WO2016179194A1

WO2016179194A1 - Lilra3 and method of using the same

Info

Publication number: WO2016179194A1
Application number: PCT/US2016/030609
Authority: WO
Inventors: Ryan PHENNICIE; Jamie WONG
Original assignee: Jounce Therapeutics, Inc.
Priority date: 2015-05-04
Filing date: 2016-05-03
Publication date: 2016-11-10

Abstract

Provided herein are embodiments relating to therapeutic applications of molecules that modulate the biological activity of LILRA3. In some embodiments, the molecules bind TIM-3. In some embodiments, modulation of the interaction of TIM-3 and LILRA3 stimulates the release of pro-inflammatory cytokines; e.g., myeloid-associated pro -inflammatory cytokines.

Description

LILRA3 AND METHOD OF USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/156,852, filed May 4, 2015, and U.S. Provisional Patent Application No. 62/157,908, filed May 6, 2015; the disclosure of each of which is hereby incorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 739512000240SEQLISTING.txt, date recorded: May 3, 2016, size: 41 KB).

FIELD OF THE INVENTION

[0003] The present invention relates to methods of using molecules that modulate the biological activity of LILRA3. Such biological activity includes stimulating the secretion of myeloid-associated cytokines. Such methods include, but are not limited to, methods of treating cancer.

BACKGROUND

[0004] According to the World Health Organization, cancer is a global pandemic that causes nearly 7 million deaths each year worldwide. That number is expected to reach 10 million by the year 2020. Traditionally, cancer is treated using a variety of modalities including surgery, radiation therapy, and chemotherapy. The choice of treatment depends upon the type, location, and dissemination of the cancer. However, these modalities have proven to be relatively ineffective.

[0005] The T-cell immunoglobulin mucin (TIM) family regulates T-cell activation and tolerance. See Kane, L.P. T Cell Ig and Mucin Domain Proteins and Immunity, Immunol. (2010) 184:2743-2749; Freeman et al., TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity, Immunol Rev (2010) 235: 172-89; and Zhu, C. TIM-3 and its regulatory role in immune responses. Curr Top Microbiol Immunol. (2009) 350: 1-15. There are eight predicted tim genes in the murine genome (on mouse chromosome 1 IB 1.1), four of which are known to encode 4 known functional proteins: TIM-1 (T-cell immunoglobulin and mucin domain-containing protein 1 or Hepatitis A virus cellular receptor 1/HAVCRl homolog), TIM-2 (T-cell immunoglobulin and mucin domain-containing protein 2/TEVID-2), TIM-3 (T-cell immunoglobulin and mucin domain-containing protein 3 or Hepatitis A virus cellular receptor 2/HAVCR2 homolog) and TIM-4 (T-cell immunoglobulin and mucin domain-containing protein 4/TIMD-4), as well as four putative proteins TIM-5, TIM-6, TIM-7 and TIM-8. In contrast to mice, the human genome (on human chromosome 5q33.2) contains only three TIM genes, all encoding functional proteins, TIM-1 (HAVCR1), TIM-3 (HAVCR2) and TIM-4. See Kane, L.P. T Cell Ig and Mucin Domain Proteins and Immunity, Immunol. (2010) 184:2743-2749. TIM family members are expressed on a wide variety of innate and adaptive immune cells and have been implicated in regulating normal immune responses, and in diseases like autoimmunity, cancer and asthma. See Kuchroo V. J. et ah, TIM family of genes in immunity and tolerance. Adv Immunol. (2006) 91:227- 49; Kane, L.P. Immune regulation by the TIM Gene family Immunologic Research (2006) 36(1-3): 147-155; Kane, L.P. T Cell Ig and Mucin Domain Proteins and Immunity, J Immunol. (2010) 184:2743-2749; and Zhu, C. TIM-3 and its regulatory role in immune responses. Curr Top Microbiol Immunol. (2009) 350: 1-15.

[0006] TIM family members also belong to the immunoglobulin superfamily. Members of the TIM family are type I transmembrane proteins, and contain a characteristic N-terminal immunoglobulin- V-like (IgV) domain, a mucin domain with O-linked glycosylation sites, membrane proximal N-linked glycosylation sites, a single transmembrane domain, and a cytoplasmic region with tyrosine kinase phosphorylation motif(s) (except TIM-4 which does not have a tyrosine kinase phosphorylation motif in its cytoplasmic region). The length of the mucin domain is variable, and depends on the family member, with TIM-3 bearing the shortest length. See Freeman, G.J. TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunological Reviews (2010) 235: 172- 189; Kane, L.P. Immune regulation by the TIM Gene family, Immunologic Research (2006) 36(1-3): 147-155; Kane, L.P. T Cell Ig and Mucin Domain Proteins and Immunity, Immunol. (2010) 184:2743-2749 and Zhu, C. TIM-3 and its regulatory role in immune responses. Curr Top Microbiol Immunol. (2009) 350: 1-15. The N- terminal IgV domain has a deep binding pocket (called the metal ion-dependent ligand-binding site (MILIBS)) that is flanked by two hydrophobic loops which extend to the membrane. The IgV domain is composed of two anti-parallel β-sheets with particularly short β-strands. See Freeman, G.J. et al., TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunological Reviews (2010) 235: 172-189. This domain also possesses six invariant cysteines, two (the first and sixth cysteines) of which form disulphide bonds bridging the two β-sheets, as in all immunoglobulin superfamily members. See Cao, E. et al. T cell immunoglobulin Mucin-3 crystal structure reveals a galactin-9-independent ligand-binding surface. Immunity (2007) 26:311-321. Without wishing to be bound by theory, these bonds stabilize the IgV domain of TIM-3 and reorient the CC loop so that it is in close proximity to the FG loop resulting in formation of a "cleft" or "pocket" structure in TIM-3 as well as other TIM proteins. This unique cleft structure is not found in other IgSF proteins and has been predicted to be involved in ligand binding. In the cytoplasmic region of both human and mouse TIM-3, there is a highly conserved region containing five tyrosine residues. Galectin-9 binding to TIM-3 results in tyrosine phosphorylation of these residues, indicating that some, if not all, of these tyrosines may be involved in TIM-3 signaling. Otherwise, protein sequence analysis does not reveal any other homology to known inhibitory domains such as an immunoreceptor tyrosine-based inhibitory motif or immunoreceptor tyrosine-based switch motif. See Zhu, C. et al., TIM-3 and its regulatory role in immune responses. Curr Top Microbiol Immunol (2011) 350: 1-15.

[0007] TIM-3 differs both structurally and in terms of spatial expression patterns from other TIM family members, which suggests that it might have distinct functions compared to other TIM family members. For example, whereas TIM-1 is expressed exclusively on T-helper 2 (Th2) cells, and TIM-4 is expressed on antigen presenting cells (APC), TIM-3 is expressed on T-helper 1 (Thl) cells, T-helper 17 (Thl7) cells, IFN-γ producing CD8+ cytotoxic T 1 (Tel) cells, as well as on dendritic cells (DC), macrophages, natural killer (NK) cells, natural killer T (NKT) cells and human monocytes. When present on DC, TIM-3 mediates uptake of apoptotic cells. TIM-3 expression is regulated by T-bet, a Thl transcription factor. See Freeman, G.J. et al., TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunological Reviews (2010) 235: 172-189. TIM-3 is hypothesized to be a negative regulator of T cell responses. For example, binding of TIM-3 to its putative ligand, galectin-9, on Thl cells, results in Thl cell death. Further, blockade of TIM-3 increases IFN-γ secreting T cells. See Zhu, C. et al. The TIM-3 ligand galactin-9 negatively regulates T helper type 1 immunity. Nat Immunol. (2005) 6: 1245-1252 and Sabatos, C.A. et al. Interaction of TIM-3 and TIM-3 ligand regulated T helper type 1 responses and induction of peripheral tolerance. Nat. Immunol. (2003) 4: 1102-1110. Additionally, co-blockade of TIM-3 and another of its putative ligands, CEACAM1, leads to enhancement of anti-tumor immune responses with improved elimination of tumors in mouse colorectal cancer models. See Huang, et al. CEACAM1 regulates TIM-3 -mediated tolerance and exhaustion. Nature (2014).

[0008] Several ligands and/or co-receptors for TIM-3 have been identified, including HMGB l, Galectin 9 and phosphatidylserine. See Hang Li et al., TIM- 3/galectin-9 signaling pathway mediates T-cell dysfunction. Hepatology (2012) 56(4): 1342-1351, Shigeki, K et al., Galectin-9 inhibits CD44-hyluronan interaction and suppresses a murine model of allergic asthma. Am L Respir Crit Care Med (2007) 176:27-35; Kang, R. et al., HMGB l in Cancer. Clin Cancer Res (2013) (PMID: 23723299), Kane, L.P. T cell Ig and mucin domain proteins and immunity. Immunol (2010) 184:2743-2749, and Zhu, C. et al, TIM-3 and its regulatory role in immune responses. Curr Top Microbiol Immunol (2011) 350: 1-15. Chiba, S., et al., Tumor- infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB l, Nat. Immunol (2012) 13(9):832-842.

[0009] Given TIM-3 's negative regulation of T cell responses, TIM-3 was initially hypothesized to regulate antitumor responses, and exploited by tumors to evade immune clearance. See Ngiow, S.F. et al. Prospects for TIM-3 -targeted anti-tumor Immunotherapy. Cancer Research. (2011) 71:6567-6571. However, subsequent studies showed that TIM-3 expression on innate cells contributed to pro-inflammatory responses. See Leavy O. TIM-3: dual role in immunity. Nature Reviews Immunology (2008) 8:4; and Anderson, A.C. et al., Promotion of tissue inflammation by the immune receptor TIM-3 expressed on innate immune cells Science (2007) 318(5853): 1141-1143. On innate cells, where TIM-3 is expressed constitutively in both humans and mice, TIM-3 synergizes with Toll-like receptors (TLR) and promotes Thl immunity, by increasing the production of pro-inflammatory cytokines by DCs. This disparate and dual functionality of TIM-3 is hypothesized to occur as a result of differences in TIM-3 expression, with inhibitory functions attributed to its expression on T cells, and stimulatory/pro-inflammatory functions attributed to its expression on innate cells. It is also hypothesized that differences in the proximal signaling pathways induced by TIM-3 might account for the differences in TIM-3 's effect on innate and adaptive immune cells. Thus, TIM-3 has been implicated in either promoting or terminating Thl immunity, and without being bound by theory, has paradoxical roles in modulating immune responses by providing costimulatory and/or coinhibitory signals. See Anderson, A.C. et al., Promotion of tissue inflammation by the immune receptor TIM-3 expressed on innate immune cells Science (2007) 318(5853): 1141-1143.

[0010] TIM-3 is hypothesized to have paradoxical roles in modulating immune responses by providing costimulatory or coinhibitory signals depending on its binding to different receptors and/or its spatial expression on different immune cells. For example, blockade of TIM-3 signaling during induction of experimental autoimmune encephalitis leads to macrophage expansion and activation resulting in a more severe clinical phenotype. See Monney et al., Thl-specific cell surface protein TIM-3 regulates macrophage activation and severity of an autoimmune disease. (2002) Nature 415:536-541; and Anderson, D.E. Expert Opin Ther Targets. (2007) Aug; 11(8): 1005-9. In contrast, TIM-3 also acts synergistically with Toll-like receptors to increase pro-inflammatory TNFa secretion from dendritic cells, which may in turn promote T effector responses. See Anderson et al., Promotion of tissue inflammation by the immune receptor TIM-3 expressed on innate immune cells. (2007) Science 318: 1141-1143. Thus, TIM-3 has been implicated in either promoting or terminating Thl immunity. Although the biological role of TIM-3 signaling in T cell activation and in modulating immune responses is still being unraveled, it is clear that TIM-3 is an important target in cancer therapy.

