Y-crystallin and human ubiq protein scaffold comprises a full IgG. Such protein scaffolds uitin (affilins): PDZ domains; scorpion toxinkunitz type will be capable of being linked to other protein domains, for domains of human protease inhibitors; Armadillo repeat pro example protein domains which have antigen-binding sites, teins, thioredoxin, and fibronectin (adnectin); which has been for example epitope-binding domains or Schv domains. Subjected to protein engineering in order to obtain binding to 0316 A “domain” is a folded protein structure which has a ligand other than the natural ligand. tertiary structure independent of the rest of the protein. Gen 03.19 Loops corresponding to CDRs of antibodies can be erally, domains are responsible for discrete functional prop substituted with heterologous sequence to confer different erties of proteins and in many cases may be added, removed binding properties i.e. Evibodies. For further details see Jour or transferred to other proteins without loss of function of the nal of Immunological Methods 248 (1-2), 31-45 (2001) remainder of the protein and/or of the domain. A “single Lipocalins are a family of extracellular proteins which trans antibody variable domain” is a folded polypeptide domain port Small hydrophobic molecules such as steroids, billins, comprising sequences characteristic of antibody variable retinoids and lipids. They have a rigid secondary structure domains. It therefore includes complete antibody variable with a number of loops at the open end of the conical structure domains and modified variable domains, for example, in which can be engineered to bind to different target antigens. which one or more loops have been replaced by sequences Anticalins are between 160-180 amino acids in size, and are which are not characteristic of antibody variable domains, or derived from lipocalins. For further details see Biochim Bio antibody variable domains which have been truncated or phys Acta 1482:337-350 (2000), U.S. Pat. No. 7,250,297B1 comprise N- or C-terminal extensions, as well as folded frag and US20070224633. An affibody is a scaffold derived from ments of variable domains which retain at least the binding Protein A of Staphylococcus aureus which can be engineered activity and specificity of the full-length domain. to bind to antigen. The domain consists of a three-helical 0317. The phrase “immunoglobulin single variable bundle of approximately 58 amino acids. Libraries have been domain” refers to an antibody variable domain (VH, V-HH, generated by randomisation of surface residues. For further V-L) that specifically binds an antigen or epitope indepen details see Protein Eng. Des. Sel. 17, 455-462 (2004) and dently of a different V region or domain. An immunoglobulin EP1641818A1 Avimers are multidomain proteins derived single variable domain can be presentina format (e.g., homo from the A-domain scaffold family. The native domains of or hetero-multimer) with other, different variable regions or approximately 35 amino acids adopt a defined disulphide variable domains where the other regions or domains are not bonded structure. Diversity is generated by shuffling of the required for antigen binding by the single immunoglobulin natural variation exhibited by the family of A-domains. For variable domain (i.e., where the immunoglobulin single Vari further details see Nature Biotechnology 23(12), 1556-1561 able domain binds antigen independently of the additional (2005) and Expert Opinion on Investigational Drugs 16(6), variable domains). A “domain antibody' or “dAb’ is the same 909-917 (June 2007) A transferrin is a monomeric serum as an “immunoglobulin single variable domain which is transport glycoprotein. Transferrins can be engineered to capable of binding to an antigen as the term is used herein. An bind different target antigens by insertion of peptide US 2014/0294765 A1 Oct. 2, 2014 24 sequences in a permissive Surface loop. Examples of engi causes the ADC to bind to the target cancer cells. Often the neered transferrin scaffolds include the Trans-body. For fur ADC is then internalized by the cell and the drug is released ther details see J. Biol. Chem. 274, 24.066-24073 (1999). into the cell. Because of the targeting, the side effects are 0320 Designed Ankyrin Repeat Proteins (DARPins) are lower and give a wider therapeutic window. Hydrophilic link derived from Ankyrin which is a family of proteins that medi ers (e.g., PEG4Ma1) help prevent the drug being pumped out ate attachment of integral membrane proteins to the cytosk of resistant cancer cells through MDR (multiple drug resis eleton. A single ankyrin repeat is a 33 residue motif consisting tance) transporters. of two alpha helices; -beta turn. They can be engineered to 0328. In another aspect, the present invention features bind different target antigens by randomising residues in the immunoconjugates comprising an anti-LSR antibody, or a first alpha-helix and a beta-turn of each repeat. Their binding fragment thereof, conjugated to a therapeutic agent, such as a interface can be increased by increasing the number of mod cytotoxin, a drug (e.g., an immunosuppressant) or a ules (a method of affinity maturation). For further details see radiotoxin. Such conjugates are referred to herein as “immu J. MoI. Biol. 332, 489-503 (2003), PNAS100(4), 1700-1705 noconjugates’ Immunoconjugates that include one or more (2003) and J. MoI. Biol. 369, 1015-1028 (2007) and cytotoxins are referred to as “immunotoxins. A cytotoxin or US2004O132O28A1. cytotoxic agent includes any agent that is detrimental to (e.g., 0321 Fibronectin is a scaffold which can be engineered to kills) cells. Examples include taxol, cytochalasin B, gramici bind to antigen. Adnectins consists of a backbone of the din D, ethidium bromide, emetine, mitomycin, etoposide, natural amino acid sequence of the 10th domain of the 15 tenoposide, Vincristine, vinblastine, colchicin, doxorubicin, repeating units of human fibronectin type 111 (FN3). Three daunorubicin, dihydroxy anthracin dione, mitoxantrone, loops at one end of the beta; -sandwich can be engineered to mithramycin, actinomycin D, 1-dehydrotestosterone, gluco enable an Adnectin to specifically recognize a therapeutic corticoids, procaine, tetracaine, lidocaine, propranolol, and target of interest. For further details see Protein Eng. Des. Sel. puromycin and analogs or homologs thereof. Therapeutic 18, 435-444 (2005), US200801 39791, WO2005056764 and agents also include, for example, antimetabolites (e.g., meth U.S. Pat. No. 6,818,418B1. otrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 0322 Peptide aptamers are combinatorial recognition 5-fluorouracil decarbazine), alkylating agents (e.g., mechlo molecules that consist of a constant scaffold protein, typically rethamine, thioepa chlorambucil, melphalan, carmustine thioredoxin (TrxA) which contains a constrained variable (BSNU) and lomustine (CCNU), cyclothosphamide, busul peptide loop inserted at the active site. For further details see fan, dibromomannitol, Streptozotocin, mitomycin C, and cis Expert Opin. Biol. Ther. 5. 783-797 (2005). dichlorodiamine platinum (II) (DDP) cisplatin), anthracy 0323 Microbodies are derived from naturally occurring clines (e.g., daunorubicin (formerly daunomycin) and microproteins of 25-50 amino acids in length which contain doxorubicin), antibiotics (e.g., dactinomycin (formerly acti 3-4 cysteine bridges—examples of microproteins include nomycin), bleomycin, mithramycin, and anthramycin KalataBI and conotoxin and knottins. The microproteins have (AMC)), and anti-mitotic agents (e.g., Vincristine and Vin a loop which can be engineered to include upto 25 amino blastine). acids without affecting the overall fold of the microprotein. 0329. Other preferred examples of therapeutic cytotoxins For further details of engineered knottin domains, see that can be conjugated to an antibody according to at least WO2O08098.796. Some embodiments of the invention include duocarmycins, 0324. Other epitope binding domains include proteins calicheamicins, maytansines and auristatins, and derivatives which have been used as a scaffold to engineer different target thereof. An example of a calicheamicin antibody conjugate is antigen binding properties include human beta-crystallin and commercially available (MylotargTM; Wyeth). human ubiquitin (affilins), kunitz type domains of human 0330 Cytotoxins can be conjugated to antibodies accord protease inhibitors, PDZ-domains of the Ras-binding protein ing to at least Some embodiments of the invention using linker AF-6, Scorpion toxins (charybdotoxin), C-type lectin domain technology available in the art. Examples of linker types that (tetranectins) are reviewed in Chapter 7-Non-Antibody Scaf have been used to conjugate a cytotoxin to an antibody folds from Handbook of Therapeutic Antibodies (2007, include, but are not limited to, hydrazones, thioethers, esters, edited by Stefan Dubel) and Protein Science 15:14-27 (2006). disulfides and peptide-containing linkers. A linker can be Epitope binding domains of the present invention could be chosen that is, for example, Susceptible to cleavage by low pH derived from any of these alternative protein domains. within the lysosomal compartment or Susceptible to cleavage 0325 Conjugates or Immunoconjugates by proteases, such as proteases preferentially expressed in 0326. The present invention encompasses conjugates for tumor tissue such as cathepsins (e.g., cathepsins B, C, D). use in immune therapy comprising the LSR antigen and 0331. For further discussion of types of cytotoxins, linkers soluble portions thereof including the ectodomain orportions and methods for conjugating therapeutic agents to antibodies, or variants thereof. For example the invention encompasses see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199 conjugates wherein the ECD of the LSRantigen is attached to 215: Trail, P. A. et al. (2003) Cancer Immunol. Immunother. an immunoglobulin or fragment thereof. The invention con 52:328-337: Payne, G. (2003) Cancer Cell 3:207-212: Allen, templates the use thereof for promoting or inhibiting LSR T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and antigen activities such as immune costimulation and the use Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089 thereof in treating transplant, autoimmune, and cancer indi 1091; Senter, P. D. and Springer, C.J. (2001) Adv. Drug Deliv. cations described herein. Rev. 53:247-264. 0327. In another aspect, the present invention features 0332 Antibodies of the present invention also can be con antibody-drug conjugates (ADCs), used for example for jugated to a radioactive isotope to generate cytotoxic radiop treatment of cancer, consisting of an antibody (or antibody harmaceuticals, also referred to as radioimmunoconjugates. fragment such as a single-chain variable fragment (ScPV) Examples of radioactive isotopes that can be conjugated to linked to a payload drug (often cytotoxic). The antibody antibodies for use diagnostically or therapeutically include, US 2014/0294765 A1 Oct. 2, 2014

but are not limited to, iodine 131, indium 111, yttrium 90 and Such as another antibody, antibody fragment, peptide orbind lutetium 177. Methods for preparing radioimmunconjugates ing mimetic, such that a bispecific molecule results. In certain are established in the art. Examples of radioimmunoconju embodiments, one of the binding specificities of the bispecific gates are commercially available, including Zevalin (IDEC antibodies is for LSR and the other is for any other antigen. In Pharmaceuticals) and Bexxar. (Corixa Pharmaceuticals), and certain embodiments, bispecific antibodies may bind to two similar methods can be used to prepare radioimmunoconju different epitopes of LSR. Bispecific antibodies may also be gates using the antibodies according to at least some embodi used to localize cytotoxic agents to cells which express LSR. ments of the invention. Bispecificantibodies can be prepared as full length antibodies 0333. The antibody conjugates according to at least some or antibody fragments. embodiments of the invention can be used to modify a given 0337. A bispecific antibody according to at least some biological response, and the drug moiety is not to be con embodiments of the invention, is an antibody which can bind Strued as limited to classical chemical therapeutic agents. For simultaneously to two targets which are of different structure. example, the drug moiety may be a protein or polypeptide Bispecific antibodies (bsAb) and bispecific antibody frag possessing a desired biological activity. Such proteins may ments (bsFab) according to at least Some embodiments of the include, for example, an enzymatically active toxin, or active invention, have at least one arm that specifically binds to a fragment thereof. Such as abrin, ricin A, pseudomonas exo B-cell antigen or epitope and at least one other arm that toxin, or diphtheria toxin; a protein Such as tumor necrosis specifically binds a targetable conjugate. factor or interferon-gamma.; or, biological response modifi 0338 According to at least some embodiments the inven erS Such as, for example, lymphokines, interleukin-1 (IL tion encompasses also a fusion antibody protein, which is a 1'), interleukin-2 (IL-2), interleukin-6 (“IL-6”), granulo recombinantly produced antigen-binding molecule in which cyte macrophage colony stimulating factor (“GM-CSF), two or more different single-chain antibody orantibody frag granulocyte colony stimulating factor (“G-CSF), or other ment segments with the same or different specificities are growth factors. linked. A variety of bispecific fusion antibody proteins can be 0334 Techniques for conjugating Such therapeutic moiety produced using molecular engineering. In one form, the to antibodies are well known, see, e.g., Amon et al., “Mono bispecific fusion antibody protein is monovalent, consisting clonal Antibodies For Immunotargeting Of Drugs. In Cancer of for example, a sent with a single binding site for one Therapy”, in Monoclonal Antibodies And Cancer Therapy, antigen and a Fab fragment with a single binding site for a Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); second antigen. In another form, the bispecific fusion anti Hellstrom et al., “Antibodies For Drug Delivery”, in Con body protein is divalent, consisting of, for example, an IgG trolled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. with two binding sites for one antigen and two schv with two 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Car binding sites for a second antigen. riers Of Cytotoxic Agents. In Cancer Therapy: A Review', in Monoclonal Antibodies 84: Biological And Clinical Appli 0339 According to at least some embodiments the inven cations, Pinchera et al. (eds.), pp. 475-506 (1985): 'Analysis, tion encompasses also engineered antibodies with three or Results, And Future Prospective Of The Therapeutic Use Of more functional antigen binding sites, including "Octopus Radiolabeled Antibody In Cancer Therapy”, in Monoclonal antibodies” (see, e.g. US 2006/0025576A1). Antibodies For Cancer Detection And Therapy, Baldwin et al. 0340 According to at least some embodiments the inven (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., tion encompasses also a “Dual Acting FAb’ or “DAF com “The Preparation And Cytotoxic Properties Of Antibody prising an antigen binding site that binds to LSR as well as Toxin Conjugates. Immunol. Rev., 62:119-58 (1982). another, different antigen (see e.g. US 2008/0069820). 0335 Bispecific Molecules 0341. Accordingly, the present invention includes bispe 0336 According to at least some embodiments the inven cific molecules comprising at least one first binding specific tion encompasses also a multispecific antibody. Multispecific ity for LSR and a second binding specificity for a second antibodies are monoclonal antibodies that have binding target epitope. According to at least some embodiments of the specificities for at least two different sites. In another aspect, invention, the second target epitope is an Fc receptor, e.g., the present invention features bispecific molecules compris human Fc gamma R1 (CD64) or a human Fc alpha receptor ing an anti-LSR antibody, or a fragment thereof, according to (CD89). Therefore, the invention includes bispecific mol at least some embodiments of the invention. An antibody ecules capable of binding both to Fc gamma. R. Fc alpha Ror according to at least Some embodiments of the invention, or Fc epsilon R expressing effector cells (e.g., monocytes, mac antigen-binding portions thereof, can be derivatized or linked rophages or polymorphonuclear cells (PMNs)), and to target to another functional molecule, e.g., another peptide or pro cells expressing LSR, respectively. These bispecific mol tein (e.g., another antibody or ligand for a receptor) to gen ecules target LSR expressing cells to effector cell and trigger erate a bispecific molecule that binds to at least two different Fc receptor-mediated effector cell activities, such as phago binding sites or target molecules. The antibody according to cytosis of an LSR expressing cells, antibody dependent cell at least Some embodiments of the invention may in fact be mediated cytotoxicity (ADCC), cytokine release, or genera derivatized or linked to more than one other functional mol tion of Superoxide anion. ecule to generate multispecific molecules that bind to more 0342. According to at least some embodiments of the than two different binding sites and/or target molecules; Such invention in which the bispecific molecule is multispecific, multispecific molecules are also intended to be encompassed the molecule can further include a third binding specificity, in by the term “bispecific molecule' as used herein. To create a addition to an anti-Fc binding specificity. In one embodiment, bispecific molecule according to at least some embodiments the third binding specificity is an anti-enhancement factor of the invention, an antibody can be functionally linked (e.g., (EF) portion, e.g., a molecule which binds to a surface protein by chemical coupling, genetic fusion, noncovalent associa involved in cytotoxic activity and thereby increases the tion or otherwise) to one or more other binding molecules, immune response against the target cell. US 2014/0294765 A1 Oct. 2, 2014 26

0343. The “anti-enhancement factor portion' can be an 0348 Fc.alpha.RI (CD89) is constitutively expressed on antibody, functional antibody fragment or a ligand that binds monocytes/macrophages, eosinophilic and neutrophilic to a given molecule, e.g., an antigen or a receptor, and thereby granulocytes, but not on non-effector cell populations. Fc results in an enhancement of the effect of the binding deter alpha R1 has medium affinity (Approximately 5x107 M-1) minants for the Fc receptor or target cell antigen. The “anti for both IgA1 and IgA2, which is increased upon exposure to enhancement factor portion' can bind an Fc receptor or a cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. target cell antigen. Alternatively, the anti-enhancement factor (1996) Critical Reviews in Immunology 16:423-440). Four portion can bind to an entity that is different from the entity to FcaRI-specific monoclonal antibodies, identified as A3, A59, which the first and second binding specificities bind. For A62 and A77, which bind Fc.alpha.RI outside the IgA ligand example, the anti-enhancement factor portion can bind a binding domain, have been described (Monteiro, R. C. et al. cytotoxic T-cell (e.g., via CD2, CD3, CD8, CD28, CD4, (1992) J. Immunol. 148:1764). CD40, ICAM-1 or other immune cell that results in an 0349 Fc. alpha. RI and Fc gamma. RI are preferred trigger increased immune response against the target cell). receptors for use in the bispecific molecules according to at 0344. According to at least some embodiments of the least some embodiments of the invention because they are (1) invention, the bispecific molecules comprise as a binding expressed primarily on immune effector cells, e.g., mono specificity at least one antibody, or an antibody fragment cytes, PMNS, macrophages and dendritic cells; (2) expressed thereof, including, e.g., an Fab, Fab', F(ab'). Sub.2, Fv, or a at high levels (e.g., 5,000-100,000 per cell); (3) mediators of single chain Fv. The antibody may also be a light chain or cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate heavy chain dimer, or any minimal fragment thereof such as enhanced antigen presentation of antigens, including self a Fv or a single chain construct as described in Ladner et al. antigens, targeted to them. U.S. Pat. No. 4,946,778, the contents of which is expressly 0350 While human monoclonal antibodies are preferred, incorporated by reference. other antibodies which can be employed in the bispecific 0345. In one embodiment, the binding specificity for an Fc molecules according to at least Some embodiments of the gamma receptor is provided by a monoclonal antibody, the invention are murine, chimeric and humanized monoclonal binding of which is not blocked by human immunoglobulin G antibodies. (IgG). As used herein, the term “IgG receptor refers to any of 0351. The bispecific molecules of the present invention the eight.gamma.-chain genes located on chromosome 1. can be prepared by conjugating the constituent binding speci These genes encode a total of twelve transmembrane or ficities, e.g., the anti-FcR and anti-LSR binding specificities, soluble receptor isoforms which are grouped into three using methods known in the art. For example, each binding Fc.gamma. receptor classes: Fc gamma R1 (CD64), Fc specificity of the bispecific molecule can be generated sepa gamma RII (CD32), and Fc gamma.RIII (CD 16). In one rately and then conjugated to one another. When the binding preferred embodiment, the Fc gamma. receptor a human high specificities are proteins or peptides, a variety of coupling or affinity Fc.gamma RI. The human Fc gammaR1 is a 72 kDa cross-linking agents can be used for covalent conjugation. molecule, which shows high affinity for monomeric IgG (10 Examples of cross-linking agents include protein A, carbodi 8-10-9 M-1). imide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'- 0346. The production and characterization of certain pre dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimale ferred anti-Fc gamma. monoclonal antibodies are described imide (oPDM), N-succinimidyl-3-(2-pyridyld-ithio) by Fangeretal. in PCT Publication WO 88/00052 and in U.S. propionate (SPDP), and sulfosuccinimidyl 4-(N- Pat. No. 4,954,617, the teachings of which are fully incorpo maleimidomethyl)cyclohaxane-1-carboxylate (sulfo rated by reference herein. These antibodies bind to an epitope SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: of Fc.gamma.R1, FcyRII or FcyRIII at a site which is distinct 1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA from the Fc.gamma. binding site of the receptor and, thus, 82:8648). Other methods include those described in Paulus their binding is not blocked substantially by physiological (1985) Behring Ins. Mitt. No. 78, 118-132: Brennan et al. levels of IgG. Specific anti-Fc.gamma. RI antibodies useful in (1985) Science 229:81-83), and Glennie et al. (1987) J. this invention are mab 22, mAb 32, mAb 44, mAb 62 and Immunol. 139: 2367-2375). Preferred conjugating agents are mAb 197. The hybridoma producing mAb 32 is available SATA and sulfo-SMCC, both available from Pierce Chemical from the American Type Culture Collection, ATCC Acces Co. (Rockford, Ill.). sion No. HB9469. In other embodiments, the anti-Fcy recep 0352. When the binding specificities are antibodies, they tor antibody is a humanized form of monoclonal antibody 22 can be conjugated via Sulfhydryl bonding of the C-terminus (H22). The production and characterization of the H22 anti hinge regions of the two heavy chains. In a particularly pre body is described in Graziano, R. F. et al. (1995) J. Immunol. ferred embodiment, the hinge region is modified to contain an 155 (10): 4996-5002 and PCT Publication WO94/10332. The odd number of sulfhydryl residues, preferably one, prior to H22 antibody producing cell line is deposited at the American conjugation. Type Culture Collection under the designation HAO22CLI 0353 Alternatively, both binding specificities can be and has the accession no. CRL 11177. encoded in the same vector and expressed and assembled in 0347 In still other preferred embodiments, the binding the same host cell. This method is particularly useful where specificity for an Fc receptor is provided by an antibody that the bispecific molecule is a mAbXm Ab, mAbXFab, FabXF binds to a human IgA receptor, e.g., an Fc-alpha receptor (Fc (ab')2 or ligandXFab fusion protein. A bispecific molecule alpha.R1 (CD89)), the binding of which is preferably not according to at least some embodiments of the invention can blocked by human immunoglobulin A (IgA). The term “IgA be a single chain molecule comprising one single chain anti receptor is intended to include the gene product of one body and a binding determinant, or a single chain bispecific alpha.-gene (Fc alpha.R1) located on chromosome 19. This molecule comprising two binding determinants. Bispecific gene is known to encode several alternatively spliced trans molecules may comprise at least two single chain molecules. membrane isoforms of 55 to 10 kDa. Methods for preparing bispecific molecules are described for US 2014/0294765 A1 Oct. 2, 2014 27 example in U.S. Pat. No. 5,260.203; U.S. Pat. No. 5,455,030; of the disease, reducing the duration of such episodes, reduc U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. ing the severity of Such episodes, slowing/reducing cancer No. 5,091,513: U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013, cell growth or proliferation, slowing progression of at least 653: U.S. Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858. one symptom, ameliorization of at least one measurable 0354 Techniques for making multispecific antibodies physical parameter and the like. include, but are not limited to, recombinant co-expression of 0358 "Mammal’ for purposes of treatment refers to any two immunoglobulin heavy chain-light chain pairs having animal classified as a mammal, including humans, domestic different specificities (see Milstein and Cuello, Nature 305: and farm animals, and Zoo, sports, or pet animals, such as 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. dogs, horses, cats, cows, etc. Preferably, the mammal is 10: 3655 (1991)), and “knob-in-hole' engineering (see, e.g., human. Preferably the mammal is a human which is diag U.S. Pat. No. 5,731,168). Multi-specific antibodies may also nosed with one of the disease, disorder or conditions be made by engineering electrostatic steering effects for mak described hereinabove, or alternatively is predisposed to at ing antibody Fc-heterodimeric molecules (WO 2009/ least one type of cancer. 089004A1); controlled Fab-arm exchange (see Labrijn et al., 0359. The term “therapeutically effective amount” refers PNAS110(13):5145-50 (2013)); cross-linking two or more to an amount of agent according to the present invention that antibodies or fragments (see, e.g., U.S. Pat. No. 4,676.980, is effective to treat a disease or disorder in a mammal. and Brennan et al., Science, 229: 81 (1985)); using leucine 0360. The therapeutic agents of the present invention can Zippers to produce bi-specific antibodies (see, e.g., Kostelny be provided to the Subject alone, or as part of a pharmaceutical et al., J. Immunol., 148(5):1547-1553 (1992)); using “dia composition where they are mixed with a pharmaceutically body’ technology for making bispecific antibody fragments acceptable carrier. (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 0361 Anti LSR antibody, a fragment, a conjugate thereof 90:6444-6448 (1993)); and using single-chain Fv (sEv) and/or a pharmaceutical composition comprising same, dimers (see, e.g. Gruber et al., J. Immunol. 152:5368 according to at least Some embodiments of the present inven (1994)); and preparing trispecific antibodies as described, tion also can be administered in combination therapy, i.e., e.g., in Tutt et al. J. Immunol. 147: 60 (1991). combined with other potentiating agents and/or other thera 0355 Binding of the bispecific molecules to their specific pies. According to at least Some embodiments, the anti LSR targets can be confirmed by, for example, enzyme-linked antibody could be used in combination with any of the known immunosorbent assay (ELISA), radioimmunoassay (RIA), in the art standart of care cancer treatment (as can be found, FACS analysis, bioassay (e.g., growth inhibition), or Western for example, in http://www.cancer.govic ancertopics). Blot assay. Each of these assays generally detects the pres 0362 For example, the combination therapy can include ence of protein-antibody complexes of particular interest by an anti LSR antibody, a fragment, a conjugate thereof and/or employing a labeled reagent (e.g., an antibody) specific for a pharmaceutical composition comprising same, combined the complex of interest. For example, the FcR-antibody com with at least one other therapeutic or immune modulatory plexes can be detected using e.g., an enzyme-linked antibody agent, other compounds or immunotherapies, or immuno orantibody fragment which recognizes and specifically binds stimulatory strategy, including, but not limited to, tumor vac to the antibody-FcR complexes. Alternatively, the complexes cines, adoptive T cell therapy, Treg depletion, antibodies (e.g. can be detected using any of a variety of other immunoassays. bevacizumab, erbituX, Ipilimumab), peptides, pepti-bodies, For example, the antibody can be radioactively labeled and Small molecules, chemotherapeutic agents such as cytotoxic used in a radioimmunoassay (RIA) (see, for example, Wein and cytostatic agents (e.g. paclitaxel, cisplatin, Vinorelbine, traub, B., Principles of Radioimmunoassays, Seventh Train docetaxel, gemcitabine, temozolomide, irinotecan, 5FU, car ing Course on Radioligand Assay Techniques, The Endocrine boplatin), immunological modifiers such as interferons and Society, March, 1986, which is incorporated by reference interleukins, immunostimulatory antibodies, growth hor herein). The radioactive isotope can be detected by such mones or other cytokines, folic acid, vitamins, minerals, aro means as the use of a gamma. counter or a Scintillation matase inhibitors, RNAi. Histone Deacetylase Inhibitors, counter or by autoradiography. proteasome inhibitors, and so forth. In another example, the 0356. Uses of Antibodies and Pharmaceutical Composi combination therapy can include an anti-LSR antibody or tions Thereof Cancer LSR modulating agent according to at least Some embodi 0357. “Treatment” refers to both therapeutic treatment ments of the present invention, such as a soluble polypeptide and prophylactic or preventative measures, which in this conjugate containing the ectodomain of the LSR antigen or a Example relates to treatment of cancer; however, also as Small molecule Such as a peptide, ribozyme, aptamer, siRNA, described below, uses of antibodies and pharmaceutical com or other drug that binds LSR, combined with at least one other positions are also provided for treatment of infectious disease therapeutic or immune modulatory agent. and/or autoimmune conditions. Those in need of treatment 0363 According to at least some embodiments of the include those already with cancer as well as those in which the present invention, therapeutic agents that can be used in com cancer is to be prevented. Hence, the mammal to be treated bination with anti-LSR antibodies, are potentiating agents herein may have been diagnosed as having the cancer or may that enhance anti-tumor responses. be predisposed or susceptible to the cancer. As used herein the 0364 Various strategies are available for combining an term “treating refers to preventing, delaying the onset of anti-LSR blocking antibody with potentiating agents for can curing, reversing, attenuating, alleviating, minimizing, Sup cer immunotherapy. According to at least Some embodiments pressing, halting the deleterious effects or stabilizing of dis of the present invention, anti-LSRantibody for cancer immu cernible symptoms of the above-described cancerous dis notherapy is used in combination with potentiating agents eases, disorders or conditions. It also includes managing the that are primarily geared to increase endogenous anti-tumor cancer as described above. By "manage' it is meant reducing responses, such as Radiotherapy, Cryotherapy, Conventional/ the severity of the disease, reducing the frequency of episodes classical chemotherapy potentiating anti-tumor immune US 2014/0294765 A1 Oct. 2, 2014 28 responses, Targeted therapy potentiating anti-tumor immune icity; certain tyrosine kinase inhibitors (TKIs) that promote responses, Anti-angiogenic therapy, Therapeutic agents tar cancer-directed immune responses by increasing MHC class geting immunosuppressive cells Such as Tregs and MDSCs, II expression, decreased levels of tumor infiltrating immuno Immunostimulatory antibodies, Cytokine therapy. Therapeu Suppressive cells—Tregs and MDScs, reducing the expres tic cancer Vaccines, Adoptive cell transfer. sion of the immunosuppressive enzyme IDO by tumor cells, 0365. The scientific rationale behind the combined use and/or inhibition of DC functions: Histone deacetylase with some chemotherapy or anti-cancer conventional drugs is (HDAC) inhibitors which were found increase the expression that cancer cell death, a consequence of the cytotoxic action of NK-activating receptor ligands on the Surface of cancer of most chemotherapeutic compounds, may result in cells, thereby facilitating tumor cell recognition by NK cells, increased levels of tumor antigen leading to enhanced antigen while proteasome inhibitors were found to sensitize tumor presentation and stimulation of anti-tumor immune responses cells to CTL-mediated or NK-mediated cell lysis. According (ie immunogenic cell death), resulting in potentiating effects to at least Some embodiments of the invention, Targeted thera with the anti LSR antibody (Zitvogel etal 2008, Galluzzi etal pies used as agents for combination with anti LSR antibodies 2012). Other combination therapies that may potentiate anti for treatment of cancer are selected from the group consisting tumor responses through tumor cell death are radiotherapy, of but not limited to: histone deacetylase (HDAC) inhibitors, Cryotherapy, Surgery, and hormone deprivation. Each of Such as Vorinostat, romidepsin, panobinostat, belinostat, these cancer therapies creates a source of tumor antigen in the mocetinostat, abexinostat, entinostat, resminostat, givinostat, host. quisinostat, sodium butyrate; Proteasome inhibitors, such as 0366 According to at least some embodiments of the bortezomib, carfilzomib, disulfuram; mTOR pathway inhibi invention, classical chemotherapies and conventional anti tors, such as temsirolimus, rapamycin, everolimus; PI3K cancer therapies as agents potentiating anti-tumor immune inhibitors, such as perifosine, CAL 101, PX-866, IPI-145, responses for combination with anti LSR antibodies are BAY 80-6946; B-raf inhibitors such as Vemurafenib, sor selected from the group consistin of but not limited to: Plati afenib; JAK2 inhibitors, such as lestaurtinib, pacritinib; num based compounds Such as oxaliplatin, cisplatin, carbo Tyrosine kinase inhibitors (TKIs), such as erlotinib, imatinib, platin; Antibiotics with anti-cancer activity, such as dactino Sunitinib, lapatinib, gefitinib, Sorafenib, nilotinib, toceranib, mycin, bleomycin, mitomycin-C, mithramycin and boSutinib, neratinib, Vatalanib, regorafenib, cabozantinib; Anthracyclines, such as doxorubicin, daunorubicin, epirubi other Protein kinase inhibitors, such as crizotinib, Inhibitors cin, idarubicin; Anthracenediones, such as mitoxantrone; of serine/threonine kinases for example Ras/Raf signalling Alkylating agents. Such as dacarbazine, melphalan, cyclo inhibitors such as farnesyltransferase inhibitors; Inhibitors of phosphamide, temozolomide, chlorambucil, buSulphan, serine proteases for example matriptase, hepsin, urokinase; nitrogen mustard, nitrosoureas; Antimetabolites, such as Inhibitors of intracellular signaling Such as tipifarnib, peri fluorouracil, raltitrexed, gemcitabine, cytosine arabinoside, fosine; Inhibitors of cell signalling through MEK and/or AKT hydroxyurea and Folate antagonists, such as methotrexate, kinases; aurora kinase inhibitors such as AZD1152, trimethoprim, pyrimethamine, pemetrexed; Antimitotic PH73.9358, VX-680, MLN8054, R763, MP235, MP529, agents such as polokinase inhibitors and Microtubule inhibi VX-528, AX39459; Cyclindependent kinase inhibitors such tors. Such as Taxanes and Taxoids, Such as paclitaxel, doc as CDK2 and/or CDK4 inhibitors: Inhibitors of survival sig etaxel; Vinca alkaloids such as Vincristine, vinblastine, Vin naling proteins including Bcl-2, Bcl-XL, such as ABT-737; desine, Vinorelbine; Topoisomerase inhibitors, such as HSP90 inhibitors: Therapeutic monoclonal antibodies, such etoposide, teniposide, amsacrine, topotecan, irinotecan, as anti-EGFR mAbs cetuximab, panitumumab, nimotu camptothecin, Cytostatic agents including Antioestrogens Zumab, anti-ERBB2 mAbs trastuzumab, pertuzumab, anti Such as tamoxifen, fulvestrant, toremifene, raloxifene, CD20 mAbs such as rituximab, ofatumumab, veltuzumab droloxifene, iodoxy fene, Antiandrogens Such as bicaluta and mAbs targeting other tumor antigens Such as alemtu mide, flutamide, nilutamide and cyproterone acetate, Zumab, labetuZumab, adecatumumab, oregovomab, onartu Progestogens Such as megestrol acetate. Aromatase inhibitors Zumab; TRAIL pathway agonists, such as dulanermin Such as anastroZole, letrozole, Vorazole, exemestane; GnRH (soluble rhTRAIL), apomab, mapatumumab, lexatumumab, analogs, such as leuprorelin, goserelin, buserelin, degarelix; conatumumab, tigatuZumab; Antibody fragments, bi-specific inhibitors of 5C.-reductase such as finasteride. antibodies and bi-specific T-cell engagers (BiTEs). Such as 0367. According to at least some embodiments of the catumaXomab, blinatumomab; Antibody drug conjugates present invention, anti-LSR antibody for cancer immuno (ADC) and other immunoconjugates, such as ibritumomab therapy is used in combination with Bisphosphonates, espe triuxetan, to situmomab, brentuximab vedotin, gemtuzumab cially amino-bisphosphonates (ABP), which have shown to oZogamicin, clivatuZumab tetraxetan, pemtumomab, trastu have anti-cancer activity. Some of the activities associated Zumab emtansine; Anti-angiogenic therapy Such as bevaci with ABPs are on human YöT cells that straddle the interface Zumab, etaracizumab, Volocliximab, ramucirumab, afliber of innate and adaptive immunity and have potent anti-tumour cept, Sorafenib, Sunitinib, regorafenib, axitinib, nintedanib, activity. Targeted therapies can also stimulate tumor-specific motesanib, paZopanib, cediranib; Metalloproteinase inhibi immune response by inducing the immunogenic death of tors such as marimastat; Inhibitors of urokinase plasminogen tumor cells or by engaging immune effector mechanisms activator receptor function; Inhibitors of cathepsin activity. (Galluzzi et al 2012, Vanneman and Dranoff 2012). In addi 0368 Other cancer immunotherapies that also increase tion, according to at least some embodiments of the present endogenous anti-tumor responses could also potentiate the invention, anti-LSR antibody for cancer immunotherapy is effect of the anti LSR antibody by enhancing immune effector used in combination with any of the following: certain thera mechanisms, such as Adoptive T cell therapy, Therapeutic peutic monoclonal antibodies, trastuzumab, that favor the cancer vaccines, reduced immune Suppressive cells and their generation of tumor-specific cytotoxic CD8 T cells, and NK function, Cytokine therapy, or Immunostimulatory antibod cells infiltration to the tumor and NK cell mediated cytotox 1CS US 2014/0294765 A1 Oct. 2, 2014 29