[0011] Members of the group 1 Leukocyte immunoglobulin-like receptor (LILR) family include the inhibitory LILRB 1 and LILRB2 and the activating LILRA1, LILRA2 and LILRA3 receptors. LILRB 1, also known as ILT-2 and LIR-1, is the most ubiquitously expressed and perhaps most thoroughly studied member of this family. It has been shown that the Dl domain of LILRB 1 binds to the non- polymorphic α3 domain of HLA-A and that the D2 domain binds a secondary site on the conserved p2-Microglobulin (Willcox et al. 2003, Nature Immunology 4(9):913). Because both a3 and β2Μ are conserved among all classical MHC-1 (HLA-A, HLA- B and HLA-C), and non-classical MHC (including HLA-E, HLA-F and HLA-G), and because the regions of LILRB l that bind to these domains are highly conserved among all group 1 LILRs, it is presumed that all group 1 LILRs bind to all classical and non-classical MHC-1 molecules and act to either suppress or activate the cell expressing the LILR through this interaction. The binding of the inhibitory LILRB l and LILRB2 proteins to various HLAs have been examined by many groups and the data are summarized in Brown et al. 2004 Tissue Antigens 64:215. LILRA3, also known as ILT6, is unique among the family members in that there is no cytoplasmic or transmembrane domains encoded by the gene, resulting in a soluble protein without signaling domains. The amino acid sequence of LILRA3 is approximately 85% identical to the extracellular domain of LILRB2, but unlike LILRB2 which is expressed by most myelo-monocitic cells, LILRA3 is known only to be expressed by monocytes and not macrophages or dendritic cells. LILRA3 is considered an activating receptor because it competes with inhibitory group 1 receptors binding to MHC-1 (Myongchol et al, 2011, PLOS ONE 6(4):el9245). Two transcript variants encoding different isoforms have been found for this gene including variant 1 (GenBank Accession No. NP_006856.3; SEQ ID NO:7 and GenBank Accession No. NM_006865.4; SEQ ID NO:8) and variant 2 (GenBank Accession No. NP_001166125.1; SEQ ID NO:9 and GenBank Accession No. NM_001172654.2; SEQ ID NO: 10). Variant 2 uses an alternate in-frame splice site in the central coding region, and is missing a 64 amino acid segment compared to variant 1. TEVI- 3/LILRB2 interactions are disclosed in U.S. Patent Application No. 14/987,703, and PCT/US2016/012094, both filed January 7, 2016 and both incorporated herein by reference in their entireties.

[0012] There remains a need for more effective treatments of cancer utilizing immunotherapy. More particularly, there remains a need for novel compositions and therapeutic agents, and methods comprising the same that modulate TIM-3 activity which are capable of enhancing the host immune response against tumors for treating cancer. SUMMARY OF THE INVENTION

[0013] The invention provides molecules which modulate the biological activity of LILRA3. Such biological activity includes stimulating the secretion of myeloid - associated cytokines. In some embodiments, such molecules stimulate the secretion of a myeloid-associated cytokines through interactions with TIM-3.

[0014] In some aspects, the invention provides molecules which specifically bind TIM-3, wherein the molecules modulate the biological activity of LILRA3. In some embodiments, the molecules are LILRA3. In some embodiments, the molecules are LILRA3-Fc. In some embodiments, LILRA3 is human LILRA3. In some embodiments, the human LILRA3 is a functional variant of human LILRA3. In some embodiments, the LILRA3 is a fragment of human LILRA3. In some embodiments, the molecules are TIM-3 antibodies.

[0015] In some embodiments of any of the above embodiments, the TIM-3 is human TIM-3. In some embodiments, the TIM-3 comprises the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3. In other embodiments, the amino acid sequence of the TIM-3 is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:3.

[0016] In some embodiments of any of the above embodiments, the LILRA3 is human LILRA3. In some embodiments, the LILRA3 comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9. In other embodiments, the amino acid sequence of the LILRA3 is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9.

[0017] In some embodiments, the molecule of the invention competes with LILRA3-Fc for binding human TIM-3. In some embodiments, the molecule of the invention competes with LILRA3-Fc for binding human TIM-3 and stimulates the secretion of one or more myeloid-associated cytokines in an individual. In some embodiments, the molecule is an anti-TIM-3 antibody. In some embodiments, binding of the anti-TIM-3 antibody blocks binding of LILRB2 to TIM-3. In some embodiments, the myeloid associated cytokine is a pro-inflammatory cytokine. In some embodiments, the myeloid associated cytokine is one or more of IL-2, TNFa, IL-Ιβ, GM-CSF or IL-6. In some embodiments, the myeloid associated cytokine is one or more of TNFa, IL-Ιβ, GM-CSF or IL-6. In some embodiments, the myeloid associated cytokines are TNFa, IL-Ιβ, GM-CSF and IL-6. In some embodiments, the molecule suppresses the secretion of a myeloid-associated cytokine in an individual. In some embodiments, secretion of myeloid associated cytokine IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed. In some embodiments, secretion of one or more of myeloid associated cytokines IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed. In some embodiments, secretion of myeloid associated cytokine IL-10, CCL2, or CCL5 is suppressed. In some embodiments, secretion of myeloid associated cytokine IL-10 or CCL5 is suppressed. In some embodiments, secretion of IL-10 is suppressed. In some embodiments, secretion of CCL2 is suppressed. In some embodiments, secretion of CCL3 is suppressed. In some embodiments, secretion of CCL4 is suppressed. In some embodiments, secretion of CCL5 is suppressed. In some embodiments, the molecule suppresses the secretion of an immunosuppressive myeloid-associated cytokine in an individual.

[0018] In some aspects, the invention provides an antibody that binds TIM-3, wherein the antibody stimulates the secretion of one or more myeloid-associated cytokines in an individual. In some embodiments, binding of the anti-TIM-3 antibody blocks LILRB2 binding to TIM-3. In some embodiments, the myeloid associated cytokine is pro-inflammatory cytokine. In some embodiments, the myeloid associated cytokine is one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6. In some embodiments, the myeloid associated cytokine is one or more of TNFa, IL- Ιβ or IL-6. In some embodiments, the myeloid associated cytokines are TNFa, IL-Ιβ and IL-6. In some embodiments, the antibody stimulates the secretion of one or more myeloid- associated cytokines in an individual to a greater extent than the stimulation of secretion of the cytokine by antibody F38-2E2. In some embodiments, the antibody stimulates the secretion of a myeloid-associated cytokine in an individual to greater than about any one of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% the stimulation of secretion of the cytokine by antibody F38-2E2. In some embodiments, the antibody suppresses the secretion of a myeloid-associated cytokine in an individual. In some embodiments, secretion of myeloid associated cytokine IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed. In some embodiments, secretion of myeloid associated cytokine IL-10, CCL2, or CCL5 is suppressed. In some embodiments, secretion of myeloid associated cytokine IL-10 or CCL5 is suppressed. In some embodiments, secretion of IL-10 is suppressed. In some embodiments, secretion of CCL2 is suppressed. In some embodiments, secretion of CCL3 is suppressed. In some embodiments, secretion of CCL4 is suppressed. In some embodiments, secretion of CCL5 is suppressed. In some embodiments, the antibody suppresses the secretion of an immunosuppressive myeloid- associated cytokine in an individual. In some embodiments, the antibody suppresses the secretion of one or more myeloid-associated cytokines in an individual to a greater extent than the suppression of secretion of the cytokine by antibody F38-2E2. In some embodiments, the antibody suppresses the secretion of a myeloid-associated cytokine in an individual to greater than about any one of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% the stimulation of secretion of the cytokine by antibody F38-2E2.

[0019] In some embodiments of the any of the above embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In other embodiments, the antibody is humanized. In yet other embodiments, the antibody is a human antibody. In some embodiments, the antibody is an antigen binding fragment of an antibody. In some embodiments, the antibody is an antibody fragment selected from a Fab, Fab', Fv, scFv or (Fab')2 fragment.

[0020] In some aspects, the invention provides a pharmaceutical composition comprising the molecules of any the above embodiments and a pharmaceutically acceptable carrier.

[0021] In some aspects, the invention provides methods of stimulating the secretion of one or more myeloid-associated cytokines in an individual, comprising administering to the individual a therapeutically effective amount of a molecule that stimulates the secretion of one or more myeloid-associated cytokine. In some embodiments, the molecule is a fusion molecule. In some embodiments, the molecule is a LILRA3 or functional variant thereof. In some embodiments, the molecule is a fusion molecule comprising a LILRA3 or functional variant thereof. In some embodiments, the molecule is in a pharmaceutical composition. In some embodiments, the myeloid-associated cytokine is a pro-inflammatory cytokine. In some embodiments, the myeloid-associated cytokine is one or more of IL-12, TNFa, IL-Ιβ, GM-CSF, or IL-6. In some embodiments, the myeloid-associated cytokine is one or more of TNFa, IL-Ιβ, GM-CSF or IL-6. In some embodiments, the myeloid- associated cytokines are TNFa, IL-Ιβ, GM-CSF and IL-6. In some embodiments, administration of the molecule to the individual preferentially stimulates the secretion of cytokines from macrophages. In some embodiments, administration of the molecule suppresses the secretion of one or more myeloid-associated cytokines in an individual. In some embodiments, secretion of one or more of myeloid associated cytokines IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed by administration of the molecule. In some embodiments, secretion of one or more of myeloid associated cytokines IL-10, CCL2, or CCL5 is suppressed by administration of the molecule. In some embodiments, secretion of one or more of myeloid associated cytokines IL-10 or CCL5 is suppressed by administration of the molecule. In some embodiments, secretion of one or more immunosurpressive cytokines is suppressed by administration of the molecule. In some embodiments, the individual has cancer. In some embodiments, the cytokine is secreted in a tumor. In some embodiments, the individual is a human.

[0022] In some aspects, the invention provides methods for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the molecule as described herein. In some embodiments, the molecule is in a pharmaceutical composition. In some embodiments, the individual is a human.

[0023] In some embodiments, the invention provides methods for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the molecule as described herein wherein the molecule is administered in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD- Ll antibody. In some embodiments, administration of the molecule of the invention and the additional therapeutic agent is simultaneous or sequential. In some embodiments, the molecule of the invention is administered first followed by administration of the additional agent. In other embodiments, the additional agent is administered first followed by administration of the molecule of the invention.

[0024] In some embodiments, the invention provides an isolated nucleic acid encoding a molecule that modulates the biological activity of LILRA3, including stimulating the secretion of myeloid-associated cytokines, as described herein. In some embodiments, the invention provides a vector comprising the nucleic acid encoding the molecule. In some embodiments, the invention provides a host cell comprising the nucleic acid or the vector. In some embodiments, the invention provides a host cell that produces a molecule as described herein.

[0025] In some aspects, the invention provides methods for making a molecule that modulates the biological activity of LILRA3 by culturing a host cell comprising a nucleic acid encoding the molecule under conditions suitable for expression of the nucleic acid encoding the molecule that modulates the biological activity of LILRA3.

[0026] In some embodiments, the invention provides methods for making an antibody that modulates the activity of LILRA3 by culturing a host cell comprising the nucleic acid encoding the antibody under conditions suitable for expression of the nucleic acid encoding the antibody that modulates the activity of LILRA3. In further embodiments the method further comprises recovering the antibody produced by the host cell.

[0027] In some embodiments, the invention provides the use of a molecule (e.g. , an antibody) that modulates the activity of LILRA3 for stimulating the secretion of one or more myeloid-associated cytokines in an individual in need thereof. In some embodiments, the invention provides the use of a molecule as described herein in the manufacture of a medicament for stimulating the secretion of one or more myeloid- associated cytokines in an individual in need thereof. In some embodiments, the molecule is in a pharmaceutical composition. In some embodiments, the myeloid- associated cytokine is a proinflammatory cytokine. In some embodiments, the myeloid-associated cytokine is one or more of IL- 12, TNFa, IL- Ιβ, GM-CSF or IL-6. In some embodiments, the myeloid-associated cytokine is one or more of TNFa, IL- 1β, GM-CSF or IL-6. In some embodiments, the myeloid-associated cytokines are TNFa, IL- Ιβ, GM-CSF and IL-6. In some embodiments, the molecule suppresses the secretion of a myeloid-associated cytokine in an individual. In some embodiments, secretion of myeloid associated cytokine IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed. In some embodiments, secretion of myeloid associated cytokine IL- 10, CCL2, or CCL5 is suppressed. In some embodiments, secretion of myeloid associated cytokine IL- 10 or CCL5 is suppressed. In some embodiments, secretion of IL- 10 is suppressed. In some embodiments, secretion of CCL2 is suppressed. In some embodiments, secretion of myeloid associated cytokine CCL3 is suppressed. In some embodiments, secretion of CCL4 is suppressed. In some embodiments, secretion of CCL5 is suppressed. In some embodiments, secretion of an immunosuppressive cytokine is suppressed. In some embodiments, the individual has cancer. In some embodiments, the individual is human.

[0028] In some embodiments, the invention provides the use of a molecule (e.g., an antibody) for treating cancer in an individual. In some embodiments, the invention provides the use of a molecule that modulates that activity of LILRA3 in the manufacture of a medicament for treating cancer in an individual. In some embodiments, the molecule is in a pharmaceutical formulation.