0369 According to at least some embodiments of the 0374. According to at least some embodiments of the present invention, anti-LSR antibody for cancer immuno present invention, anti-LSR antibody for cancer immuno therapy is used in combination with Therapeutic agents tar therapy is used in combination with Immunostimulatory anti geting regulatory immunosuppressive cells Such as regula bodies as agents potentiating anti-tumor immune responses tory T cells (Tregs) and myeloid derived suppressor cells (Pardoll 2012): (MDSCs). A number of commonly used chemotherapeutics 0375. Immunostimulatory antibodies promote anti-tumor exert non-specific targeting of Tregs and reduce the number or immunity by directly modulating immune functions, i.e. the immunosuppressive capacity of Tregs or MDSCs (Fac blocking other inhibitory targets or enhancing costimulatory ciabene etal 2012; Byrne etal 2011; Gabrilovich and Nagaraj proteins. According to at least Some embodiments of the 2009). In this regard, metronomic therapy with some chemo present invention, anti-LSR antibody for cancer immuno therapy drugs results in immunostimulatory rather than therapy is used in combination with antagonistic antibodies immunosuppressive effects, via modulation of regulatory targeting immune checkpoints including Anti-CTLA4 mAbs, cells. Thus, according to at least some embodiments of the such as ipilimumab, tremelimumab; Anti-PD-1 such as niv present invention, anti-LSR antibody for cancer immuno olumab BMS-936558/MDX-1106/ONO-4538, AMP224, therapy is used in combination with drugs selected from but CT-011, MK-3475; Anti-PDL-1 antagonists such as BMS not limited to cyclophosphamide, gemcitabine, mitox 936559/MDX-1105, MEDI4736, RG-7446/MPDL3280A: antrone, fludarabine, fludarabine, docetaxel, paclitaxel, tha Anti-LAG-3 such as IMP-321), Anti-TIM-3, Anti-BTLA, lidomide and thalidomide derivatives. Anti-B7-H4, Anti-B7-H3, Anti-VISTA: Agonistic antibodies 0370. In addition, according to at least some embodiments targeting immunostimulatory proteins, including Anti-CD40 of the present invention, anti-LSR antibody for cancer immu mAbs such as CP-870,893, lucatumumab, dacetuzumab, notherapy is used in combination with novel Treg-specific Anti-CD137 mAbs such as BMS-663513 urelumab, targeting agents including: 1) depleting or killing antibodies PF-05082566; Anti-OX40 mAbs, such as Anti-OX40; Anti that directly target Tregs through recognition of Treg cell GITR mAbs such as TRX518; Anti-CD27 mAbs, such as surface receptors such as anti-CD25 mAbs daclizumab, basil CDX-1127; Anti-ICOS mAbs. iximab or 2) ligand-directed toxins such as denileukin diftitox 0376 Cytokines are molecular messengers that allow the (Ontak)—a fusion protein of human IL-2 and diphtheria cells of the immune system to communicate with one another toxin, or LMB-2 a fusion between an schv against CD25 to generate a coordinated, robust, but self-limited response to and the pseudomonas exotoxin. 3) antibodies targeting Treg a target antigen. Cytokine-based therapies embody a direct cell surface receptors such as CTLA4, PD-1, OX40 and attempt to stimulate the patients own immune system to GITR. reject cancer. The growing interest over the past two decades 0371. According to at least some embodiments of the inharnessing the immune system to eradicate cancer has been present invention, anti-LSR antibody for cancer immuno accompanied by heightened efforts to characterize cytokines therapy is used in combination with any of the options and exploit their vast signaling networks to develop cancer described below for disrupting Treg induction and/or func treatments. Cytokines directly stimulate immune effector tion, including TLR (toll like receptors) agonists; agents that cells and stromal cells at the tumor site and enhance tumor interfere with the adenosinergic pathway, Such as ectonucle cell recognition by cytotoxic effector cells. Numerous animal otidase inhibitors, or inhibitors of the A2A adenosine recep tumor model studies have demonstrated that cytokines have tor; TGF-B inhibitors, such as fresolimumab, lerdelimumab, broad anti-tumor activity and this has been translated into a metelimumab, trabedersen, LY2157299, LY210976; block number of cytokine-based approaches for cancer therapy ade of Tregs recruitment to tumor tissues including chemok (Lee and Margolin 2011). A number of cytokines are in pre ine receptor inhibitors, such as the CCR4/CCL2/CCL22 clinical or clinical development as as agents potentiating anti pathway. tumor immune responses for cancer immunotherapy, includ ing among others: IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and 0372 According to at least some embodiments of the IL-21, IL23, IL-27, GM-CSF, IFNo. (interferon alpha), IFNB, present invention, anti-LSR antibody for cancer immuno IFNy. therapy is used in combination with any of the options 0377 Several cytokines have been approved for therapy of described below for inhibiting the immunosuppressive tumor cancer and many more are under development. However, microenvironment, including inhibitors of cytokines and therapeutic efficacy is often hampered by severe side effects enzymes which exert immunosuppressive activities, such as and poor pharmacokinetic properties. Thus, in addition to IDO (indoleamine-2,3-dioxygenase) inhibitors; inhibitors of systemic administration of cytokines, a variety of strategies anti-inflammatory cytokines which promote an immunosup can be employed for the delivery of therapeutic cytokines and pressive microenvironment, such as IL-10, IL-35, IL-4 and their localization to the tumor site, in order to improve their IL-13; Bevacizumab which reduces Tregs and favors the dif pharmacokinetics, as well as their efficacy and/or toxicity, ferentiation of DCs. including antibody-cytokine fusion molecules (immunocy 0373 According to at least some embodiments of the tokines), chemical conjugation to polyethylene glycol (PE present invention, anti-LSR antibody for cancer immuno Gylation), transgenic expression of cytokines in autologous therapy is used in combination with any of the options whole tumor cells, incorporation of cytokine genes into DNA described below for targeting MDSCs, including promoting vaccines, recombinant viral vectors to deliver cytokine genes, their differentiation into mature myeloid cells that do not have etc. In the case of immunocytokines, fusion of cytokines to suppressive functions By Vitamin D3, or VitaminA metabo tumor-specific antibodies or antibody fragments allows for lites, such as retinoic acid, all-trans retinoic acid (ATRA); targeted delivery and therefore improved efficacy and phar inhibition of MDSCs suppressive activity by COX2 inhibi macokinetics, and reduced side effects. tors, phosphodiesterase 5 inhibitors like sildenafil. ROS 0378. According to at least some embodiments of the inhibitors such as nitroaspirin. present invention, anti-LSR antibody for cancer immuno US 2014/0294765 A1 Oct. 2, 2014 30 therapy is used in combination with Cytokine therapy, involv immune response to the cancer cells. Dendritic cells can also ing the use of cytokines as agents potentiating anti-tumor be primed in vivo by injection of patients with irradiated immune responses, including cytokines such as IL-2, IL-7, whole tumor cells engineered to secrete stimulating cytokines IL-12, IL-15, IL-17, IL-18 and IL-21, IL23, IL-27, GM-CSF, (such as GM-CSF). Similar approaches can be carried out IFNC (interferon alpha), IFNC-2b, IFNB, IFNY, and their with monocytes. Sipuleucel-T (Provenge), a therapeutic can different strategies for delivery, as described above. cer vaccine which has been approved for treatment of 0379 Cancer vaccines are used to treat existing cancer advanced prostate cancer, is an example of a dendritic cell (therapeutic) or prevent the development of cancer in certain vaccine. high-risk individuals (prophylactic). Therapeutic cancer vac 0386 Thus, according to at least some embodiments of the cines allow for improved priming of T cells and improved present invention, anti-LSR antibody for cancer immuno antigen presentation, and can be used as therapeutic agents therapy is used in combination with Therapeutic cancer vac for potentiating anti-tumor immune responses (Mellman etal cines. Non limiting examples of such therapeutic cancer vac 2011; Schlom 2012). cines include Whole tumor cell vaccines, Tumor antigen 0380. Several types of therapeutic cancer vaccines are in vaccines, Vector-based vaccines. Oncolytic virus Vaccines, preclinical and clinical development. These include for Dendritic-cell vaccines, as described above. example: 0387. One approach to cancer immunotherapy is based on 0381 1) Whole tumor cell vaccines, in which cancer cells adoptive T cell therapy or adoptive cell transfer (ACT), which removed during Surgery are treated to enhance their immu involves the ex vivo identification and expansion of autolo nogenicity, and injected into the patient to induce immune gous naturally occurring tumor specific T cells, which are responses againstantigens in the tumor cells. The tumor cell then adoptively transferred back into the cancer patient (Res vaccine can be autologous, i.e. a patients own tumor, or tifo etal 2012). Cells that are infused back into a patient after allogeneic which typically contain two or three established ex vivo expansion can traffic to the tumor and mediate its and characterized human tumor cell lines of a given tumor destruction. Prior to this adoptive transfer, hosts can be immu type, such as the GVAX vaccine platforms. nodepleted by irradiation and/or chemotherapy. The combi 0382 2) Tumorantigenvaccines, in which a tumorantigen nation of lymphodepletion, adoptive cell transfer, and a T cell (or a combination of a few tumor antigens), usually proteins growth factor (such as IL-2), can lead to prolonged tumor or peptides, are administered to boost the immune system eradication in tumor patients. A more novel approach (possibly with an adjuvant and/or with immune modulators or involves the ex vivo genetic modification of normal periph attractants of dendritic cells such as GM-CSF). The tumor eral blood T cells to confer specificity for tumor-associated antigens may be specific for a certain type of cancer, but they antigens. For example, clones of TCRs of T cells with par are not made for a specific patient. ticularly good anti-tumor responses can be inserted into viral 0383 3) Vector-based tumor antigen vaccines and DNA expression vectors and used to infect autologous T cells from vaccines can be used as a way to provide a steady Supply of the patient to be treated. Another option is the use of chimeric antigens to stimulate an anti-tumor immune response. Vectors antigen receptors (CARS) which are essentially a chimeric encoding for tumor antigens are injected into the patient immunoglobulin-TCR molecule, also known as a T-body. (possibly with proinflammatory or other attractants such as CARs have antibody-like specificities and recognize MHC GM-CSF), taken up by cells in vivo to make the specific nonrestricted structures on the Surface of target cells (the antigens, which would then provoke the desired immune extracellular target-binding module), grafted onto the TCR response. Vectors may be used to deliver more than one tumor intracellular domains capable of activating T cells (Restifo et antigen at a time, to increase the immune response. In addi all 2012, Shietal 2013). tion, recombinant virus, bacteria or yeast vectors should trig 0388 According to at least some embodiments of the ger their own immune responses, which may also enhance the present invention, anti-LSR antibody for cancer immuno overall immune response. therapy is used in combination with Adoptive cell transfer to 0384 4) Oncolytic virus vaccines, such as OncoVex/T- potentiate anti-tumor immune responses, including geneti VEC, which involves the intratumoral injection of replica cally modified T cells, as described above. tion-conditional herpes simplex virus which preferentially 0389. The LSR specific antibodies, and/or alternative infects cancer cells. The virus, which is also engineered to scaffolds and/or multispecific and bispecific molecules and express GM-CSF, is able to replicate inside a cancer cell immunoconjugates, compositions comprising same accord causing its lysis, releasing new viruses and an array of tumor ing to at least some embodiments of the present invention can antigens, and secreting GM-CSF in the process. Thus, Such be co-administered together with one or more other therapeu oncolytic virus vaccines enhance DCS function in the tumor tic agents, which acts in conjunction with or synergistically microenvironment to stimulate anti-tumor immune with the composition according to at least Some embodiments responses. of the present invention to treat or prevent the cancer. The 0385 5) Dendritic cell vaccines (Palucka and Banchereau LSR related therapeutic agents and the one or more other 2012): Dendritic cells (DCs) phagocytose tumor cells and therapeutic agents can be administered in either order or present tumor antigens to tumor specific T cells. In this simultaneously. The other therapeutic agents are for example, approach, DCs are isolated from the cancer patient and a cytotoxic agent, a radiotoxic agent or an immunosuppres primed for presenting tumor-specific T cells. To this end sive agent. The composition can be linked to the agent (as an several methods can be used: DCs are loaded with tumor cells immunocomplex) or can be administered separately from the or lysates; DCs are loaded with fusion proteins or peptides of agent. In the latter case (separate administration), the com tumor antigens; coupling of tumor antigens to DC-targeting position can be administered before, after or concurrently mAbs. The DCs are treated in the presence of a stimulating with the agent or can be co-administered with other known factor (such as GM-CSF), activated and matured ex vivo, and therapies, e.g., an anti-cancer therapy, e.g., radiation. Such then re-infused back into the patient in order provoke an therapeutic agents include, among others, anti-neoplastic US 2014/0294765 A1 Oct. 2, 2014 agents such as doxorubicin (adriamycin), cisplatin bleomycin IgM which bind complement, can also be used in the presence Sulfate, carmustine, chlorambucil, and cyclophosphamide of complement. In one embodiment, ex vivo treatment of a hydroxyurea which, by themselves, are only effective at lev population of cells comprising target cells with a binding els which are toxic or Subtoxic to a patient. Cisplatin is intra agent according to at least some embodiments of the present venously administered as a 100 mg/dose once every four invention and appropriate effector cells can be supplemented weeks and adriamycin is intravenously administered as a by the addition of complement or serum containing comple 60-75 mg/ml dose once every 21 days. Co-administration of ment. Phagocytosis of target cells coated with a binding agent the human anti-LSRantibodies, orantigenbinding fragments according to at least Some embodiments of the present inven and/or alternative scaffolds thereof, according to at least some tion can be improved by binding of complement proteins. In embodiments of the present invention with chemotherapeutic another embodiment target cells coated with the composi agents provides two anti-cancer agents which operate via tions (e.g., human antibodies, multispecific and bispecific different mechanisms which yield a cytotoxic effect to human molecules) according to at least Some embodiments of the tumor cells. Such co-administration can solve problems due present invention can also be lysed by complement. In yet to development of resistance to drugs or a change in the another embodiment, the compositions according to at least antigenicity of the tumor cells which would render them Some embodiments of the present invention do not activate unreactive with the antibody. In other embodiments, the sub complement. ject can be additionally treated with an agent that modulates, 0394 The therapeutic compositions (e.g., human antibod e.g., enhances or inhibits, the expression or activity of Fcy or ies, alternative scaffolds multispecific and bispecific mol Fcy receptors by, for example, treating the Subject with a ecules and immunoconjugates) according to at least some cytokine. Preferred cytokines for administration during treat embodiments of the present invention can also be adminis ment with the multispecific molecule include of granulocyte tered together with complement. Thus, according to at least colony-stimulating factor (G-CSF), granulocyte-macrophage Some embodiments of the present invention there are compo colony-stimulating factor (GM-CSF), interferon-gamma sitions, comprising human antibodies, multispecific or bispe (IFN-gamma), and tumor necrosis factor (TNF). cific molecules and serum or complement. These composi 0390 Target-specific effector cells, e.g., effector cells tions are advantageous in that the complement is located in linked to compositions (e.g., human antibodies, multispecific close proximity to the human antibodies, multispecific or and bispecific molecules) according to at least some embodi bispecific molecules. Alternatively, the human antibodies, ments of the present invention can also be used as therapeutic multispecific or bispecific molecules according to at least agents. Effector cells for targeting can be human leukocytes some embodiments of the present invention and the comple Such as macrophages, neutrophils or monocytes. Other cells ment or serum can be administered separately. include eosinophils, natural killer cells and other IgG- or 0395. A “therapeutically effective dosage” of an anti-LSR IgA-receptor bearing cells. If desired, effector cells can be antibody according to at least some embodiments of the obtained from the subject to be treated. The target-specific present invention preferably results in a decrease in severity effector cells can be administered as a suspension of cells in of disease symptoms, an increase in frequency and duration a physiologically acceptable solution. The number of cells of disease symptom-free periods, an increase in lifepan, dis administered can be in the order of 10-8 to 10-9 but will vary ease remission, or a prevention or reduction of impairment or depending on the therapeutic purpose. In general, the amount disability due to the disease affliction. For example, for the will be sufficient to obtain localization at the target cell, e.g., treatment of LSR positive tumors, a “therapeutically effective a tumor cell expressing LSR proteins, and to effect cell killing dosage' preferably inhibits cell growth or tumor growth by at by, e.g., phagocytosis. Routes of administration can also vary. least about 20%, more preferably by at least about 40%, even 0391 Therapy with target-specific effector cells can be more preferably by at least about 60%, and still more prefer performed in conjunction with other techniques for removal ably by at least about 80% relative to untreated subjects. The of targeted cells. For example, anti-tumor therapy using the ability of a compound to inhibit tumor growth can be evalu compositions (e.g., human antibodies, multispecific and ated in an animal model system predictive of efficacy in bispecific molecules) according to at least some embodi human tumors. Alternatively, this property of a composition ments of the present invention and/or effector cells armed can be evaluated by examining the ability of the compound to with these compositions can be used in conjunction with inhibit, such inhibition in vitro by assays known to the skilled chemotherapy. Additionally, combination immunotherapy practitioner. Atherapeutically effective amount ofatherapeu may be used to direct two distinct cytotoxic effector popula tic compound can decrease tumor size, or otherwise amelio tions toward tumor cell rejection. For example, anti-LSR rate symptoms in a subject. antibodies linked to anti-Fc-gamma R1 or anti-CD3 may be 0396. One of ordinary skill in the art would be able to used in conjunction with IgG- or IgA-receptor specific bind determine a therapeutically effective amount based on such ing agents. factors as the subjects size, the severity of the subjects 0392 Bispecific and multispecific molecules according to symptoms, and the particular composition or route of admin at least Some embodiments of the present invention can also istration selected. be used to modulate FcgammaR or FcgammaR levels on 0397. The anti-LSR antibodies, according to at least some effector cells, such as by capping and elimination of receptors embodiments of the present invention, can be used as neu on the cell surface. Mixtures of anti-Fc receptors can also be tralizing antibodies. A Neutralizing antibody (Nabs), is an used for this purpose. antibody that is capable of binding and neutralizing or inhib 0393. The therapeutic compositions (e.g., human antibod iting a specific antigen thereby inhibiting its biological effect, ies, alternative scaffolds multispecific and bispecific mol for example by blocking the receptors on the cell or the virus, ecules and immunoconjugates) according to at least some inhibiting the binding of the virus to the host cell. NAbs will embodiments of the present invention which have comple partially or completely abrogate the biological action of an ment binding sites, such as portions from IgG1, -2, or -3 or agent by either blocking an important Surface molecule US 2014/0294765 A1 Oct. 2, 2014 32 needed for its activity or by interfering with the binding of the 936.558. The scientific rationale behind the combined use of agent to its receptor on a target cell. LSR blockade and chemotherapy is that cell death, that is a 0398 As used herein “therapeutic agent” is any one of the consequence of the cytotoxic action of most chemotherapeu monoclonal and/or polyclonal antibodies, and/or antigen tic compounds, should result in increased levels of tumor binding fragments, and/or conjugates containing same, and/ antigen in the antigen presentation pathway. Other combina or alternative scaffolds, thereof comprising an antigen bind tion therapies that may result in synergy with LSR blockade ing site that binds specifically to any one of the LSR polypep through cell death are radiotherapy, cryotherapy, Surgery, and tides or an epitope thereof, adopted for treatment of cancer, as hormone deprivation. Other additional combination therapies recited herein. with additional immunomodulatory molecules will Synergis 0399. According to an additional aspect of the present tically contribute to the stimulation of the immune system to invention the therapeutic agents can be used to prevent patho eradicate the cancer. Each of these protocols creates a source logic inhibition of T cell activity, Such as that directed against of tumorantigen in the host. Angiogenesis inhibitors may also cancer cells. be combined with LSR blockade. Inhibition of angiogenesis 0400. According to an additional aspect of the present leads to tumor cell death which may feed tumor antigen into invention the therapeutic agents can be used to inhibit T cell host antigen presentation pathways. activation, as can be manifested for example by T cell prolif 04.05 LSR blocking antibodies can also be used in com eration and cytokine secretion. bination with bispecific antibodies that target Fc alpha or Fc 04.01 Thus, according to an additional aspect of the Y receptor-expressing effectors cells to tumor cells (see, e.g., present invention there is provided a method of treating can U.S. Pat. Nos. 5,922,845 and 5,837.243). Bispecificantibod cer as recited herein, and/or for promoting immune stimula ies can be used to target two separate antigens. For example tion mediated by the LSR polypeptide in a subject by admin anti-Fc receptor/anti tumor antigen (e.g., Her-2/neu) bispe istering to a Subject in need thereofan effective amount of any cific antibodies have been used to target macrophages to sites one of the therapeutic agents and/or a pharmaceutical com of tumor. This targeting may more effectively activate tumor position comprising any of the therapeutic agents and further specific responses. The T cell arm of these responses would comprising a pharmaceutically acceptable diluent or carrier. by augmented by the use of LSR blockade. Alternatively, 0402 Atherapeutic agent or pharmaceutical composition antigen may be delivered directly to DCs by the use of bispe according to at least some embodiments of the present inven cific antibodies which bind to tumor antigen and a dendritic tion may also be administered in conjunction with other com cell specific cell Surface marker. pounds or immunotherapies. For example, the combination therapy can include a compound of the present invention 0406 Tumors evade host immune surveillance by a large combined with at least one other therapeutic or immune variety of mechanisms. Many of these mechanisms may be modulatory agent, or immunostimulatory strategy, including, overcome by the inactivation of proteins which are expressed but not limited to, tumor vaccines, adoptive T cell therapy, by the tumors and which are immunosuppressive. These Treg depletion, antibodies (e.g. bevacizumab, erbituX), pep include among others TGF-beta (Kehrl, J. et al. (1986) J. Exp. tides, pepti-bodies, Small molecules, chemotherapeutic Med. 163: 1037-1050), IL-10 (Howard, M. & O'Garra, A. agents such as cytotoxic and cytostatic agents (e.g. paclitaxel, (1992) Immunology Today 13: 198-200), and Fas ligand cisplatin, vinorelbine, docetaxel, gemcitabine, temozolo (Hahne, M. et al. (1996) Science 274: 1363-1365). Antibod mide, irinotecan, 5FU, carboplatin), immunological modifi ies to each of these entities may be used in combination with ers such as interferons and interleukins, immunostimulatory anti-LSR to counteract the effects of the immunosuppressive antibodies, growth hormones or other cytokines, folic acid, agent and favor tumor immune responses by the host. vitamins, minerals, aromatase inhibitors, RNAi, Histone 0407. Other antibodies which may be used to activate host Deacetylase Inhibitors, proteasome inhibitors, and so forth. immune responsiveness can be used in combination with 0403. According to at least some embodiments, immune anti-LSR. These include molecules on the surface of dendritic cells, preferably T cells, can be contacted in vivo or ex vivo cells which activate DC function and antigen presentation. with the therapeutic agents to modulate immune responses. Anti-CD40 antibodies are able to substitute effectively for T The T cells contacted with the therapeutic agents can be any cell helper activity (Ridge, J. et al. (1998) Nature 393: 474 cell which expresses the T cell receptor, including C/Bandy/8 478) and can be used in conjunction with LSR antibodies (Ito, T cell receptors. T-cells include all cells which express CD3. N. et al. (2000) Immunobiology 201 (5) 527–40). Activating including T-cell subsets which also express CD4 and CDS. antibodies to T cell costimulatory molecules such as OX-40 T-cells include both naive and memory cells and effector cells (Weinberg, A. etal. (2000) Immunol 164: 2160-2169), 4-1BB such as CTL. T-cells also include cells such as Th1, Tcl, Th2, (Melero, I. et al. (1997) Nature Medicine3: 682-685 (1997), Tc2. Th3, Th17, Th22, Treg, and Tr1 cells. T-cells also and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) as include NKT-cells and similar unique classes of the T-cell well as antibodies which block the activity of negative lineage. costimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 0404 LSR blockade may also be combined with standard 5,811,097, implimumab) or BTLA (Watanabe, N. et al. cancer treatments. LSR blockade may be effectively com (2003) Nat Immunol 4:670-9), B7-H4 (Sica, G. Letal. (2003) bined with chemotherapeutic regimes. In these instances, it Immunity 18:849-61) PD-1 (may also provide for increased may be possible to reduce the dose of chemotherapeutic levels of T cell activation. Bone marrow transplantation is reagent administered. An example of such a combination is an currently being used to treat a variety of tumors of hemato anti-LSR antibody in combination with Temsirolimus for the poietic origin. While graft versus host disease is a conse treatment of late stage renal cell cancer. Another example of quence of this treatment, therapeutic benefit may be obtained Such a combination is an anti-LSR antibody in combination from graft vs. tumor responses. LSR blockade can be used to with interleukin-2 (IL-2) for the treatment of late stage renal increase the effectiveness of the donor engrafted tumor spe cell cancer.as well as combination with Ipilimumab or BMS cific T cells. US 2014/0294765 A1 Oct. 2, 2014

0408. There are also several experimental treatment pro create fusion proteins between two unrelated sequences (i.e. tocols that involve ex vivo activation and expansion of antigen bcr-abl in the Philadelphia chromosome), or idiotype from B specific T cells and adoptive transfer of these cells into recipi cell tumors. ents in order to antigen-specific T cells against tumor (Green 0413. Other tumor vaccines may include the proteins from berg, R. & Riddell, S. (1999) Science 285: 546-51). These viruses implicated in human cancers such a Human Papil methods may also be used to activate T cell responses to lomaViruses (HPV), Hepatitis Viruses (HBV and HCV) and infectious agents such as CMV. Ex vivo activation in the Kaposi's Herpes Sarcoma Virus (KHSV). Another form of presence of anti-LSRantibodies may be expected to increase tumor specificantigen which may be used in conjunction with the frequency and activity of the adoptively transferred T LSR blockade is purified heat shock proteins (HSP) isolated cells. from the tumor tissue itself. These heat shock proteins contain 04.09 Optionally, antibodies to LSR can be combined with fragments of proteins from the tumor cells and these HSPs are an immunogenic agent, such as cancerous cells, purified highly efficient at delivery to antigen presenting cells for tumor antigens (including recombinant proteins, peptides, eliciting tumor immunity (Suot, R & Srivastava, P (1995) and carbohydrate molecules), cells, and cells transfected with Science 269:1585-1588; Tamura, Y. et al. (1997) Science genes encoding immune stimulating cytokines (He et al 278: 117-120). (2004) J. Immunol. 173:4919-28). Non-limiting examples of 0414 Dendritic cells (DC) are potent antigen presenting tumor vaccines that can be used include peptides of MUC1 cells that can be used to prime antigen-specific responses. for treatment of colon cancer, peptides of MUC-1/CEA/TRI DC's can be produced ex vivo and loaded with various protein COM for the treatment of ovary cance, or tumor cells trans and peptide antigens as well as tumor cell extracts (Nestle, F. fected to express the cytokine GM-CSF (discussed further et al. (1998) Nature Medicine 4:328-332). DCs may also be below). transduced by genetic means to express these tumor antigens 0410. In humans, some tumors have been shown to be as well. DCs have also been fused directly to tumor cells for immunogenic Such as RCC. It is anticipated that by raising the purposes of immunization (Kugler, A. etal. (2000) Nature the threshold of T cell activation by LSR blockade, we may Medicine 6:332-336). As a method of vaccination, DC immu expect to activate tumor responses in the host. nization may be effectively combined with LSR blockade to 0411 LSR blockade is likely to be most effective when activate more potent anti-tumor responses. combined with a vaccination protocol. Many experimental strategies for vaccination against tumors have been devised Use of the Therapeutic Agents According to at Least Some (see Rosenberg, S., 2000, Development of Cancer Vaccines, Embodiments of the Invention as Adjuvant for Cancer ASCO Educational Book Spring: 60-62; Logothetis, C. Vaccination: 2000, ASCO Educational Book Spring: 300-302: Khayat, D. 0415 Immunization against tumor-associated antigens 2000, ASCO Educational Book Spring: 414-428: Foon, K. (TAAS) is a promising approach for cancer therapy and pre 2000, ASCO Educational Book Spring: 730-738; see also vention, but it faces several challenges and limitations, such Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. as tolerance mechanisms associated with self-antigens 3023-3043 in DeVita, V. etal. (eds.), 1997, Cancer: Principles expressed by the tumor cells. Costimulatory molecules Such and Practice of Oncology. Fifth Edition). In one of these as B7.1 (CD80) and B7.2 (CD86) have improved the efficacy strategies, a vaccine is prepared using autologous or alloge of gene-based and cell-based vaccines in animal models and neic tumor cells. These cellular vaccines have been shown to are under investigation as adjuvant in clinical trials. This be most effective when the tumor cells are transduced to adjuvant activity can be achieved either by enhancing the express GM-CSF. GM-CSF has been shown to be a potent costimulatory signal or by blocking inhibitory signal that is activator of antigen presentation for tumor vaccination (Dra transmitted by negative costimulators expressed by tumor noffetal. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43). cells (Neighbors et al., 2008J Immunother; 31(7):644-55). 0412. The study of gene expression and large scale gene 0416. According to at least some embodiments of the expression patterns in various tumors has led to the definition invention, any one of polyclonal or monoclonal antibody of so-called tumor specific antigens (Rosenberg, SA (1999) and/or antigen binding fragments and/or conjugates contain Immunity 10: 281-7). In many cases, these tumor specific ing same, and/or alternative scaffolds, specific to any one of antigens are differentiation antigens expressed in the tumors LSR proteins, can be used as adjuvant for cancer vaccination. and in the cell from which the tumor arose, for example According to at least Some embodiments, the invention pro melanocyte antigens gp100, MAGE antigens, and Trp-2. vides methods for improving immunization against TAAS, More importantly, many of these antigens can be shown to be comprising administering to a patient an effective amount of the targets of tumor specific T cells found in the host. LSR any one of polyclonal or monoclonal antibody and/or antigen blockade may be used in conjunction with a collection of binding fragments and/or conjugates containing same, and/or recombinant proteins and/or peptides expressed in a tumor in alternative scaffolds, specific to any one of LSR proteins. order to generate an immune response to these proteins. These proteins are normally viewed by the immune system as Use of the Therapeutic Agents According to at Least Some self antigens and are therefore tolerant to them. The tumor Embodiments of the Invention for Immunoenhancement antigen may also include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and 1. Treatment of Cancer which is expressed in more than 85% of human cancers and in only a limited number of somatic tissues (Kim, Netal. (1994) 0417. The therapeutic agents provided herein are gener Science 266: 2011-2013). (These somatic tissues may be ally useful in vivo and ex vivo as immune response-stimulat protected from immune attack by various means). Tumor ing therapeutics. In general, the disclosed therapeutic agent antigen may also be "neo-antigens' expressed in cancer cells compositions are useful for treating a Subject having or being because of Somatic mutations that alter protein sequence or predisposed to any disease or disorder to which the subjects US 2014/0294765 A1 Oct. 2, 2014 34 immune system mounts an immune response. The ability of or a derivative thereof; e.g. 40-O-(2-hydroxy)ethyl-rapamy therapeutic agents to modulate LSR immune signals enable a cin, a lymphocyte homing agent, e.g. FTY720 or an analog more robust immune response to be possible. The therapeutic thereof, corticosteroids; cyclophosphamide; azathioprene; agents according to at least some embodiments of the inven methotrexate; leflunomide or an analog thereof mizoribine; tion are useful to stimulate or enhance immune responses mycophenolic acid, mycophenolate mofetil: 15-deoxysper involving immune cells, such as T cells. gualine or an analog thereof; immunosuppressive mono 0418. The therapeutic agents according to at least some clonal antibodies, e.g., monoclonal antibodies to leukocyte embodiments of the invention are useful for stimulating or receptors, e.g., MHC, CD2, CD3, CD4, CD11a/CD18, CD7, enhancing an immune response in host for treating cancer by CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, administering to a subject an amount of a therapeutic agent CD150 (SLAM), OX40, 4-1BB or their ligands; or other effective to stimulate T cells in the subject. immunomodulatory compounds, e.g. CTLA4-Ig (abatacept, ORENCIAR, belatacept), CD28-Ig, B7-H4-Ig, or other 2. Use of the Therapeutic Agents in Vaccines costimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or low molecular weight inhibitors including LFA-1 0419. The therapeutic agents according to at least some antagonists, Selectin antagonists and VLA-4 antagonists, or embodiments of the invention, are administered alone or in another immunomodulatory agent. combination with any other suitable treatment. In one embodiment the therapeutic agents can be administered in 0425 Thus, treatment of multiple sclerosis using the conjunction with, or as a component of a vaccine composition agents according to at least Some embodiments of the present as described above. The therapeutic agents according to at invention may be combined with, for example, any known least some embodiments of the invention can be administered therapeutic agent or method for treating multiple Sclerosis. prior to, concurrently with, or after the administration of a Non-limiting examples of Such known therapeutic agent or vaccine. In one embodiment the therapeutic agents is admin method for treating multiple sclerosis include interferon istered at the same time as administration of a vaccine. class, IFN-beta-1a (REBIFR). AVONEX(R) and CIN NOVEX(R) and IFN-beta-1b (BETASERONR), EXTAVIAR), Use of Antibodies and Pharmaceutical Compositions for BETAFERONR), ZIFERONR); glatirameracetate (COPAX Treatment of Autoimmune Disease ONE(R), a polypeptide; natalizumab (TYSABRIR); and mitoxantrone (NOVANTRONER), a cytotoxic agent, Fam 0420 According to at least some embodiments, antibodies pridine (AMPYRAR). Other drugs include corticosteroids, and pharmaceutical compositions as described herein may methotrexate, cyclophosphamide, azathioprine, and intrave optionally be used for treating an immune system related nous immunoglobulin (IVIG), inosine, Ocrelizumab disease. (R1594), Mylinax (Caldribine), alemtuzumab (Campath), 0421 Optionally, the immune system related condition daclizumab (Zenapax), Panaclar/dimethyl fumarate (BG comprises an immune related condition, autoimmune dis 12), Teriflunomide (HMR1726), fingolimod (FTY720), eases as recited herein, transplant rejection and graft versus laquinimod (ABR216062), as well as Haematopoietic stem host disease and/or for blocking or promoting immune cell transplantation, Neurovax, Rituximab (Rituxan) BCG costimulation mediated by LSR, immune related diseases as vaccine, low dose naltrexone, helminthic therapy, angio recited herein and/or for immunotherapy (promoting or plasty, Venous stents, and alternative therapy, such as Vitamin inhibiting immune costimulation). D. polyunsaturated fats, medical marijuana. 0422 Optionally the immune condition is selected from 0426. Thus, treatment of rheumatoid arthritis, using the autoimmune disease, transplant rejection, or graft versus host agents according to at least Some embodiments of the present disease. invention may be combined with, for example, any known 0423 Optionally the treatment is combined with another therapeutic agent or method for treating rheumatoid arthritis. moiety useful for treating immune related condition. Non-limiting examples of Such known therapeutic agents or 0424 Optionally the moiety is selected from the group methods for treating rheumatoid arthritis include glucocorti consisting of immunosuppressants such as corticosteroids, coids, nonsteroidal anti-inflammatory drug (NSAID) such as cyclosporin, cyclophosphamide, prednisone, azathioprine, salicylates, or cyclooxygenase-2 inhibitors, ibuprofen and methotrexate, rapamycin, tacrolimus, leflunomide oranana naproxen, diclofenac, indomethacin, etodolac Disease-modi log thereof, mizoribine; mycophenolic acid, mycophenolate fying antirheumatic drugs (DMARDs)-Oral DMARDs: mofetil: 15-deoxyspergualine orananalog thereof biological Auranofin (Ridaura), Azathioprine (Imuran), Cyclosporine agents such as TNF-alpha blockers or antagonists, or any (Sandimmune, Gengraf. Neoral, generic), D-Penicillamine other biological agent targeting any inflammatory cytokine, (Cuprimine), Hydroxychloroquine (Plaquenil), IMgold Gold nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, Sodium thiomalate (Myochrysine) Aurothioglucose (Solga hydroxychloroquine, SulphaSalazopryine, gold salts, etaner nal), Leflunomide (Arava), Methotrexate (Rheumatrex), cept, infliximab, mycophenolate mofetil, basiliximab, ataci Minocycline (Minocin), Staphylococcal protein A immu cept, rituximab, cytoxan, interferon beta-1a, interferon beta noadsorption (Prosorba column), Sulfasalazine (AZulfidine). 1b, glatirameracetate, mitoxantrone hydrochloride, anakinra Biologic DMARDS: TNF-C. blockers including Adalimumab and/or other biologics and/or intravenous immunoglobulin (Humira), Etanercept (Enbrel), Infliximab (Remicade), goli (IVIG), interferons such as IFN-beta-1a (REBIFR). mumab (Simponi), certolizumab pegol (Cimzia), and other AVONEX(R) and CINNOVEX(R) and IFN-beta-1b (BETASE Biological DMARDs, such as Anakinra (Kineret), Rituximab RONR); EXTAVIAR), BETAFERONR), ZIFERONR); glati (Rituxan), Tocilizumab (Actemra), CD28 inhibitor including ramer acetate (COPAXONE(R), a polypeptide; natalizumab Abatacept (Orencia) and Belatacept. (TYSABRIR), mitoxantrone (NOVANTRONE(R), a cyto 0427 Thus, treatment of IBD, using the agents according toxic agent, a calcineurin inhibitor, e.g. cyclosporin A or to at least some embodiments of the present invention may be FK506; an immunosuppressive macrollide, e.g. rapamycine combined with, for example, any known therapeutic agent or US 2014/0294765 A1 Oct. 2, 2014 method for treating IBD. Non-limiting examples of such neret(R), Abatacept (OrenciaR), Diamyd, alefacept known therapeutic agents or methods for treating IBD include (Ameviv(R), Otelixizumab, DiaPep277 (Hsp60 derived pep immunosuppression to control the symptom, Such as pred tide), Alpha 1-Antitrypsin, Prednisone, azathioprine, nisone, Mesalazine (including Asacol, Pentasa, Lialda, Ciclosporin, E1-INT (an injectable islet neogenesis therapy Aspiro),azathioprine (Imuran), methotrexate, or 6-mercap comprising an epidermal growth factor analog and a gastrin topurine, steroids, Ondansetron, TNF-C. blockers (including analog), statins including Zocor R, Simlup(R), Simcard(R), infliximab, adalimumab golimumab, certolizumab pegol), Simvacor R, Sitagliptin (dipeptidyl peptidase (DPP-4) inhibi Orencia (abatacept), ustekinumab (Stelara(R), Briakinumab tor), Anti-CD3 mAb (e.g., Teplizumab); CTLA4-Ig (abata (ABT-874), Certolizumab pegol (CimziaR), ITF2357 (givi cept), Anti IL-1Beta (Canakinumab), Anti-CD20 mAb (e.g. nostat), Natalizumab (Tysabri), Firategrast (SB-683699), rituximab). Remicade (infliximab), vedolizumab (MLNO002), other 0430 Thus, treatment of uveitis, using the agents accord drugs including GSK1605786 CCX282-B (Traficet-EN), ing to at least some embodiments of the present invention may AJM300, Stelara (ustekinumab), Semapimod (CNI-1493) be combined with, for example, any known therapeutic agent tasocitinib (CP-690550), LMW Heparin MMX, Budesonide or method for treating uveitis. Non-limiting examples of such MMX, Simponi (golimumab), MultiStemR, Gardasil HPV known therapeutics for treating uveitis include corticoster vaccine, Epaxal Berna (virosomal hepatitis. A vaccine), Sur oids, topical cycloplegics, such as atropine or homatropine, or gery, such as bowel resection, strictureplasty or a temporary injection of PSTTA (posterior subtenon triamcinolone or permanent colostomy or ileostomy; antifungal drugs such acetate), antimetabolite medications, such as methotrexate, as nyStatin (a broad spectrum gut antifungal) and eitheritra TNF-C. blockers (including infliximab, adalimumab, etaner conazole (Sporanox) or fluconazole (Diflucan); alternative cept, golimumab, certolizumab pegol). medicine, prebiotics and probiotics, cannabis, Helminthic therapy or ova of the Trichuris suis helminth. 0431. Thus, treatment for Sjogren's syndrome, using the 0428 Thus, treatment of psoriasis, using the agents agents according to at least Some embodiments of the present according to at least some embodiments of the present inven invention may be combined with, for example, any known tion may be combined with, for example, any known thera therapeutic agent or method for treating for Sjogren's Syn peutic agent or method for treating psoriasis. Non-limiting drome. Non-limiting examples of Such known therapeutics examples of Such known therapeutics for treating psoriasis for treating for Sjogren's syndrome include Cyclosporine, include topical agents, typically used for mild disease, pho pilocarpine (Salagen) and cevimeline (EVOxac), Hydroxy totherapy for moderate disease, and systemic agents for chloroquine (Plaquenil), cortisone (prednisone and others) severe disease. Non-limiting examples of topical agents: bath and/or azathioprine (Imuran) or cyclophosphamide (Cy Solutions and moisturizers, mineral oil, and petroleum jelly; toxan). Dexamethasone, Thalidomide, Dehydroepiandroster ointment and creams containing coal tar, dithranol (anthra one, NGX267, Rebamipide, FID 1 14657, Etanercept, Raptiv lin), corticosteroids like desoximetasone (Topicort), a, Belimumab, MabThera (rituximab); Anakinra, intravenous Betamethasone, fluocinonide, vitamin D3 analogues (for immune globulin (IVIG), Allogeneic Mesenchymal Stem example, calcipotriol), and retinoids. Non-limiting examples Cells (AlloMSC), Automatic neuro-electrostimulation by of phototherapy: sunlight; wavelengths of 311-313 nm, pso “Salliwell Crown. ralen and ultraviolet A phototherapy (PUVA). Non-limiting 0432. Thus, treatment for systemic lupus erythematosus, examples of systemic agents: Biologics, such as interleukin using the agents according to at least some embodiments of antagonists, TNF-C. blockers including antibodies such as the present invention may be combined with, for example, infliximab (Remicade), adalimumab (Humira), golimumab, any known therapeutic agent or method for treating for sys certolizumab pegol, and recombinant TNF-C. decoy receptor, temic lupus erythematosus. Non-limiting examples of Such etanercept (Enbrel); drugs that target T cells, such as efali known therapeutics for treating for systemic lupus erythema Zumab (Xannelim/Raptiva), alefacept (Ameviv), dendritic tosus include corticosteroids and Disease-modifying anti cells such Efalizumab; monoclonal antibodies (MAbs) tar rheumatic drugs (DMARDs), commonly anti-malarial drugs geting cytokines, including anti-IL-12/IL-23 (ustekinumab Such as plaquenil and immunosuppressants (e.g. methotrex (brand name Stelara)) and anti-Interleukin-17: Briakinumab ate and azathioprine) Hydroxychloroquine, cytotoxic drugs (ABT-874); small molecules, including but not limited to (e.g., cyclophosphamide and mycophenolate), Hydroxychlo ISA247: Immunosuppressants, such as methotrexate, roquine (HCO), Benlysta (belimumab), nonsteroidal anti cyclosporine; vitamin A and retinoids (synthetic forms of inflammatory drugs, Prednisone, Cellcept, Prograf, Ataci Vitamin A); and alternative therapy, such as changes in diet cept, Lupuzor, Intravenous Immunoglobulins (IVIGs), and lifestyle, fasting periods, low energy diets and vegetarian CellCept (mycophenolate mofetil), Orencia, CTLA4-IgG4m diets, diets supplemented with fish oil rich in Vitamin A and (RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO Vitamin D (such as cod liver oil), Fish oils rich in the two 136, Sifalimumab (MEDI-545), A-623 (formerly AMG 623), omega-3 fatty acids eicosapentaenoic acid (EPA) and docosa AMG 557, Rontalizumab, paguinimod (ABR-215757), hexaenoic acid (DHA) and contain Vitamin E. Ichthyo LY2127399, CEP-33457, Dehydroepiandrosterone, therapy, Hypnotherapy, cannabis. Levothyroxine, abetimus sodium (LJP 394), Memantine, 0429 Thus, treatment of type 1 diabetes, using the agents Opiates, Rapamycin, Renal transplantation, stem cell trans according to at least some embodiments of the present inven plantation. tion may be combined with, for example, any known thera 0433. The therapeutic agents and/or a pharmaceutical peutic agent or method for treating type laiabetes. Non-lim composition comprising same, as recited herein, according to iting examples of Such known therapeutics for treating type 1 at least Some embodiments of the invention, may be admin diabetes include insulin, insulin analogs, islet transplantation, istered as the sole active ingredient or together with other stem cell therapy including PROCHYMALR, non-insulin drugs in immunomodulating regimens or other anti-inflam therapies such as il-1beta inhibitors including Anakinra (Ki matory agents e.g. for the treatment or prevention of alto- or US 2014/0294765 A1 Oct. 2, 2014 36

Xenograft acute or chronic rejection or inflammatory or any of multiple Sclerosis, rheumatoid arthritis, type I diabetes, autoimmune disorders, or to induce tolerance. psoriasis, systemic lupus erythematosus, inflammatory bowel 0434. The term “autoimmune disease' as used herein disease, uveitis, or Sjogren's syndrome. should be understood to encompass any autoimmune disease 0436. As used herein, "multiple sclerosis' comprises one and chronic inflammatory conditions. According to at least or more of multiple Sclerosis, benign multiple Sclerosis, Some embodiments of the invention, the autoimmune dis relapsing remitting multiple Sclerosis, secondary progressive eases should be understood to encompass any disease disor multiple Sclerosis, primary progressive multiple Sclerosis, der or condition selected from the group including but not progressive relapsing multiple Sclerosis, chronic progressive limited to multiple Sclerosis, including relapsing-remiting multiple Sclerosis, transitional/progressive multiple Sclerosis, multiple Sclerosis, primary progressive multiple Sclerosis, rapidly worsening multiple Sclerosis, clinically-definite mul and secondary progressive multiple Sclerosis; psoriasis; rheu tiple Sclerosis, malignant multiple Sclerosis, also known as matoid arthritis; psoriatic arthritis, systemic lupus erythema Marburg's Variant, and acute multiple sclerosis. Optionally, tosus (SLE); ulcerative colitis; Crohn's disease; benign lym “conditions relating to multiple Sclerosis' include, e.g., phocytic angiitis, thrombocytopenic purpura, idiopathic Devic's disease, also known as Neuromyelitis Optica; acute thrombocytopenia, idiopathic autoimmune hemolytic ane disseminated encephalomyelitis, acute demyelinating optic mia, pure red cell aplasia, Sjogren's syndrome, rheumatic neuritis, demyelinative transverse myelitis, Miller-Fisher disease, connective tissue disease, inflammatory rheumatism, syndrome, encephalomyelradiculoneuropathy, acute demy degenerative rheumatism, extra-articular rheumatism, juve elinative polyneuropathy, tumefactive multiple Sclerosis and nile rheumatoid arthritis, arthritis uratica, muscular rheuma Balo's concentric Sclerosis. tism, chronic polyarthritis, cryoglobulinemic vasculitis, 0437. As used herein, "rheumatoid arthritis’ comprises ANCA-associated vasculitis, antiphospholipid syndrome, one or more of rheumatoid arthritis, gout and pseudo-gout, myasthenia gravis, autoimmune haemolytic anaemia, Guil juvenile idiopathic arthritis, juvenile rheumatoid arthritis, lian-Barre Syndrome, chronic immune polyneuropathy, Still's disease, ankylosing spondylitis, rheumatoid vasculitis. autoimmune thyroiditis, insulin dependent diabetes mellitus, Optionally, conditions relating to rheumatoid arthritis type I diabetes. Addison's disease, membranous glomerulo include, e.g., osteoarthritis, sarcoidosis, Henoch-Schönlein nephropathy, Goodpasture's disease, autoimmune gastritis, purpura, Psoriatic arthritis, Reactive arthritis, Spondyloarthr autoimmune atrophic gastritis, pernicious anaemia, pemphi opathy, septic arthritis, Haemochromatosis, Hepatitis, vascu gus, pemphigus Vulgarus, cirrhosis, primary biliary cirrhosis, litis, Wegener's granulomatosis, Lyme disease, Familial dermatomyositis, polymyositis, fibromyositis, myogelosis, Mediterranean fever, Hyperimmunoglobulinemia D with celiac disease, immunoglobulin A nephropathy, Henoch recurrent fever, TNF receptor associated periodic syndrome, Schonlein purpura, Evans syndrome, atopic dermatitis, pso and Enteropathic arthritis associated with inflammatory riasis, psoriasis arthropathica, Graves disease, Graves oph bowel disease. thalmopathy, Scleroderma, systemic scleroderma, 0438. As used herein, “Uveitis' comprises one or more of progressive systemic scleroderma, asthma, allergy, primary uveitis, anterior uveitis (or iridocyclitis), intermediate uveitis biliary cirrhosis, Hashimoto's thyroiditis, primary myxe (pars planitis), posterior uveitis (or chorioretinitis) and the dema, sympathetic ophthalmia, autoimmune uveitis, hepati panuveitic form. tis, chronic action hepatitis, collagen diseases, ankylosing 0439. As used herein, “inflammatory bowel disease” com spondylitis, periarthritis humeroScapularis, panarteritis prises one or more of inflammatory bowel disease Crohn's nodosa, chondrocalcinosis, Wegener's granulomatosis, disease, ulcerative colitis (UC), Collagenous colitis, Lym microscopic polyangiitis, chronic urticaria, bullous skin dis phocytic colitis, Ischaemic colitis, Diversion colitis, Behgets orders, pemphigoid, atopic eczema, Devic's disease, child disease, Indeterminate colitis. hood autoimmune hemolytic anemia, Refractory or chronic 0440. As used herein, psoriasis' comprises one or more Autoimmune Cytopenias, Prevention of development of of psoriasis, Nonpustular Psoriasis including Psoriasis Vul Autoimmune Anti-Factor VIII Antibodies in Acquired Hemo garis and Psoriatic erythroderma (erythrodermic psoriasis), philia A, Cold Agglutinin Disease, Neuromyelitis Optica, Pustular psoriasis including Generalized pustular psoriasis Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis, (pustular psoriasis of Von Zumbusch), Pustulosis palmaris et myocarditis, vasculitis, gastritis, gout, gouty arthritis, and plantaris (persistent palmoplantarpustulosis, pustular psoria inflammatory skin disorders, selected from the group consist sis of the Barber type, pustular psoriasis of the extremities), ing of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, Annular pustular psoriasis, Acrodermatitis continua, Impe and acne, normocomplementemic urticarial vasculitis, peri tigo herpetiformis. Optionally, conditions relating to psoria carditis, myositis, anti-synthetase syndrome, Scleritis, mac sis include, e.g., drug-induced psoriasis, Inverse psoriasis, rophage activation syndrome, Bechet's Syndrome, PAPA Napkin psoriasis, Seborrheic-like psoriasis, Guttate psoria Syndrome, Blau's Syndrome, gout, adult and juvenile Stills sis, Nail psoriasis, Psoriatic arthritis. disease, cryropyrinopathy, Muckle-Wells syndrome, familial 0441. As used herein, “type 1 diabetes’ comprises one or cold-induced auto-inflammatory syndrome, neonatal onset more of type 1 diabetes, insulin-dependent diabetes mellitus, multisystemic inflammatory disease, familial Mediterranean idiopathic diabetes, juvenile type laiabetes, maturity onset fever, chronic infantile neurologic, cutaneous and articular diabetes of the young, latent autoimmune diabetes in adults, syndrome, systemic juvenile idiopathic arthritis, Hyper Ig) gestational diabetes. Conditions relating to type 1 diabetes syndrome, Schnitzler's syndrome, autoimmune retinopathy, include, neuropathy including polyneuropathy, mononeur age-related macular degeneration, atherosclerosis, chronic opathy, peripheral neuropathy and autonomicneuropathy; prostatitis and TNF receptor-associated periodic syndrome eye complications: glaucoma, cataracts, retinopathy. (TRAPS). 0442. As used herein, "Sjogren's syndrome' comprises 0435. Optionally and preferably, the autoimmune disease one or more of Sjogren's syndrome, Primary Sjogren's Syn includes but is not limited to any of the types and subtypes of drome and Secondary Sjogren's syndrome, as well as condi US 2014/0294765 A1 Oct. 2, 2014 37 tions relating to Sjogren's syndrome including connective Use of Antibodies and Pharmaceutical Compositions for tissue disease, Such as rheumatoid arthritis, systemic lupus Treatment of Infectious Disease erythematosus, or Scleroderma. Other complications include 0446. According to at least some embodiments, antibodies pneumonia, pulmonary fibrosis, interstitial nephritis, inflam and pharmaceutical compositions as described herein may mation of the tissue around the kidney's filters, glomerulone optionally be used for treating infectious disease. phritis, renal tubular acidosis, carpal tunnel syndrome, 0447 Chronic infections are often characterized by vary peripheral neuropathy, cranial neuropathy, primary biliary ing degrees of functional impairment of virus-specific T-cell cirrhosis (PBC), cirrhosis, Inflammation in the esophagus, responses, and this defect is a principal reason for the inability stomach, pancreas, and liver (including hepatitis), Polymyo of the host to eliminate the persisting pathogen. Although sitis, Raynaud's phenomenon, Vasculitis, Autoimmune thy functional effector T cells are initially generated during the roid problems, lymphoma. early stages of infection, they gradually lose function during the course of the chronic infection as a result of persistant 0443) As used herein, “systemic lupus erythematosus', exposure to foreign antigen, giving rise to T cell exhaustion. comprises one or more of systemic lupus erythematosus, Exhausted T cells express high levels of multiple co-inhibi discoid lupus, lupus arthritis, lupus pneumonitis, lupus tory receptors such as CTLA-4, PD-1, and LAG3 (Crawford nephritis. Conditions relating to systemic lupus erythemato et al., Curr Opin Immunol. 2009; 21:179-186: Kaufmann et SuS include osteoarticular tuberculosis, antiphospholipid al., J. Immunol 2009; 182:5891-5897, Sharpe et al., Nat antibody syndrome, inflammation of various parts of the Immunol 2007; 8:239-245). PD-1 overexpression by heart, Such as pericarditis, myocarditis, and endocarditis, exhausted T cells was observed clinically in patients suffering Lung and pleura inflammation, pleuritis, pleural effusion, from chronic viral infections including HIV, HCV and HBV chronic diffuse interstitial lung disease, pulmonary hyperten (Crawford et al., Curr Opin Immunol 2009; 21:179-186: Sion, pulmonary emboli, pulmonary hemorrhage, and shrink Kaufmann et al., JImmunol 2009; 182:5891-5897, Sharpe et ing lung syndrome, lupus headache, Guillain-Barré syn al., Nat Immunol 2007; 8:239-245). There has been some drome, aseptic meningitis, demyelinating syndrome, investigation into this pathway in additional pathogens, mononeuropathy, mononeuritis multiplex, myasthenia including other viruses, bacteria, and parasites (Hofineyer et gravis, myelopathy, cranial neuropathy, polyneuropathy, vas al., J Biomed Biotechnol. Vol 2011, Art. ID 451694, Bhadraet culitis. al., Proc Natl Acad. Sci. 2011; 108(22): 9196-201). For example, the PD-1 pathway was shown to be involved in 0444 The term “immune related disease (or disorder or controlling bacterial infection using a sepsis model induced condition) as used herein should be understood to encom by the standard cecal ligation and puncture method. The pass any disease disorder or condition selected from the group absence of PD-1 in knockout mice protected from sepsis including but not limited to autoimmune diseases, inflamma induced death in this model (Huang et al., PNAS 2009: 106: tory disorders and immune disorders associated with graft 6303-6308). transplantation rejection, Such as acute and chronic rejection 0448 T cell exhaustion can be reversed by blocking co of organ transplantation, allogenic stem cell transplantation, inhibitory pathways such as PD-1 or CTLA-4 (Rivas et al., J. autologous stem cell transplantation, bone marrow tranplan Immunol. 2009; 183:4284-91; Golden-Mason et al., J. Virol. tation, and graft versus host disease. 2009; 83.9122-30; Hofineyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694), thus allowing restoration of anti viral 0445. As used herein the term “inflammatory disorders' immune function. The therapeutic potential of co-inhibition and/or “inflammation', used interchangeably, includes blockade for treating viral infection was extensively studied inflammatory abnormalities characterized by disregulated by blocking the PD-1/PD-L1 pathway, which was shown to immune response to harmful stimuli. Such as pathogens, dam be efficacious in several animal models of infection including aged cells, or irritants. Inflammatory disorders underlie a vast acute and chronic simian immunodeficiency virus (SIV) variety of human diseases. Non-immune diseases with etio infection in rhesus macaques (Valu et al., Nature 2009; 458: logical origins in inflammatory processes include cancer, ath 206-210) and in mouse models of chronic viral infection, erosclerosis, and ischaemic heart disease. Examples of disor such as lymphocytic choriomeningitis virus (LCMV) (Barber ders associated with inflammation include: Chronic et al., Nature. 2006: 439:682-7), and Theiler's murine prostatitis, Glomerulonephritis, Hypersensitivities, Pelvic encephalomyelitis virus (TMEV) model in SJL/J mice (Dun inflammatory disease, Reperfusion injury, Sarcoidosis, Vas can and Miller PLoS One. 2011; 6:e18548). In these models culitis, Interstitial cystitis, normocomplementemic urticarial PD-1/PD-L1 blockade improved antiviral responses and pro vasculitis, pericarditis, myositis, anti-synthetase syndrome, moted clearance of the persisting viruses. In addition, PD-1/ Scleritis, macrophage activation syndrome, Bechet's Syn PD-L1 blockade increased the humoral immunity manifested drome, PAPA Syndrome, Blau's Syndrome, gout, adult and as elevated production of specific anti-virus antibodies in the juvenile Still's disease, cryropyrinopathy, Muckle-Wells syn plasma, which in combination with the improved cellular drome, familial cold-induced auto-inflammatory syndrome, responses leads to decrease in plasma viral loads and neonatal onset multisystemic inflammatory disease, familial increased Survival. Mediterranean fever, chronic infantile neurologic, cutaneous 0449. As used herein the term “infectious disorder and/or and articular syndrome, systemic juvenile idiopathic arthritis, disease' and/or “infection', used interchangeably, includes Hyper Ig|D syndrome, Schnitzler's syndrome, TNF receptor any disorder, disease and/or condition caused by presence associated periodic syndrome (TRAPSP), gingivitis, peri and/or growth of pathogenic biological agent in an individual odontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vas host organism. As used herein the term “infection' comprises culitis, gastritis, gout, gouty arthritis, and inflammatory skin the disorder, disease and/or condition as above, exhibiting disorders, selected from the group consisting of psoriasis, clinically evident illness (i.e., characteristic medical signs atopic dermatitis, eczema, rosacea, urticaria, and acne. and/or symptoms of disease) and/or which is asymtomatic for US 2014/0294765 A1 Oct. 2, 2014

much or all of it course. As used herein the term “infection' rhea, Meningitis in infants, Traveller's diarrhea, Hemor also comprises disorder, disease and/or condition caused by rhagic colitis, Hemolytic-uremic syndrome, Tularemia, Pep persistence of foreign antigen that lead to exhaustion T cell tic ulcer, Gastric and Duodenal ulcers, Legionnaire's phenotype characterized by impaired functionality which is Disease, Pontiac fever, Leptospirosis, Listeriosis, Leprosy manifested as reduced proliferation and cytokine production. (Hansen's disease), Tuberculosis, Gomorrhea, Ophthalmia As used herein the term “infectious disorder and/or disease' neonatorum, Septic arthritis, Meningococcal disease includ and/or “infection, further includes any of the below listed ing meningitis, Waterhouse-Friderichsen syndrome, infectious disorders, diseases and/or conditions, caused by a Pseudomonas infection, Rocky mountain spotted fever, bacterial infection, viral infection, fungal infection and/or Typhoid fever type salmonellosis, Salmonellosis with gastro parasite infection. As used herein the term “viral infection comprises any infection caused by a virus, optionally includ enteritis and enterocolitis, Bacillary dysentery/Shigellosis, ing but not limited to Retroviridae (e.g., human immunode Coagulase-positive Staphylococcal infections: Localized skin ficiency viruses, such as HIV-1 or HIV-2, acquired immune infections including Diffuse skin infection (Impetigo), Deep deficiency (AIDS) also referred to as HTLV-III, LAV or localized infections, Acute infective endocarditis, Septice HTLV-III/LAV, or HIV-III; and other isolates, such as HIV mia, Necrotizing pneumonia, Toxinoses such as Toxic shock LP, Picornaviridae (e.g., polio viruses, hepatitis. A virus; syndrome and Staphylococcal food poisoning, Cystitis, enteroviruses, human coxsackie viruses, rhinoviruses, echo Endometritis, Otitis media, Streptococcal pharyngitis, Scar viruses); Calciviridae (e.g., strains that cause gastroenteritis); let fever, Rheumatic fever, Puerperal fever, Necrotizing fas Togaviridae (e.g., equine encephalitis viruses, rubella ciitis, Cholera, Plague (including Bubonic plague and Pneu viruses); Flaviridae (e.g., dengue viruses, encephalitis monic plague), as well as any infection caused by a bacteria viruses, yellow fever viruses); Coronaviridae (e.g., coronavi selected from but not limited to Helicobacterpyloris, Boreliai ruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, burgdorferi, Legionella pneumophilia, Mycobacteria sps rabies viruses); Filoviridae (e.g., ebola viruses); Paramyx (e.g., M. tuberculosis, M. avium, M. Intracellulare, M. kan oviridae (e.g., parainfluenza viruses, mumps virus, measles Saii, M. gordonae), Staphylococcus aureus, Neisseria gonor virus, respiratory syncytial virus); Orthomyxoviridae (e.g., rhoeae, Neisseria meningitidis, Listeria monocytogenes, influenza viruses); Bungaviridae (e.g., Hantaan viruses, Streptococcus pyogenes (Group A Streptococcus), Strepto bunga viruses, phleboviruses and Nairo viruses); Arena viri coccus agalactiae (Group B Streptococcus), Streptococcus dae (hemorrhagic fever virus); Reoviridae (e.g., reoviruses, (viridans group), Streptococcus faecalis, Streptococcus orbiviruses and rotaviruses); Birnaviridae: Hepadnaviridae bovis, Streptococcus (anaerobic sps.). Streptococcus pneu (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviri moniae, pathogenic Campylobacter sp., Enterococcus sp., dae (papilloma viruses, polyoma viruses); Adenoviridae Haemophilus influenzae, Bacillus antracis, corynebacterium (most adenoviruses); Herperviridae (herpes simplex virus diphtheriae, corynebacterium sp., Erysipelothrix rhusio (HSV) 1 and 2, varicella Zoster virus, cytomegalovirus pathiae, Clostridium perfingers, Clostridium tetani, Entero (CMV), herpesviruses); Poxyiridae (variola virsues, vaccinia bacter erogenes, Klebsiella pneuomiae, Pasturella multi viruses, pox viruses); and Iridoviridae (e.g., African Swine coda, Bacteroides sp., Fusobacterium nucleatum, fever virus); and unclassified viruses (e.g., the etiological Sreptobacillus moniliformis, Treponema pallidium, Tre agents of Spongiform encephalopathies, the agent of delta ponema pertenue, Leptospira, and Actinomeyces israeli. hepatitides (thought to be a defective satellite of hepatitis B 0453 Nonlimiting examples of infectious disorder and/or virus), the agents of non-A, non-B hepatitis (class 1-inter disease caused by virus is selected from the group consisting nally transmitted; class 2-parenterally transmitted (i.e., Hepa of but not limited to acquired immune deficiency (AIDS), titis C); Norwalk and related viruses, and astroviruses) as well West Nile encephalitis, coronavirus infection, rhinovirus as Severe acute respiratory syndrome virus and respiratory infection, influenza, dengue, hemorrhagic fever, an otologi syncytial virus (RSV). cal infection; severe acute respiratory syndrome (SARS), 0450. As used herein the term “fungal infection' com acute febrile pharyngitis, pharyngoconjunctival fever, epi prises any infection caused by a fungi, optionally including demic keratoconjunctivitis, infantile gastroenteritis, infec but not limited to Cryptococcus neoformans, Histoplasma tious mononucleosis, Burkitt lymphoma, acute hepatitis, capsulatum, Coccidioides immitis, Blastomyces dermatitidis, chronic hepatitis, hepatic cirrhosis, hepatocellular carci Chlamydia trachomatis, Candida albicans. noma, primary HSV-1 infection, (gingivostomatitis in chil 0451. As used herein the term “parasite infection' com dren, tonsillitis & pharyngitis in adults, keratoconjunctivitis), prises any infection caused by a parasite, optionally including latent HSV-1 infection (herpes labialis, cold sores), aseptic but not limited to protozoa, such as Amebae, Flagellates, meningitis, Cytomegalovirus infection, Cytomegalic inclu Plasmodium falciparum, Toxoplasma gondii, Ciliates, Coc sion disease, Kaposi sarcoma, Castleman disease, primary cidia, Microsporidia, Sporozoa, helminthes, Nematodes effusion lymphoma, influenza, measles, encephalitis, postin (Roundworms), Cestodes (Tapeworms), Trematodes fectious encephalomyelitis, Mumps, hyperplastic epithelial (Flukes), Arthropods, and aberrant proteins known as prions. lesions (common, flat, plantarandanogenital warts, laryngeal 0452. An infectious disorder and/or disease caused by papillomas, epidermodysplasia Verruciformis), croup, pneu bacteria may optionally comprise one or more of Sepsis, monia, bronchiolitis, Poliomyelitis, Rabies, bronchiolitis, septic shock, sinusitis, skin infections, pneumonia, bronchi pneumonia, German measles, congenital rubella, Hemor tis, meningitis, Bacterial vaginosis, Urinary tract infection rhagic Fever, Chickenpox, Dengue, Ebola infection, Echovi (UCI), Bacterial gastroenteritis, Impetigo and erysipelas, rus infection, EBV infection, Fifth Disease, Filovirus, Fla Erysipelas, Cellulitis, anthrax, whooping cough, lyme dis vivirus, Hand, foot & mouth disease, Herpes Zoster Virus ease, Brucellosis, enteritis, acute enteritis, Tetanus, diphthe (Shingles), Human Papilloma Virus Associated Epidermal ria, Pseudomembranous colitis, Gas gangrene, Acute food Lesions, Lassa Fever, Lymphocytic choriomeningitis, Parain poisoning, Anaerobic cellulitis, Nosoconial infections, Diar fluenza Virus Infection, Paramyxovirus, Parvovirus B19 US 2014/0294765 A1 Oct. 2, 2014 39