[0029] In some embodiments, the invention provides a pharmaceutical composition for treating cancer in an individual comprising a therapeutically effective amount of a molecule (e.g., an antibody) that modulates the activity of LILRA3 as described herein and a pharmaceutically acceptable carrier. In some embodiments, the invention provides a pharmaceutical composition for treating cancer in an individual comprising a therapeutically effective amount of a molecule that modulates the activity of LILRA3 as described herein and a pharmaceutically acceptable carrier.

[0030] In some embodiments, the invention provides kits for stimulating the secretion of myeloid-associated cytokines in an individual, comprising the molecule (e.g., an antibody) that modulates the activity of LILRA3. In some embodiments, the molecule is in a pharmaceutical formulation. In some embodiments, the myeloid- associated cytokine is a proinflammatory cytokine. In some embodiments, the myeloid-associated cytokine is one or more of IL- 12, TNFa, IL- Ιβ, GM-CSF or IL-6. In some embodiments, the myeloid-associated cytokine is one or more of TNFa, IL- 1β, or IL-6. In some embodiments, the myeloid-associated cytokines are TNFa, IL- 1β, GM-CSF and IL-6. In some embodiments, the molecule of the kit suppresses the secretion of a myeloid-associated cytokine in an individual. In some embodiments, secretion of myeloid associated cytokine IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed. In some embodiments, secretion of IL- 10 is suppressed. In some embodiments, secretion of CCL2 is suppressed. In some embodiments, secretion of myeloid associated cytokine CCL3 is suppressed. In some embodiments, secretion of CCL4 is suppressed. In some embodiments, secretion of CCL5 is suppressed. In some embodiments, secretion of an immunosuppressive cytokine is suppressed. In some embodiments, the individual has cancer. In some embodiments, the invention provides kits for treating cancer in an individual, comprising the molecule that modulates the activity of LILRA3. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1A is a graph showing IL-2 secretion by Staphylococcal enterotoxin B (SEB)-activated whole blood samples treated with no antibody, an isotype control antibody, an anti-PD-Ll antibody with an IgGl isotype control antibody, antibody F38-2E2, or antibody F38-2E2 and anti-PD-Ll. ** p<0.01; **** p<0.0001. Fig.lB. shows diverse bins of anti-TEVI-3 antibodies when arranged according to their ability to cross-block one another in binding plate -bound TIM-3 protein.

[0032] FIGs. 2A and 2B show that SEB induction of TIM-3 on monocyte/macrophages has different kinetics than on T cells. Fig. 2A is a graph showing a time course of expression of PD-1 on the surface of indicated cells from time = 0 to four days in culture. Fig. 2B is a graph showing a time course of expression of TIM-3 on the surface of indicated cells from time = 0 to four days in culture. Circles represent CD4+ T cells, squares represent CD8+ T cells, triangles represent CD14+ monocytes/macrophages, and diamonds represent CDl lc+ dendritic cells (DCs).

[0033] FIGs. 3A-30 show SEB induction of innate inflammatory cytokines and IL-2 can be measured before TIM-3 is upregulated on T cells. SEB-activated PBMC were treated with a control isotype antibody (circles), an anti-PD-Ll antibody (squares), an anti-TIM-3 antibody (triangles) or an anti-PD-Ll antibody and an anti- TEVI-3 antibody (inverted triangles). Fig. 3A shows expression of IL-2 over the four day time course. Fig. 3B shows expression of TNFa over the four day time course. Fig. 3C shows expression of IL-Ιβ over the four day time course. Fig. 3D-30 show the expression of other cytokines as indicated over the four day time course.

[0034] FIGs. 4A and 4B show that TIM-3 is more strongly associated with myeloid cells (monocytes/macrophages and dendritic cells) than T cells in human cancers. Fig. 4A and Fig. 4B show graphs representing the correlation of TIM-3 expression and the T cell marker CD3g (Fig. 4A) or the myeloid cell marker CD l ib (Fig. 4B) in tumor samples from a breast cancer (BRCA), a lung adenocarcinoma (LUAD), an ovarian cancer (OV), and a prostate adenocarcinoma (PRAD). X and Y axes represent normalized level of mRNA expression, Corr(S) stands for Spearman correlation coefficient, Pval(S) denotes p-value of the correlation.

[0035] FIG. 5 is a graph showing that LILRA3 binds TIM-3. [0036] FIG. 6 shows graphs demonstrating dose curve release of GM-CSF, IL-Ιβ, CCL5, IL-6 and TNFa from macrophages stimulated by LPS following treatment with different doses of LILRA3-Fc (diamonds), mlgl isotype (circles) served as a negative control.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Embodiments provided herein relate to molecules that modulate the activity of LILRA3 (e.g., modulate the interaction of TIM-3 and LILRA3) and their use in various methods to determine and/or deliver appropriate cancer therapies and/or methods for increasing production of cytokines and/or increasing cytokine secretion and/or methods for increasing T-cell proliferation. In some embodiments, the molecules bind TIM-3 and modulate the interaction of TIM-3 with LILRA3. In some embodiments, the molecules bind TIM-3 and modulate the interaction of TIM-3 with LILRA3 such that binding of LILRB2 to TIM-3 is inhibited.

Definitions and Various Embodiments:

[0038] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0039] All references cited herein, including patent applications, patent publications, and Genbank Accession numbers are herein incorporated by reference, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

[0040] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et ah, eds., 1994); Current Protocols in Immunology (J. E. Coligan et ah, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et ah, eds., J.B. Lippincott Company, 1993); and updated versions thereof.

[0041] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include pluralities and plural terms shall include the singular. For any conflict in definitions between various sources or references, the definition provided herein will control.

[0042] It is understood that embodiments of the invention described herein include "consisting" and/or "consisting essentially of embodiments. As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise. Use of the term "or" herein is not meant to imply that alternatives are mutually exclusive.

[0043] In this application, the use of "or" means "and/or" unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim.

[0044] As is understood by one skilled in the art, reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0045] The phrase "reference sample", "reference cell", or "reference tissue", denote a sample with at least one known characteristic that can be used as a comparison to a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to establish a level of protein and/or mRNA that is present in, for example, healthy tissue, in contrast to a level of protein and/or mRNA present in the sample with unknown characteristics. In some embodiments, the reference sample comes from the same subject, but is from a different part of the subject than that being tested. In some embodiments, the reference sample is from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but is a sample from a subject known to have, or not to have, a disorder in question (for example, a particular cancer or TIM-3 related disorder). In some embodiments, the reference sample is from the same subject, but from a point in time before the subject developed cancer. In some embodiments, the reference sample is from a benign cancer sample (for example, benign breast cancer sample), from the same or a different subject. When a negative reference sample is used for comparison, the level of expression or amount of the molecule in question in the negative reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is no and/or a low level of the molecule. When a positive reference sample is used for comparison, the level of expression or amount of the molecule in question in the positive reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is a level of the molecule.

[0046] The terms "benefit", "clinical benefit", "responsiveness", and "therapeutic responsiveness" as used herein in the context of benefiting from or responding to administration of a therapeutic agent, can be measured by assessing various endpoints, e.g., inhibition, to some extent, of disease progression, including slowing down and complete arrest; reduction in the number of disease episodes and/or symptoms; reduction in lesion size; inhibition (that is, reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; inhibition (that is, reduction, slowing down or complete stopping) of disease spread; decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; relief, to some extent, of one or more symptoms associated with the disorder; increase in the length of disease-free presentation following treatment, for example, progression-free survival; increased overall survival; higher response rate; and/or decreased mortality at a given point of time following treatment. A subject or cancer that is "non-responsive" or "fails to respond" is one that has failed to meet the above noted requirements to be "responsive".

[0047] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.

[0048] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0049] "TIM-3" as used herein, refers to a type I transmembrane protein belonging to the TIM family, alternatively known as Hepatitis A virus cellular receptor 2 (HAVCR2), T cell immunoglobulin and mucin domain-containing protein-3 (TIMD- 3), or Kidney Injury Molecule-3 (KIM-3). TIM-3 is expressed on, at least, T-helper 1 (Thl) cells, T-helper 17 (Thl7) cells, IFN-γ producing CD8+ cytotoxic T 1 (Tel) cells, as well as some dendritic cells (DC), macrophages, natural killer (NK) cells, natural killer T (NKT) cells and human monocytes. See Freeman et al., TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. (2010) Immunol. Rev. 235: 172-189.)·

[0050] Human TIM-3 is believed to be 301 amino acids long with residues 1 - 21 encoding a signal peptide; residues 22-202 encoding the TIM-3 extracellular domain; residues 203-223 encoding a helical, transmembrane domain; and residues 224-301 encoding the cytoplasmic portion of TIM-3 (all residue numbers refer to SEQ ID NO: l). Within the extracellular domain, it is believed that residues 22-124 encode an Ig-like V-type (IgV) domain followed by the mucin domain (starting at about residue 125 and ending at about residue 182) and the stalk domain (starting at about residue 183 and ending at about residue 202) (all residue numbers refer to SEQ ID NO: l). Also within the extracellular domain, the cleft and/or FG loop domain (where residues 50, 62, 69, 112, and 121 are predicted to be involved in ligand binding) is predicted to start at about residue 49 and extend to about residue 122 (all residue numbers refer to SEQ ID NO: l). See 84868 (Entrez); ENSG00000135077 (Ensemble); Q8TDQ0 (UniProt); and NM_032782.4 (human RNA sequence) and NP_116171 (human polypeptide sequence) (NCBI); and Cao, E. et al. T cell immunoglobulin Mucin-3 crystal structure reveals a galactin-9-independent ligand-binding surface. Immunity (2007) 26:311-321, each of which is herein incorporated by reference in its entirety for all purposes.

[0051] The TIM-3 gene is believed to be located at chromosome 5 (156.51-156.57 Mb). Two isoforms or alternatively spliced forms of the human TIM-3 have been reported: Isoform 1 (UniProt: Q8TDQ0-1) and Isoform 2 (UniProt: Q8TDQ0-2). Several additional natural human TIM-3 variants have also been reported. In one variation of TIM-3 isoform 1, as an alternative sequence is found at residues 132-142. The residues AKVTPATTRQT in isoform 1 are replaced by residues GEWTGFACHLYE in isoform 2. Amino acids at residues 143-301 of isoform 1 are missing in isoform 2. A natural variant occurs at residue 140 of isoform 1 where a R to L substitution may occur (Monney, L. Nature (2002) 415:536-541). Accordingly, the present invention, in some aspects and embodiments, relates to therapeutic agents (e.g. antibodies, including bi-specific or multispecific antibodies and antibodies that competitively inhibit and/or bind the same epitope as a TIM-3 antibody disclosed herein) that bind to one, some or all of the human TIM-3 isoforms, alternatively spliced polypeptides and/or natural variants (e.g. including, without limitation, therapeutic agents (e.g. antibodies) that bind Isoform 1 or Isoform 2; or Isoforms 1 and 2) that may be specifically expressed in tumors or non-tumor cells.

[0052] Murine TIM-3 (NCBI Reference Sequence: NM_134250.2; SEQ ID NOs:9 and 10) is believed to be approximately 343 amino acids long with residues 1 - 22 encoding a signal peptide. See 102657 (Entrez); ENSMUSG00000020399 (Ensemble); Q6U7R4 (UniProt); and NM_134520 (murine RNA sequence) and NP_599011 (murine polypeptide sequence) (NCBI), each of which is herein incorporated by reference in its entirety for all purposes. The murine gene is believed to be located at chromosome 11 (46.45-46.48 Mb). TIM-3 is a highly conserved molecule, bearing 63% sequence homology between mice and humans.

[0053] "LILRA3" or "ILT6" as used herein refers to "Leukocyte immunoglobulin- like receptor subfamily A member 3." LILRA3 is also known as CD85 antigen-like family member E, CD85e, CD85E, ILT-6, Immunoglobulin-like transcript 6, Leukocyte immunoglobulin-like receptor 4, Leukocyte immunoglobulin-like receptor subfamily A member 3, LIR-4, HM31, HM43, e3. LILRA3 is a protein that in humans is encoded by the LILRA3 gene. LILRA3 is a member of the leukocyte immunoglobulin-like receptor (LIR) family, and the gene encoding LILRA3 is found in a gene cluster at chromosomal region 19ql3.4. Multiple transcript variants encoding different isoforms have been found for this gene including isoform 1 (GenBank Accession Nos.: NM_006865.4 and NP_006856.3), isoform 2 (GenBank Accession Nos.: NM_001172654.2 and NP_001166125.1).

[0054] The term "specifically binds" to an antigen or epitope is a term that is well understood in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, a fusion protein or an antibody that specifically or preferentially binds to a TIM-3 epitope is fusion protein or an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other TIM-3 epitopes or non-TIM-3 epitopes. It is also understood by reading this definition that; for example, a fusion protein or an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind an antigen.

[0055] As used herein, the term "modulate" with regard to the activity of LILRA3 refers to a change in the activity of LILRA3. In some examples, "modulate" refers to an increase in LILRA3 activity (e.g., an increase in binding to TEVI-3 and/or an increase in the stimulation of cytokine release) compared to LILRA3 in the absence of the modulator. Modulation may be the result of direct interaction with LILRA3 or may be indirect effect caused by LILRA3 by modulating upstream or downstream activities associated with LILRA3 (e.g., stimulation of cytokine release). Modulation of the activity of LILRA3 may result in the interrupting or inhibiting the binding of LILRB2 to TIM-3. In some embodiments, a molecule that modulates the activity of LILRA3 mimics LILRA3.

[0056] As used herein, "substantially pure" refers to material which is at least 50% pure (that is, free from contaminants), for example, at least 90% pure, at least 95% pure, yet more preferably, at least 98% pure, and most preferably, at least 99% pure.

[0057] As used herein, the term "epitope" refers to a site on a target molecule (for example, an antigen, such as a protein, nucleic acid, carbohydrate or lipid) to which an antigen-binding molecule (for example, an antibody, antibody fragment, or scaffold protein containing antibody binding regions) binds. Epitopes often include a chemically active surface grouping of molecules such as amino acids, polypeptides or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed both from contiguous and/or juxtaposed noncontiguous residues (for example, amino acids, nucleotides, sugars, lipid moiety) of the target molecule. Epitopes formed from contiguous residues (for example, amino acids, nucleotides, sugars, lipid moiety) typically are retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding typically are lost on treatment with denaturing solvents. An epitope may include but is not limited to at least 3, at least 5 or 8- 10 residues (for example, amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (for example, amino acids or nucleotides) in length, less than 15 residues or less than 12 residues. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen. In some embodiments, an epitope can be identified by a certain minimal distance to a CDR residue on the antigen-binding molecule. In some embodiments, an epitope can be identified by the above distance, and further limited to those residues involved in a bond (for example, a hydrogen bond) between an antibody residue and an antigen residue. An epitope can be identified by various scans as well, for example an alanine or arginine scan can indicate one or more residues that the antigen-binding molecule can interact with. Unless explicitly denoted, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antibody. Rather, the presence of such a set designates a minimal series (or set of species) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope designates a minimal epitope of relevance for the antigen, rather than an exclusive list of residues for an epitope on an antigen.

[0058] A "nonlinear epitope" or "conformational epitope" comprises noncontiguous polypeptides, amino acids and/or sugars within the antigenic protein to which an antibody specific to the epitope binds. In some embodiments, at least one of the residues will be noncontiguous with the other noted residues of the epitope; however, one or more of the residues can also be contiguous with the other residues.

[0059] A "linear epitope" comprises contiguous polypeptides, amino acids and/or sugars within the antigenic protein to which an antibody specific to the epitope binds. It is noted that, in some embodiments, not every one of the residues within the linear epitope need be directly bound (or involved in a bond) with the antibody. In some embodiments, linear epitopes can be from immunizations with a peptide that effectively consisted of the sequence of the linear epitope, or from structural sections of a protein that are relatively isolated from the remainder of the protein (such that the antibody can interact, at least primarily), just with that sequence section.

[0060] The term "fusion protein" or "fusion polypeptide" herein is used to refer to a protein having at least two heterologous polypeptides covalently linked, either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C- terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. In some examples, the heterologous polypeptide may be a full-length native protein or a fragment or variant thereof (e.g., a functional fragment or a functional variant). In some examples, the fusion protein comprises an IgG Fc region (e.g., an IgG Fc region fused to a LILRA3 protein).

[0061] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific (such as Bi- specific T-cell engagers) and trispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0062] The term antibody includes, but is not limited to, fragments that are capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody) and (Fab')₂ (including a chemically linked F(ab')₂). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc. Furthermore, for all antibody constructs provided herein, variants having the sequences from other organisms are also contemplated. Thus, if a human version of an antibody is disclosed, one of skill in the art will appreciate how to transform the human sequence based antibody into a mouse, rat, cat, dog, horse, etc. sequence. Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments also include nanobodies (sdAb, an antibody having a single, monomeric domain, such as a pair of variable domains of heavy chains, without a light chain). An antibody fragment can be referred to as being a specific species in some embodiments (for example, human scFv or a mouse scFv). This denotes the sequences of at least part of the non-CDR regions, rather than the source of the construct.

[0063] The term "monoclonal antibody" refers to an antibody of a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0064] The term "CDR" denotes a complementarity determining region as defined by at least one manner of identification to one of skill in the art. In some embodiments, CDRs can be defined in accordance with any of the Chothia numbering schemes, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and/or the contact definition. Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-Hl, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The AbM definition can include, for example, CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-Hl, CDR-H2, and CDR-H3) at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, H26- H35B of HI, 50-58 of H2, and 95-102 of H3. The Contact definition can include, for example, CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-Hl, CDR-H2, and CDR-H3) at amino acid residues 30-36 of LI, 46-55 of L2, 89-96 of L3, 30-35 of HI, 47-58 of H2, and 93-101 of H3. The Chothia definition can include, for example, CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-Hl, CDR-H2, and CDR-H3) at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 26-32...34 of HI, 52-56 of H2, and 95-102 of H3. CDRs can also be provided as shown in any one or more of the accompanying figures. With the exception of CDR1 in V_H, CDRs generally comprise the amino acid residues that form the hypervariable loops. The various CDRs within an antibody can be designated by their appropriate number and chain type, including, without limitation as: a) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3; b) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3; c) LCDR-1, LCDR-2, LCDR-3, HCDR-1, HCDR-2, and HCDR-3; or d) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3; etc. The term "CDR" is used herein to also encompass HVR or a "hyper variable region", including hypervariable loops. Exemplary hypervariable loops occur at amino acid residues 26- 32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).)

[0065] The term "heavy chain variable region" as used herein refers to a region comprising at least three heavy chain CDRs. In some embodiments, the heavy chain variable region includes the three CDRs and at least FR2 and FR3. In some embodiments, the heavy chain variable region includes at least heavy chain HCDR1, framework (FR) 2, HCDR2, FR3, and HCDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of an FRl and/or at least a portion of an FR4.

[0066] The term "heavy chain constant region" as used herein refers to a region comprising at least three heavy chain constant domains, C_HI, C_H2, and C_H3. Of course, non-function-altering deletions and alterations within the domains are encompassed within the scope of the term "heavy chain constant region," unless designated otherwise. Nonlimiting exemplary heavy chain constant regions include γ, δ, and a. Nonlimiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an a constant region is an IgA antibody. Further, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgGl (comprising a γι constant region), IgG2 (comprising a γ₂ constant region), IgG3 (comprising a γ₃ constant region), and IgG4 (comprising a γ₄ constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an ai constant region) and IgA2 (comprising an a₂ constant region) antibodies; and IgM antibodies include, but are not limited to, IgMl and IgM2.

[0067] The term "heavy chain" as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0068] The term "light chain variable region" as used herein refers to a region comprising at least three light chain CDRs. In some embodiments, the light chain variable region includes the three CDRs and at least FR2 and FR3. In some embodiments, the light chain variable region includes at least light chain LVR1, framework (FR) 2, LVR2, FR3, and LVR3. For example, a light chain variable region may comprise light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a light chain variable region also comprises at least a portion of an FR1 and/or at least a portion of an FR4.

[0069] The term "light chain constant region" as used herein refers to a region comprising a light chain constant domain, C_L- Nonlimiting exemplary light chain constant regions include λ and κ. Of course, non-function-altering deletions and alterations within the domains are encompassed within the scope of the term "light chain constant region," unless designated otherwise.

[0070] The term "light chain" as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0071] An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (V_L) framework or a heavy chain variable domain (V_H) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the V_L acceptor human framework is identical in sequence to the V_L human immunoglobulin framework sequence or human consensus framework sequence.

[0072] "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_d). Affinity can be measured by common methods known in the art (such as, for example, ELISA K_D, KinExA and/or surface plasmon resonance devices (such as a BIAcore® device), including those described herein.

[0073] The term "K_D", as used herein, refers to the equilibrium dissociation constant of an antibody- antigen interaction.

[0074] In some embodiments, the "K_D," "K_d," "Kd" or "Kd value" of the antibody is measured by using surface plasmon resonance assays using a BIACORE^®-2000 or a BIACORE^®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 °C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΕ/ηιίηυίε to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, serial dilutions of polypeptide, for example, full length antibody, are injected in PBS with 0.05% TWEEN-20™ surfactant (PBST) at 25 °C at a flow rate of approximately 25 μΕ/ηιίη. Association rates (k_on) and dissociation rates (k₀ff) are calculated using a simple one-to-one Langmuir binding model (BIACORE^® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K_d) is calculated as the ratio k₀ff/k_on. See, for example, Chen et al, J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M^'V¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25 °C of a 20 nM anti-antigen antibody in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0075] In some embodiments, the difference between said two values (for example, K_d values) is substantially the same, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.

[0076] In some embodiments, the difference between said two values (for example, K_d values) is substantially different, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

[0077] "Surface plasmon resonance" denotes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al. (1993) Ann. Biol. Clin. 51 : 19-26.

[0078] The term "k_on", as used herein, refers to the rate constant for association of an antibody to an antigen. Specifically, the rate constants (k_m and k_0ff) and equilibrium dissociation constants are measured using Fab antibody fragments (that is, univalent) and TEVI-3. "K_on", "k_on", "association rate constant", or "k_a", are used interchangeably herein. The value indicates the binding rate of a binding protein to its target antigen or the rate of complex formation between an antibody and antigen, shown by the equation: Antibody("Ab")+Antigen("Ag")- Ab-Ag.

[0079] The term "k_0ff", as used herein, refers to the rate constant for dissociation of an antibody from the antibody/antigen complex. k_0ff is also denoted as "K_0ff" or the "dissociation rate constant". This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation: Ab+Ag - Ab-Ag. [0080] The term "biological activity" refers to any one or more biological properties of a molecule (whether present naturally as found in vivo, or provided or enabled by recombinant means). Biological properties include, but are not limited to, binding a receptor, inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis, and enzymatic activity.

[0081] The term "LILRA3 activity" or "biological activity" of LILRA3, as used herein, includes any biological effect or at least one of the biologically relevant functions of the LILRA3 protein. In certain embodiments, LILRA3 activity includes the ability of LILRA3 to interact or bind to a substrate or receptor. In certain embodiments, the biological activity of LILRA3 is the ability of LILRA3 to bind to TIM-3. In certain embodiments, the biological activity of LILRA3 is the ability of LILRA3 to inhibit the binding of LILRB2 to TEVI-3. In certain embodiments, LILRA3 binds to and catalyzes a reaction involving TIM-3. In certain embodiments, biological activity of LILRA3 includes any biological activity resulting from TIM-3 signaling.

[0082] The phrase "TIM-3 activity" indicates at least one of the biologically relevant functions of the TIM-3 protein. In some embodiments, this can be mediated by through the binding of the TIM-3 protein to a TIM-3 ligand. Examples of known TDVI-3 ligands include but are not limited to LILRB2, Gal-9, and CEACAM. In some embodiments, TIM-3 activity of the present invention is the TIM-3 activity that is mediated through its binding to LILRB2.

[0083] As used herein, the term "myeloid-associated cytokine" refers to cytokines produced by and/or that interact with cells of myeloid lineage; for example, cytokines produced by or that interact with monocytes and/or macrophages and/or dendritic cells. In some nonlimiting examples, a myeloid-associated cytokine that interacts with a macrophage and/or dendritic cell binds to or activates the macrophage or dendritic cells.

[0084] An "agonist" or "activating" antibody is one that increases and/or activates a biological activity of the protein e.g. , a TIM-3 or LILRA3 protein. In some embodiments, the agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0085] An "antagonist", a "blocking" or "neutralizing" antibody is one that decreases and/or inactivates a biological activity of the protein; e.g. , a TDVI-3 or LILRA3 protein. In some embodiments, the neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85% 90%, 95%, 99% or more. In some embodiments, the antibody competes with LILRA3 for binding TIM-3, wherein antibody binding to TIM-3 inhibits binding of TIM-3 to LILRB2.