Infection, Picornavirus, Poxviruses infection, Rotavirus diar transcriptase inhibitor including Purine analogue guanine rhea, Rubella, Rubeola, Varicella, Variola infection. (Acicloviril/Valacyclovir, Ganciclovir/Valganciclovir, Penci 0454. An infectious disorder and/or disease caused by clovir/Famciclovir) and adenine (Vidarabine), Pyrimidine fungi optionally includes but is not limited to Allergic bron analogue, uridine (Idoxuridine, Trifluridine, Edoxudine), chopulmonary aspergillosis, Aspergilloma, Aspergillosis, thymine Basidiobolomycosis, Blastomycosis, Candidiasis, Chronic 0459 (Brivudine), cytosine (Cytarabine); Foscarnet; pulmonary aspergillosis, Chytridiomycosis, Coccidioidomy Nucleoside analogues/NARTIs: Entecavir, Lamivudine, Tel cosis, Conidiobolomycosis, Covered Smut (barley), Crypto bivudine, Clevudine; Nucleotide analogues/NtRTIs: Ade coccosis, Dermatophyte, Dermatophytid, Dermatophytosis, fovir, Tenofovir, Nucleic acid inhibitors such as Cidofovir, Endothrix, Entomopathogenic fungus, Epizootic lymphangi Interferon Interferon alfa-2b, Peginterferon alfa-2a: Ribavi tis, Epizootic ulcerative syndrome, Esophageal candidiasis, rinii/Taribavirin; antiretroviral drugs including zidovudine, Exothrix, Fungemia, Histoplasmosis, Lobomycosis, Massos lamivudine, abacavir, lopinavir, ritonavir, tenofoviriemtricit pora cicadina, Mycosis, Mycosphaerella fragariae, Myringo abine, efavirenz, each of them alone or a various combina mycosis, Paracoccidioidomycosis, Pathogenic fungi, Penicil tions, gp41 (Enfluvirtide), Raltegravir, protease inhibitors liosis, Thousand cankers disease, Tinea, Zeaspora, Such as Fosamprenavir, Lopinavir and Atazanavir, Methisa Zygomycosis.Non limiting examples of infectious disorder Zone, Docosanol, Fomivirsen, Tromantadine. and/or disease caused by parasites is selected from the group 0460. The therapeutic agents and/or a pharmaceutical consisting of but not limited to Acanthamoeba, Amoebiasis, composition comprising same, as recited herein, can be Ascariasis, Ancylostomiasis, Anisakiasis, Babesiosis, Balan administered in combination with one or more additional tidiasis, Baylisascariasis, Blastocystosis, Candiru, Chagas therapeutic agents used for treatment of fungal infections, disease, Clonorchiasis, Cochliomyia, Coccidia, Chinese including, but not limited to, antifungal drugs of the Polyene Liver Fluke Cryptosporidiosis, Dientamoebiasis, Diphyllo antifungals, Imidazole, triazole, and thiazole antifungals, bothriasis, Dioctophyme renalis infection, Dracunculiasis, Allylamines, Echinocandins or other anti fungal drugs. Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Pharmaceutical Compositions Hymenolepiasis, HalZoun Syndrome, Isosporiasis, Katayama fever, Leishmaniasis, lymphatic filariasis, Malaria, 0461. In another aspect, the present invention provides a Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pri composition, e.g., a pharmaceutical composition, containing mary amoebic meningoencephalitis, Parasitic pneumonia, one or a combination of the therapeutic agent, according to at Paragonimiasis, Scabies, Schistosomiasis, Sleeping sickness, least some embodimants of the invention. Strongyloidiasis, Sparganosis, Rhinosporidiosis, River 0462. Thus, the present invention features a pharmaceuti blindness, Taeniasis (cause of Cysticercosis), Toxocariasis, cal composition comprising a therapeutically effective Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis, amount of a therapeutic agent according to at least some Trypanosomiasis, Tapeworm infection. embodiments of the present invention. 0455. A preferred example of infectious disease is a dis 0463 The pharmaceutical composition according to at ease caused by any of hepatitis B, hepatitis C, infectious least some embodiments of the present invention is further mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV preferably used for the treatment of cancer, as recited herein. 2, tuberculosis, malaria and Schistosomiasis. 0464 "Treatment” refers to both therapeutic treatment 0456. According to at least some embodiments of the and prophylactic or preventative measures. Those in need of present invention, there is provided use of a combination of treatment include those already with the disorder as well as thetherapeutic agents and/or a pharmaceutical composition those in which the disorder is to be prevented. Hence, the comprising same, as recited herein, and a known therapeutic mammal to be treated herein may have been diagnosed as agent effective for treating infection. having the disorder or may be predisposed or Susceptible to 0457. The therapeutic agents and/or a pharmaceutical the disorder. “Mammal’ for purposes of treatment refers to composition comprising same, as recited herein, can be any animal classified as a mammal, including humans, administered in combination with one or more additional domestic and farm animals, and Zoo, sports, or pet animals, therapeutic agents used for treatment of bacterial infections, Such as dogs, horses, cats, cows, etc. Preferably, the mammal including, but not limited to, antibiotics including Aminogly is human. cosides, Carbapenems, Cephalosporins, Macrollides, Lin 0465. The term “therapeutically effective amount” refers cosamides, Nitrofurans, penicillins, Polypeptides, Quinolo to an amount of agent according to the present invention that nes, Sulfonamides, Tetracyclines, drugs against is effective to treat a disease or disorder in a mammal. mycobacteria including but not limited to Clofazimine, 0466. The therapeutic agents of the present invention can Cycloserine, Cycloserine, Rifabutin, Rifapentine, Strepto be provided to the Subject alone, or as part of a pharmaceutical mycin and other antibacterial drugs such as Chlorampheni composition where they are mixed with a pharmaceutically col, Fosfomycin, Metronidazole, Mupirocin, and Timidazole. acceptable carrier. 0458. The therapeutic agents and/or a pharmaceutical 0467. A composition is said to be a “pharmaceutically composition comprising same, as recited herein, can be acceptable carrier if its administration can be tolerated by a administered in combination with one or more additional receipient patient. As used herein, “pharmaceutically accept therapeutic agents used for treatment of viral infections, able carrier includes any and all solvents, dispersion media, including, but not limited to, antiviral drugs such as oselta coatings, antibacterial and antifungal agents, isotonic and mivir (brand name Tamiflu) and Zanamivir (brand name absorption delaying agents, and the like that are physiologi Relenza) Arbidol—adamantane derivatives (Amantadine, cally compatible. Preferably, the carrier is suitable for intra Rimantadine)—neuraminidase inhibitors (Oseltamivir, Lani venous, intramuscular, Subcutaneous, parenteral, spinal or namivir, Peramivir, Zanamivir) nucleotide analog reverse epidermal administration (e.g., by injection or infusion). US 2014/0294765 A1 Oct. 2, 2014 40

0468. Such compositions include sterile water, buffered compositions. In addition, prolonged absorption of the inject saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic able pharmaceutical form may be brought about by the inclu strength and optionally additives Such as detergents and Sol sion of agents which delay absorption Such as aluminum ubulizing agents (e.g., Polysorbate 20, Polysorbate 80), anti monostearate and gelatin. oxidants (e.g. ascorbic acid, Sodium metabisulfite), preserva 0472 Pharmaceutically acceptable carriers include sterile tives (e.g. Thimersol, benzyl alcohol) and blulking aqueous Solutions or dispersions and sterile powders for the Substances (e.g., lactose, manitol). Non-aqueoes solvents or extemporaneous preparation of sterile injectable solutions or vehicles may also be used as detailed below. dispersion. The use of such media and agents for pharmaceu 0469 Examples of Suitable aqueous and nonaqueous car tically active Substances is known in the art. Except insofar as riers that may be employed in the pharmaceutical composi any conventional media or agent is incompatible with the tions according to at least some embodiments of the invention active compound, use thereof in the pharmaceutical compo include water, ethanol, polyols (such as glycerol, propylene sitions according to at least Some embodiments of the inven glycol, polyethylene glycol, and the like), and Suitable mix tion is contemplated. Supplementary active compounds can tures thereof, vegetable oils, such as olive oil, and injectable also be incorporated into the compositions. organic esters, such as ethyl oleate. Proper fluidity can be 0473. Therapeutic compositions typically must be sterile maintained, for example, by the use of coating materials, such and stable under the conditions of manufacture and storage. as lecithin, by the maintenance of the required particle size in The composition can beformulated as a solution, microemul the case of dispersions, and by the use of surfactants. Depend Sion, liposome, or other ordered structure Suitable to high ing on the route of administration, the active compound, i.e., drug concentration. The carrier can be a solvent or dispersion monoclonal or polyclonal antibodies and antigen binding medium containing, for example, water, ethanol, polyol (for fragments and conjugates containing same, and/or alternative example, glycerol, propylene glycol, and liquid polyethylene scaffolds, that specifically bind any one of LSR proteins, or glycol, and the like), and suitable mixtures thereof. The bispecific molecule, may be coated in a material to protect the proper fluidity can be maintained, for example, by the use of compound from the action of acids and other natural condi a coating Such as lecithin, by the maintenance of the required tions that may inactivate the compound. The pharmaceutical particle size in the case of dispersion and by the use of Sur compounds according to at least Some embodiments of the factants. In many cases, it will be preferable to include iso invention may include one or more pharmaceutically accept tonic agents, for example, Sugars, polyalcohols such as man able salts. A “pharmaceutically acceptable salt” refers to a salt nitol, Sorbitol, or Sodium chloride in the composition. that retains the desired biological activity of the parent com Prolonged absorption of the injectable compositions can be pound and does not impart any undesired toxicological brought about by including in the composition an agent that effects (see e.g., Berge, S.M., et al. (1977).J. Pharm. Sci. 66: delays absorption, for example, monostearate salts and gela 1-19). Examples of such salts include acid addition salts and tin. Sterile injectable solutions can be prepared by incorpo base addition salts. Acid addition salts include those derived rating the active compound in the required amount in an from nontoxic inorganic acids, such as hydrochloric, nitric, appropriate solvent with one or a combination of ingredients phosphoric, Sulfuric, hydrobromic, hydroiodic, phosphorous enumerated above, as required, followed by sterilization and the like, as well as from nontoxic organic acids such as microfiltration. Generally, dispersions are prepared by incor aliphatic mono- and dicarboxylic acids, phenyl-substituted porating the active compound into a sterile vehicle that con alkanoic acids, hydroxy alkanoic acids, aromatic acids, ali tains a basic dispersion medium and the required other ingre phatic and aromatic Sulfonic acids and the like. Base addition dients from those enumerated above. In the case of sterile salts include those derived from alkaline earth metals, such as powders for the preparation of sterile injectable solutions, the Sodium, potassium, magnesium, calcium and the like, as well preferred methods of preparation are vacuum drying and as from nontoxic organic amines, such as N,N'-dibenzyleth freeze-drying (lyophilization) that yield a powder of the ylenediamine, N-methylglucamine, chloroprocaine, choline, active ingredient plus any additional desired ingredient from diethanolamine, ethylenediamine, procaine and the like. a previously sterile-filtered solution thereof. 0470 A pharmaceutical composition according to at least 0474 Sterile injectable solutions can be prepared by Some embodiments of the invention also may include a phar incorporating the active compound in the required amount in maceutically acceptable anti-oxidant. Examples of pharma an appropriate solvent with one or a combination of ingredi ceutically acceptable antioxidants include: (1) water soluble ents enumerated above, as required, followed by sterilization antioxidants, such as ascorbic acid, cysteine hydrochloride, microfiltration. Generally, dispersions are prepared by incor sodium bisulfate, sodium metabisulfite, sodium sulfite and porating the active compound into a sterile vehicle that con the like; (2) oil-soluble antioxidants, such as ascorbyl palmi tains a basic dispersion medium and the required other ingre tate, butylated hydroxyanisole (BHA), butylated hydroxy dients from those enumerated above. In the case of sterile toluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and powders for the preparation of sterile injectable solutions, the the like; and (3) metal chelating agents, such as citric acid, preferred methods of preparation are vacuum drying and ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric freeze-drying (lyophilization) that yield a powder of the acid, phosphoric acid, and the like. active ingredient plus any additional desired ingredient from 0471. These compositions may also contain adjuvants a previously sterile-filtered solution thereof. Such as preservatives, wetting agents, emulsifying agents and 0475. A composition of the present invention can be dispersing agents. Prevention of presence of microorganisms administered via one or more routes of administration using may be ensured both by Sterilization procedures, Supra, and one or more of a variety of methods known in the art. As will by the inclusion of various antibacterial and antifungal be appreciated by the skilled artisan, the route and/or mode of agents, for example, paraben, chlorobutanol, phenol Sorbic administration will vary depending upon the desired results. acid, and the like. It may also be desirable to include isotonic Preferred routes of administration for therapeutic agents agents, such as Sugars, sodium chloride, and the like into the according to at least Some embodiments of the invention US 2014/0294765 A1 Oct. 2, 2014

include intravascular delivery (e.g. injection or infusion), 0479. In certain embodiments, the anti-LSR antibodies intravenous, intramuscular, intradermal, intraperitoneal, Sub can be formulated to ensure proper distribution in vivo. For cutaneous, spinal, oral, enteral, rectal, pulmonary (e.g. inha example, the blood-brain bather (BBB) excludes many highly lation), nasal, topical (including transdermal, buccal and Sub hydrophilic compounds. To ensure that the therapeutic com lingual), intravesical, intravitreal, intraperitoneal, vaginal, pounds according to at least some embodiments of the inven brain delivery (e.g. intra-cerebroventricular, intra-cerebral, tion cross the BBB (if desired), they can be formulated, for and convection enhanced diffusion), CNS delivery (e.g. example, in liposomes. For methods of manufacturing lipo intrathecal, perispinal, and intra-spinal) or parenteral (includ somes, see, e.g., U.S. Pat. Nos. 4.522,811; 5,374,548; and ing Subcutaneous, intramuscular, intravenous and intrader 5,399.331. The liposomes may comprise one or more moi mal), transmucosal (e.g., Sublingual administration), admin eties which are selectively transported into specific cells or istration or administration via an implant, or other parenteral organs, thus enhance targeted drug delivery (see, e.g., V. V. routes of administration, for example by injection or infusion, Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary tar or other delivery routes and/or forms of administration known geting moieties include folate or biotin (see, e.g., U.S. Pat. in the art. The phrase “parenteral administration” as used No. 5,416,016 to Low et al.); mannosides (Umezawa et al., herein means modes of administration other than enteral and (1988) Biochem. Biophys. Res. Commun. 153:1038): anti topical administration, usually by injection, and includes, bodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. without limitation, intravenous, intramuscular, intraarterial, Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); intrathecal, intracapsular, intraorbital, intracardiac, intrader surfactant protein A receptor (Briscoe et al. (1995) Am. J. mal, intraperitoneal, transtracheal, Subcutaneous, Subcuticu Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. lar, intraarticular, Subcapsular, Subarachnoid, intraspinal, epi Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen dural and intrasternal injection and infusion. In a specific (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) embodiment, a protein, a therapeutic agent or a pharmaceu Immunomethods 4:273. tical composition according to at least some embodiments of 0480. The anti-LSR antibodies, according to at least some the present invention can be administered intraperitoneally or embodiments of the present invention, can be used as neu intravenously. tralizing antibodies. A Neutralizing antibody (Nabs), is an 0476 Alternatively, an LSR specific antibody or can be antibody that is capable of binding and neutralizing or inhib administered via a non-parenteral route, such as a topical, iting a specific antigen thereby inhibiting its biological effect, epidermal or mucosal route of administration, for example, for example by blocking the receptors on the cell or the virus, intranasally, orally, vaginally, rectally, Sublingually or topi inhibiting the binding of the virus to the host cell. NAbs will cally. partially or completely abrogate the biological action of an agent by either blocking an important Surface molecule 0477 The active compounds can be prepared with carriers needed for its activity or by interfering with the binding of the that will protect the compound against rapid release. Such as agent to its receptor on a target cell. a controlled release formulation, including implants, trans 0481. In yet another embodiment, immunoconjugates of dermal patches, and microencapsulated delivery systems. the invention can be used to target compounds (e.g., thera Biodegradable, biocompatible polymers can be used, such as peutic agents, labels, cytotoxins, radiotoxins immunosup ethylene vinyl acetate, polyanhydrides, polyglycolic acid, pressants, etc.) to cells which have LSR cell surface receptors collagen, polyorthoesters, and polylactic acid. Many methods by linking Such compounds to the antibody. Thus, the inven for the preparation of such formulations are patented or gen tion also provides methods for localizing ex vivo or in vivo erally known to those skilled in the art. See, e.g., Sustained cells expressing LSR (e.g., with a detectable label. Such as a and Controlled Release Drug Delivery Systems, J. R. Robin radioisotope, a fluorescent compound, an enzyme, or an son, ed., Marcel Dekker, Inc., New York, 1978. enzyme co-factor). Alternatively, the immunoconjugates can 0478. Therapeutic compositions can be administered with be used to kill cells which have LSR cell surface receptors by medical devices known in the art. For example, in a preferred targeting cytotoxins or radiotoxins to LSR antigen. embodiment, a therapeutic composition according to at least 0482. As used herein, “pharmaceutically acceptable car some embodiments of the invention can be administered with rier includes any and all solvents, dispersion media, coat a needles hypodermic injection device, such as the devices ings, antibacterial and antifungal agents, isotonic and absorp disclosed in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335; tion delaying agents, and the like that are physiologically 5,064,413: 4,941,880; 4,790,824; or 4,596,556. Examples of compatible. Preferably, the carrier is suitable for intravenous, well-known implants and modules useful in the present intramuscular, Subcutaneous, parenteral, spinal or epidermal invention include: U.S. Pat. No. 4.487,603, which discloses administration (e.g., by injection or infusion). Depending on an implantable micro-infusion pump for dispensing medica the route of administration, the active compound, i.e., soluble tion at a controlled rate; U.S. Pat. No. 4,486,194, which polypeptide conjugate containing the ectodomain of the LSR discloses atherapeutic device for administering medicaments antigen, antibody, immunoconjugate, alternative scaffolds, through the skin; U.S. Pat. No. 4,447.233, which discloses a and/or bispecific molecule, may be coated in a material to medication infusion pump for delivering medication at a pre protect the compound from the action of acids and other cise infusion rate; U.S. Pat. No. 4,447.224, which discloses a natural conditions that may inactivate the compound. The variable flow implantable infusion apparatus for continuous pharmaceutical compounds according to at least some drug delivery; U.S. Pat. No. 4,439,196, which discloses an embodiments of the present invention may include one or osmotic drug delivery system having multi-chamber com more pharmaceutically acceptable salts. A "pharmaceutically partments; and U.S. Pat. No. 4,475,196, which discloses an acceptable salt” refers to a salt that retains the desired bio osmotic drug delivery system. These patents are incorporated logical activity of the parent compound and does not impart herein by reference. Many other such implants, delivery sys any undesired toxicological effects (see e.g., Berge, S. M., et tems, and modules are known to those skilled in the art. al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts US 2014/0294765 A1 Oct. 2, 2014 42 include acid addition salts and base addition salts. Acid addi glycol, and the like), and suitable mixtures thereof. The tion salts include those derived from nontoxic inorganic proper fluidity can be maintained, for example, by the use of acids, such as hydrochloric, nitric, phosphoric, Sulfuric, a coating Such as lecithin, by the maintenance of the required hydrobromic, hydroiodic, phosphorous and the like, as well particle size in the case of dispersion and by the use of Sur as from nontoxic organic acids such as aliphatic mono- and factants. In many cases, it will be preferable to include iso dicarboxylic acids, phenyl-substituted alkanoic acids, tonic agents, for example, Sugars, polyalcohols such as man hydroxy alkanoic acids, aromatic acids, aliphatic and aro nitol, Sorbitol, or Sodium chloride in the composition. matic sulfonic acids and the like. Base addition salts include Prolonged absorption of the injectable compositions can be those derived from alkaline earth metals, such as Sodium, brought about by including in the composition an agent that potassium, magnesium, calcium and the like, as well as from delays absorption, for example, monostearate salts and gela nontoxic organic amines, such as N,N'-dibenzylethylenedi tin. Sterile injectable solutions can be prepared by incorpo amine, N-methylglucamine, chloroprocaine, choline, dietha rating the active compound in the required amount in an nolamine, ethylenediamine, procaine and the like. appropriate solvent with one or a combination of ingredients 0483 A pharmaceutical composition according to at least enumerated above, as required, followed by sterilization Some embodiments of the present invention also may include microfiltration. Generally, dispersions are prepared by incor a pharmaceutically acceptable anti-oxidant. Examples of porating the active compound into a sterile vehicle that con pharmaceutically acceptable antioxidants include: (1) water tains a basic dispersion medium and the required other ingre soluble antioxidants, such as ascorbic acid, cysteine hydro dients from those enumerated above. In the case of sterile chloride, sodium bisulfate, sodium metabisulfite, sodium powders for the preparation of sterile injectable solutions, the Sulfite and the like; (2) oil-soluble antioxidants, such as ascor preferred methods of preparation are vacuum drying and byl palmitate, butylated hydroxyanisole (BHA), butylated freeze-drying (lyophilization) that yield a powder of the hydroxytoluene (BHT), lecithin, propyl gallate, alpha-toco active ingredient plus any additional desired ingredient from pherol, and the like; and (3) metal chelating agents, such as a previously sterile-filtered solution thereof. citric acid, ethylenediamine tetraacetic acid (EDTA), sorbi 0487 Sterile injectable solutions can be prepared by tol, tartaric acid, phosphoric acid, and the like. Examples of incorporating the active compound in the required amount in Suitable aqueous and nonaqueous carriers that may be an appropriate solvent with one or a combination of ingredi employed in the pharmaceutical compositions according to at ents enumerated above, as required, followed by sterilization least some embodiments of the present invention include microfiltration. Generally, dispersions are prepared by incor water, ethanol, polyols (such as glycerol, propylene glycol, porating the active compound into a sterile vehicle that con polyethylene glycol, and the like), and Suitable mixtures tains a basic dispersion medium and the required other ingre thereof, vegetable oils, such as olive oil, and injectable dients from those enumerated above. In the case of sterile organic esters, such as ethyl oleate. Proper fluidity can be powders for the preparation of sterile injectable solutions, the maintained, for example, by the use of coating materials, such preferred methods of preparation are vacuum drying and as lecithin, by the maintenance of the required particle size in freeze-drying (lyophilization) that yield a powder of the the case of dispersions, and by the use of Surfactants. active ingredient plus any additional desired ingredient from 0484 These compositions may also contain adjuvants a previously sterile-filtered solution thereof. Such as preservatives, wetting agents, emulsifying agents and 0488. The amount of active ingredient which can be com dispersing agents. Prevention of presence of microorganisms bined with a carrier material to produce a single dosage form may be ensured both by Sterilization procedures, Supra, and will vary depending upon the Subject being treated, and the by the inclusion of various antibacterial and antifungal particular mode of administration. The amount of active agents, for example, paraben, chlorobutanol, phenol Sorbic ingredient which can be combined with a carrier material to acid, and the like. It may also be desirable to include isotonic produce a single dosage form will generally be that amount of agents, such as Sugars, sodium chloride, and the like into the the composition which produces a therapeutic effect. Gener compositions. In addition, prolonged absorption of the inject ally, out of one hundred percent, this amount will range from able pharmaceutical form may be brought about by the inclu about 0.01 percent to about ninety-nine percent of active sion of agents which delay absorption Such as aluminum ingredient, preferably from about 0.1 percent to about 70 monostearate and gelatin. percent, most preferably from about 1 percent to about 30 0485 Pharmaceutically acceptable carriers include sterile percent of active ingredient in combination with a pharma aqueous Solutions or dispersions and sterile powders for the ceutically acceptable carrier. extemporaneous preparation of sterile injectable solutions or 0489 Dosage regimens are adjusted to provide the opti dispersion. The use of such media and agents for pharmaceu mum desired response (e.g., a therapeutic response). For tically active Substances is known in the art. Except insofar as example, a single bolus may be administered, several divided any conventional media or agent is incompatible with the doses may be administered over time or the dose may be active compound, use thereof in the pharmaceutical compo proportionally reduced or increased as indicated by the exi sitions according to at least Some embodiments of the present gencies of the therapeutic situation. It is especially advanta invention is contemplated. Supplementary active compounds geous to formulate parenteral compositions in dosage unit can also be incorporated into the compositions. form for ease of administration and uniformity of dosage. 0486 Therapeutic compositions typically must be sterile Dosage unit form as used herein refers to physically discrete and stable under the conditions of manufacture and storage. units Suited as unitary dosages for the Subjects to be treated; The composition can beformulated as a solution, microemul each unit contains a predetermined quantity of active com Sion, liposome, or other ordered structure Suitable to high pound calculated to produce the desired therapeutic effect in drug concentration. The carrier can be a solvent or dispersion association with the required pharmaceutical carrier. The medium containing, for example, water, ethanol, polyol (for specification for the dosage unit forms according to at least example, glycerol, propylene glycol, and liquid polyethylene some embodiments of the present invention are dictated by US 2014/0294765 A1 Oct. 2, 2014 and directly dependent on (a) the unique characteristics of the duration of the treatment, other drugs, compounds and/or active compound and the particular therapeutic effect to be materials used in combination with the particular composi achieved, and (b) the limitations inherent in the art of com tions employed, the age, sex, weight, condition, general pounding Such an active compound for the treatment of sen health and prior medical history of the patient being treated, sitivity in individuals. and like factors well known in the medical arts. 0490 For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually Formulations for Parenteral Administration 0.01 to 5 mg/kg, of the host body weight. For example dos ages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 0494. In a further embodiment, compositions disclosed mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body herein, including those containing peptides and polypeptides, weight or within the range of 1-10 mg/kg. An exemplary are administered in an aqueous Solution, by parenteral injec treatment regime entails administration once per week, once tion. The formulation may also be in the form of a Suspension every two weeks, once every three weeks, once every four or emulsion. In general, pharmaceutical compositions are weeks, once a month, once every 3 months or once every three provided including effective amounts of a peptide or polypep to 6 months. Preferred dosage regimens for an antibody tide, and optionally include pharmaceutically acceptable according to at least some embodiments of the present inven diluents, preservatives, solubilizers, emulsifiers, adjuvants tion include 1 mg/kg body weight or 3 mg/kg body weight via and/or carriers. Such compositions optionally include one or intravenous administration, with the antibody being given more for the following: diluents, sterile water, buffered saline using one of the following dosing schedules: (i) every four of various buffer content (e.g., Tris-HCl, acetate, phosphate), weeks for six dosages, then every three months; (ii) every pH and ionic strength; and additives Such as detergents and three weeks; (iii) 3 mg/kg body weight once followed by 1 solubilizing agents (e.g., TWEEN 20 (polysorbate-20), mg/kg body weight every three weeks. TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., water 0491. In some methods, two or more monoclonal antibod soluble antioxidants such as ascorbic acid, sodium met ies with different binding specificities are administered abisulfite, cysteine hydrochloride, sodium bisulfate, sodium simultaneously, in which case the dosage of each antibody metabisulfite, sodium sulfite; oil-soluble antioxidants, such administered falls within the ranges indicated. Antibody is as ascorbyl palmitate, butylated hydroxyanisole (BHA), usually administered on multiple occasions. Intervals butylated hydroxytoluene (BHT), lecithin, propyl gallate, between single dosages can be, for example, weekly, alpha-tocopherol; and metal chelating agents, such as citric monthly, every three months or yearly. Intervals can also be acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tar irregular as indicated by measuring blood levels of antibody taric acid, phosphoric acid), and preservatives (e.g., Thimer to the target antigen in the patient. In some methods, dosage Sol, benzyl alcohol) and bulking Substances (e.g., lactose, is adjusted to achieve a plasma antibody concentration of mannitol). Examples of non-aqueous solvents or vehicles are about 1-1000 mug?ml and in some methods about 25-300 ethanol, propylene glycol, polyethylene glycol, vegetable microgram/ml. oils, such as olive oil and corn oil, gelatin, and injectable 0492 Alternatively, therapeutic agent can be administered organic esters such as ethyl oleate. The formulations may be as a Sustained release formulation, in which case less frequent freeze dried (lyophilized) or vacuum dried and redissolved/ administration is required. Dosage and frequency vary resuspended immediately before use. The formulation may depending on the half-life of the therapeutic agent in the be sterilized by, for example, filtration through a bacteria patient. In general, human antibodies show the longest half retaining filter, by incorporating sterilizing agents into the life, followed by humanized antibodies, chimeric antibodies, compositions, by irradiating the compositions, or by heating and nonhuman antibodies. The half-life for fusion proteins thecompositions. may vary widely. The dosage and frequency of administration 0495 Formulations for Topical Administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low 0496 LSR polypeptides, fragments, fusion polypeptides, dosage is administered at relatively infrequent intervals over nucleic acids, and vectors disclosed herein can be applied a long period of time. Some patients continue to receive topically. Topical administration does not work well for most treatment for the rest of their lives. In therapeutic applica peptide formulations, although it can be effective especially if tions, a relatively high dosage at relatively short intervals is applied to the lungs, nasal, oral (Sublingual, buccal), vaginal, Sometimes required until progression of the disease is or rectal mucosa. reduced or terminated, and preferably until the patient shows 0497 Compositions can be delivered to the lungs while partial or complete amelioration of symptoms of disease. inhaling and traverse across the lung epithelial lining to the Thereafter, the patient can be administered a prophylactic blood stream when delivered either as an aerosol or spray regime. dried particles having an aerodynamic diameter of less than 0493 Actual dosage levels of the active ingredients in the about 5 microns. A wide range of mechanical devices pharmaceutical compositions of the present invention may be designed for pulmonary delivery of therapeutic products can varied so as to obtainanamount of the active ingredient which be used, including but not limited to nebulizers, metered dose is effective to achieve the desired therapeutic response for a inhalers, and powder inhalers, all of which are familiar to particular patient, composition, and mode of administration, those skilled in the art. Some specific examples of commer without being toxic to the patient. The selected dosage level cially available devices are the Ultravent nebulizer (Mallinck will depend upon a variety of pharmacokinetic factors includ rodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest ing the activity of the particular compositions of the present Medical Products, Englewood, Colo.); the Ventolin metered invention employed, or the ester, salt or amide thereof, the dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and route of administration, the time of administration, the rate of the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). excretion of the particular compound being employed, the Nektar, Alkermes and Mannkind all have inhalable insulin US 2014/0294765 A1 Oct. 2, 2014 44 powder preparations approved or in clinical trials where the can then be linked to certain disease symptoms. Alternatively, technology could be applied to the formulations described the antibodies can be used to inhibit or block LSR function herein. which, in turn, can be linked to the prevention oramelioration 0498 Formulations for administration to the mucosa will of cancer. This can be achieved by contacting a sample and a typically be spray dried drug particles, which may be incor control sample with the anti-LSR antibody under conditions porated into a tablet, gel, capsule, Suspension or emulsion. that allow for the formation of a complex between the corre Standard pharmaceutical excipients are available from any sponding antibody and LSR. Any complexes formed between formulator. Oral formulations may be in the form of chewing the antibody and LSR are detected and compared in the gum, gel Strips, tablets or lozenges. Transdermal formula sample and the control. tions may also be prepared. These will typically be ointments, 0505 According to at least some embodiments of the lotions, sprays, or patches, all of which can be prepared using present invention, the antibodies (e.g., human antibodies, standard technology. Transdermal formulations will require multispecific and bispecific molecules and compositions) can the inclusion of penetration enhancers. be initially tested for binding activity associated with thera 0499 Controlled Delivery Polymeric Matrices peutic or diagnostic use in vitro. For example, compositions 0500 LSR polypeptides, fragments, fusion polypeptides, according to at least Some embodiments of the present inven nucleic acids, and vectors disclosed herein may also be tion can be tested using low cytometric assays. administered in controlled release formulations. Controlled 0506 Also within the scope of the present invention are release polymeric devices can be made for long term release kits comprising the LSR specific antibody according to at systemically following implantation of a polymeric device least some embodiments of the present invention (e.g., human (rod, cylinder, film, disk) or injection (microparticles). The antibodies, alternative scaffolds, bispecific or multispecific matrix can be in the form of microparticles Such as micro molecules, or immunoconjugates) and instructions for use. spheres, where peptides are dispersed within a Solid poly The kit can further contain one or more additional reagents, meric matrix or microcapsules, where the core is of a different Such as an immunosuppressive reagent, a cytotoxic agent or a material than the polymeric shell, and the peptide is dispersed radiotoxic agent, or one or more additional human antibodies or Suspended in the core, which may be liquid or Solid in according to at least Some embodiments of the present inven nature. Unless specifically defined herein, microparticles, tion (e.g., a human antibody having a complementary activity microspheres, and microcapsules are used interchangeably. which binds to an epitope in the antigen distinct from the first Alternatively, the polymer may be cast as a thin slab or film, human antibody). ranging from nanometers to four centimeters, a powder pro duced by grinding or other standard techniques, or even a gel 0507. The antibodies according to at least some embodi Such as a hydrogel. Either non-biodegradable or biodegrad ments of the present invention can also be used to target cells able matrices can be used for delivery of polypeptides or expressing Fc gamma R or LSR for example for labeling Such nucleic acids encoding the polypeptides, although biodegrad cells. For Such use, the binding agent can be linked to a able matrices are preferred. These may be natural or synthetic molecule that can be detected. Thus, the present invention polymers, although synthetic polymers are preferred due to provides methods for localizing ex vivo or in vitro cells the better characterization of degradation and release profiles. expressing Fc receptors, such as Fc gamma R, or LSR anti The polymer is selected based on the period over which gen. The detectable label can be, e.g., a radioisotope, a fluo release is desired. In some cases linear release may be most rescent compound, an enzyme, or an enzyme co-factor. useful, although in others a pulse release or “bulk release' 0508. In a particular embodiment, the present invention may provide more effective results. The polymer may be in provides methods for detecting the presence and/or level of the form of a hydrogel (typically in absorbing up to about LSR antigen in a sample, or measuring the amount of LSR 90% by weight of water), and can optionally be crosslinked antigen, respectively, comprising contacting the sample, and with multivalent ions or polymers. a control sample, with an antibody, or an antigen binding 0501. The matrices can be formed by solvent evaporation, portion thereof, which specifically binds to LSR, under con spray drying, Solvent extraction and other methods known to ditions that allow for formation of a complex between the those skilled in the art. Bioerodible microspheres can be antibody or portion thereof and LSR. The formation of a prepared using any of the methods developed for making complex is then detected, wherein a difference complex for microspheres for drug delivery, for example, as described by mation between the sample compared to the control sample is Mathiowitz and Langer, J. Controlled Release, 5:13-22 indicative the presence of LSR antigen in the sample. As (1987); Mathiowitz, et al., Reactive Polymers, 8: 275-283 noted the present invention in particular embraces assays for (1987); and Mathiowitz, et al., J. Appl Polymer ScL,35:755 detecting LSR antigen in vitro and in vivo Such as immunoas 774 (1988). says, radioimmunoassays, radioassays, radioimaging assays, 0502. The devices can be formulated for local release to ELISAs, Western blot, FACS, slot blot, immunohistochemi treat the area of implantation or injection which will typi cal assays, and other assays well known to those skilled in the cally deliver a dosage that is much less than the dosage for art. treatment of an entire body—or systemic delivery. These can 0509. In yet another embodiment, immunoconjugates of be implanted or injected Subcutaneously, into the muscle, fat, the present invention can be used to target compounds (e.g., or Swallowed. therapeutic agents, labels, cytotoxins, radiotoxins immuno 0503 Diagnostic Uses of Anti-LSR Antibodies suppressants, etc.) to cells which have LSR cell surface recep 0504. According to at least some embodiments of the tors by linking such compounds to the antibody. Thus, the present invention, the antibodies (e.g., human monoclonal present invention also provides methods for localizing eX antibodies, multispecific and bispecific molecules and com vivo or in vivo cells expressing LSR (e.g., with a detectable positions) can be used to detect levels of LSR or levels of cells label. Such as a radioisotope, a fluorescent compound, an which contain LSR on their membrane surface, which levels enzyme, or an enzyme co-factor). Alternatively, the immuno US 2014/0294765 A1 Oct. 2, 2014

conjugates can be used to kill cells which have LSR cell variants include, but are not limited to, enhanced GFP Surface receptors by targeting cytotoxins or radiotoxins to (EGFP), destabilized EGFP, the GFP variants described in LSR antigen. Doan et al., Mol. Microbiol. 55:1767-1781 (2005), the GFP 0510. According to at least some embodiments, the variant described in Crameri et al., Nat. Biotechnol., 14:315 present invention provides a method for imaging an organ or 319 (1996), the cerulean fluorescent proteins described in tissue, the method comprising: (a) administering to a subject Rizzo et al., Nat. Biotechnol, 22:445 (2004) and Tsien, Annu. in need of Such imaging, a labeled polypeptide; and (b) Rev. Biochem, 67:509 (1998), and the yellow fluorescent detecting the labeled polypeptide to determine where the protein described in Nagai et al., Nat. Biotechnol. 20:87-90 labeled polypeptide is concentrated in the subject. When used (2002). Dsked variants are described in, e.g., Shaner et al., in imaging applications, the labeled polypeptides according Nat. Biotechnol., 22:1567-1572 (2004), and include mStraw to at least some embodiments of the present invention typi berry, mCherry, morange, mBanana, mHoneydew, and mTan cally have an imaging agent covalently or noncovalently gerine. Additional DSRed variants are described in, e.g., attached thereto. Suitable imaging agents include, but are not Wang et al., Proc. Natl. Acad. Sci. U.S.A., 101: 16745-16749 limited to, radionuclides, detectable tags, fluorophores, fluo (2004) and include mRaspberry and mPlum. Further rescent proteins, enzymatic proteins, and the like. One of skill examples of DsRed variants include mRFPmars described in in the art will be familiar with other methods for attaching Fischer et al., FEBS Lett., 577:227-232 (2004) and mRF imaging agents to polypeptides. For example, the imaging Pruby described in Fischer et al., FEBS Lett., 580:2495-2502 agent can be attached via site-specific conjugation, e.g., cova (2006). lent attachment of the imaging agent to a peptide linker Such 0514. In other embodiments, the imaging agent that is as a polyarginine moiety having five to seven arginines bound to a polypeptide according to at least Some embodi present at the carboxyl-terminus of and Fc fusion molecule. ments of the present invention comprises a detectable tag The imaging agent can also be directly attached via non-site Such as, for example, biotin, avidin, streptavidin, or neutra specific conjugation, e.g., covalent attachment of the imaging vidin. In further embodiments, the imaging agent comprises agent to primary amine groups present in the polypeptide. an enzymatic protein including, but not limited to, luciferase, One of skill in the art will appreciate that an imaging agent chloramphenicol acetyltransferase, B-galactosidase, B-glu can also be bound to a protein via noncovalent interactions (e.g., ionic bonds, hydrophobic interactions, hydrogen bonds, curonidase, horseradish peroxidase, Xylanase, alkaline phos Van der Waals forces, dipole-dipole bonds, etc.). phatase, and the like. 0511. In certain instances, the polypeptide is radiolabeled 0515. Any device or method known in the art for detecting with a radionuclide by directly attaching the radionuclide to the radioactive emissions of radionuclides in a Subject is the polypeptide. In certain other instances, the radionuclide is suitable for use in the present invention. For example, meth bound to a chelating agent or chelating agent-linker attached ods such as Single Photon Emission Computerized Tomog to the polypeptide. Suitable radionuclides for direct conjuga raphy (SPECT), which detects the radiation from a single tion include, without limitation, 18 F, 124 I, 125 I, 131 I, and photongamma-emitting radionuclide using a rotating gamma mixtures thereof. Suitable radionuclides for use with a chelat camera, and radionuclide Scintigraphy, which obtains an ing agent include, without limitation, 47 Sc. 64 Cu, 67 Cu, 89 image or series of sequential images of the distribution of a Sr,86Y.87Y,90Y,105 Rh, 111 Ag, 111 In, 117m Sn, 149 Pm, radionuclide in tissues, organs, or body systems using a scin 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, and tillation gamma camera, may be used for detecting the radia mixtures thereof. Preferably, the radionuclide bound to a tion emitted from a radiolabeled polypeptide of the present chelating agent is 64 Cu, 90Y, 111 In, or mixtures thereof. invention. Positron emission tomography (PET) is another Suitable chelating agents include, but are not limited to, Suitable technique for detecting radiation in a Subject. Min DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their phos iature and flexible radiation detectors intended for medical phonate analogs, and mixtures thereof. One of skill in the art use are produced by Intra-Medical LLC (Santa Monica, will be familiar with methods for attaching radionuclides, Calif.). Magnetic Resonance Imaging (MRI) or any other chelating agents, and chelating agent-linkers to polypeptides imaging technique known to one of skill in the art is also of the present invention. In particular, attachment can be Suitable for detecting the radioactive emissions of radionu conveniently accomplished using, for example, commer clides. Regardless of the method or device used, such detec cially available bifunctional linking groups (generally hetero tion is aimed at determining where the labeled polypeptide is bifunctional linking groups) that can be attached to a func concentrated in a subject, with Such concentration being an tional group present in a non-interfering position on the indicator of disease activity. polypeptide and then further linked to a radionuclide, chelat 0516 Non-invasive fluorescence imaging of animals and ing agent, or chelating agent-linker. humans can also provide in vivo diagnostic information and 0512 Non-limiting examples of fluorophores or fluores be used in a wide variety of clinical specialties. For instance, cent dyes Suitable for use as imaging agents include Alexa techniques have been developed over the years for simple Fluorr dyes (Invitrogen Corp.; Carlsbad, Calif.), fluorescein, ocular observations following UV excitation to sophisticated fluorescein isothiocyanate (FITC), Oregon GreenTM: spectroscopic imaging using advanced equipment (see, e.g., rhodamine, Texas red, tetrarhodamine isothiocynate Andersson-Engels et al., Phys. Med. Biol. 42:815-824 (TRITC), CyDyeTM fluors (e.g., Cy2, Cy3, Cy5), and the like. (1997)). Specific devices or methods known in the art for the 0513. Examples of fluorescent proteins suitable for use as in vivo detection of fluorescence, e.g., from fluorophores or imaging agents include, but are not limited to, green fluores fluorescent proteins, include, but are not limited to, in vivo cent protein, red fluorescent protein (e.g., DSRed), yellow near-infrared fluorescence (see, e.g., Frangioni, Curr. Opin. fluorescent protein, cyan fluorescent protein, blue fluorescent Chem. Biol. 7:626-634 (2003)), the MaestroTM in vivo fluo protein, and variants thereof (see, e.g., U.S. Pat. Nos. 6,403, rescence imaging system (Cambridge Research & Instrumen 374, 6,800,733, and 7,157.566). Specific examples of GFP tation, Inc., Woburn, Mass.), in vivo fluorescence imaging US 2014/0294765 A1 Oct. 2, 2014 46 using a flying-spot Scanner (see, e.g., Ramanujam et al., IEEE “true positives'). Diseased individuals not detected by the Transactions on Biomedical Engineering, 48:1034-1041 assay are “false negatives. Subjects who are not diseased and (2001), and the like. who test negative in the assay are termed “true negatives.” The 0517. Other methods or devices for detecting an optical “specificity of a diagnostic assay is 1 minus the false positive response include, without limitation, visual inspection, CCD rate, where the “false positive' rate is defined as the propor cameras, video cameras, photographic film, laser-scanning tion of those without the disease who test positive. While a devices, fluorometers, photodiodes, quantum counters, epif particular diagnostic method may not provide a definitive luorescence microscopes, Scanning microscopes, flow diagnosis of a condition, it suffices if the method provides a cytometers, fluorescence microplate readers, or signal ampli positive indication that aids in diagnosis. fication using photomultiplier tubes. 0522. As used herein the term "diagnosis” refers to the 0518. According to some embodiments, the sample taken process of identifying a medical condition or disease by its from a Subject (patient) to perform the diagnostic assay signs, symptoms, and in particular from the results of various according to at least some embodiments of the present inven diagnostic procedures, including e.g. detecting the expres tion is selected from the group consisting of a body fluid or sion of the nucleic acids or polypeptides according to at least secretion including but not limited to blood, serum, urine, Some embodiments of the invention in a biological sample plasma, prostatic fluid, seminal fluid, semen, the external (e.g. in cells, tissue or serum, as defined below) obtained from secretions of the skin, respiratory, intestinal, and genitouri an individual. Furthermore, as used herein the term “diagno nary tracts, tears, cerebrospinal fluid, synovial fluid, sputum, sis' encompasses screening for a disease, detecting a pres saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, secre ence or a severity of a disease, providing prognosis of a tions of the breast ductal system (and/or lavage thereof), disease, monitoring disease progression or relapse, as well as broncho alveolar lavage, lavage of the reproductive system assessment of treatment efficacy and/or relapse of a disease, and lavage of any other part of the body or system in the body; disorder or condition, as well as selecting a therapy and/or a samples of any organ including isolated cells or tissues, treatment for a disease, optimization of a given therapy for a wherein the cell or tissue can be obtained from an organ disease, monitoring the treatment of a disease, and/or predict selected from, but not limited to lung, colon, ovarian and/or ing the Suitability of a therapy for specific patients or Sub breast tissue; stool or a tissue sample, or any combination populations or determining the appropriate dosing of a thera thereof. In some embodiments, the term encompasses peutic product in patients or subpopulations. The diagnostic samples of in vivo cell culture constituents. Prior to be sub procedure can be performed in vivo or in vitro. jected to the diagnostic assay, the sample can optionally be 0523. In some embodiments, the phrase "qualitative” diluted with a suitable eluant. when in reference to differences in expression levels of a 0519 In some embodiments, the phrase “marker” in the polynucleotide or polypeptide as described herein, refers to context of the present invention refers to a nucleic acid frag the presence versus absence of expression, or in some ment, a peptide, or a polypeptide, which is differentially embodiments, the temporal regulation of expression, or in present in a sample taken from patients (subjects) having one Some embodiments, the timing of expression, or in some of the herein-described diseases or conditions, as compared to embodiments, any post-translational modifications to the a comparable sample taken from Subjects who do not have expressed molecule, and others, as will be appreciated by one one the above-described diseases or conditions. skilled in the art. In some embodiments, the phrase "quanti 0520. In some embodiments, the phrase “differentially tative' when in reference to differences in expression levels of present refers to differences in the quantity or quality of a a polynucleotide or polypeptide as described herein, refers to marker present in a sample taken from patients having one of absolute differences in quantity of expression, as determined the herein-described diseases or conditions as compared to a by any means, known in the art, or in other embodiments, comparable sample taken from patients who do not have one relative differences, which may be statistically significant, or of the herein-described diseases or conditions. For example, a in Some embodiments, when viewed as a whole or over a nucleic acid fragment may optionally be differentially prolonged period of time, etc., indicate a trend in terms of present between the two samples if the amount of the nucleic differences in expression. acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for 0524. In some embodiments, the term “diagnosing refers example as measured by hybridization and/or NAT-based to classifying a disease or a symptom, determining a severity assays. A polypeptide is differentially present between the of the disease, monitoring disease progression, forecasting an two samples if the amount of the polypeptide in one sample is outcome of a disease and/or prospects of recovery. The term significantly different from the amount of the polypeptide in “detecting may also optionally encompass any of the above. the other sample. It should be noted that if the marker is 0525 Diagnosis of a disease according to the present detectable in one sample and not detectable in the other, then invention can, in some embodiments, be affected by deter such a marker can be considered to be differentially present. mining a level of a polynucleotide or a polypeptide of the Optionally, a relatively low amount of up-regulation may present invention in a biological sample obtained from the serve as the marker, as described herein. One of ordinary skill subject, wherein the level determined can be correlated with in the art could easily determine such relative levels of the predisposition to, or presence or absence of the disease. It markers; further guidance is provided in the description of should be noted that a “biological sample obtained from the each individual marker below. Subject may also optionally comprise a sample that has not 0521. In some embodiments, the phrase “diagnostic' been physically removed from the subject, as described in means identifying the presence or nature of a pathologic greater detail below. condition. Diagnostic methods differ in their sensitivity and 0526 In some embodiments, the term “level” refers to specificity. The “sensitivity of a diagnostic assay is the per expression levels of RNA and/or protein or to DNA copy centage of diseased individuals who test positive (percent of number of a marker of the present invention. US 2014/0294765 A1 Oct. 2, 2014 47

0527 Typically the level of the marker in a biological nucleic acid molecules (see, e.g., P. D. Fahrlander and A. sample obtained from the subject is different (i.e., increased Klausner, Bio/Technology 6:1165 (1988)). Quantitation of or decreased) from the level of the same marker in a similar the signal is achieved by, e.g., Scintillation counting, densito sample obtained from a healthy individual (examples of bio metry, or flow cytometry. logical samples are described herein). 0535 Exemplary detectable labels, optionally and prefer 0528 Numerous well known tissue or fluid collection ably for use with immunoassays, includebut are not limited to methods can be utilized to collect the biological sample from magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., the subject in order to determine the level of DNA, RNA horse radish peroxide, alkaline phosphatase and others com and/or polypeptide of the marker of interest in the subject. monly used in an ELISA), and calorimetric labels such as 0529. Examples include, but are not limited to, fine needle colloidal gold or colored glass or plastic beads. Alternatively, biopsy, needle biopsy, core needle biopsy and Surgical biopsy the marker in the sample can be detected using an indirect (e.g., brain biopsy), and lavage. Regardless of the procedure assay, wherein, for example, a second, labeled antibody is employed, once a biopsy/sample is obtained the level of the used to detect bound marker-specific antibody, and/or in a marker can be determined and a diagnosis can thus be made. competition or inhibition assay wherein, for example, a 0530 Determining the level of the same marker in normal monoclonal antibody which binds to a distinct epitope of the tissues of the same origin is preferably effected along-side to marker are incubated simultaneously with the mixture. detect an elevated expression and/or amplification and/or a 0536 “Immunoassay” is an assay that uses an antibody to decreased expression, of the marker as opposed to the normal specifically bind an antigen. The immunoassay is character tissues. ized by the use of specific binding properties of a particular 0531. In some embodiments, the term “test amount of a antibody to isolate, target, and/or quantify the antigen. marker refers to an amount of a marker in a Subject's sample 0537. The phrase “specifically (or selectively) binds” to an that is consistent with a diagnosis of a particular disease or antibody or “specifically (or selectively) immunoreactive condition. A test amount can be either in absolute amount with.” or “specifically interacts or binds” when referring to a (e.g., microgram/ml) or a relative amount (e.g., relative inten protein or peptide (or other epitope), refers, in Some embodi sity of signals). ments, to a binding reaction that is determinative of the pres 0532. In some embodiments, the term “controlamount of ence of the protein in a heterogeneous population of proteins a marker can be any amount or a range of amounts to be and other biologics. Thus, under designated immunoassay compared against a test amount of a marker. For example, a conditions, the specified antibodies bind to a particular pro control amount of a marker can be the amount of a marker in tein at least two times greater than the background (non a patient with a particular disease or condition or a person specific signal) and do not substantially bind in a significant without Such a disease or condition. A control amount can be amount to other proteins present in the sample. Specific bind either in absolute amount (e.g., microgram/ml) or a relative ing to an antibody under Such conditions may require an amount (e.g., relative intensity of signals). antibody that is selected for its specificity for a particular 0533. In some embodiments, the term “detect refers to protein. For example, polyclonal antibodies raised to seminal identifying the presence, absence or amount of the object to basic protein from specific species such as rat, mouse, or be detected. human can be selected to obtain only those polyclonal anti 0534. In some embodiments, the term “label includes any bodies that are specifically immunoreactive with seminal moiety or item detectable by spectroscopic, photo chemical, basic protein and not with other proteins, except for polymor biochemical, immunochemical, or chemical means. For phic variants and alleles of seminal basic protein. This selec example, useful labels include 32P 35S, fluorescent dyes, tion may beachieved by Subtracting out antibodies that cross electron-dense reagents, enzymes (e.g., as commonly used in react with seminal basic protein molecules from other an ELISA), biotin-streptavadin, dioxigenin, haptens and pro species. A variety of immunoassay formats may be used to teins for which antisera or monoclonal antibodies are avail selectantibodies specifically immunoreactive with a particu able, or nucleic acid molecules with a sequence complemen lar protein. For example, Solid-phase ELISA immunoassays tary to a target. The label often generates a measurable signal, are routinely used to selectantibodies specifically immunore Such as a radioactive, chromogenic, or fluorescent signal, that active with a protein (see, e.g., Harlow & Lane, Antibodies, A can be used to quantify the amount of bound label in a sample. Laboratory Manual (1988), for a description of immunoassay The label can be incorporated in or attached to a primer or formats and conditions that can be used to determine specific probe either covalently, or through ionic, van der Waals or immunoreactivity). Typically a specific or selective reaction hydrogen bonds, e.g., incorporation of radioactive nucle will be at least twice background signal or noise and more otides, or biotinylated nucleotides that are recognized by typically more than 10 to 100 times background. streptavadin. The label may be directly or indirectly detect 0538. In another embodiment, this invention provides a able. Indirect detection can involve the binding of a second method for detecting the polypeptides of this invention in a label to the first label, directly or indirectly. For example, the biological sample, comprising: contacting a biological label can be the ligand of a binding partner, Such as biotin, sample with an antibody specifically recognizing a polypep which is a binding partner for streptavadin, or a nucleotide tide according to the present invention and detecting said sequence, which is the binding partner for a complementary interaction; wherein the presence of an interaction correlates sequence, to which it can specifically hybridize. The binding with the presence of a polypeptide in the biological sample. partner may itself be directly detectable, for example, an 0539. In some embodiments of the present invention, the antibody may be itself labeled with a fluorescent molecule. polypeptides described herein are non-limiting examples of The binding partner also may be indirectly detectable, for markers for diagnosing a disease and/or an indicative condi example, a nucleic acid having a complementary nucleotide tion. Each marker of the present invention can be used alone sequence can be a part of a branched DNA molecule that is in or in combination, for various uses, including but not limited turn detectable through hybridization with other labeled to, prognosis, prediction, Screening, early diagnosis, determi US 2014/0294765 A1 Oct. 2, 2014 48 nation of progression, therapy selection and treatment moni herein, sensitivity relates to the number of positive (diseased) toring of a disease and/or an indicative condition. samples detected out of the total number of positive samples 0540 Each polypeptide/polynucleotide of the present present; specificity relates to the number of true negative invention can be used alone or in combination, for various (non-diseased) samples detected out of the total number of uses, including but not limited to, prognosis, prediction, negative samples present. Preferably, the method distin screening, early diagnosis, determination of progression, guishes a disease or condition with a sensitivity of at least therapy selection and treatment monitoring of disease and/or 80% at a specificity of at least 90% when compared to normal an indicative condition, as detailed above. subjects. More preferably, the method distinguishes a disease 0541. Such a combination may optionally comprise any or condition with a sensitivity of at least 90% at a specificity Subcombination of markers, and/or a combination featuring of at least 90% when compared to normal subjects. Also more at least one other marker, for example a known marker. Fur preferably, the method distinguishes a disease or condition thermore. Such a combination may optionally and preferably with a sensitivity of at least 70% at a specificity of at least 85% be used as described above with regard to determining a ratio when compared to Subjects exhibiting symptoms that mimic between a quantitative or semi-quantitative measurement of disease or condition symptoms. any marker described herein to any other marker described 0549. A marker panel may be analyzed in a number of herein, and/or any other known marker, and/or any other fashions well known to those of skill in the art. For example, marker. each member of a panel may be compared to a “normal” 0542. In some embodiments of the present invention, there value, or a value indicating a particular outcome. A particular are provided of methods, uses, devices and assays for the diagnosis/prognosis may depend upon the comparison of diagnosis of a disease or condition. Optionally a plurality of each marker to this value; alternatively, if only a subset of markers may be used with the present invention. The plurality markers is outside of a normal range, this Subset may be of markers may optionally include a markers described indicative of a particular diagnosis/prognosis. The skilled herein, and/or one or more known markers. The plurality of artisan will also understand that diagnostic markers, differ markers is preferably then correlated with the disease or ential diagnostic markers, prognostic markers, time of onset condition. For example, Such correlating may optionally markers, disease or condition differentiating markers, etc., comprise determining the concentration of each of the plu may be combined in a single assay or device. Markers may rality of markers, and individually comparing each marker also be commonly used for multiple purposes by, for concentration to a threshold level. Optionally, if the marker example, applying a different threshold or a different weight concentration is above or below the threshold level (depend ing factor to the marker for the different purposes. ing upon the marker and/or the diagnostic test being per 0550. In one embodiment, the panels comprise markers formed), the marker concentration correlates with the disease for the following purposes: diagnosis of a disease; diagnosis or condition. Optionally and preferably, a plurality of marker of disease and indication if the disease is in an acute phase concentrations correlates with the disease or condition. and/or if an acute attack of the disease has occurred; diagnosis 0543. Alternatively, such correlating may optionally com of disease and indication if the disease is in a non-acute phase prise determining the concentration of each of the plurality of and/or if a non-acute attack of the disease has occurred; markers, calculating a single index value based on the con indication whether a combination of acute and non-acute centration of each of the plurality of markers, and comparing phases or attacks has occurred; diagnosis of a disease and the index value to a threshold level. prognosis of a Subsequent adverse outcome; diagnosis of a 0544. Also alternatively, such correlating may optionally disease and prognosis of a Subsequent acute or non-acute comprise determining a temporal change in at least one of the phase or attack; disease progression (for example for cancer, markers, and wherein the temporal change is used in the Such progression may include for example occurrence or correlating step. recurrence of metastasis). 0545 Also alternatively, such correlating may optionally 0551. The above diagnoses may also optionally include comprise determining whether at least “X” number of the differential diagnosis of the disease to distinguish it from plurality of markers has a concentration outside of a prede other diseases, including those diseases that may feature one termined range and/or above or below a threshold (as or more similar or identical symptoms. described above). The value of “X” may optionally be one 0552. In certain embodiments, one or more diagnostic or marker, a plurality of markers or all of the markers; alterna prognostic indicators are correlated to a condition or disease tively or additionally, rather than including any marker in the by merely the presence or absence of the indicators. In other count for “X”, one or more specific markers of the plurality of embodiments, threshold levels of a diagnostic or prognostic markers may optionally be required to correlate with the indicators can be established, and the level of the indicators in disease or condition (according to a range and/or threshold). a patient sample can simply be compared to the threshold 0546. Also alternatively, such correlating may optionally levels. The sensitivity and specificity of a diagnostic and/or comprise determining whether a ratio of marker concentra prognostic test depends on more than just the analytical tions for two markers is outside a range and/or above or below “quality” of the test—they also depend on the definition of a threshold. Optionally, if the ratio is above or below the what constitutes an abnormal result. In practice, Receiver threshold level and/or outside a range, the ratio correlates Operating Characteristic curves, or “ROC curves, are typi with the disease or condition. cally calculated by plotting the value of a variable versus its 0547 Optionally, a combination of two or more these cor relative frequency in “normal” and “disease' populations, relations may be used with a single panel and/or for correlat and/or by comparison of results from a subject before, during ing between a plurality of panels. and/or after treatment. 0548. Optionally, the method distinguishes a disease or 0553. The present invention also relates to kits based upon condition with a sensitivity of at least 70% at a specificity of Such diagnostic methods or assays. Also within the scope of at least 85% when compared to normal subjects. As used the present invention are kits comprising LSR conjugates or US 2014/0294765 A1 Oct. 2, 2014 49 antibody compositions of the invention (e.g., human antibod of an antibody-marker complex can optionally be determined ies, bispecific or multispecific molecules, or immunoconju by comparing to a standard. As noted above, the test amount gates) and instructions for use. The kit can further contain one of marker need not be measured in absolute units, as long as or more additional reagents, such as an immunosuppressive the unit of measurement can be compared to a control amount reagent, a cytotoxic agent or a radiotoxic agent, or one or and/or signal. more additional human antibodies according to at least some 0562 Radio-immunoassay (RIA): In one version, this embodiments of the invention (e.g., a human antibody having method involves precipitation of the desired substrate and in a complementary activity which binds to an epitope in the the methods detailed herein below, with a specific antibody antigen distinct from the first human antibody). and radiolabeled antibody binding protein (e.g., protein A 0554. Immunoassays labeled with 1125) immobilized on a precipitable carrier such 0555. In another embodiment of the present invention, an as agarose beads. The number of counts in the precipitated immunoassay can be used to qualitatively or quantitatively pellet is proportional to the amount of Substrate. detect and analyze markers in a sample. This method com 0563. In an alternate version of the RIA, a labeled sub prises: providing an antibody that specifically binds to a strate and an unlabelled antibody binding protein are marker; contacting a sample with the antibody; and detecting employed. A sample containing an unknown amount of Sub the presence of a complex of the antibody bound to the marker strate is added in varying amounts. The decrease in precipi in the sample. tated counts from the labeled substrate is proportional to the 0556. To prepare an antibody that specifically binds to a amount of Substrate in the added sample. marker, purified protein markers can be used. Antibodies that 0564. Enzyme linked immunosorbent assay (ELISA): specifically bind to a protein marker can be prepared using This method involves fixation of a sample (e.g., fixed cells or any Suitable methods known in the art. a proteinaceous Solution) containing a protein Substrate to a 0557. After the antibody is provided, a marker can be Surface Such as a well of a microtiter plate. A Substrate spe detected and/or quantified using any of a number of well cific antibody coupled to an enzyme is applied and allowed to recognized immunological binding assays. Useful assays bind to the substrate. Presence of the antibody is then detected include, for example, an enzyme immune assay (EIA) Such as and quantitated by a colorimetric reaction employing the enzyme-linked immunosorbent assay (ELISA), a radioim enzyme coupled to the antibody. Enzymes commonly mune assay (RIA), a Western blot assay, or a slot blot assay employed in this method include horseradish peroxidase and see, e.g., U.S. Pat. Nos. 4.366,241; 4,376,110; 4,517,288; and alkaline phosphatase. If well calibrated and within the linear 4.837,168). Generally, a sample obtained from a subject can range of response, the amount of substrate present in the be contacted with the antibody that specifically binds the sample is proportional to the amount of color produced. A marker. Substrate standard is generally employed to improve quanti 0558 Optionally, the antibody can be fixed to a solid Sup tative accuracy. port to facilitate washing and Subsequent isolation of the 0565 Western blot: This method involves separation of a complex, prior to contacting the antibody with a sample. Substrate from other protein by means of an acrylamide gel Examples of solid Supports includebut are not limited to glass followed by transfer of the substrate to a membrane (e.g., or plastic in the form of, e.g., a microtiter plate, a Stick, a bead, nylon or PVDF). Presence of the substrate is then detected by or a microbead. Antibodies can also be attached to a solid antibodies specific to the substrate, which are in turn detected Support. by antibody binding reagents. Antibody binding reagents 0559. After incubating the sample with antibodies, the may be, for example, protein A, or other antibodies. Antibody mixture is washed and the antibody-marker complex formed binding reagents may be radiolabeled or enzyme linked as can be detected. This can be accomplished by incubating the described hereinabove. Detection may be by autoradiogra washed mixture with a detection reagent. Alternatively, the phy, colorimetric reaction or chemiluminescence. This marker in the sample can be detected using an indirect assay, method allows both quantitation of an amount of Substrate wherein, for example, a second, labeled antibody is used to and determination of its identity by a relative position on the detect bound marker-specific antibody, and/or in a competi membrane which is indicative of a migration distance in the tion or inhibition assay wherein, for example, a monoclonal acrylamide gel during electrophoresis. antibody which binds to a distinct epitope of the marker are 0566 Immunohistochemical analysis: This method incubated simultaneously with the mixture. involves detection of a substrate in situ in fixed cells by 0560. Throughout the assays, incubation and/or washing substrate specific antibodies. The substrate specific antibod steps may be required after each combination of reagents. ies may be enzyme linked or linked to fluorophores. Detec Incubation steps can vary from about 5 seconds to several tion is by microscopy and Subjective evaluation. If enzyme hours, preferably from about 5 minutes to about 24 hours. linked antibodies are employed, a colorimetric reaction may However, the incubation time will depend upon the assay be required. format, marker, Volume of Solution, concentrations and the 0567 Fluorescence activated cell sorting (FACS): This like. Usually the assays will be carried out at ambient tem method involves detection of a substrate in situ in cells by perature, although they can be conducted over a range of substrate specific antibodies. The substrate specific antibod temperatures, such as 10°C. to 40°C. ies are linked to fluorophores. Detection is by means of a cell 0561. The immunoassay can be used to determine a test sorting machine which reads the wavelength of light emitted amount of a marker in a sample from a subject. First, a test from each cell as it passes through a light beam. This method amount of a marker in a sample can be detected using the may employ two or more antibodies simultaneously. immunoassay methods described above. If a marker is 0568 Radio-Imaging Methods present in the sample, it will form an antibody-marker com 0569. These methods include but are not limited to, plex with an antibody that specifically binds the marker under positron emission tomography (PET) single photon emission suitable incubation conditions described above. The amount computed tomography (SPECT). Both of these techniques US 2014/0294765 A1 Oct. 2, 2014 50 are non-invasive, and can be used to detect and/or measure a by standardized assays (including but not limited to tradi wide variety of tissue events and/or functions, such as detect tional clinical chemistry, immunoassay, cell-based technolo ing cancerous cells for example. Unlike PET, SPECT can gies, nucleic acid tests and imaging modalities). optionally be used with two labels simultaneously. SPECT 0576. The therapeutic compositions (e.g., human antibod has some other advantages as well, for example with regard to ies, multispecific and bispecific molecules and immunocon cost and the types of labels that can be used. For example, jugates) according to at least some embodiments of the inven U.S. Pat. No. 6,696,686 describes the use of SPECT for tion which have complement binding sites, such as portions detection of breast cancer, and is hereby incorporated by from IgG1, -2, or -3 or IgM which bind complement, can also reference as if fully set forth herein. be used in the presence of complement. In one embodiment, 0570. Theranostics: ex vivo treatment of a population of cells comprising target 0571. The term theranostics describes the use of diagnos cells with a binding agent according to at least Some embodi tic testing to diagnose the disease, choose the correct treat ments of the invention and appropriate effector cells can be ment regime according to the results of diagnostic testing Supplemented by the addition of complement or serum con and/or monitor the patient response to therapy according to taining complement. Phagocytosis of target cells coated with the results of diagnostic testing. Theranostic tests can be used a binding agent according to at least Some embodiments of the to select patients for treatments that are particularly likely to invention can be improved by binding of complement pro benefit them and unlikely to produce side-effects. They can teins. In another embodiment target cells coated with the also provide an early and objective indication of treatment compositions (e.g., human antibodies, multispecific and efficacy in individual patients, so that (if necessary) the treat bispecific molecules) according to at least some embodi ment can be altered with a minimum of delay. For example: ments of the invention can also be lysed by complement. In DAKO and Genentech together created HercepTest and Her yet another embodiment, the compositions according to at ceptin (trastuzumab) for the treatment of breast cancer, the least some embodiments of the invention do not activate first theranostic test approved simultaneously with a new complement. therapeutic drug. In addition to HercepTest (which is an 0577. The therapeutic compositions (e.g., human antibod immunohistochemical test), other theranostic tests are in ies, multispecific and bispecific molecules and immunocon development which use traditional clinical chemistry, immu jugates) according to at least some embodiments of the inven noassay, cell-based technologies and nucleic acid tests. tion can also be administered together with complement. PPGx's recently launched TPMT (thiopurine S-methyltrans Thus, according to at least Some embodiments of the inven ferase) test, which is enabling doctors to identify patients at tion there are compositions, comprising human antibodies, risk for potentially fatal adverse reactions to 6-mercaptopu multispecific or bispecific molecules and serum or comple rine, an agent used in the treatment of leukemia. Also, Nova ment. These compositions are advantageous in that the Molecular pioneered SNP genotyping of the apolipoprotein E complement is located in close proximity to the human anti gene to predict Alzheimer's disease patients’ responses to bodies, multispecific or bispecific molecules. Alternatively, cholinomimetic therapies and it is now widely used in clinical the human antibodies, multispecific or bispecific molecules trials of new drugs for this indication. Thus, the field of according to at least Some embodiments of the invention and theranostics represents the intersection of diagnostic testing the complement or serum can be administered separately. information that predicts the response of a patient to a treat (0578. The present invention is further illustrated by the ment with the selection of the appropriate treatment for that following examples. This information and examples is illus particular patient. trative and should not be construed as further limiting. The 0572. As described herein, the term “theranostic' may contents of all figures and all references, patents and pub optionally refer to first testing the Subject, such as the patient, lished patent applications cited throughout this application for a certain minimum level of LSR, for example optionally in are expressly incorporated herein by reference. the cancerous tissue and/or in the immune infiltrate, as described herein as a sufficient level of LSR expression. Test EXAMPLES ing may optionally be performed ex vivo, in which the sample is removed from the subject, or in vivo. Example 1 0573. If the cancerous tissue and/or the immune infiltrate have been shown to have the minimum level of LSR, then an Cloning of LSR Proteins anti-LSR antibody, alone or optionally with other treatment 0579. A. Cloning of LSR T1 P5a ORF modalities as described herein, may optionally be adminis 0580) Cloning of LSR T1 P5a open reading frame (ORF) tered to the subject. (SEQID NO: 154) was performed by PCR to generate LSR 0574) Surrogate Markers: P5a protein (SEQID NO: 11), as described below. 0575. A surrogate marker is a marker, that is detectable in 0581 A PCR reaction was performed using Pful Jltra II a laboratory and/or according to a physical sign or symptom Fusion HS DNA Polymerase (Agilent, Catalog no. 600670) on the patient, and that is used in therapeutic trials as a under the following conditions: 50 ng of plRES puro3 Substitute for a clinically meaningful endpoint. The Surrogate LSR T1 P5a Flag construct described above served as a marker is a direct measure of how a patient feels, functions, or template for a PCR reaction with 0.5 microliter of each of the survives which is expected to predict the effect of the therapy. primers 200 369 LSR Kozak Nhel (SEQID NO: 147) and The need for Surrogate markers mainly arises when Such 200-372 LSR BamHI Rev (SEQ ID NO: 152) in a total markers can be measured earlier, more conveniently, or more reaction volume of 25 ul. The reaction conditions were 5 frequently than the endpoints of interest in terms of the effect minutes at 98° C.: 35 cycles of: 20 seconds at 98° C., 30 of a treatment on a patient, which are referred to as the clinical seconds at 55° C. and 1.5 minutes at 72°C.; then 10 minutes endpoints. Ideally, a Surrogate marker should be biologically at 72° C. All of the primers that were used include gene plausible, predictive of disease progression and measurable specific sequences, restriction enzyme sites and Kozak US 2014/0294765 A1 Oct. 2, 2014