[0086] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more CDRs compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0087] A "chimeric antibody" as used herein refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while at least a part of the remainder of the heavy and/or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species. The chimeric construct can also be a functional fragment, as noted above.

[0088] A "humanized antibody" as used herein refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is an antibody fragment, such as Fab, an scFv, a (Fab')₂, etc. The term humanized also denotes forms of non-human (for example, murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) that contain minimal sequence of non-human immunoglobulin. Humanized antibodies can include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are substituted by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (CDR LI, CDR L2, CDR L3, CDR HI, CDR H2, and/or CDR H3) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily denote the process by which the antibody was created.

[0089] A "CDR-grafted antibody" as used herein refers to a humanized antibody in which one or more complementarity determining regions (CDRs) of a first (non- human) species have been grafted onto the framework regions (FRs) of a second (human) species.

[0090] A "human antibody" as used herein encompasses antibodies produced in humans, antibodies produced in non-human animals that comprise human immunoglobulin genes, such as XenoMouse^® mice, and antibodies selected using in vitro methods, such as phage display (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al, 1998, Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581), wherein the antibody repertoire is based on a human immunoglobulin sequence. The term "human antibody" denotes the genus of sequences that are human sequences. Thus, the term is not designating the process by which the antibody was created, but the genus of sequences that is relevant. [0091] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding; CDC; ADCC; phagocytosis; down regulation of cell surface receptors (for example B-cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.

[0092] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0093] A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. In some embodiments, the variant Fc region herein will possess at least about 80% sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide, at least about 90% sequence identity therewith, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith.

[0094] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcyR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0095] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al).

[0096] "Effector functions" refer to biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation.

[0097] "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcyRIII and perform ADCC effector function(s). Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T-cells, and neutrophils. The effector cells may be isolated from a native source, for example, from blood.

[0098] "Antibody-dependent T-cell-mediated cytotoxicity" and "ADCC" refer to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (for example NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased ADCC activity are described, for example, in U.S. Pat. No. 7,923,538, and U.S. Pat. No. 7,994,290.

[0099] "Complement dependent cytotoxicity" and "CDC" refer to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, for example, as described in Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased Clq binding capability are described, for example, in U.S. Pat. No. 6,194,551 B l, U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290 and WO 1999/51642. See also, for example, Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0100] A polypeptide variant with "altered" FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. The polypeptide variant which "displays increased binding" to an FcR binds at least one FcR with better affinity than the parent polypeptide. The polypeptide variant which "displays decreased binding" to an FcR, binds at least one FcR with lower affinity than a parent polypeptide. Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, for example, 0-20% binding to the FcR compared to a native sequence IgG Fc region.

[0101] The polypeptide variant which "mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells more effectively" than a parent antibody is one which in vitro or in vivo is more effective at mediating ADCC, when the amounts of polypeptide variant and parent antibody used in the assay are essentially the same. Generally, such variants will be identified using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, for example in an animal model etc., are contemplated.

[0102] The term "substantially similar" or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two or more numeric values such that one of skill in the art would consider the difference between the two or more values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said value. In some embodiments the two or more substantially similar values differ by no more than about any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0103] The phrase "substantially different," as used herein, denotes a sufficiently high degree of difference between two numeric values such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. In some embodiments, the two substantially different numeric values differ by greater than about any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0104] The phrase "substantially reduced," as used herein, denotes a sufficiently high degree of reduction between a numeric value and a reference numeric value such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. In some embodiments, the substantially reduced numeric values is reduced by greater than about any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

[0105] The term "leader sequence" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence can be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences can be natural or synthetic, and they can be heterologous or homologous to the protein to which they are attached. [0106] A "native sequence" polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide found in nature. Thus, a native sequence polypeptide can have the amino acid sequence of naturally occurring polypeptide from any mammal. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (for example, an extracellular domain sequence), naturally occurring variant forms (for example, alternatively spliced forms) and naturally occurring allelic variants of the polypeptide.

[0107] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant will have at least about 80% amino acid sequence identity. In some embodiments, a variant will have at least about 90% amino acid sequence identity. In some embodiments, a variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.

[0108] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [0109] Amino acid substitutions may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Table 1

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe. [0110] Non-conservative substitutions will entail exchanging a member of one of these classes for a member from another class.

[0111] The term "vector" is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, β-galactosidase). The term "expression vector" refers to a vector that is used to express a polypeptide of interest in a host cell.

[0112] A "host cell" refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6^® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) as provided herein.

[0113] The term "isolated" as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be "isolating" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as "isolated".

[0114] The terms "individual" or "subject" are used interchangeably herein to refer to an animal; for example a mammal. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to receive the treatment can be a patient, designating the fact that the subject has been identified as having a disorder of relevance to the treatment, or being at adequate risk of contracting the disorder.

[0115] A "disease" or "disorder" as used herein refers to a condition where treatment is needed and/or desired.

[0116] The term "tumor cell", "cancer cell", "cancer", "tumor", and/or "neoplasm", unless otherwise designated, are used herein interchangeably and refer to a cell (or cells) exhibiting an uncontrolled growth and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. The terms "cancer" and "tumor" encompass solid and hematological/lymphatic cancers and also encompass malignant, pre-malignant, and benign growth, such as dysplasia. Also, included in this definition are cells having abnormal proliferation that is not impeded (e.g. immune evasion and immune escape mechanisms) by the immune system (e.g. virus infected cells). Exemplary tumor cells include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g. , small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS -related lymphoma; and Waldenstrom's Macro globulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasts leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0117] The term "non-tumor cell" as used herein refers to a normal cell or tissue. Exemplary non-tumor cells include, but are not limited to: T-cells, B-cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, interstitial kidney cells, fibroblast-like synoviocytes, osteoblasts, and cells located in the breast, skeletal muscle, pancreas, stomach, ovary, small intestines, placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term "a cell or tissue located in the periphery" as used herein refers to non-tumor cells not located near tumor cells and/or within the tumor microenvironment.

[0118] The term "cells or tissue within the tumor microenvironment" as used herein refers to the cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell. Exemplary cells or tissue within the tumor microenvironment include, but are not limited to: tumor vasculature; tumor- infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T-cells; macrophages; neutrophils; and other immune cells located proximal to a tumor. Methods for identifying tumor cells, and/or cells/tissues located within the tumor microenvironment are well known in the art, as described herein, below.

[0119] As used herein, the phrase "inhibiting or reducing T cell activation" refers to decreasing the activity of a target T cell subpopulation(s), as measured using a suitable in vitro, cellular, or in vivo assay. In particular, "reducing" or "inhibiting" can mean decreasing a (relevant or intended) biological activity of a target T cell subpopulation(s), as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target involved), by: at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, inclusive, compared to activity of the target in the same assay under the same conditions but without the presence of an agent. A "decrease" refers to a statistically significant decrease. For the avoidance of doubt, an decrease will be at least about 10% relative to a reference, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, inclusive. As will be clear to the skilled person, "inhibiting" can also involve effecting a change in affinity, avidity, specificity and/or selectivity of a target or antigen, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; effecting a change and/or decrease in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.); and/or cellular proliferation or cytokine production compared to the same conditions but without the presence of an antibody, bispecific or multispecific polypeptide agent. This can be determined in any suitable manner and/or using any suitable assay known per se or described herein, depending on the target involved.

[0120] As used herein, the term "tolerance" or "tolerance to a tumor" refers to tumor- induced tolerance and/or immune suppression caused by the tumor. In particular immunological tolerance refers to a state of immune unresponsiveness specific to a particular tumor antigen or a set of tumor antigens. The phrase can refer to decreasing the activity of immune cell populations or subpopulations, as measured using a suitable in vitro, cellular, or in vivo assay to determine "change or modulation" of the activity and/or population of immune cells within the tumor and/or tumor microenvironment. In particular, "change or modulation" can mean increasing or decreasing a (relevant or intended) biological activity of a target T-cell subpopulation(s), as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target involved), by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, inclusive, compared to activity of the target in the same assay under the same conditions but without the presence of an agent.

[0121] An "increase or decrease" refers to a statistically significant increase or decrease respectively. As will be clear to the skilled person, "modulating" can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.); and/or cellular proliferation or cytokine production, compared to the same conditions but without the presence of an antibody, bispecific or multispecific polypeptide agent. This can be determined in any suitable manner and/or using any suitable assay known per se or described herein, depending on the target involved.

[0122] As used herein, "an immune response" is meant to encompass cellular and/or humoral immune responses that are sufficient to inhibit or prevent onset or ameliorate the symptoms of disease (for example, cancer or cancer metastasis). "An immune response" can encompass aspects of both the innate and adaptive immune systems.

[0123] The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokine secretions, and traditional polypeptide hormones. Included among the cytokines are, for example, growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and tumor necrosis factor-β; mullerian-inhibiting substance; mouse gonadotropin- associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-a; platelet- growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteo inductive factors; interferons such as, for example, interferon-a, interferon-β and interferon-γ (and interferon type I, II, and III), colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as, for example, IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12; a tumor necrosis factor such as, for example, TNFa or TNF-β; and other polypeptide factors including, for example, LIF and kit ligand (KL); chemokine (C-C motif) ligands (CCLs) such as CCL1, CCL2 CCL3, CCL4, and CCL5. As used herein, the term cytokine includes proteins obtained from natural sources or produced from recombinant bacterial, eukaryotic or mammalian cell culture systems and biologically active equivalents of the native sequence cytokines.

[0124] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. "Treatment" as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by "treatment" is a reduction of pathological consequence of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder. [0125] "Ameliorating" means a lessening or improvement of one or more symptoms as compared to not administering; for example administering a TIM-3 antibody compared to not administering a TIM-3 antibody. "Ameliorating" also includes shortening or reduction in duration of a symptom.

[0126] The term "biological sample" means a quantity of a substance from a living thing or formerly living thing. Such substances include, but are not limited to, blood, (for example, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

[0127] The term "control" refers to a composition known to not contain an analyte ("negative control") or to contain analyte ("positive control"). A positive control can comprise a known concentration of analyte. "Control," "positive control," and "calibrator" may be used interchangeably herein to refer to a composition comprising a known concentration of analyte. A "positive control" can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (for example, analytes).

[0128] "Predetermined cutoff and "predetermined level" refer generally to an assay cutoff value that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the predetermined cutoff/level, where the predetermined cutoff/level already has been linked to or associated with various clinical parameters (for example, severity of disease, progression/nonprogression/improvement, etc.). While the present disclosure may provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (for example, antibodies employed, etc.). It further is well within the skill of one of ordinary skill in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific cutoff values for those other immunoassays based on this disclosure. Whereas the precise value of the predetermined cutoff/level may vary between assays, correlations as described herein (if any) may be generally applicable.

[0129] The terms "inhibition" or "inhibit" refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic; for example the interaction of TIM-3 and LILRA3. To "reduce" or "inhibit" is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. In some embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 1% or greater. In some embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 10% or greater. In some embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 50% or greater. In some embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time.

[0130] The terms "stimulate" refer to an induction or stimulation of any phenotypic characteristic or to the induction or increase in the incidence, degree, or likelihood of that characteristic; for example the expression of pro-inflammatory cytokines. To "stimulate" is to increase or induce an activity, function, and/or amount as compared to a reference. In some embodiments, by "stimulate" or "increase" is meant the ability to cause an overall increase of 1% or greater. In some embodiments, by "stimulate" or "increase" is meant the ability to cause an overall increase of 10% or greater. In some embodiments, by "stimulate" or "increase" is meant the ability to cause an overall increase of 50% or greater. In some embodiments, by "stimulate" or "increase" is meant the ability to cause an overall increase of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amount noted above is stimulated or increased over a period of time, relative to a control dose (such as a placebo) over the same period of time.

[0131] As used herein, "delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0132] "Preventing," as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms "reduce", "inhibit", or "prevent" do not denote or require complete prevention over all time.

[0133] As used herein, to "suppress" a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.

[0134] A "therapeutically effective amount" of a substance/molecule, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and/or prophylactic result.

[0135] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0136] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile.

[0137] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0138] A "sterile" formulation is aseptic or essentially free from living microorganisms and their spores.

[0139] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[0140] The term "concurrently" is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered with a time separation of no more than about a specified number of minutes.

[0141] The term "sequentially" is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s). For example, administration of the two or more therapeutic agents are administered with a time separation of more than about a specified number of minutes.

[0142] As used herein, "in conjunction with" refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0143] As used herein, the term "immune checkpoint inhibitor" refers to an agent that blocks certain proteins (e.g. , checkpoint proteins) made by some types of immune system cells, such as T cells, and some cancer cell that keep immune responses in check and can keep T cells from killing cells (e.g., cancer cells). When these proteins are blocked, the "brakes" on the immune system are released and T cells are able to kill cells (e.g., cancer cells) better. Examples of checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7- 1/B7-2. In some embodiments, an immune checkpoint inhibitor is an agent (e.g. , an antibody) that inhibits the activity of checkpoint proteins, including but not limited to PD-1/PD-L1 and CTLA-4/B7- 1/B7-2. [0144] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0145] An "article of manufacture" is any manufacture (for example, a package or container) or kit comprising at least one reagent, for example, a medicament for treatment of a disease or disorder (for example, cancer), or a probe for specifically detecting a biomarker described herein. In some embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0146] The terms "label" and "detectable label" mean a moiety attached to an antibody or its analyte to render a reaction (for example, binding) between the members of the specific binding pair, detectable. The labeled member of the specific binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" refers to a protein with a label incorporated that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (for example, 3 H, 14 C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ^mIn, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); chromogens, fluorescent labels (for example, FITC, rhodamine, lanthanide phosphors), enzymatic labels (for example, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, for example, acridinium compounds, and moieties that produce fluorescence, for example, fluorescein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Methods and Compositions Relating to LILRA3

[0147] The interaction between cancer and the immune system is complex and multifaceted. See de Visser et ah, Nat. Rev. Cancer (2006) 6:24-37. While many cancer patients appear to develop an anti-tumor immune response, cancers also develop strategies to evade immune detection and destruction. Recently, immunotherapy has been developed for the treatment and prevention of cancer and other disorders. Immunotherapy provides the advantage of cell specificity that other treatment modalities lack. As such, methods for enhancing the efficacy of immune based therapies can be clinically beneficial.

[0148] The therapeutic molecules modulate the activity of LILRA3. Modulating LILRA3 activity may lead to the secretion of myeloid- associated pro-inflammatory cytokines; for example, cytokines produced by or that interact with macrophages. In some embodiments, the molecules modulate LILRA3 activity such that macrophages are preferentially activated. In some embodiments, the molecules modulate LILRA3 activity such that dendritic cells are preferentially activated. In some embodiments, the molecules modulate LILRA3 activity such that macrophages and dendritic cells are preferentially activated.

[0149] In some embodiments, the molecule modulates the interaction of TIM-3 and LILRA3. In some embodiments, the molecule is a LILRA3 polypeptide or functional variant thereof {e.g., a functional fragment of LILRA3). In some embodiments, the molecule binds TIM-3. In some embodiments, the molecule is an antibody that binds TIM-3. In some embodiments, the modulation of the interaction is an increase in the interaction of TIM-3 and LILRA3; for example, an increase in the binding of TIM-3 and LILRA3. Increasing the interaction of TIM-3 and LILRA3 may lead to the secretion of myeloid-associated pro-inflammatory cytokines; for example, cytokines produced by or that interact with macrophages. In some embodiments, the molecules are antibodies that increase the interaction of TIM-3 and LILRA3 such that macrophages are preferentially activated. In some embodiments, the molecules are antibodies that decrease the interaction of TIM-3 and LILRB2 such that macrophages are preferentially activated. In some embodiments, the antibodies increase the interaction of TIM-3 and LILRA3 such that dendritic cells are preferentially activated. In some embodiments, the molecules are antibodies that decrease the interaction of TIM-3 and LILRB2 such that dendritic cells are preferentially activated. In some embodiments, the antibodies increase the interaction of TIM-3 and LILRA3 such that macrophages and dendritic cells are preferentially activated. In some embodiments, the antibody competes with LILRA3 for binding TIM-3 and inhibits binding of LILRB2 to TIM-3 such that macrophages and dendritic cells are preferentially activated.

[0150] In some embodiments, the molecules (e.g., an LILRA3 polypeptide or functional variant thereof or an antibody) of the invention inhibit, block and/or reduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; or stimulate, induce, and/or increase cell death of a pro-tumor T cell. T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferative and effector functions, culminating in clonal deletion (See, e.g., Virgin et al. (2009) Cell 138:30-50). Accordingly, as used herein the term "a pro-tumor T cell" refers to a state of T cell dysfunction that arises during many chronic infections and cancer. This dysfunction is defined by poor proliferative and/or effector functions, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. See Wherry, J. W. T cell exhaustion. Nat Immunol (2011) 12:492-499. In addition, as used herein, the term "an anti-tumor CD8+ and/or CD4+ T cell" refers to T cells that can mount an immune response to a tumor. Exemplary pro-tumor T cells include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells and Thl7 cells. The term "checkpoint inhibitory receptors", as used herein, refers to receptors (e.g. CTLA-4, B7-H3, B7-H4, PD-1, TIM-3) expressed on immune cells that prevent or inhibit uncontrolled immune responses. See Stagg, J. et al., Immunotherapeutic approach in triple-negative breast cancer. Ther Adv Med Oncol. (2013) 5(3): 169- 181. Thus, in some embodiments, inhibition of TIM-3 activity can include reducing the level of and/or preventing the inhibition of T cell proliferation. In some embodiments, this can also be described as restoring and/or increasing T cell proliferation. In some embodiments, the inhibition of TIM-3 activity can also be described as restoring and/or increasing myeloid cell proliferation, activation and/or differentiation; for example, activation of monocytes, macrophages, and/or dendritic cells.

[0151] In some embodiments, the modulation of LILRA3 activity is an increase in the binding of TIM-3 and LILRA3. An increase in the interaction of TIM-3 and LILRA3 leads to the secretion of myeloid-associated pro-inflammatory cytokines; for example, cytokines produced by or that interact with macrophages. In some embodiments, the molecules increase the interaction of TIM-3 and LILRA3 such that macrophages are preferentially activated. In some embodiments, the molecules increase the interaction of TIM-3 and LILRA3 such that dendritic cells are preferentially activated. In some embodiments, the molecules increase the interaction of TIM-3 and LILRA3 such that macrophages and dendritic cells are preferentially activated.

[0152] Despite recent advances, a need has been identified for more effective treatments of cancer utilizing immunotherapy. More particularly, a need has been identified for novel anti-TIM-3 antibodies or antibodies that inhibit the interaction of TIM-3, its ligands and methods that modulate TIM-3 activity which are capable of enhancing the host immune response against tumors for treating cancer. For example, to allow for increased T-cell proliferation, for example, for the treatment of cancer.

Methods of Treating Diseases using Molecules that Modulate LILRA3 Activity

[0153] In some aspects, the invention provides methods of stimulating the secretion of a myeloid-associated cytokine in an individual. The method comprises administering to the individual an effective amount of a molecule that modulates the activity of LILRA3. In some embodiments, the modulation of the activity of LILRA3 may lead to the activation of monocytes; e.g. , macrophages, which leads to the secretion of pro -inflammatory cytokines. In some embodiments, the molecule is an antibody that binds TIM-3. In some embodiments, the molecule is an antibody competes with LILRA3 for binding to TIM-3 and inhibits binding of LILRB2 binding to TIM-3. In some embodiments, activation of LILRA3 leads to the preferential activation of macrophages and/or the preferential secretion of pro-inflammatory myeloid-associated cytokines. In some embodiments, activation of LILRA3 leads to the preferential activation of dendritic cells and/or the preferential secretion of proinflammatory myeloid-associated cytokines. In some embodiments, activation of LILRA3 leads to the preferential activation of macrophages and dendritic cells and/or the preferential secretion of pro-inflammatory myeloid-associated cytokines. In some embodiments, the individual is human.

[0154] In some embodiments, the molecule is a LILRA3; for example but not limited to, a LILRA3 fusion protein, a functional variant of a LILRA3 polypeptide, a fusion protein comprising a LILRA3 functional variant. In some embodiments, the molecule binds TIM-3. In some embodiments, the molecule that binds TIM-3 is an antibody.

[0155] In some aspects, the invention provides methods of stimulating the secretion of a myeloid-associated cytokine in an individual. The method comprises administering to the individual, an effective amount of a molecule that modulates the activity of LILRA3. In some embodiments, the modulation of the activity LILRA3 is an increase in the interaction of TIM-3 and LILRA3; for example, by increasing the binding of TIM-3 and LILRA3. The increase in the interaction of TIM-3 and LILRA3 may lead to the activation of monocytes; e.g. , macrophages, which leads to the secretion of pro-inflammatory cytokines. In some embodiments, the molecule is an antibody that binds TIM-3. In some embodiments, binding of the antibody to TIM-3 leads to the preferential activation of macrophages and/or the preferential secretion of pro-inflammatory myeloid-associated cytokines. In some embodiments, binding of the antibody to TIM-3 leads to the preferential activation of dendritic cells and/or the preferential secretion of pro-inflammatory myeloid-associated cytokines. In some embodiments, binding of the antibody to TIM-3 leads to the preferential activation of macrophages and dendritic cells and/or the preferential secretion of proinflammatory myeloid-associated cytokines. In some embodiments, the individual is human.

[0156] In some embodiments, the pro-inflammatory cytokine is IL- 12, TNFa, IL-Ιβ, GM-CSF, or IL-6. In some embodiments, any one, any two, any three, any four, or all five cytokines are secreted by monocytes or macrophages following administration of a molecule that modulates the activity of LILRA3. In some embodiments, the one or more pro -inflammatory cytokine is IL- 12, TNFa, IL- Ιβ, GM-CSF, or IL-6 and is secreted by or interacts with monocytes following administration of a molecule that modulates the activity of LILRA3. In some embodiments, secretion of proinflammatory cytokines following administration of a molecule of the invention is increased compared to secretion of pro-inflammatory cytokines following administration of antibody F38-2E2. In some embodiments, the secretion of proinflammatory cytokines (e.g. , IL- 12, TNFa, IL- Ιβ, GM-CSF, or IL-6) is at least about any of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold following administration of a molecule of the invention compared to secretion of proinflammatory cytokines following administration of antibody F38-2E2. In some embodiments, activation of macrophages is increased by at least about any of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold following administration of a molecule of the invention compared to activation of macrophages following administration of antibody F38-2E2. In some embodiments, activation of dendritic cells is increased by at least about any of 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, or 10-fold following administration of a molecule of the invention compared to activation of dendritic cells following administration of antibody F38-2E2. In some embodiments, activation of macrophages and dendritic cells is increased following administration of a molecule of the invention compared to activation of macrophages and dendritic cells following administration of antibody F38-2E2.

[0157] In some embodiments, treatment with a molecule of the invention suppresses secretion of cytokines. In some embodiments, treatment with a molecule of the invention suppresses expression of one or more of IL- 10, CCL2, CCL3, CCL4 or CCL5. In some embodiments, treatment with a molecule of the invention suppresses expression of one or more of IL- 10, CCL2, or CCL5. In some embodiments, treatment with a molecule of the invention suppresses expression of one or more of IL- 10 or CCL5. In some embodiments, treatment with a molecule of the invention suppresses expression of one or more immunosuppressive cytokines. In some embodiments, secretion of any one, any two, any three, any four, or all five cytokines by monocytes or macrophages is suppressed following administration of a molecule that activates LILRA3. In some embodiments, secretion of cytokines following administration of a molecule of the invention is suppressed compared to suppression of cytokines following administration of antibody F38-2E2. In some embodiments, the secretion cytokines (e.g. , IL- 10, CCL2, CCL3, CCL4 or CCL5) is at least about any of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold suppressed following administration of a molecule of the invention compared to suppression of secretion cytokines following administration of antibody F38-2E2.

[0158] In some embodiments, the invention provides methods of stimulating the secretion of a myeloid- associated cytokine in an individual with cancer, wherein the method comprises administering to the individual, an effective amount of a molecule that modulates the activity of LILRA3. In some embodiments, the cytokines are secreted in a tumor; for example, pro-inflammatory cytokines are secreted by a monocyte, a macrophage or a dendritic cell located in or near a tumor. In some embodiments, the individual is human.