sequence. The PCR product was separated on 1% agarose gel. constructs were verified by sequence (SEQ ID No:201) and After verification of the expected band size, the PCR product Subsequently used for transfections and stable pool genera was purified using QIAquickTM Gel Extraction kit as tion as described below. described above. 0582. The purified PCR product was digested with NheI 4. Cloning of Mouse LSR-WT Flag Construct and BamHI restriction enzymes (New England Biolabs, Bev erly, Mass., U.S.A.). After digestion, the DNA was separated 0592 Full length cDNA of mouse WT LSR (SEQ ID on a 1% agarose gel. The expected band size was excised and NO:202) was synthesized with a Flag tag at the C-terminus, extracted from the gel as described above. The digested DNA cloned into puC57 vector by GenScript, and subcloned into a was then ligated into pIRESpuro3 vector that was digested mammalian expression vector, pcDNA3.1, to create an with Nhe and BamHI as described above, incubated with expression construct, as described below. Antarctic Phosphatase (New England Biolabs, Beverly, 0593 cDNA was digested with Nhel and BamHI restric Mass., U.S.A., Catalog no. MO289L) for 30 minutes at 37° C. tion enzymes and ligated to pcDNA3.1+ mammalian expres and purified from 1% agarose gel using QIAquickTM Gel sion vector previously digested with the same enzymes. The Extraction kit as described above. The ligation reaction was resulting expression constructs were verified by sequence performed with T4 DNA Ligase (Promega; Catalog no. (SEQ ID No:202) and subsequently used for transfections M180A). and stable pool generation as described below. 0583 Sequence verification of both tagged and untagged 5. Cloning of cyno LSR WTORF constructs described above was performed (Hylabs, Rehovot, 0594 Cloning of cyno LSR WT open reading frame Israel). Two nucleotide mismatches were identified, as fol (ORF) (SEQID NO:203) was performed by PCR to generate lows: G to Aat nucleic acid position 119 of SEQID NO: 154, cyno LSR WT protein (SEQ ID NO: 203), as described and A to G at nucleic acid position 626 from SEQID NO: 154, below. resulting in a nucleic sequence set forth in SEQID NO: 145 0595 A PCR reaction was performed using GO Taq DNA for the untagged construct, and SEQ ID NO: 146 for the Polymerase (Promega, Catalog no. M3001) under the follow tagged construct; yielding a polypeptide having an amino ing conditions: Pool of Monkey clNA (Biochain, Cat. No. acid mismatch I to Minamino acid position 11, resulting in a C8534502-Cy, C8534501-Cy) served as a template for 2 dif protein having amino acid sequence set forth in SEQID NO: ferent PCR reactions. The first with 1 microliter of each of the 143 for the untagged construct and SEQID NO: 144 for the primers 200 403 cILSR Kozak Nhel (SEQ ID NO:204) tagged construct. and 200-407 cILSR Rev (SEQ ID NO:205) and the second 0584) The above recombinant plasmids were processed with 1 microliter of each of the primers 200 404 cISR for stable pool generation as described below. Flag EcoRI (SEQ ID NO:206) and 200-406 cLSR. For 0585 2. Cloning of LSR WT ORF (SEQ ID NO: 207), both in a total reaction volume of 50 ul. 0586 Cloning of LSR WT open reading frame (ORF) 0596. The reaction conditions were 5 minutes at 95°C.; 40 was performed by substitution of Alanine at position 627 to cycles of 30 seconds at 95°C., 30 seconds at 55° C. and 1 Glycine by one-step site-directed mutagenesis PCR to gen minute at 72°C.; then 5 minutes at 72°C. All of the primers erate LSR WT protein (SEQ ID NO: 154), as described that were used include gene specific sequences, restriction below. enzyme sites and Kozak sequence. The PCR product was 0587. A PCR reaction was performed using Pful Jltra II separated on 1% agarose gel. After verification of the Fusion HS DNA Polymerase (Agilent, Catalog no. 600670) expected band size, the PCR products were purified using under the following conditions: 20 ng of plRES puro3 QIAquickTM Gel Extraction kit as described above. These LSR T1 P5a Flag m construct described above (SEQ ID purified PCR products used as a template for a PCR reaction NO: 145), served as a template for a PCR reaction with 2.5 under the following conditions: 5 minutes at 95°C.; 40 cycles microliter of each of the primers 200 398 (SEQID NO: 199) of 30 seconds at 95°C., 30 seconds at 55° C. and 1.5 minutes and 200-399 (SEQID NO:200) in a total reaction volume of at 72° C.; then 5 minutes at 72° C., using GO Taq DNA 50ul. The reaction conditions were 3 minutes at 95°C.; 12 polymerase (Promega, Catalog no. M3001). The PCR prod cycles of 1 minute at 95°C., 1 minute at 55° C. and 3 minutes uct was loaded on 1% agarose gel and the product was puri at 72° C.; then 10 minutes at 72° C. 2u1 DpnI were added to fied the same way as described above. the PCR reaction and incubate for 2 h at 37° C. 0597. The purified PCR product was digested with NheI 0588 Sequence verification of tagged construct described and EcoRI restriction enzymes (New England Biolabs, Bev above was performed (Hylabs, Rehovot, Israel). erly, Mass., U.S.A.). After digestion, the DNA was separated 0589. The above recombinant plasmid was processed for on a 1% agarose gel. The expected band size was excised and stable pool generation as described below. extracted from the gel as described above. The digested DNA was then ligated into pIRESpuro3 vector that was digested 3. Cloning of LSR SKIP4 ORF with Nhe and EcoRI as described above, incubated with 0590 Full length cDNA of human-LSR (SEQ ID NO: Antarctic Phosphatase (New England Biolabs, Beverly, 201) variant skipping exon 4 was synthesized with a Flag tag Mass., U.S.A., Catalog no. MO289L) for 30 minutes at 37° C. at the C-terminus, and cloned in puC57 vector by GenWiz and purified from 1% agarose gel using QIAquickTM Gel (USA). This cDNA was subsequently cloned in the pRp3 Extraction kit as described above. The ligation reaction was mammalian expression vector, pcDNA3.1, to create an performed with T4 DNA Ligase (Promega; Catalog no. expression construct, as described below. M180A). 0591 cDNA was digested with Nhel and BamHI restric 0598. Sequence verification of tagged construct described tion enzymes and ligated to pIRESpuro3 (pRp3) mammalian above was performed (Hylabs, Rehovot, Israel). expression vector (Clontech, Cat No: 631619) previously 0599. The above recombinant plasmid was processed for digested with the same enzymes. The resulting expression stable pool generation as described below. US 2014/0294765 A1 Oct. 2, 2014 52

6. Cloning of cyno LSR SKIP4 ORF with 5 g\ml puromycin (Sigma, catalog number P8833). 0600 Cloning of cyno LSR skip4 open reading frame Cells were placed in an incubator, and the medium was (ORF) (SEQID NO:208) was performed by PCR to generate replaced every 3-4 days, until clone formation was observed. cyno LSR skip4 protein (SEQ ID NO:208), as described 0609 2. Generation of Stable Transfectant Pools Express below. ing Human and cyno Wt and Skip4 LSR Proteins 0601 A PCR reaction was performed using GO Taq DNA 0610 HEK-293T (ATCC, CRL-11268) cells were trans Polymerase (Promega, Catalog no. M3001) under the follow fected with the human and cyno LSR (SEQID NOs: 154, 201, ing conditions: Pool of Monkey clNA (Biochain, Cat. No. 203, 208)LSR pRp3 constructs described above or with the C8534502-Cy, C8534501-Cy) served as a template for PCR empty vector (pRp3) as negative control, using Fugene6 reaction. transfection reagent (Roche, Cat No: 111-988-387). Puromy 0602 1 microliter of each of the primers 200 403 cISR cin resistant colonies were selected for stable pool generation. Kozak Nhel (SEQ ID NO:204) and 200-404 cILSR Flag Formouse LSRWT (SEQID NO:202), a different expression EcoRI (SEQID NO: 206) in a total reaction volume of 50 ul. vector and other cell lines were used for generation of stable 0603. The reaction conditions were 5 minutes at 95°C.; 40 transfectants pools (see below). cycles of 30 seconds at 95°C., 30 seconds at 55° C. and 1.45 0611 3. Generation of Stable Transfectant Pools Express minutes at 72°C.; then 5 minutes at 72°C. All of the primers ing Mouse WT Protein that were used include gene specific sequences, restriction 0612 Stable transfectant cell pools expressing the WT enzyme sites and Kozak sequence. The PCR product was Mouse LSR-flag protein (SEQID NO: 202) were generated at separated on 1% agarose gel. After verification of the GeneScript (USA Inc). The mouse WT LSR sequence (SEQ expected band size, the PCR products were purified using ID NO: 202) with the Flag tag at the C-terminus was synthe QIAquickTM Gel Extraction kit as described above. The puri sized, cloned into puC57 vector, and sub-cloned into a mam fied PCR product was digested with Nhe and EcoRI restric malian expression vector pcDNA3.1. The recombinant plas tion enzymes (New England Biolabs, Beverly, Mass., U.S. mid was transfected into CHO-K1 (ATCC, cat #CCL-61) and A.). After digestion, the DNA was separated on a 1% agarose into HEK-293 (ATCC cat #CRL-1573TM) cells. Cell pools of gel. The expected band size was excised and extracted from stable transfectants were screened using G418 and analyzed the gel as described above. The digested DNA was then by western blot using anti-flag Ab. ligated into pIRESpuro3 vector that was digested with Nhel and EcoRI as described above, incubated with Antarctic Example 3 Phosphatase (New England Biolabs, Beverly, Mass., U.S.A., Catalog no. MO289L) for 30 minutes at 37° C. and purified Expression Validation from 1% agarose gel using QIAquickTM Gel Extraction kit as 0613 A. Analysis of the Ectopic Expression of LSR P5a described above. The ligation reaction was performed with FLAG M in Stably-Transfected HEK293T Cells T4 DNA Ligase (Promega; Catalog no. M180A). 0614. The expression of LSR P5a Flag m (SEQID NO: 0604 Sequence verification of tagged construct described 144) instably-transfected HEK293T cells was determined by above was performed (Hylabs, Rehovot, Israel). Western blot analysis of the cell lysates, using anti LSR 0605. The above recombinant plasmid was processed for Antibodies and anti flag antibody as indicated in Table 1. stable pool generation as described below. 0615 Cells were dissociated from the plate using Cell Example 2 Dissociation Buffer Enzyme-Free PBS-Based (Gibco; 13151-014), washed in Dulbecco's Phosphate Buffered Establishment of Stable Pools of Recombinant Cells Saline (PBS) (Biological Industries, 02*023-1A) and centri Expressing LSR Proteins fuged at 1200 g for 5 minutes. Whole cell extraction was performed by resuspending the cells in 50 mM Tris-HCl 0606 1. Establishment of a Stable Pool of Recombinant pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, Hek293T Cells Expressing LSR P5a FLAG M Protein supplemented with 25x complete EDTA free protease inhibi 0607 HEK-293T cells were stably transfected with LSR tor cocktail (Roche, 11873 580 001) and vortexing for 20 T1: P5a Flag m (SEQID NO: 146) and plRESpuro3 empty seconds. Cell extracts were collected following centrifuga vector plasmids as follows: tion at 20000 g for 20 minutes at 4°C. and protein concen 0608 HEK-293T (ATCC, CRL-11268) cells were plated tration was determined with Bradford Biorad Protein Assay in a sterile 6 well plate Suitable for tissue culture, containing (Biorad catfis00-0006). Equal protein amounts were ana 2 ml pre-warmed of complete media, DMEMDulbecco's lyzed by SDS-PAGE (Invitrogen NuPAGE 4-12% NuPAGE modified Eagle's Media, Biological Industries (Beit Bis Tris, Cath NP0335, NP0322) and transferred to Nitrocel HaEmek, Israel, catalog number: 01-055-1A)+10% FBS lulose membrane (BA83, 0.2 um, Schleicher & Schuell, Fetal Bovine Serum, Biological Industries (Beit HaEmek, CathA01385). The membrane was blocked with TTBS (Bi Israel, catalog number: 04-001-1A)+4 mM L-Glutamine olab, Cath: 20892323)/10% skim milk (Difco, Cath232100) (Biological Industries (Beit HaEmek, Israel), catalog num and incubated with the indicated primary antibodies (FIG. 1) ber: 03-020-1A).500,000 cells per well were transfected with diluted in TTBS/5% BSA (Sigma-Aldrich, A4503) at the 2 ug of DNA construct using 6 ul FuGENE 6 reagent (Roche, indicated concentrations (Table 2), for 16 hours at 4°C. After catalog number: 11-814-443-001) diluted into 94u1 DMEM. 3 washes with TTBS, The membrane was further incubated The mixture was incubated at room temperature for 15 min for 1 hour at Room Temperature with the secondary-conju utes. The complex mixture was added dropwise to the cells. gated antibodies as indicated, diluted in TTBS. Chemilumi The cells were placed in an incubator maintained at 37° C. nescence reaction was performed with ECL Western Blotting with 5% CO2 content. 48 hours after the transfection, the cells Detection Reagents (GE Healthcare, Cat # RPN2209) and the were transferred to a 75 cm2 tissue culture flask containing 15 membrane was exposed to Super RX Fuji X-Ray film (Cata ml of selection medium: complete medium Supplemented log no. 4741008389). US 2014/0294765 A1 Oct. 2, 2014

0616 FIG. 1 demonstrates the expression of LSR P5a as a negative control, whereas lane #2 represents HEK293T Flag m protein (SEQ ID: 144) in recombinant HEK293T pIRESpuro3 human LSR skip4 Flag transfected cells. cells at the expected band size ~70 kDa, as detected with anti 0623 FIG. 4 presents detection of the cyno LSR WT Flag (Sigma cathi A8592) (FIG. 1A) and anti LSR antibodies (lane 2) and LSR skip4 (lane 3) proteins, both compared to as follow: Abnova, catiH00051599-B01P (FIG. 1B) Abcam, the empty vector cells lysate (lane 1). cat ab59646 (FIG. 1C) and Sigma cath HPAO07270 (FIG. 0624 FIG. 5 presents detection by anti-flag Abs of mouse 1D). LSR WT protein in CHO-K1 cells (lane 2) and in HEK293 0617 B. Expression Validation of Human, Cyno and cells (lane 4) compared to the relevant empty vector cells Mouse LSR Wt and LSR SKIP4 in Stably-Transfected lysate (lanes 1 and 3 respectively). HEK293T Cells or in CHO-K1 Cells 0618. The expression of human, cyno and mouse LSR WT and LSR skip4 instably-transfected HEK293T cells or in TABLE 1 CHO-K1 cells was determined by Western blot analysis of the Primary and secondary antibodies cell lysates, using anti LSR Antibodies and anti-flag antibody Dilu as indicated in Table 1. Antibody Application tion 0619 Cells were dissociated from the plate using Cell Mouse Anti FLAG-Cy3 (Sigma catalog number: IF :200 Dissociation Buffer Enzyme-Free PBS-Based (Gibco; A9594) 13151-014), washed in Dulbecco's Phosphate Buffered Mouse Anti FLAG-HRP (Sigma Catalog no. A8592) WB :2OOO Saline (PBS) (Biological Industries, 02*023-1A) and centri Rabbit Anti LSR (Abcam catalog number: ab59646) IF :500 fuged at 1200 g for 5 minutes. Whole cell extraction was WB :4OOO Rabbit Anti LSR (Sigma catalog number: IF :100 performed by resuspending the cells in 50 mM Tris-HCl HPAO07270) WB :2SOO pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, Mouse Anti LSR (Abnova catalog number: IF :500 supplemented with 25x complete EDTA free protease inhibi H00051599-B01P) WB :1000 tor cocktail (Roche, 11873 580 001) and vortexing for 20 Mouse Anti GAPDH (Abcam catalog number: WB :1000 ab9484) seconds. Cell extracts were collected following centrifuga Donkey Anti Rabbit Cy3 (Jackson ImmunoResearch IF :200 tion at 20000 g for 20 minutes at 4°C. and protein concen Laboratories Inc. catalog no. 711-165-152) tration was determined with Bradford Biorad Protein Assay Donkey Anti Mouse Dylight 549 (Jackson IF :100 (Biorad cati500-0006). Equal protein amounts were ana mmunoResearch Laboratories Inc. catalog no. 715 506-150) lyzed by SDS-PAGE (Invitrogen NuPAGE 4-12% NuPAGE Peroxidase conjugated affinity purified Goat Anti WB :1OOOO Bis Tris, Cath NP0335, NP0322) and transferred to Nitrocel Rabbit IgG (Jackson ImmunoResearch Laboratories lulose membrane (BA83, 0.2 um, Schleicher & Schuell, inc. catalog no. 111-035-003) CathA01385). The membrane was blocked with TTBS (Bi Peroxidase conjugated affinity purified Goat Anti- WB :1OOOO olab, Cath: 20892323)/5% skim milk (Difico, Cath232100) Mouse IgG (Jackson ImmunoResearch Laboratories and incubated with the indicated primary antibodies (FIG. 1) inc. catalog no. 115-035-146) diluted in TTBS/5% BSA (Sigma-Aldrich, A4503) at the indicated concentrations (Table 1), for 1 hour at Room Tem 0625 C. Analysis of the Expression of Endogenous LSR perature. After 3 washes with TTBS, The membrane was Protein in Various Cell Lines further incubated for 1 hour at Room Temperature with the 0626. The expression of endogenous LSR protein in vari secondary-conjugated antibodies Goat anti-Rabbit IgG-Per ous cell lines was analyzed by Western Blotting as described oxidase (Jackson, Cat. No. 111-035-003) diluted 1:20,000 in below. TTBS. Chemiluminescence reaction was performed with ECL Western Blotting Detection Reagents (GE Healthcare, 0627 SK-OV3 (ATCC no. HTB-77) Caov3 (ATCC no. Cati RPN2209) and the membrane was exposed to Super RX HTB-75), OVCAR3 (ATCC no. HTB-161), ES-2 (ATCC no. Fuji X-Ray film (Catalog no. 4741008389). CRL-1978), OV-90 (ATCC no. CRL-11732), TOV112D 0620 FIGS. 2, 3, 4, 5 present western blot analysis results (ATCC no. CRL-11731) and Hep G2 (ATCC no. HB-8065) demonstrating the expression of human (FIGS. 2, 3), cyno cell extracts were prepared as described above. (FIG. 4) and mouse (FIG. 5) LSR WT and LSR skip4 pro 0628. HeLa (catalog no. sc-2200), MCF-7 (catalog no. teins (SEQ ID NOS:11, 13, 211, 212, 31) in recombinant sc-2206), CaCo2 (catalog no. sc-2262) and SkBR3 (catalog HEK293T cells transfected with both LSR WT and LSR no. sc-2218) cell extracts were purchased from SantaCruz skip4 constructs of human and cyno LSR) and in HEK293 Biotechnology. and CHO-K1 cells transfected with WT construct of mouse 0629 Equal protein amounts were analyzed by SDS LSR, at the expected band size ~70 kDa, as detected with PAGE and transferred to Nitrocellulose membrane as anti-Flag (Sigma cathi A8592) (FIG. 5) and anti LSR antibod described above. The membrane was blocked with TTBS ies (Abcam, cat ab59646) (FIGS. 2, 3, 4). (Biolab, Cath: 20892323)/10% skim milk (Difco, 0621 FIG.2 presents detection of human LSR WT with 3 Catil 232100) and incubated with anti LSR antibodies (Ab different commercial Abs against LSR: 2A refers to SIGMA cam.cathab59646) diluted in TTBS/5% BSA (Sigma-Ald Ab, whereas 2B to Abcam Ab and 2C to Abnova Ab (as rich, A4503) at the indicated concentrations (Table 2), for 16 detailed in Table 1). Lane #1 represents HEK293T pIRE hours at 4°C. After 3 washes with in TTBS, The membrane Spuro3 empty vector transfected cells which were used as a was further incubated for 1 hour at Room Temperature with negative control, whereas lane #2 represents HEK293T p the secondary-conjugated antibodies as indicated (Table 2), RESpuro3 human WT LSR Flag transfected cells. diluted in TBS. Chemiluminescence reaction was performed 0622 FIG. 3 presents detection of the human LSR skip4 with ECL Western Blotting Detection Reagents (GE Health protein using Abcam Ab. Lane #1 represents HEK293T care, Cat it RPN2209) and the membrane was exposed to pIRESpuro3 empty vector transfected cells which were used Super RX Fuji X-Ray film (Catalog no. 4741008389). US 2014/0294765 A1 Oct. 2, 2014 54