[0159] In some aspects, the invention provides methods for treating cancer in an individual. The method comprises administering to the individual an effective amount of a molecule that modulates the activity LILRA3. In some embodiments, the modulation of the activity of LILRA3 is an increase in the interaction of TIM-3 and LILRA3; for example, by increasing the binding of TIM-3 and LILRA3. The increase in the interaction of TIM-3 and LILRA3 may lead to the activation of monocytes; e.g. , macrophages, which leads to the secretion of pro-inflammatory cytokines. In some embodiments, the molecule binds TIM-3. In some embodiments, the molecule is a LILRA3; for example but not limited to, a LILRA3 fusion protein, a functional variant of a LILRA3 polypeptide, a fusion protein comprising a LILRA3 functional variant, or an antibody. In some embodiments, binding of the molecule to TIM-3 leads to the preferential activation of macrophages and/or the preferential secretion of pro-inflammatory cytokines by macrophages. In some embodiments, binding of the molecule to TIM-3 leads to the preferential activation of dendritic cells and/or the preferential secretion of pro-inflammatory cytokines by dendritic cells. In some embodiments, binding of the molecule to TIM-3 leads to the preferential activation of macrophages and dendritic cells and/or the preferential secretion of proinflammatory cytokines by macrophages and dendritic cells. In some embodiments, binding of the molecule to TIM-3 leads to the preferential secretion of proinflammatory cytokines that interact with macrophages. In some embodiments, binding of the molecule to TIM-3 leads to the preferential secretion of proinflammatory cytokines that interact with dendritic cells. In some embodiments, binding of the molecule to TIM-3 leads to the preferential secretion of proinflammatory cytokines that interact with macrophages and dendritic cells. In some embodiments, the cytokines are secreted in a tumor; for example, pro-inflammatory cytokines are secreted by a monocyte, a macrophage or a dendritic cell located in or near a tumor. In some embodiments, the individual is human.

[0160] Molecules and compositions comprising molecules are provided for use in methods of treatment for humans or animals. Methods of treating disease comprising administering molecules that modulate the activity of LILRA3 are also provided. Nonlimiting exemplary diseases that can be treated with molecules that modulate the activity of LILRA3 include, but are not limited to, various forms of cancer. In some embodiments, the molecule is a LILRA3, a LILRA3 fusion, a functional variant of a LILRA3, or an antibody that binds TEVI-3.

[0161] The molecules that modulate the activity of LILRA3 can be administered as needed to subjects. Determination of the frequency of administration can be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like. In some embodiments, an effective dose of an antibody is administered to a subject one or more times. In some embodiments, an effective dose of an antibody is administered to the subject once a month, more than once a month, such as, for example, every two months or every three months. In some embodiments, an effective dose of an antibody is administered less than once a month, such as, for example, every two weeks or every week. An effective dose of an antibody is administered to the subject at least once. In some embodiments, the effective dose of an antibody may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.

[0162] In some embodiments, pharmaceutical compositions are administered in an amount effective for treatment of (including prophylaxis of) cancer. The therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated. In general, molecules that modulate the activity of LILRA3 may be administered in an amount in the range of about 0.05 mg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, molecules that modulate the activity of LILRA3 may be administered in an amount in the range of about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.05 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.05 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 5 mg/kg body weight or lower, for example less than 4, less than 3, less than 2, or less than 1 mg/kg of the antibody.

[0163] In some embodiments, pharmaceutical compositions are administered in an amount effective for treatment of cancer and/or encouraging T-cell proliferation. The therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated. In general, molecules that modulate the activity of LILRA3 may be administered in an amount in the range of about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 5 mg/kg body weight per dose.

[0164] Below is an outline of further embodiments and particulars for performing the above noted methods, as well as further methods. The placement of the embodiments below is to clarify that it is contemplated that any of the embodiments provided herein can be combined with any of the other aspects listed herein.

[0165] In some embodiments, the molecule that modulates the activity of LILRA3 is given concurrently with a second therapeutic agent, for example, a PD-1 therapy. Nonlimiting exemplary PD-1 therapies include nivolumab (anti-PD-1 antibody; BMS- 936558, MDX-1106, ONO-4538; OPDIVO^®; Bristol-Myers Squibb); pidilizumab (anti-PD-1 antibody, CureTech), pembrolizumab (anti-PD-1 antibody; KEYTRUDA^®, MK-3475, lambrolizumab); durvalumab (anti-PD-Ll antibody, MEDI-4736; AstraZeneca/Medlmmune); RG-7446; MSB-0010718C; AMP-224; BMS-936559 (an anti-PD-Ll antibody; Bristol-Myers Squibb); AMP-514; MDX- 1105; ANB-011; anti-LAG-3/PD- 1 ; anti-PD-1 Ab (CoStim); anti-PD-1 Ab (Kadmon Pharm.); anti-PD-1 Ab (Immunovo); anti-TIM-3/PD-l Ab (AnaptysBio); anti-PD-Ll Ab (CoStim/Novartis); atezolizumab (an anti-PD-Ll antibody, Genentech/Roche); avelumab (an anti-PD-Ll antibody, MSB0010718C, Pfizer); KD-033, PD-1 antagonist (Agenus); STI-A1010; STI-A1110; TSR-042; and other antibodies that are directed against programmed death- 1 (PD-1) or programmed death ligand 1 (PD-L1).

[0166] In some embodiments, the therapeutic treatment involving the use of a molecule that modulates the activity of LILRA3 is achieved by T-cell modulation. In some embodiments, increasing T-cell proliferation inhibits growth of the cancer. In some embodiments, inhibition of growth of the cancer is further enhanced by antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, inhibition of growth of the cancer does not occur by ADCC. In some embodiments, inhibition of growth of the cancer does not occur by ADC (antibody-drug conjugate). In some embodiments, inhibition of growth of the cancer occurs by allowing the host's immune system to properly act on the cancer. In some embodiments, T-cell proliferation is a result of T-cell activation. In some embodiments, the use of a molecule that modulates the activity of LILRA3 based therapy in one of the methods provided herein restores the subject's endogenous immune response to the cancer. In some embodiments, the subject's endogenous immune response is sufficient to slow the progression of or remove the cancer. In some embodiments, any of the methods provided herein can further comprise assaying an amount of TIM-3 present in a cancer in the subject. In some embodiments, the subject can be identified as one that has previously received no significant improvement from a PD-1 therapy. In some embodiments, the subject is one that received a detectable level of improvement from the PD-1 therapy, but an additional amount of improvement is beneficial or desired for the subject. Any method of detecting the level of a protein in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of proteins being detected. Nonlimiting exemplary such methods include immunohistochemistry, ELISA, Western blotting, multiplex analyte detection (using, for example, Luminex technology), mass spectrometry, etc. Similarly, any method of detecting the level of an mRNA in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of mRNAs being detected. Nonlimiting exemplary such methods include RT-PCR, quantitative RT-PCR and microarray-based methods, etc.

[0167] In some embodiments, the method of treatment or inducing T-cell proliferation described herein can further include administering: radiation therapy, chemotherapy, vaccination, targeted tumor therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, an antisense agent against a tumor target, a siRNA agent against a tumor target, a microRNA agent against a tumor target or an anti-cancer/tumor agent.

[0168] As will be appreciated by one of skill in the art, in some embodiments, any of the herein disclosed methods can be used separately or in combination for one or more of: treatment of cancer, increasing production of cytokines and/or increasing cytokine secretion, and/or increasing T-cell proliferation. Thus, any of the methods directed to any of these three areas (treatment of cancer, increasing production of cytokines and/or increasing cytokine secretion, and/or increasing T-cell proliferation) is contemplated as being alternative methods for the other two areas (treatment of cancer, increasing production of cytokines and/or increasing cytokine secretion, and/or increasing T-cell proliferation).

[0169] In some embodiments, the methods provided herein allow for one to increase production of cytokines and/or increase cytokine secretion. In some embodiments, any cytokine level can be increased. In some embodiments, the cytokine that has its level increased is at least one of IL-Ιβ, TNFa and/or IL-12.

Molecules that modulate the activity LILRA3

LILRA3 molecules

[0170] In some embodiments, the invention provides molecules that modulate the activity of LILRA3. Examples of molecules that modulate the activity of LILRA3 include but are not limited to polypeptides such as LILRA3 polypeptides and variants thereof, molecules that bind LILRA3, small molecules, nucleic acids (e.g. , nucleic acids that modulate the expression of LILRA3 such as siRNA, miRNA, shRNA, etc.).

[0171] In some embodiments, the molecule that modulates the activity of LILRA3 is a LILRA3 polypeptide. In some embodiments, the LILRA3 polypeptide is a human LILRA3 polypeptide. In some embodiments, the LILRA3 polypeptide is a LILRA3 isoform 1 polypeptide. In some embodiments, the LILRA3 polypeptide is a LILRA3 isoform 2 polypeptide. In some embodiments the LILRA3 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9. In some embodiments the LILRA3 polypeptide comprises an amino acid sequence that is more than about any of 80%, 85%, 90%, 95%, 99% identical to the sequence set forth in SEQ ID NO:7 or SEQ ID NO:9 while maintaining the activity of LILRA3 (e.g., binds to TIM-3 and/or stimulates pro-inflammatory cytokines).

[0172] In some embodiments the molecule that modulates LILRA3 activity is fused to another polypeptide. In some embodiments, the molecule that modulates LILRA3 activity is a LILRA3 polypeptide or functional variant thereof, fused to an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region. In some embodiments, the molecule that modulates LILRA3 activity is a human LILRA3 polypeptide or functional variant thereof fused to a human IgGl Fc region.

Antibodies

[0173] In some embodiments, the invention provides antibodies that modulate the activity of LILRA3 such that cells of myeloid lineage, particularly macrophages, are stimulated to secrete pro-inflammatory cytokines. In some embodiments the antibody increases the interaction of TIM-3 and LILRA3; for example, by increasing the binding of TIM-3 to LILRA3.

[0174] In some embodiments, the antibody specifically binds TIM-3 such that binding of TIM-3 to LILRA3 is increased. In some embodiments, the binding of TEVI-3 to LILRA3 is stimulated by at least about any one of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the binding of TIM-3 to LILRA3 is increased by any one of about 1% to about 10%, about 10% to about 25%, 10% to about 50%, 10% to about 75%, about 10% to about 100%, about 25% to about 50%, about 25% to about 75%, about 25% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%.

[0175] In some embodiments, the antibody specifically competes with LILRB2 for binding to TIM-3. Methods to determine competition for binding are known in the art; for example, by using the OctetRED 96 system as demonstrated in Example 6 below. Other examples include but are not limited to competitive binding in a flow- cytometric assay to a molecule displayed on the surface of a cell or bead or by ELISA where the molecule is bound to a plate and competition is demonstrated by competitive binding. In some embodiments, the antibody competes with LILRB2 for binding to TIM-3 such that binding of LILRB2 to TIM-3 is inhibited by at least about any one of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the binding of TIM-3 to LILRB2 is inhibited by any one of about 1% to about 10%, about 10% to about 25%, 10% to about 50%, 10% to about 75%, about 10% to about 100%, about 25% to about 50%, about 25% to about 75%, about 25% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%.

[0176] In some embodiments, the antibody is from a human, a mouse or a rat. In some embodiments, the TIM-3 is an isoform 1 TIM-3. In other embodiments, the TIM-3 is an isoform 2 TIM-3. In some embodiments, the TIM-3 comprises the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3 or SEQ ID NO:9. In some embodiments, the TIM-3 is a variant of TIM-3 isoform 1 or TIM-3 isoform 2. In some embodiments, the TIM-3 comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-25 or 25-50 amino acid substitutions of the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3 or SEQ ID NO:9, while maintaining TIM-3 activity. In some embodiments, the TIM-3 comprises an amino acid sequence that is at least about any of 60%, 70%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3 or SEQ ID NO:9.

[0177] In some aspects, the invention provides an antibody that modulates the activity of LILRA3. In some embodiments, the modulation of activity of LILRA3 is an increase in the interaction of TIM-3 and LILRA3; for example, by increasing the binding of TIM-3 and LILRA3. The increase in the interaction of TIM-3 and LILRA3 may lead to the activation of cells of monocyte/macrophage lineages; e.g. , macrophages, which leads to the secretion of pro-inflammatory cytokines. In some embodiments, the antibody binds TIM-3. In some embodiments, binding of the antibody to TIM-3 leads to the preferential activation of macrophages. In some embodiments, binding of the antibody to TIM-3 leads to the preferential activation of dendritic cells. In some embodiments, binding of the antibody to TIM-3 leads to the preferential activation of macrophages and dendritic cells. In some embodiments, binding of the antibody to TIM-3 competes with binding to LILRA3 to TIM-3 and inhibits the binding of LILRB2 to TIM-3. In some embodiments, binding of the antibody to TIM-3 leads to the preferential secretion of pro-inflammatory cytokines by macrophages. In some embodiments, binding of the antibody to TIM-3 leads to the preferential secretion of pro-inflammatory cytokines by dendritic cells. In some embodiments, binding of the antibody to TIM-3 leads to the preferential secretion of pro-inflammatory cytokines by macrophages and dendritic cells. In some embodiments, binding of the antibody to TIM-3 leads to the preferential secretion of pro-inflammatory cytokines that interact with macrophages. In some embodiments, binding of the antibody to TIM-3 leads to the preferential secretion of proinflammatory cytokines that interact with dendritic cells. In some embodiments, binding of the antibody to TIM-3 leads to the preferential secretion of proinflammatory cytokines that interact with macrophages and dendritic cells. In some embodiments, the individual is human.