0630 FIG. 6 demonstrates the endogenous expression of (lane 1), indicating specific knockdown of the ectopic expres LSR in various cell lines. A band at 72 kDa corresponding to sion of the Human WT LSR-flag protein. LSR was detected with anti LSR antibody in extracts of SK OV3, Caov3, OVCAR3, OV-90, Hep G2, HeLa, CaCo2, and Example 4B SkBR3 (FIG. 6A). Anti GAPDH (Abcam cathi ab9484) served as a loading control (FIG. 6B). Knock Down of LSR in Endogenous Cell Lines 0634 Knockdown of the endogenous expression of LSR TABLE 2 protein (SEQ ID NO:11) in HT29 cells or in HepG2/C3A Primary and secondary antibodies cells was carried out by transient transfection of siRNA spe cific to LSR (SEQID NO:11). Cells were transfected with 30 Applica umol (10 nM) (for HT29 cells) and with 50 umol (for HepG2/ Antibody tion Dilution C3A) LSR specific siRNA (Thermo Scientific CathM Mouse Anti FLAG-Cy3 (Sigma catalog number: A9594) IF :200 009672-00-0005) and scrambled siRNA (Thermo Scientific Mouse Anti FLAG-HRP (Sigma Catalog no. A8592) WB :2OOO Catil D-001810-01-05) as a negative control, using Lipo Rabbit Anti LSR (Abcam catalog number: ab59646) IF :500 fectamineR RNAiMAX Transfection Reagent (Invitrogen, WB :4OOO Rabbit Anti LSR (Sigma catalog number: HPAO07270) IF :100 Catil 13778-150). Following incubation of 72 hr (for HT29 WB :2SOO cells) or 48 hr (for HepG2/C3A cells) cells were analyzed by Mouse Anti LSR (Abnova catalog number: HO0051599- IF :500 FACS using hybridoma sup of the anti-LSR (SEQID NO:11) B01P) WB :1000 mAb 8C8 or by Western blot using commercial anti-LSR Mouse Anti GAPDH (Abcam catalog number: ab9484) WB :1000 Donkey Anti Rabbit Cy3 (Jackson ImmunoResearch IF :200 polyclonal antibodies, as described above in Table 1. Laboratories Inc. catalog no. 711-165-152) 0635 FIG. 16 demonstrates a specific knockdown of Donkey Anti Mouse Dylight 549 (Jackson IF :100 endogenous LSR (SEQ ID NO:11) protein expression ana mmunoResearch Laboratories Inc. catalog no. 715-506 lyzed by Western blot. 50) Peroxidase conjugated affinity purified Goat Anti Rabbit WB :1OOOO 0636 FIG. 17 demonstrates FACS analysis results of gG (Jackson ImmunoResearch Laboratories Inc. catalog HT29 (FIG. 17A) and HepG2/C3A (FIG. 17B) cells follow no. 111-035-003) ing transient transfection with the LSR-specific siRNA Peroxidase conjugated affinity purified Goat Anti-Mouse WB :1OOOO (Thermo Scientific CathM-009672-00-0005). gG (Jackson ImmunoResearch Laboratories Inc. catalog 0637 Specific knockdown of endogenous LSR (SEQ ID no. 115-035-146) NO:11) protein surface expression (arrow#1) is shown, as compared to cells transfected with scrambled-siRNA (Thermo Scientific CathD-001810-01-05) (arrowi2) as nega Example 4A tive control. Knock Down of LSR Protein Expression by siRNA Example 5 0631. In order to verify the performance of siRNA (Thermo Scientific CathM-009672-00-0005) specific to the Determination of the Subcellular Localization of the human LSR WT (SEQID NO:11) protein, and to validate its Ectopic LSR Proteins in the Transfected Cells specificity, knock down of LSR protein was performed on the 0638 A. Determination of the Sub Cellular Localization HEK293T cells stably expressing the Human LSR WT(SEQ of the Ectopic LSR P5a FLAG M in HEK293T Cells ID NO:11). 0639. The subcellular localization of the LSR P5a 0632 Stably transfected recombinant HEK293T cells Flag m protein (SEQID NO: 144) was determined in stably expressing human LSR WT (SEQ ID NO:11) described transfected cells by confocal microscopy. above were plated in 6 wells plate in 2 ml Opti-MEMO I (0640 Stably transfected recombinant HEK293T cells Reduced Serum Medium (Gibco, catfi3 1985-047) containing expressing a LSR P5a Flag m (SEQID NO: 144) described 10% FBS 24 hr prior the siRNA transfection. For each trans above were plated on coverslips pre-coated with Poly-L- fection sample, oligomer and lipofectamine 2000 transfec Lysine (Sigma; Catalogue no. P4.832). After 24 hrs the cells tion reagent (Invitrogen cathi 11668019) complexes were pre were processed for immunostaining and analyzed by confo pared and added as follow: 100 umol siRNA oligomer and cal microscopy. The cover slip was washed in phosphate 5ul of the transfection reagent were mixed in a final volume buffered saline (PBS), then fixed for 15 minutes in a solution of 250ul optimum without serum. The above complexes were of PBS/3.7% paraformaldehyde (PFA) (EMS, catalog num incubated at RT for 20 minutes and added to each well con ber: 15710)/3% glucose (Sigma, catalog number: G5767). taining cells and medium. The plate was mixed gently and The PFA was Quenched with PBS/3 mM Glycine (Sigma, incubated for 48 hr at 37°C. in a CO, incubator, then the cells catalog number: G7126) for 5 minutes. After two 5-minute were collected from the plate using Cell Dissociation Buffer washes in PBS, the cells were permeabilized with PBS/0.1% Enzyme-Free PBS-Based (Gibco: 13151-014), washed in Triton-X100 for 5 minutes at Room Temperature and washe Dulbecco's Phosphate Buffered Saline (PBS) (Biological twice in PBS. Then, blocking of non-specific regions was Industries, 02*023-1A) and centrifuged at 1200 g for 5 min performed with PBS/5% Bovine Serum Albumin (BSA) utes. Whole cell extraction and western analysis were per (Sigma, catalog number: A4503) for 20 minutes. The cover formed as mentioned above. slip was then incubated in a humid chamber for 1 hour with 0633. The results shown in FIG.7 demonstrate a dramatic each of the primary antibodies antibodies diluted in PBS/5% decrease in the signal intensity of the ~70kDa band following BSA as indicated, followed by three 5-minute washes in PBS. transfection by the LSR specific siRNA (Thermo Scientific The coverslips were then incubated for 30 minutes with the CathM-009672-00-0005) (lane 2), as compared to the corresponding secondary antibody diluted in PBS/2.5% BSA scrambled siRNA (Thermo Scientific Cati D-001810-01-05) at the indicated dilution. The antibodies and the dilutions that US 2014/0294765 A1 Oct. 2, 2014

were used are specified in Table 2. After a prewash in Hank’s 11A, 12A and 13A) and anti LSR antibodies as follows: Balanced Salt Solutions w/o phenol red (HBSS) (Biological Abcam, cat ab59646 (FIG. 9C) and Sigma cath HPAO07270 Industries Catalog no. 02-016-1), the coverslip was incubated (FIGS. 9B, 10B, 11B, 12B and 13B). All recombinant cells with WGA-Alexa 488 (Invitrogen, catalog number W11261) expressing LSR proteins were compared to the empty vector diluted 1:200 in HBSS for 10 min, washed in HBSS and cells which were used as a negative control (numbered as A-1, incubated in BISBENZIMIDE H 33258 (Sigma, catalog B-1 and C-1 in all figures). Arrows indicate membrane stain number: 14530) diluted 1:1000 in HBSS. The coverslip was 1ng. then mounted on a slide with Gel Mount Aqueous medium (0646 Human WT LSR (SEQ ID NO:11) was observed (Sigma, catalog number: G0918) and cells were observed for mainly in intracellular regions with both the anti-Flag (Sigma the presence of fluorescent product using confocal micros cati A9594) (FIG. 9A-2) and anti-LSR antibodies (Sigma copy. cath HPAO07270, Abcam, catab59646 FIGS.9B-2 and 9C-2 0641. The subcellular localization of LSR P5a Flag mis respectively), with a low percentage of cells demonstrating demonstrated in FIG. 8, LSR P5a Flag m (SEQ ID NO: membrane localization. 144) is localized mainly to the cell cytoplasm, but can also be (0647. The cyno WT LSR (SEQ ID NO:211) protein was detected on the cell surface as detected with anti Flag (Sigma also observed mainly in intracellular regions, including in the cati A9594) (FIG. 8A) and anti LSR antibodies as follows: ER and golgi, using an anti-flag antibody (FIG.10A-2). How Abcam, cat ab59646 (FIG. 8B) Abnova, catiH00051599 ever, the same cells expressing the cyno WT LSR (SEQ ID B01P (FIG. 8C) and Sigma catt HPAO07270 (FIG. 8D). NO:211) protein showed a more pronounced membrane 0642 B. Determination of the Subcellular Localization of staining when using an anti-LSR antibody (Sigma cathi the Ectopic Human, Cyno and Mouse LSR WT and LSR HPAO07270) (FIG.10B-2). Interestingly, using both antibod SKIP4 in HEK293T and CHO-K1 Cells ies, the expression on the cell Surface was more pronounced in (0643. The subcellular localization of the LSR WT (SEQ the recombinant cells expressing the cyno Skip4 LSR-flag ID NOS:11,211,31) and skip4 protein (SEQIDNOs: 13,212) variant (SEQID NO:212) as compared to recombinant cells were determined in stably-transfected cells by confocal expressing cyno WT LSR-flag protein (SEQ ID NO:211) microscopy. (FIGS. 12A-2 and 12B-2). (0644 Stably transfected recombinant HEK293T cells 0648 Recombinant HEK293 cells expressing the mouse expressing human and cyno LSR WT (SEQID NOs: 11, 211) WT LSR (SEQ ID NO:31) protein show a signal with the and LSR skip4 (SEQ ID NOS:13, 212) and CHO-Kt cells anti-flag antibody, albeit very weak (FIG. 11A), and a weak expressing mouse LSR WT (SEQ ID NO:31), described signal on the cell membrane using the specific anti-LSR anti above were plated on coverslips pre-coated with Poly-L- body (Sigma cath HPAO07270) (FIG. 11B). Recombinant Lysine (Sigma; Catalogue no. P4832). After 24 hrs the cells CHO-K1 cells expressing the mouse LSR-flag protein (SEQ were processed for immunostaining and analyzed by confo ID NO:31) (FIGS. 13A and 13B) demonstrate ER localiza cal microscopy. The cover slip was washed in phosphate tion with both antibodies. buffered saline (PBS), then fixed for 15 minutes in a solution Example 6 of PBS/3.7% paraformaldehyde (PFA) (EMS, catalog num ber: 15710)/3% glucose (Sigma, catalog number: G5767). 0649. A. Validation of Cell Surface Expression of Human The PFA was Quenched with PBS/3 mM Glycine (Sigma, and Cyno LSR Proteins by Facs Analysis of Stably Express catalog number: G7126) for 5 minutes. After two 5-minute ing Cells washes in PBS, blocking of non-specific regions was per 0650 HEK293T and CHO K1 stably transfected cells formed with PBS/5% Bovine Serum Albumin (BSA) (Sigma, over expressing the various LSR proteins (WT and skip4) catalog number: A4503) for 20 minutes. The coverslip was (SEQ ID NOS:11, 13, 211, 212 and 31), were analyzed by then incubated in a humid chamber for 1 hour with each of the flow cytometry (FACS) using the 8C8 hybridoma clone. As primary antibodies antibodies diluted in PBS/5% BSA as shown in FIG. 14, binding of the 8C8 mAb with different indicated, followed by three 5-minute washes in PBS. The mAb concentrations to cells stably expressing the LSR pro coverslips were then incubated for 30 minutes with the cor teins (human WT (SEQ ID NO:11), cyno WT (SEQ ID responding secondary antibody diluted in PBS/2.5% BSA at NO:211), mouse WT (SEQ ID NO:31), human and cyno the indicated dilution. The antibodies and the dilutions that Skip4 variants (SEQID NOS:13, 212 respectively) was con were used are specified in Table 1. After a prewash in Hank’s siderably higher than that observed with cells transfected Balanced Salt Solutions w/o phenol red (HBSS) (Biological with the empty vector, or cells stained with the control culture Industries Catalog no. 02-016-1), the coverslip was incubated medium, indicating cell membrane expression of the five with WGA-Alexa 488 (Invitrogen, catalog number W11261) LSR proteins. diluted 1:200 in HBSS for 10 min, washed in HBSS and 0651 B. Analysis of the Expression of Endogenous LSr incubated in BISBENZIMIDE H 33258 (Sigma, catalog Protein in Various Cell Lines number: 14530) diluted 1:1000 in HBSS. The coverslip was 0652 The expression of endogenous LSR protein in vari then mounted on a slide with Gel Mount Aqueous medium ous cancer lines (derived from ovary, liver, breast, cervix and (Sigma, catalog number G0918) and cells were observed for colon, described in Table 3) was analyzed by Western Blot the presence of fluorescent product using confocal micros ting as described below. copy. 0653) Whole cell extracts (50-75 ug for the cancer cell 0645. The subcellular localization of human, cyno and lines, and 30 ug for the ectopically expressing cell lines), were mouse LSR WT(SEQID NOS:11,211,31, respectively) and analyzed by SDS-PAGE and transferred to Nitrocellulose cyno LSR skip4(SEQID NO:212) is demonstrated in FIGS. membrane as described above. The membrane was blocked 9, 10, 11, 12. LSR protein is localized mainly to the cell with TTBS (Biolab, Cath: 20892323)/5% skim milk (Difco, cytoplasm, but can also be detected on the cell Surface as Catil 232100) and incubated with anti LSR antibodies (Ab detected with anti Flag (Sigma cathi A9594) (FIGS.9A, 10A, cam.cathab596460R Sigma, cath HPAO07270) diluted in US 2014/0294765 A1 Oct. 2, 2014 56

TTBS/5% BSA (Sigma-Aldrich, A4503) at the indicated con 15, 16, 17), breast cancer cell line SK-BR-3 (lanei2) and centrations (Table 1), for 1 hour at Room Temperature. After ovarian cancer cell lines SKOV-3, OVCAR-3, ES-2, Caov-3 3 washes with in TTBS, the membrane was further incubated (lanes 8, 9, 10, 12). for 1 hour at Room Temperature with the secondary-conju gated antibodies as indicated (Table 1), diluted in TTBS. Chemiluminescence reaction was performed with ECL West Example 7 ern Blotting Detection Reagents (GE Healthcare, Cat it RPN2209) and the membrane was exposed to Super RX Fuji Generation of Mouse Monoclonal Antibodies X-Ray film (Catalog no. 4741008389). Directed Against LSR TABLE 3 0655 Production of murine monoclonal antibodies against the extra-cellular domain of human LSR protein Lane No. in (SEQID NO:10) was performed at BIOTEM (Parc d'activite FIG. Bievre Dauphine, 885 rue Alphonse Gourju, 38140 15 Cell line ATCC No. Morphology Source Disease APPRIEU, France), using a peptide immunization strategy. 2 SK-BR-3 HTB-30, Epithelial Breast Adeno The peptides that were used for the immunization were taken ATCC carcinoma from the extra cellular domain of the human LSR protein 3 MCF7 HTB-22, Epithelial Breast Adeno ATCC carcinoma (SEQ ID NO:10), and are disclosed below. 5 HeLa CCL-2, Epithelial Cervix Adeno ATCC carcinoma 0656. The first phase of the project to raise anti-LSRmAbs 6 Caco-2 HTB-37, Epithelial Colon colorectal included immunization of 3 BALB/c mice using two peptides ATCC adeno derived from the ECD region of the LSR protein (SEQ ID carcinoma 22 HT-29 HTB-38, Epithelial Colon colorectal NO:10) as follows: peptide 1: KSFCRDRIADAFSPASVD, ATCC adeno corresponding to amino acid residues 81-98 of the SEQ ID carcinoma NO:10, as set forth in SEQID NO:215, and peptide 2: CQDS 7 TOV- CRL-11731, Epithelial Ovary Primary 112D ATCC malignant VRTVRVVATKOGNA, corresponding to amino acid resi adeno dues 118-135 of SEQ ID NO:10, as set forth in SEQ ID carcinoma; NO:216. The amino acid positions are counted from the sec endometrioid carcinoma ond Met in the open reading frame. 8 SK-OV-3 HTB-77, Epithelial Ovary Adeno ATCC carcinoma 0657 The second phase of the protocol included fusion of 9 OVCAR3 HTB-161, Epithelial Ovary Adeno the lymphocytes from the immunized mice with Sp2/O-Ag14 ATCC carcinoma myeloma cells and plating out on 10 microtiter 96-well plates. 1O OV-90 CRL-11732, Epithelial Ovary Malignant ATCC papillary 0658. Mature clones were screened by ELISA using the SeOS adeno human LSR fusion protein (SEQ ID NO:236), the peptides carcinoma used for immunization (SEQ ID NOS: 215, 216), and the 11 ES2 CRL-1978, Epithelia Ovary clear cell recombinant HEK293T cells expressing human WT LSR ATCC carcinoma flag protein. FACS analysis was Subsequently carried out with 12 Caow-3 HTB-75, Epithelia Ovary Adeno ATCC carcinoma purified monoclonal Ab, 8C8, using a goat anti mouse-Alexa 4, 15 HepG2 HB-8065, Epithelia Liver Hepatocellular Fluor 488 (Invitrogen cathi A10667) as secondary Ab for ATCC carcinoma detection. 16 HepG2/ CRL-10741, Epithelia Liver Hepatocellular C3A ATCC carcinoma 0659. The third phase includes hybridoma cloning by lim 17 Hep 3B HB-8064, Epithelia Liver Hepatocellular iting dilution and stabilization, and further processing for ATCC carcinoma 18 SNU182 CRL-2235, Epithelia Liver Hepatocellular production and purification. ATCC carcinoma 0660 Culture supernatants of the hybridoma clones were 19 SKHEP-1 HTB-52, Epithelia Liver Adeno ATCC carcinoma analyzed by ELISA. As shown in Table 4, one positive clone 20 PLC/PRF5 CRL-8024, Epithelia Liver Hepatoma (8C8) was identified that showed specific binding to the ATCC human LSR fusion protein (SEQ ID NO:236) and to 21 Chang 330139, CLS Epithelia Liver Normal liver HEK293T cells over expressing the human WT LSR protein, and not to non-relevant human IgG1 fusion protein or HEK293T cells transfected with empty pRp3 vector. This 0654 FIG. 15 demonstrates endogenous expression of clone showed binding to peptide 1 (SEQID NO:215), but not LSR in various cell lines. A proteinband corresponding to the to peptide 2 (SEQID NO:216). expected ~70 kDa LSR was observed in the positive control 0661 The purified mAb, 8C8, of the positive clone was cells (lanes 1 & 14) and was also detected in several cell lines, further analyzed by flow cytometry (FACS) using HEK293T pointing to endogenous expression of LSR in liver cancer cell transfected cells over expressing the various LSR proteins lines HepE3, HepG2 and its derivative HepG2/C3A (lanes 4, (SEQID NOs: 11, 13, 211, 212, 31). US 2014/0294765 A1 Oct. 2, 2014 57

TABLE 4 Absorbances against the following antigens:

CGEN Cells Cells 15021-Fc Protein X-Fc HEK293T HEK293T. Hybridoma Peptide 1 Peptide 2 Human IgG1 Human IgG1 No antigen CGEN15022 pRp3 (Dilution of supernatant) Pure /10 Pure /10 Pure /10 Pure /10 Pure /10 Pure /10 Pure /10 8C8 1824 2.085 0.041. O.O41 1.048 1187 O.047 O.O41 0.043 O.O39 O.310 0.281 0.1OO O.134

0662. The results presented in FIG. 14 point to the speci- CDR2, CDR3 are set forth in SEQ ID NOs: 230, 231, 232, ficity of the 8C8 mAb and its suitability for FACS analysis, respectively. The corresponding amino acid sequences of 8C8 shown by the detection of the ectopic cell surface expression antibody Light chain CDR1, CDR2, CDR3 are set forth in of LSR proteins using the mab 8C8, as compared to the SEQ ID NOS: 233,234, 235, respectively. negative controls—mouse IgG2a (isotype control) and cells transfected with the empty vector. The specificity of the 8C8 SEQID NO: 217, 8C8 Heavy chain: DNA sequence (420 bp) hybridoma clone to LSR was further confirmed using siRNA Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 mediated specific knockdown of endogenous LSR cell Sur face expression on HT29 colon cancer cells endogenously, as described and shown above. ATGAACTTCGGGCTCAGATTGATTTTCCTTGTCCTTGTTTTAAAAGG

Example 8 TGTCCTGTGTGACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGA Monoclonal Antibody Sequencing AGCTTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACT 0663 Total RNA was extracted from frozen hybridoma TCAGTAGCTATTACAGCTTGGGTTCGCCAGACTCCAGAGAAGAG cells following the technical manual of TRIZolR Plus RNA GCTGGAGTTGGTCGCAGCCATAAAGTAATGGGGTAGCACCTAC Purification System (Invitrogen, Cat. No.: 15596-026). The total RNA was analyzed by agarose gel electrophoresis. Total ACCAGACACGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCC RNA was reverse transcribed into cDNA using isotype-spe cific anti-sense primers or universal primers following the AAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACAC technical manual of SuperScriptTM III First-Strand Synthesis System (Invitrogen, Cat. No. 18080-051). RT-PCR was then AGCCTTGTATTACTGTGCAAGACATGATTACTACGGTAGTAGCTTTG performed to amplify the heavy and light chains of the anti body. The antibody fragments of VH and VL were amplified CTAGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA according to the standard operating procedure of RACE of GenScript. SEQID NO: 218, 8C8 Heavy chain. Amino acids sequence 0664 Amplified antibody fragments were separately (140 AA) cloned into a standard cloning vector using standard molecu Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 lar cloning procedures. 0665 Colony PCR screening was performed to identify clones with inserts of correct sizes. MNFGRIFWLWKGVCDWKLWESGGGLWKLGGSLKLSCAASGFTESS 0666 Ten single colonies with correct VH and VL insert YYMSWVROTPEKRLELVAAINSNGGSTYYPDTWKGRFTISRDNAKNTLYL sizes were sent for sequencing. The VH and VL genes often different clones were found nearly identical. OMSSLKSEDTALYYCARHDYYGSSFAMDYWGOGTSVTVSS 0667 The consensus sequence, shown below is the sequence of the antibody produced by the hybridoma 8C8 SEQID NO: 219, 8C8 Light chain: DNA sequence (381 bp) antibody 8C8. The DNA and amino acid sequence of the Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-1-R4 heavy chain of the 8C8 antibody is shown in SEQ ID NOs: 217 and 218, respectively. The DNA and amino acid sequence of the light chain of the 8C8 antibody is shown in SEQ ID ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG NOs: 219 and 220, respectively. The leader sequence is shown in Italic font; the sequences of CDR1, CDR2, CDR3 TACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCT are shown in bold. The constant regions FR1, FR2, FR3 and FR4 are shown in a regular font. FIG. 18A presents the CTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATAGC nucleic and amino acid sequences of the 8C8 antibody Heavy AATAAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCT chain CDRs. The nucleic acid sequences of 8C8 antibody Heavy chain CDR1, CDR2, CDR3 are set forth in SEQ ID GATCTACTACACACAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTG NOs: 224, 225, 226, respectively. The corresponding amino GCAGTGGGTCTGGGACAGATTATTCTCTCACTATTAGCAACCTGGAACAA acid sequences of 8C8 antibody Heavy chain CDR1, CDR2, CDR3 are set forth in SEQID NOs: 227, 228, 229, respec GAAGATATTGCCACTTACTTTTGCCAACAGGATAGAAGCATCCGGGAC tively. FIG. 18B presents the nucleic and amino acid sequences of the 8C8 antibody Light chain CDRs. The GTTCGGTGGAGGCACCAAGCTGGAAATCAAA. nucleic acid sequences of 8C8 antibody Light chain CDR1. US 2014/0294765 A1 Oct. 2, 2014

SEQ ID NO: 220, 8C8 Light chain: Amino acids sequence SEQ ID NO. 234, 8C8 Light chain CDR2 amino acid (127 AA) Sequence Leader sequence-FR1-CDR1-1R2-CDR2-FR3-CDR3-1- R4 YSRLHS SEQ ID NO: 235, 8C8 Light chain CDR3 amino acid MMSSAOFLGLLLLCFQGTRCDIOMTOTTSSLSASLGDRVTISCRASQDIS Sequence NYLNWYOOKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEO

EDIATYFCQQDSKHPWTFGGGTKLEIK QQDSKHPWT SEQ ID NO: 224, 8C8 Heavy chain-CDR1DNA sequence Example 9

GGATTCACTTTCAGTAGCTATTACATGTCT IHC Analysis of LSR Proteins SEQ ID NO: 225, 8C8 Heavy chain-CDR2DNA sequence 0668 Calibration Study 0669. In order to establish the correctantibody concentra tion and antigen retrieval for evaluation of LSR expression, a GCCATTAATAGTAATGGGGTAGCACCTACTATCCAGACACGTGAA calibration study was carried out. GGGC 0670) Formalin Fixed Paraffin Embedded (FFPE) sections (4 lum) of cell lines: HEK293T expressing LSR and, pRp SEQ ID NO: 226, 8C8 Heavy chain-CDR3DNA sequence Empty Vector cells and full-face tissue sections of normal liver, tumour liver, breast tumour and ovarian tumour, described in Table 5 herein, were used. CATGATTACTACGGTAGTAGCTTTGCTATGGACTAC 0671 A positive control of the detection of Von Will ebrand’s factor in sections of human colon, and a positive SEQ ID NO: 227, 8C8 Heavy chain CDR1 amino acid control cell line were included in the assay to validate the Sequence secondary antibody, LSR antibodies and detection reagents. A no primary control was included. GETFssy YMs 0672. The sections were deparaffinised, antigen retrieved and rehydrated using pH9.0 Flex-- 3-in-1 antigen retrieval SEQ ID NO: 228, 8C8 Heavy chain CDR2 amino acid buffer, in PT Link apparatus at 95°C. for 20 min Following Sequence antigen retrieval, sections were placed in Flex washbuffer for 10 min, and then loaded into a DAKO Autostainer Plus. The sections were then incubated for 10 min with Flex-- Peroxi ANSNGGSTYYPDTWKG dase Blocking reagent, rinsed twice in 50 mM Tris. HCl, 150 mMNaCl, 0.1% Tween-20, pH 7.6 (TBST), followed by a 10 SEQ ID NO: 229, 8C8 Heavy chain CDR3 amino acid min incubation with Protein Block reagent (DAKO X0909). Sequence 0673. The sections were incubated for 30 min with pri mary antibody diluted in DAKO Envision Flex antibody dilu ent (DAKO Cytomation, Cat # K8006). LSR pAb was tested HDYYGSSFAMOY at: 6, 4 and 2 ug/ml in the respective sections The negative control sections were incubated with non-immune rabbit IgG SEQ ID NO: 230, 8C8 Light CDR1DNA sequence antibodies (Dako, CAT #0936) at 6, 4 and 2 ug/ml or in DAKO Envision Flex antibody diluent (no primary control). AGGGCAAGTCAGGACATTAGCAATTATTTAAAC 0674) Following incubation with primary antibodies, the sections were then rinsed twice in FLEX buffer, incubated SEQ ID NO. 231, 8C8 Light CDR2DNA sequence with anti-mouse/rabbit Flex-- HRP for 20 min, rinsed twice in FLEX buffer and then incubated with diaminobenzidine (DAB) substrate for 10 min. The chromagenic reaction was TACACACAAGATTACACTCA stopped by rinsing the slides with distilled water. Following chromagenesis, the sections were counterstained with hae SEQ ID NO. 232, 8C8 Light CDR3DNA sequence matoxylin, dehydrated in an ascending series of ethanols (90-99-100%), cleared in three changes of xylene and cover slipped under DePeX. Stained sections were analyzed, and CAACAGGATAGTAAGCATCCGTGGACG Suitable digital images captured, using an Olympus BX51 SEQ ID NO. 233, 8C8 Light chain CDR1 amino acid microscope with a Leica DFC290 camera. Sequence IHC Tissue Microarray (TMA) 0675. The aim of the study was to determine the LSR RASQDISNYLN expression in various cancerous tissues using LSR specific antibodies according to at least Some embodiments of the US 2014/0294765 A1 Oct. 2, 2014 59 invention. The distribution of LSR informalin-fixed/paraffin Panel A, demonstrating valid antibody working conditions. embedded (FFPE) sections was examined using immunohis The adjacent no primary section (Panel B) shows no apparent tochemistry (IHC). immunoreactivity. Specific immunoreactivity was detected in 0676 Using polyclonal Rabbit antibodies, as described positive control tissues in both normal and tumour liver above, in FFPE sections of tumour and normal tissue microar samples and in breast tumour samples. The LSR antibody ray (mutli-tumour TMA) and in full face sections of normal demonstrated membrane and cytoplasmatic staining in the lymph node, tonsiland spleen from three donors, as described two normal liver donors and in one tumour donor. in Table 6 herein. The TMA comprised 11 tissue types (Table 0678 Specific LSR immunoreactivity was detected in 3): breast, colon, lymphoid and prostate (8 tumour and 2 hepatocytes and the bile duct epithelium of the two normal normal samples of each), gastric, ovary, brain, kidney, liver liversamples at 6 and 4 ug/ml. In tumour liver, membrane and and skin (4 tumour and 2 normal samples of each), and lung cytoplasmatic LSR immunoreactivity was seen, and was (8 non-Small cell tumour and 4 Small cell tumour samples, markedly intense in tumour cells compared to the normal and 4 normal samples). Additional normal tissues, sections of liver samples. In ovarian tumour (DI 16974), specific mem lymph node (n=3), tonsil (n=3) and spleen (n-3) were sec brane and cytoplasmatic LSR immunoreactivity was seen at tioned and used in this study (Table 7). FFPE sections (4 um) the highest concentration (6 ug/ml). At 4 Lug/ml, only cyto of cell line HEK293T expressing LSR, the multi-tumour plasmic immunoreactivity was noted in the tumour cells. In TMA and full-face sections of normal lymph node, tonsiland sample DI 16976, specific cytoplasmic immunoreactivity spleen were used. The sections were de-paraffinised, antigen was only observed in tumour cells at both 6 and 4 ug/ml. retrieved and rehydrated using pH9.0 Flex-- 3-in-1 antigen Non-specific immunoreactivity was detected, but only retrieval buffers, in PT Link apparatus at 95°C. for 20 min present at the highest concentration in both donor samples. with automatic heating and cooling. Following antigen 0679. No apparent LSR immunoreactivity was detected in retrieval, sections were washed in distilled water for 2x5 min pRp3 empty vector cells. Non-specific immunoreactivity was then loaded into a DAKO Autostainer Plus. The sections were detected. then incubated for 10 min with Flex-- Peroxidase Blocking 0680 LSR immunoreactive tissues have shown a varied in reagent, rinsed twice in 50 mM Tris. HCl, 150 mM. NaCl, staining pattern and the level of intensity amongst tumour 0.1% Tween-20, pH 7.6 (TBST), followed by a 10 min incu types. It was however noted that tumours of colon and liver bation with Protein Block reagent (DAKOX0909). The sec stained the most intense, while other tumours showed mod tions were incubated for 30 min with primary antibody erate staining throughout the samples. In the majority of diluted in DAKO Envision Flex antibody diluent (DAKO cores, immunoreactivity averaged 75-100%. Table 8 presents Cytomation, Cat if K8006). LSR poly clonal antibody was the full analysis of the TMA. applied at 6 ug/ml. The negative control sections were incu 0681 LSR Expression in Breast Tumors bated with non-immune rabbit IgG antibodies (Dako, CAT 0682. Within the breast tumour set, the intensity of stain i0936) at 6 ug/ml or in DAKO Envision Flex antibody diluent ing varied in the range of (1-2+) with two tumours scoring2+. (no primary control). Following incubation with primary Only one core of breast tumour within the array was 0-25% antibodies, the sections were then rinsed twice in FLEX immunoreactive, the majority were in the region of 75-100% buffer, incubated with anti-mouse/rabbit Flex-- HRP for 20 immunoreactive. The 2+ scoring tumours were invasive duc min, rinsed twice in FLEX buffer and then incubated with tal carcinomas (IDCs). diaminobenzidine (DAB) substrate for 10 min. The chroma 0683 LSR Expression in Large Bowel Cancer genic reaction was stopped by rinsing the slides with distilled 0684 Within the large bowel cohort, eight samples were water. Following chromagenesis, the sections were counter of moderately differentiated adenocarcinoma tumours. Six stained with haematoxylin, dehydrated in an ascending series samples scored a (2+ or 3+), one scored the strongest level of of ethanols (90-99-100%), and cleared in three changes of immunoreactivity of (3+), and one sample scored (0-1+). All xylene and coverslipped under DePeX. Stained sections were samples were 75-100% immunoreactive. In the one normal analyzed, and Suitable digital images captured, using an sample, specific cytoplasmic immunoreactivity was seen in Olympus BX51 microscope with a Leica DFC290 camera. the ascending portion of the mucosal epithelium, where the The sections were analysed for the intensity of the specific descending crypts exhibited a membrane-bound phenotype staining and a semi-quantitative scoring system was used. with a staining score of (2-3). The core in the tissue array with the most intense LSR immu 0685 LSR Expression in Prostate Cancer noreactivity was assigned a score of 3+and the intensities of 0686. In the prostate tumours, the level of immunoreactiv the immunoreactivity in the other cores were scored relative ity varied between (1-2+). One tumour recorded a 2+ score to that of the 3+core. The percentage of LSR immunoreactive (Gleason score 9) and two recorded a 1-2 score (Gleason tumour was estimated and recorded using the following scores 9 and 7) and three scored 1+ from a cohort of 8 ranges: 0-25%, 25-50%, 50-75% and 75-100%. adenocarcinomas. All prostate tumours appeared to be LSR immunoreactive. In these cores, immunoreactivity was cyto Results: plasmic with exceptions where a prominent membrane phe 0677. The following dataset represents the optimisation notype was observed within the tumour epithelium. Within and detection of LSR Rabbit polyclonal antibody in specified the normal prostate samples, staining was seen in the glandu positive control tissues and in an empty vector cell line. Assay lar epithelium and occasional Smooth muscle regions with positive controls demonstrating internal assay working con staining intensity of +1-+2. ditions and antibody validation are shown in FIG. 19. FIG. 19 0687 LSR Expression in Lymphoma (X60) shows sections of LSR positive cells following pH9.0 0688. Within lymphoma samples, a lower level of staining AR. Panel A shows the slides incubated with LSR antibody was observed three samples of NHL scored (1+) and five of applied at 2 g/ml. Panel B shows the adjacent no primary NHL and HLwith (0-1+), ranging in 75-100% immunoreac incubated sections. Positive LSR immunoreactivity is seen in tivity within cores (HL sample had an IHC score of 0-1). It US 2014/0294765 A1 Oct. 2, 2014 60 was however noted that in one donor (15052), a discrete cells. One donor (donor 9516) in particular, showed nuclear population of tumourcells demonstrated higher level of stain membrane staining (2+), compared to donor 13845, with a ing. In two samples of normal lymph node, cytoplasmic stain less-intense stain (0-1+). One patient with grade 2 astrocy ing was seen in one sample within the germinal centres. A few tome had an IHC Scone of 0. No correlation can be made discrete immune cells were observed to demonstrate immu between tumour type and level of immunoreactivity. In nor noreactivity within the other sample. malbrain, one sample demonstrated apparently specific cyto 0689 LSR Expression in Lung Cancer: plasmic immunoreactivity within the neuropil cells and other 0690. In the lung tumour set, eight out of twelve tumour observed neuro-fibres. samples exhibited specific LSR immunereactivity. The inten LSR Expression in Renal Tumors sity and immunoreactivity seen was varied within tumour 0699 types. In one sample of NSCL adenocarcinoma, the tumours 0700. In the renal carcinomas, three clear-cell type were strongly immunoreactive with a score of 2+-3+, where tumours and one non-clear cell carcinoma demonstrated LSR in two samples of squamous carcinoma—an intensity score immunoreactivity. The level of intensity varied between of (2+) and (1+-2+) was seen. Two samples of NSCLC had an 1+-2+ with 75-100% of tumour cells being immunoreactive intensity level of 1+-2+ and both were poorly to moderately in each core. It was also noted that clear-cell carcinomas differentiated. In three adenocarcinoma samples, two dem showed a cytoplasmic membrane-specific bound staining onstrated a staining intensity Score of 2+ where the other pattern compared to less differentiated cell types. In normal sample showed a staining score of only 1+, and was poorly kidney samples, specific cytoplasmic staining was seen in the differentiated. Most tumours were seen to be 75-100% immu collecting tubules. noreactive. In the cohort of Small cell carcinoma samples, no 0701 LSR Expression in Liver Tumors apparent immunoreactivity was seen in tumour cells; only occasional immunoreactivity was seen in putative macroph 0702. In liver tumours, all three samples demonstrated ages. In samples of normal lung, positive cytoplasmic immu prominent immunoreactivity in tumour cells, where 75-100% noreactivity was seen in the respiratory epithelium. Occa of tumour cells were stained in each core. Donor 19115 was sional pneumocytes were also seen to be immunoreactive assigned a score of 3+, (and was used to score other cores with intensity of +1 to +2. relative to its intensity level) as this was the most intensely (0691 LSR Expression in Stomach Tumor stained core in the array. It was also noted that in a Subset of 0692. In stomach tumor, four adenocarcinoma samples cells an intense level of cytoplasmic staining was observed, (moderately differentiated) demonstrated apparently LSR compared to the Surrounding tumours, with occasional mem expression within the tumours, of which, 75-100% were brane-associated immunoreactivity. In normal liver, specific immunoreactive in each core. All samples were seen to vary membrane immunoreactivity was seen. in the level of scoring with one sample scoring 2+-3+, where 0703 LSR Expression in Normal Lymphatic Tissue the rest of the samples scored 1 + or 2+. It was noted in certain tissues—occasional prominent membrane-associated Stain 0704. In full-face sections of normal lymph node, tonsil ing was seen, with infiltrating putative macrophages also and spleen, it was observed that the majority demonstrated demonstrating immunoreactivity. Within the normal stomach positive cytoplasmic staining in the germinal centres from the tissue, apparently specific cytoplasmic immunoreactivity was three tissue sets, with a few putative macrophages also show seen in the mucosal epithelium. In one particular donor ing immunoreactivity. In lymph node, some cells were (2874), cytoplasmic and nuclear staining was seen, with observed to show staining of the cytoplasmic-membrane intensity of +1 to +2. within the lymphoid follicle and occasional smooth muscle (0693 LSR Expression in Ovarian Cancer was also stained. 0694. The ovarian carcinoma cohort also demonstrated specific diffuse-cytoplasmic immunoreactivity in tumours TABLE 5 with cores 75-100% immunoreactive. Staining intensity was TMA samples characterisitcs (calibration to be variable within tumour types. Two samples of serous papillary carcinoma scored 0-1+ and 2+ respectively, with a Donor few tumour cells showing a darker-intense staining pattern Tissue ID Age Sex Clinical diagnosis Pathology (Donor 13003). In the granulosa tumour, this was scored Liver 3323 77 Female Pulmonary Sections of relatively weak (0-1+), however, the serous cystadenocarci parenchyma. embolism (COD); normal liver Pulmonary noma sample was seen to have an intense stain of 2+with infarction(COD); associated-membrane immunoreactivity (Donor 94.07). In Myocardial normal ovary tissues, some specific immunoreactivity was infarction noted in only one sample in the Stromal region with staining Liver 14453 15 Male Cerebral Sections of parenchyma. infarction(CoD); normal liver intensity of +2. Cardiomyopathy; with portal (0695 LSR Expression in Melanoma Penicillin allergy tracts 0696. In skin melanoma, staining was seen weak (0-1+), Tumour 14826 66 Female Hepatocellular Moderately liver carcinoma; differentiated with 25-50% of the tumour cells being immunoreactive. No Alcoholic hepatocellular apparent membrane-associated Staining was observed within cirrhosis: carcinoma this tissue. In normal skin, no immunoreactivity was seen Cholecystitis, within the epidermis. Only occasional dermal lymphocytes chronic were positively stained. Tumour 16974 57 Female Cystadeno- Cystadeno ovary carcinoma, ovary; carcinoma (0697. LSR Expression in Brain Tumor Heart disease: 0698 Inbrain tumour, (grade 4-astrocytoma) two samples Diabetes demonstrated immunoreactivity in 75-100% of the tumour US 2014/0294765 A1 Oct. 2, 2014

TABLE 5-continued TABLE 6-continued TMA samples characterisitcs (calibration Cancer TMA samples characteristics Donor Donor Tissue ID Age Sex Clinical diagnosis Pathology Map ID ID Tissue Age Sex Tumour 16976 49 Female Cystadeno- Cystadeno 28 5756 tumour: prostate: adenocarcinoma 68 Male Ovary carcinoma, carcinoma 29 5678 Normal: Prostate Gland 48 Male ovary; Hyper 30 3951 Normal: Prostate Gland 37 Male tension; Diabetes 31 5052 Lymphoma 45 Female Breast 13533 73 Female Breast carcinoma: Poorly 32 576.0 Tumour: lymphoma 72 Female tumour Intraductal breast differentiated 33 7549 Tumour: lymphoma 47 Male carcinoma; Grade 3 carcinoma 34 5039 Lymphoma 47 Male consistent 35 5034 Lymphoma 71 Male with 36 5037 Lymphoma 53 Female grade 3 ductal 37 7547 Lymphoma carcinoma of 38 5775 Tumour: lymphoma 75 Female breast. 39 4655 Normal: lymph-node 1 Female Breast 4957 86 Female Breast carcinoma: Sections of 40 0789 Normal: lymph-node 58. Male tumour Adenocarcinoma; normal breast 41 2053 tumour: lung 72 Male Hypertension; tissue 42 5772 Tumour: lung: non-Small cell 44 Male Leiomyoma; carcinoma Arthritis; Migraine 43 3586 tumour: lung 67 Female Hek293T- Positive Positive 44 2760 tumour: lung: squamous-cell- 64 Male LSR transfected carcinoma cells 45 9354 tumour: lung: adenocarcinoma 63 Male Hek293T- Negative Negative' 46 3473 tumour: lung: adenocarcinoma 72 Male empty 47 2765 tumour: lung: adenocarcinoma 64 Female Prp vector' cell 48 4852 tumour: lung: adenocarcinoma 56 Female lines 49 10414 small cell 74 Male 50 15055 tumour: lung: small cell 52 Male 51 15054 tumour: lung: small cell 65 Male 52 15053 tumour: lung: small cell 52 Male TABLE 6 53 1311 lung: parenchyma. 36 Female S4 141 ung: parenchyma 39 Female Cancer TMA samples characteristics 55 5767 Normal: lung: parenchyma. 45 Male 56 2649 Normal: lung: parenchyma. 37 Male Donor Map ID ID Tissue Age Sex 57 14551 tumour: stomach 83 Female 58 10656 tumour: stomach 74 Male 1 8589 tumour: breast: ductal- 46 Female 59 13200 tumour: stomach 85 Male adenocarcinoma 60 2295 tumour: stomach 66 Fema 2 8707 tumour: breast: ductal- 46 Female 61 13665 stomach: body 57 Fema adenocarcinoma 62 2874 stomach: body 53 Male 3 8723 tumour: breast: ductal- 74 Female 63 12998 tumour: ovary 78 Female adenocarcinoma 64 13003 tumour: ovary 74 Female 4 15778 tumour: breast: lobular carcinoma 52 Female 65 5739 tumour: ovary 48 Female 5 3724 tumour: breast: ductal- 82 Female 66 94.07 tumour: ovary 75 Female adenocarcinoma 67 4739 Normal: ovary 42 Female 6 2953 tumour: breast: ductal- 67 Female 68 4781 Normal: ovary 34 Female adenocarcinoma 69 5759 Tumour: skin melanoma 65 Male 7 9132 tumour: breast: ductal- 82 Female 70 5753 Tumour: skin melanoma 46 Female adenocarcinoma 8 9298 tumour: breast: ductal- 73 Female 71 5038 Tumour: skin melanoma 41 Male adenocarcinoma 72 5343 Tumour: skin melanoma 24 Male 9 5704 Normal: breast 46 Female 73 3110 Normal: skin 22 Fema 10 5347 Normal: breast 64 Female 74 5415 Normal: skin 45 Fema 11 3550 tumour: colon: adenocarcinoma 61 Male 75 5342 Tumour: brain: glioblastoma 56 Male 12 15767 Tumour: large 58. Female multiforme intestine: adenocarcinoma 76 9514 tumour: brain 17 Male 13 4537 intestine: adenocarcinoma Sigmoid 44 Female 77 3845 tumour: brain 58. Male colon carcinoma; 78 9516 tumour: brain 25 Female 14 2206 tumour: colon: adenocarcinoma 76 Female 79 2007 Normal: brain: cortex: frontal 40 Male 15 9542 tumour: colon: adenocarcinoma 73 Male 8O 4585 Normal: brain: cortex: frontal 85 Male 16 2893 tumour: colon: adenocarcinoma 62 Male 81 3266 tumour: kidney 71 Male 17 15764 Tumour: large 75 Female 82 4125 tumour: kidney 41 Male intestine: adenocarcinoma 83 4764 tumour: kidney 66 Male 18 15763 Tumour: large 69 Female 84 9043 tumour: kidney 45 Male intestine: adenocarcinoma 19 2681 Normal: colon 54 Female 85 2874 Normal: kidney: cortex 53 Male 2O 3121 Normal: colon 34 Male 86 4818 Normal: kidney: cortex 52 Female 21 15296 tumour: prostate 68 Male 87 19115 Tumour: liver 22 15295 tumour: prostate: adenocarcinoma 71 Male 88 15757 Tumour: liver 25 Male 23 15301 tumour: prostate: adenocarcinoma 51 Male 89 14826 tumour: liver 66 Female 24 15758 tumour: prostate: adenocarcinoma 74 Male 90 19114 Tumour: liver 25 15745 tumour: prostate: adenocarcinoma 52 Male 91 1991 Normal: liver: parenchyma. 79 Female 26 15777 tumour: prostate: adenocarcinoma 68 Male 92 3123 Normal: liver: parenchyma. 31 Male 27 15755 tumour: prostate: adenocarcinoma 55 Male US 2014/0294765 A1 Oct. 2, 2014 62

TABLE 7 TABLE 7-continued Lympatic organ TMA samples charateristics Lympatic organ TMA samples charateristics Clinical Clinical Tissue ID Age Sex diagnosis Pathology Report Tissue ID Age Sex diagnosis Pathology Report Lymph- 53524 73 F Subarachnoid Sections of lymph node Tonsil 1OO45 2S F Tonsillitis Normal tonsillar tissue node haemorrhage showing normal histological including epithelium and (COD); eatures with lymphoid lymphoid follicles. Hypertension; aggregates and well defined Tonsil 11024 6 M Tonsillitis, Tonsil with few neutrophils Non-insulin sinuses. DIAGNOSIS: nor chronic; in the epithelium. mal Dyspnoea dependent ymph node. Spleen 14345 60 M Tonsillitis Normal spleen. White and diabetes red pulp present. mellitus Spleen 13851 18 F Intracerebral Normal spleen with normal Lymph- 3217 36 M Intracranial Sections of lymph node haemorrhage red and white pulp node haemorrhage showing normal histological (CoD); identified. Moderate (COD); Organ features with lymphoid Hypertension; preservation donor aggregates and well defined Hyper sinuses. DIAGNOSIS: nor lipidaemia; mal Non-insulin ymph node. dependent Lymph- 9191 32 M Pulmonary A lymph node containing diabetes node arterial many macrophages which mellitus; hypertension are filled with anthracotic Arthritis and heart pigment. No significant Spleen 15947 53 M Intracranial Normal spleen. defect pathological abnormality. haemorrhage Tonsil 10821, 17 F Tonsillitis, Non dysplastic squamous (CoD); chronic epithelium overlying normal Endometriosis onsillar lymphoid tissue.

TABLE 8 cancer TMA samples IHC analysis

% Tumour tumour Map immuno immuno ID Tissue Diagnosis reactivity reactivity coments 1 tumour: breast: ductal Intra duct and 1-2+ adenocarcinoma invasive ductal carcinoma 2 tumour: breast: ductal Invasive ductal O adenocarcinoma carcinoma 3 tumour: breast: ductal Primary 2+ 75 adenocarcinoma (invasive ductal pattern) 4 tumour: breast: lobular Core loss carcinoma 5 tumour: breast: ductal 2+ 7S-100 adenocarcinoma 6 tumour: breast: ductal DCIS (ductal adenocarcinoma carcinoma in Core loss situ) 7 tumour: breast: ductal sarcomatoid Core loss adenocarcinoma ductal 8 tumour: breast: ductal Invasive ductal Core loss adenocarcinoma 9 breast Normal breast Core loss tissue 10 breast Normal breast Core loss tissue 11 tumour: colon: Differentiated 2-3+ 75-1OO tumour adenocarcinoma adenocarcinoma cells. Putative macrophages 12 Tumour: large Moderately Core loss intestine: differentiated adenocarcinoma 13 Sigmoid colon adenocarcinoma. 3+ 7S-100 carcinoma; Moderately differentiated 14 tumour: colon: invasive 2-3+ 7S-100 adenocarcinoma US 2014/0294765 A1 Oct. 2, 2014 63

TABLE 8-continued cancer TMA samples IHC analysis

% Tumour tumour Map immuno immuno ID Tissue Diagnosis reactivity reactivity connents 15 tumour: colon: Moderately 7S-100 adenocarcinoma di erentiated adenocarcinoma. 16 tumour: colon: We 2-3+ 7S-100 adenocarcinoma di erentiated adenocarcinoma 17 Tumour: large Moderately 7S-100 Core is intestine: di erentiated 75% adenocarcinoma adenocarcinoma. necrotic 18 Tumour: large Moderately 2-3+ 7S-100 intestine: di erentiated adenocarcinoma adenocarcinoma. 19 colon No rmal colon: 2-3 ful thickness. Mucosal staining - Mucosal staining - colon Fu I thickness Core loss normal colon 21 tumour: prostate A. enocarcinoma Core loss 22 tumour: prostate: A. enocarcinoma 1+ 50-75 adenocarcinoma Gleason Score 3 + 3 = 6 23 tumour: prostate: Gleason Score 1+ adenocarcinoma 3 + 4 = 7 26 tumour: prostate: Gleason Score 1+ adenocarcinoma 4 + 4 = 8 27 tumour: prostate: Gleason Score 1-2+ adenocarcinoma 3 + 4 = 7 28 tumour: prostate: Gleason Score 2+ 7S-100 adenocarcinoma 4 - 5 = 9 29 Prostate Gland No rmal 1+ prostatic tissue cytoplasmic staining in glandular epithelium. Occasional membrane stain, 1+ staining in Smooth muscle 30 Prostate Gland No rmal prostate 1+ prominent membrane staining in luminal Surface. Core 75% loss 31 Lymphoma Lymph node 1+ Discrete infiltrated by population large cell of lymphoma tumour cells - darker staining (2+). 32 Tumour: lymphoma Low Grade Non 1+ 50-75 Hodgkin's Lymphoma 33 Tumour: lymphoma 7S-100 34 Lymphoma High grade Non 7S-100 Hodgkin's Lymphoma 35 Lymphoma No n-Hodgkin's 7S-100 Lymphoma 36 Lymphoma No n-Hodgkin's 1+ 7S-100 Lymphoma US 2014/0294765 A1 Oct. 2, 2014 64

TABLE 8-continued cancer TMA samples IHC analysis

% Tumour tumour Map immuno immuno ID Tissue Diagnosis reactivity reactivity connents 37 Lymphoma 75 38 Tumour: lymphoma Hodgkin's 75 Lymphoma 39 lymph-node O-1 cytoplasmic staining. In sub-set of immune cells - macs. Staining only in these 40 lymph-node Normal lymph 1-2- node. cytoplasmic staining in germinal centres - diffuse staining. 41 tumour: lung Poorly 7S-100 differentiated non-Small cell carcinoma with Some squamoid features 42 Tumour: lung: non Poorly 1-2+ Small cell carcinoma Differentiated non-small Cell Carcinoma 43 tumour: lung Moderately to 1-2+ 7S-100 poorly differentiated Squamous carcinoma. 44 tumour: lung: Moderately well 2+ 7S-100 squamous-cell-carcinoma differentiated keratinising Squamous cell carcinoma 45 tumour: lung: large cell type 2-3+ 7S-100 adenocarcinoma showing features of an adenocarcinoma 46 tumour: lung: Poorly 1+ 7S-100 adenocarcinoma differentiated adenocarcinoma 49 tumour: lung Small cell Negative staining 50 tumour: lung Small cell Negative staining 51 tumour: lung Small cell Negative staining 52 tumour: lung Small cell Negative staining 53 ung: parenchyma. Normal lung 1+ Cytoplasmic staining in respiratory epithelium. lung: parenchyma. Normal lung terminal bronchiole columnar epithelium staining (1-2+) 55 lung: parenchyma. Normal lung Terminal bronchiole columnar epithelium US 2014/0294765 A1 Oct. 2, 2014 65

TABLE 8-continued cancer TMA samples IHC analysis

% Tumour tumour immuno immuno ID Tissue Diagnosis reactivity reactivity coments staining (1-2+). Macrophages 1+ staining. 56 lung: parenchyma. Normal lung No alveolar staining. Macrophages positive staining (0-1+) 57 tumour: Stomach Moderately 7S-100 differentiated adenocarcinoma 58 tumour: Stomach Moderately 1+ 7S-100 differentiated adenocarcinoma 59 tumour: Stomach Moderately 1+ 7S-100 Diffuse differentiated cytoplasmic adenocarcinoma staining. Macrophages (1+) staining. 60 tumour: Stomach Moderately 2+ 7S-100 differentiated adenocarcinoma 61 stomach: body Normal stomach 1+ Cytoplasmic and membrane staining in mucosal epithelium 62 stomach: body Normal stomach 1-2- Cytoplasmic and membrane staining in mucosal epithelium. Occasional nuclear staining. 63 tumour: ovary A serous 2+ 7S-100 papillary cystic carcinoma. 64 tumour: ovary Invasive serous 0-1+ 7S-100 papillary carcinoma. 65 tumour: ovary Granulosa cell 7S-100 tumour. 66 tumour: ovary Serous 7S-100 cystadenocarcinoma 67 ovary Normal ovary Granular epithelium occasional discreet cells (2+) staining. Stromal region - negative staining, 68 ovary Normal ovary No staining observed. 69 Tumour: skin Malignant NA PD Melanoma 70 Tumour: skin High grade Core loss malignant Melanoma US 2014/0294765 A1 Oct. 2, 2014 66

TABLE 8-continued cancer TMA samples IHC analysis

% Tumour tumour immuno immuno ID Tissue Diagnosis reactivity reactivity coments 71 melanoma Malignant 25-50 melanoma 72 Tumour: skin Malignant O O Melanoma 73 skin Normal skin. Epidermis negative staining 74 skin Normal skin. Epidermis negative staining. Occasional dermal lymphocytes positive (1+) staining. 75 Tumour: brain Glioblastoma Core loss multiforme 76 tumour: brain Astrocytoma; O No grade 2. staining observed 77 tumour: brain Glioblastoma 7S-100 multiforme: synonym grade 4 Astrocytoma 78 tumour: brain Astrocytoma; 7S-100 grade 4. 79 brain: cortex: frontal Normal brain O-1 Cytoplasmic staining in neuropil. brain: cortex: frontal Normal brain Negative cortex staining observed. 81 tumour: kidney We 7S-100 differentiated renal clear cell carcinoma 82 tumour: kidney Histology 1+ 7S-100 consistent with renal cell carcinoma. 83 tumour: kidney Renal cell (clear 1-2+ 50-75 cell) carcinoma 84 tumour: kidney Clear cell renal 1-2+ 7S-100 cell carcinoma of kidney. 85 kidney: cortex Normal renal 0-1+ cortex Cytoplasmic staining in collecting tubules 86 kidney: cortex Normal renal 2+ cortex. Cytoplasmic staining in collecting tubules 87 Tumour: liver Hepatocellular 3+ 7S-100 carcinoma 88 Tumour: liver Fibrolamellar 1+ Hepatocellular Carcinoma 89 tumour: liver Low Grade 2+ 7S-100 hepatocellular carcinoma 90 Tumour: liver Hepatocellular Core loss carcinoma US 2014/0294765 A1 Oct. 2, 2014 67

TABLE 8-continued cancer TMA samples IHC analysis

Tumour tumour Map immuno immuno ID Tissue Diagnosis reactivity reactivity coments 91 liver: parenchyma Normal liver O-1 Cytoplasmic membrane staining 92 liver: parenchyma Liver - normal O-1 limits. Membrane staining

Top 4Tm Tissue Microarray Study Using LSR Specific Pab: regions, infiltrating immune cells were also positively stained. The majority of tumours were mainly infiltrating (0705. The Top 4 TMA consisted of cores from 4 tissue ductal and lobular carcinomas—mixed grades. There was no types: breast (4 normal and 26 tumors), large intestine (4 observed pattern of immunoreactivity that could be specifi normal and 26 tumors), lung (4 normal and 26 tumors) and cally attributed to tumor type or grade. In normal breast, prostate (4 normal and 26 tumors). The TMA layout is shown specific cytoplasmic immunoreactivity was seen within the in Table 9. FFPE sections (4 um) of cell line HEK293T LSR, glandular acini. the Top4 multi-tumor TMA were used. Unless otherwise 0707. Within the large bowel cohort, the adenocarcinoma indicated, all incubations were carried out at room tempera samples were all immunoreactive, with the exception of one ture. The sections were de-paraffinized, antigen retrieved and sample in this study. Within the cohort, the majority of rehydrated using pH9.0Flex-3-in-1 antigen retrieval buffers, tumours were poor to well differentiated types. In eight samples, an assigned score of 3+ staining was seen within in PT Link apparatus at 95°C. for 20 min with automatic 25-100% of tumours of grades 2/3. Most of these tumours had heating and cooling. Following antigen retrieval, sections a distinct feature exhibiting a prominent cytoplasmic-mem were washed in Flex (TBST) buffer for 2x5 min then loaded brane phenotype. In ten samples, a score of 2+ was seen in into a DAKO Autostainer Plus. The sections were then incu 25-100% of reactive tumor grades 2/3. In six samples, a lower bated for 10 min with Flex-- Peroxidase Blocking reagent, score of 1 + was assigned to tumours, with the majority dem rinsed twice in 50 mM Tris. HCl, 150 mMNaCl, 0.1% Tween onstrating 0-25%-50-75% reactivity. An apparently consis 20, pH 7.6 (TBST), followed by a 10 min incubation with tent pattern of staining, relative to the assigned intensity Protein Block reagent (DAKO X0909). The sections were scores was seen in these tumor sets. Higher scores demon incubated for 30 min with primary antibody diluted in DAKO strated a prominent membrane phenotype. In normal tissue Envision Flex antibody diluent (DAKO Cytomation, Cat it samples, specific immunoreactivity was detected in mucosal K8006). Anti LSR pAb (Abcam ab169583) was applied at 6 epithelium and micro-vascular elements. ug/ml. Anti Von Willebrand factor (VWf) antibody was 0708. In the lung tumorset, specific immunoreactivity was applied at 1 ug/ml. The negative control sections were incu seen in the majority of tumours investigated, where a weak to bated with non-immune rabbit IgG antibodies (Dako, CAT moderate staining intensity was noted. The majority of i0936) at 6 and 1 lug/ml or in DAKO Envision Flex antibody tumours were non-small cell carcinomas (NSCLC)—of diluent (no primary control). Following incubation with adenocarcinoma origin, of well to poorly differentiated cell primary antibodies, the sections were then rinsed twice in types. In these tumours, six samples were assigned a maxi FLEX buffer, incubated with anti-mouse/rabbit Flex--HRP mum intensity score of 2+ staining, of which 50-100% of for 20 min, rinsed twice in FLEX buffer and then incubated tumours were immunoreactive in most respective cores. with diaminobenzidine (DAB) substrate for 10 min The chro Eighteen samples were assigned a weaker score of 1/1+ stain mogenic reaction was stopped by rinsing the slides with dis ing, within 25-100% of tumours. In one sample of small-cell tilled water. Following chromagenesis, the sections were carcinoma, specific cytoplasmic immunoreactivity was seen counterstained with haematoxylin, dehydrated in an ascend to be weakly stained (1+), within 25-100% of tumours over ing series of ethanols (90-99-100%), and cleared in three the three cores analyzed. Other notable staining features were changes of xylene and coverslipped under DePeX. Stained seen in vascular elements, and intensely-stained infiltrating sections were analyzed, and Suitable digital images captured, immune cells. In the normal lung tissue, specific immunore using an Olympus BX51 microscope with a Leica DFC290 activity was detected in a sample of bronchiole epithelium, CaCa. and free macrophages of the alveolar spaces. Negative stain 0706. Within the breast tumorset, the intensity of staining ing was seen in the alveolar septa. was heterogeneous in the majority of cases; the immunore 0709. In the set of prostate tumours, specific staining was activity seen was weak to moderate. In this cohort, two seen in most samples, where intensity of staining was weak to samples scored a maximum intensity of 3-3+within 50-100% moderate in the tumorepithelium. In one sample, a maximum of reactive tumours. Within fourteen of the samples, the stain assigned score of (3) staining was seen in 75-100% of ing intensity scored a maximum of 2+, within 25-100% of tumours, (Gleason score 4+5), with a membranous pattern of tumours (grades 2/3). Ten other samples scored a lower inten immunoreactivity. Seven other samples had a score of 2+ sity of 1+, of which most tumor samples were 25-50% reac within 25-100% of tumours. Most of these resided in tumor tive. Few other reactive tumor samples were also seen to be islands, with Gleason scores ranging from (3+4) and (4+3) 0-25% to 75-100% weakly stained. Within the stromal respectively. Lastly, seventeen tumor samples were scored a US 2014/0294765 A1 Oct. 2, 2014 68 weaker 1/1+ staining in 25-100% of tumours. In the normal glandular epithelium. In general, immunoreactivity was prostate tissues, a few samples demonstrated weak-cytoplas either blush staining or negative in epithelium. Other notable mic and occasional cytoplasmic-membrane staining in the staining was seen in putative infiltrating immune cells. TABLE 9 Top4 TMA tissue annotation Case Tissue ID AGE SEX Pathology Breas S1168 40 Norma Breas 14784 38 Norma Breas 11292 53 Norma Breas 33349 61 Norma Breas 8998 45 infiltrating Ductal Carcinoma. Grade 2/3 Breas 91OO 36 infiltrating Ductal Carcinoma. Grade 2/3 Breas 9553 53 infiltrating Ductal Carcinoma. Grade 2 Breas 26375 87 infiltrating Ductal Carcinoma. Moderately Differentiated Breas 36386 67 infiltrating Ductal Carcinoma. Grade 3 Breas 41690 70 F Medullary Carcinoma. Grade 2 Breas 898O 45 infiltrating Ductal Carcinoma. Grade 1/3 Breas 91.33 42 infiltrating Ductal Carcinoma. Grade 2 Breas 9536 44 infiltrating Ductal Carcinoma Breas 12281 33 infiltrating Ductal Carcinoma 2 Breas 33139 S4 infiltrating Ductal Carcinoma Grade III Breas 16996 74 infiltrating Ductal Carcinoma. Grade 1 Breas 33.162 75 infiltrating Ductal Carcinoma Grade III Breas 33119 43 infiltrating Ductal Carcinoma Ductal carcinoma in situ Grade III Breas 12759 49 infiltrating ductal carcinoma Ductal carcinoma in situ Grade II Breas 15537 61 infiltrating Ductal Carcinoma Poorly Differentiated Breas 16957 63 infiltrating Ductal Carcinoma Fibrocystic change. Grade 33 Breas 17916 75 infiltrating Ductal Carcinoma Breas 18381 37 infiltrating Ductal Carcinoma. Grade 3/3 Breas 27813 53 infiltrating Ductal Carcinoma of the breast. Grade 3 Breas S9919 53 infiltrating ductal carcinoma of the breast. Grade II Breas 35405 53 infiltrating Ductal Carcinoma, Lobular carcinoma of he breast. Grade 3 Breas S4741 43 infiltrating ductal mixed with other types of carcinoma of the breast. Grade II Breas 54876 51 infiltrating ductal carcinoma of the breast. Grade II Breas 55082 62 infiltrating ductal carcinoma of the breast. Grade II Breas 55238 70 infiltrating ductal and lobular carcinoma of the breast. Grade II Rectum 9533 66 M Norma Rectum 96.08 53 M Norma Rectum 16049 45 M Norma Colon 17038 49 F Norma Colon 36317 73 F Moderately differentiated: Adenocarcinoma of the large intestine Colon 19746 89 M Moderately Differentiated: Adenocarcinoma of the sigmoid colon Colon 22182 63 M Moderate to Poorly Differentiated: Adenocarcinoma of the cecum Colon 25846 66 F Moderate to Poorly Differentiated: Adenocarcinoma of the colon Colon 381.89 41 F Well to Moderately Differentiated: Adenocarcinoma of the colon Colon 41243 73 M Moderately differentiated: Adenocarcinoma of the colon Rectum 52940 74 F Moderately differentiated: Adenocarcinoma of the rectum Colon 53615 48 F Moderately differentiated: Adenocarcinoma of the colon descending Colon S4804 43 M Moderate to poorly differentiated: Adenocarcinoma of the large intestine Rectum 55076 70 M. Poorly differentiated: Adenocarcinoma of the rectum Colon 55827 57 M Moderately Differentiated: Adenocarcinoma of the large intestine Colon 9566 66 F Moderately Differentiated Adenocarcinoma of the colon Colon 11960 62 F Moderately Differentiated: Adenocarcinoma Rectum 44270 66 M Grade 3: Adenocarcinoma of the rectum US 2014/0294765 A1 Oct. 2, 2014 69

TABLE 9-continued Top4 TMA tissue annotation Case Tissue ID AGE SEX Pathology Rectum 18671 65 M Moderately Differentiated: Adenocarcinoma of the Cl Rectum 1995O 64 F Moderately Differentiated: Adenocarcinoma of the Cl Rectum 20022 53 M Moderately Differentiated: Adenocarcinoma of the Cl Rectum 20061 62 M Moderately Differentiated: Mucinous adenocarcinoma of the rectum Rectum 20560 58 M Moderately Differentiated: Adenocarcinoma of Cl Rectum 20921 75 Moderately Differentiated: Adenocarcinoma of the Cl Rectum 20990 84 Moderately Differentiated: Adenocarcinoma of Cl Colon 19948 71 M Grade2: Tubular adenocarcinoma of the ascending colon Rectum 20895 25 Moderately Differentiated Adenocarcinoma of the Cl Colon 22O72 67 M Well Differentiated Adenocarcinoma of the colon Colon 36274 61 Moderate to poorly differentiated Adenocarcinoma of the large intestine Colon 42362 58 Moderately differentiated Adenocarcinoma of the colon Lung 6692 37 M Normal Lung 7892 48 M Normal Lung 7893 75 F Normal Lung 7900 75 F Normal Lung 61215 59 M. Non-Small cell carcinoma: Moderately differentiated Adenocarcinoma of the lung Lung 62743 75 M. Non-Small cell carcinoma: Moderately differentiated Adenocarcinoma of the lung Lung 3O426 63 M. Non-Small Cell Carcinoma: Squamous Cell, Poorly differentiated Lung 40222 52 M Non-small Cell Carcinoma: Grade 3 Lung 454.09 52 M Non-Small Cell Carcinoma: Grade 3 Lung 45925 62 M. Non-Small Cell Carcinoma: Squamous cell, Moderate to poorly differentiated Lung 46417 70 M. Non-Small Cell Carcinoma: Squamous Cell, Moderately differentiated Lung 471.83 68 F Non-Small Cell Carcinoma: Squamous Cell, Moderately differentiated Lung 471,84 57 M. Non-Small Cell Carcinoma: Squamous Cell, Poorly differentiated Lung 47231 78 M. Non-Small Cell Carcinoma: Squamous Cell, Moderate o poorly differentiated Lung 62745 47 M Non-Small cell carcinoma: Adenocarcinoma, Moderate to poorly differentiated Lung 50538 56 M. Non-Small Cell Carcinoma: Squamous Cell, Keratinizing. Moderate to poorly differentiated Lung 52835 59 M Non-Small cell carcinoma: Squamous cell carcinoma, keratinizing Well to moderately differentiated Lung S9389 65 M Non-Small cell carcinoma: Papillary adenocarcinoma, Moderate to poorly differentiated Lung 55383 58 M Non-Small cell carcinoma: Large cell carcinoma, Poorly differentiated Lung 57766 62 M Non-Small cell carcinoma: Squamous cell carcinoma, arge cell, non-keratinizing, Moderate to poorly differentiated Lung 57771 61 M Non-Small cell carcinoma: Squamous cell carcinoma, arge cell, non-keratinizing, Poorly differentiated Lung 57772 59 M Non-Small cell carcinoma: Squamous cell carcinoma, keratinizing, Moderately differentiated Lung 59370 51 M Non-Small cell carcinoma: Large cell carcinoma, Ondifferentiated Lung 4457 73 M Squamous Cell Carcinoma: Moderately Differentiated Lung 3.0167 57 M Non-Small cell carcinoma: Adenocarcinoma, Poorly differentiated Lung 10891 56 M. Non-Small Cell Carcinoma: Poorly differentiated Lung 12002 74 F Non-small Cell Carcinoma: Poorly differentiated Lung 12047 60 F Adenocarcinoma: Moderate to Poorly Differentiated Lung 61076 69 M. Non-Small cell carcinoma: Poorly differentiated Adenocarcinoma of the lung