[0178] In some embodiments, the pro-inflammatory cytokine is IL- 12, TNFa, IL-Ιβ, GM-CSF, or IL-6. In some embodiments, any one, any two, any three, any four or all five cytokines are secreted by monocytes or macrophages following administration of an antibody that increases the interaction of TIM-3 and LILRA3. In some embodiments, one or more of pro-inflammatory cytokine is IL-12, TNFa, IL- Ιβ, GM- CSF or IL-6 is secreted following administration of an antibody that increases the interaction of TIM-3 and LILRA3. In some embodiments, secretion of proinflammatory cytokines following administration of an antibody of the invention is increased compared to secretion of pro-inflammatory cytokines following administration of antibody F38-2E2. In some embodiments, the secretion of proinflammatory cytokines (e.g. , IL- 12, TNFa, IL-Ιβ, GM-CSF or IL-6) is at least about any of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold following administration of an antibody of the invention compared to secretion of proinflammatory cytokines following administration of antibody F38-2E2. [0179] In some embodiments, the antibody suppresses secretion of a cytokine. In some embodiments, the cytokine is IL- 10, CCL2, CCL3, CCL4 or CCL5. In some embodiments, secretion of any one, any two, any three, any four or all five cytokines are inhibited following administration of an antibody that modulates the activity of LILRA3. In some embodiments, secretion of cytokines following administration of an antibody of the invention is suppressed compared to secretion of cytokines following administration of antibody F38-2E2. In some embodiments, the secretion of cytokines (e.g. , IL- 10, CCL2, CCL3, CCL4 or CCL5) is at least about any of 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold less following administration of an antibody of the invention compared to secretion of proinflammatory cytokines following administration of antibody F38-2E2. In some embodiments, the secretion of immunosuppressive cytokines is inhibited.

[0180] In some embodiments, the invention provides antibodies that stimulate the secretion of a myeloid-associated cytokine in an individual with cancer. In some embodiments, the cytokines are secreted in a tumor; for example, pro-inflammatory cytokines are secreted by a cell located in or near a tumor. In some embodiments, the individual is human.

[0181] In some embodiments, the antibody that modulates the activity of LILRA3 is a monoclonal antibody; for example, a monoclonal antibody that binds TIM-3. In some embodiments, the monoclonal antibody is chimeric antibody, a humanized antibody or a human antibody. In some embodiments, the monoclonal antibody is an antigen binding fragment; for example, a Fab, a Fab', an Fv, an scFv, or a (Fab')2 fragment.

[0182] In some embodiments, the antibody that modulates the activity of LILRA3 comprises a heavy chain variable region and a light chain variable region. In some embodiments, the antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. As used herein, a single-chain Fv (scFv), or any other antibody that comprises, for example, a single polypeptide chain comprising all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs. In some embodiments, the light chain is the region of the antibody that comprises the three light chain CDRs.

[0183] In some embodiments, an antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81 :6851-6855. In one example, a chimeric antibody comprises a non-human variable region (for example, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0184] In some embodiments, a chimeric antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a chimeric antibody described herein comprises a human IgG constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 constant region and a human κ light chain.

[0185] As noted above, whether or not effector function is desirable may depend on the particular method of treatment intended for an antibody. Thus, in some embodiments, when effector function is desirable, a chimeric antibody comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desirable, a chimeric antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

[0186] In some embodiments, humanized antibodies that modulate the activity of LILRA3 are provided. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can result in an immune response to an antibody therapeutic, and decreased effectiveness of the therapeutic. [0187] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (for example, the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0188] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033; US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"); Dall'Acqua et al, (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al, (2005) Methods 36:61-68 and Klimka et al, (2000) Br. J. Cancer, 83:252-260 (describing the "guided selection" approach to FR shuffling).

[0189] Human framework regions that can be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, for example, Sims et al. (1993) J. Immunol. 151 :2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633); and framework regions derived from screening FR libraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272: 10678-10684 and Rosok et al, (1996) J. Biol. Chem. Ill :22611-22618).

[0190] In some embodiments, the antibody that modulates the activity of LILRA3 is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, (2001) Curr. Opin. Pharmacol. 5:368-374 and Lonberg, (2008) Curr. Opin. Immunol. 20:450-459. In some embodiments, the human antibody is not a naturally occurring antibody. In some embodiments, the human antibody is a monoclonal antibody; thus, in some embodiments, each of the human antibodies in a set can bind to the same epitope on the antigen.

[0191] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, (2005) Nat. Biotech. 23: 1117-1125. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB^® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE^® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE^® technology. Human variable regions from intact antibodies generated by such animals may be further modified, for example, by combining with a different human constant region.

[0192] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor (1984) J. Immunol, 133: 3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, (1991) J. Immunol., 147:86). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., (2006) Proc. Natl. Acad. Sci. USA, 103:3557-3562. Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, (2006) Xiandai Mianyixue, 26(4):265- 268 (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, (2005) Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, (2005) Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-191.

[0193] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0194] Antibodies may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in the McCafferty et al, (1990) Nature 348:552-554; Clackson et al, (1991) Nature 352: 624-628; Marks et al, (1992) J. Mol. Biol 222: 581-597; Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al, (2004) J. Mol. Biol. 338(2): 299-310; Lee et al, (2004) J. Mol. Biol. 340(5): 1073-1093; Fellouse, (2004) Proc. Natl. Acad. Sci. USA 101(34): 12467-12472; and Lee et al, (2004) J. Immunol. Methods 284(1-2): 119-132 and PCT publication WO 99/10494.

[0195] In certain phage display methods, repertoires of V_H and V_L genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., (1994) Ann. Rev. Immunol., 12:433-455. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (for example, from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al, (1993) EMBO J 12:725-734. Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter (1992), J. Mol. Biol, 227:381-388. Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

[0196] In some embodiments, the antibody that modulates the activity of LILRA3 comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a human antibody described herein comprises a human IgG constant region. In some embodiments, a human antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, a human antibody described herein comprises a human IgG4 constant region and a human κ light chain.

[0197] In some embodiments, when effector function is desirable, a human antibody comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desirable, a human TIM-3 antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

[0198] As noted herein, the term "human antibody" denotes the genus of possible sequences for the antibody construct, rather than a source of the antibody.

[0199] In some embodiments, the antibodies inhibit and/or reduce a tumor intrinsic signal. In some embodiments, the tumor intrinsic signal is one or more signals selected from: a pro-survival signal; an autocrine or paracrine growth signal; a differentiation signal; a STAT-, JAK-, AKT- or PI3K-mediated signal; an anti- apoptotic signal; and a signal promoting and/or necessary for one or more of: tumor invasiveness, metastasis, epithelial-mesenchymal transition, and/or spreading from one tissue or organ to another non-adjacent tissue or organ.

[0200] In some embodiments, the antibodies inhibit or reduce immune modulation or immune tolerance to tumor cells. In some embodiments, the antibody inhibits or reduces the activity or activation of one or more cells including, but not limited to: regulatory T-cells (Tregs); myeloid suppressor cells; tumor associated neutrophils (TANs) and tumor associated macrophages (TAMs). [0201] In some embodiments, the antibodies described herein enhance, restore, promote and/or stimulate immune modulation. In some embodiments, the antibodies enhance, restore, promote and/or stimulate the activity or activation of one or more immune cells against tumor cells including, but not limited to: T-cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. Ml macrophages), macrophages, B-cells, and dendritic cells.

[0202] In some embodiments, the antibodies enhance, restore, promote and/or stimulate the activity and/or activation of T-cells, including, by way of a non-limiting example, activating, enhancing, restoring, and/or stimulation one or more T-cell intrinsic signals, including a pro-survival signal; an autocrine or paracrine growth signal; a proliferative signal; a differentiation signal; a T-cell maturation signal; a p38 MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; an anti-apoptotic signal; and/or a signal promoting and/or necessary for one or more of: cell survival, cell-cycle progression, T-cell proliferation, glucose metabolism, protein synthesis and cytokine production.

Exemplary Antibody Constant Regions

[0203] In some embodiments, an antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, an antibody described herein comprises a human IgG constant region.

[0204] Throughout the present specification and claims unless explicitly stated or known to one skilled in the art, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody.

[0205] As noted above, whether or not effector function is desirable may depend on the particular method of treatment intended for an antibody. Thus, in some embodiments, when effector function is desirable, the antibody that modulates the activity of LILRA3 comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desirable, a TIM-3 antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

[0206] In some embodiments, an antibody comprises a variant Fc region has at least one amino acid substitution compared to the Fc region of a wild-type IgG or a wild- type antibody. In some embodiments, the variant Fc region has two or more amino acid substitutions in the Fc region of the wild-type antibody. In some embodiments, the variant Fc region has three or more amino acid substitutions in the Fc region of the wild-type antibody. In some embodiments, the variant Fc region has at least one, two or three or more Fc region amino acid substitutions described herein. In some embodiments, the variant Fc region herein will possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide. In some embodiments, the variant Fc region herein will possess at least about 90% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide. In some embodiments, the variant Fc region herein will possess at least about 95% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide.

[0207] In some embodiments, an antibody is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0208] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.

[0209] In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (for example, complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, that is, between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

[0210] Antibody variants are further provided with bisected oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.) WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

[0211] Antibody variants are also provided with amino-terminal leader extensions. For example, one or more amino acid residues of the amino-terminal leader sequence are present at the amino-terminus of any one or more heavy or light chains of an antibody. An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both light chains of an antibody variant.

[0212] The in vivo or serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice, in humans, or in non- human primates to which the polypeptides with a variant Fc region are administered. See also, for example, Petkova et al. International Immunology 18(12): 1759-1769 (2006).

[0213] In some embodiments, the antibody variant mediates ADCC in the presence of human effector cells more effectively than a parent antibody. In some embodiments, the antibody variant is substantially more effective at mediating ADCC in vitro, when the amounts of polypeptide variant and parent antibody used in the assay are essentially the same. In some embodiments, the antibody variant is substantially more effective at mediating ADCC in vivo, when the amounts of polypeptide variant and parent antibody used in the assay are essentially the same. Generally, such variants will be identified using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, for example in an animal model etc. , are contemplated.

Polypeptide Expression and Production

[0214] Nucleic acid molecules comprising polynucleotides can encode a polypeptide that modulates the activity of LILRA3. The following provides methods for expression of an exemplary molecule that modulates the activity of LILRA3, an antibody. Nucleic acid comprising polynucleotides can encode one or more chains of antibodies. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.

[0215] In some embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.

[0216] In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody that modulates the activity of LILRA3 comprises a nucleotide sequence that encodes at least one CDR. In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody comprises a nucleotide sequence that encodes at least 3 CDRs. In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody comprises a nucleotide sequence that encodes at least 6 CDRs. In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.

[0217] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

Vectors

[0218] Vectors comprising polynucleotides may encode a polypeptide that modulates the activity of LILRA3. An exemplary vector provided herein encodes an antibody that modulates the activity of LILRA3; however, vectors that encode a polypeptide such as a LILRA3 polypeptide or fusion polypeptide vectors are also contemplated. Vectors that encode heavy chains and/or light chains of an antibody that modulate the activity are provided. Vectors comprising polynucleotides that encode heavy chains and/or light chains are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

[0219] In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5: 1 and 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1: 1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1:2 for the vector encoding the heavy chain and the vector encoding the light chain is used.

[0220] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, for example, in Running Deer et ah, Biotechnol. Prog. 20:880-889 (2004).

[0221] Antibodies can be screened to determine, for example, their affinity and specificity of binding to TIM-3, TIM-3 isoforms, tumor- specific TIM-3 polypeptides, post-translationally modified TIM-3 polypeptides, and/or differentially expressed, glycosylated, post-translationally modified and/or spliced TIM-3 polypeptides by using assays known in the art. For example, the assays may include competitive and noncompetitive assays. Assays of interest include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, etc. Binding assays including Biacore or Octet may also be used. For example, binding assays may use purified or semi-purified TIM-3, or alternatively may use cells that express TIM-3, e.g., cells transfected with an expression construct for TIM-3; T-cells that have been stimulated through cross-linking of CD3 and CD28; the addition of irradiated allogeneic cells, etc. As an example of a binding assay, purified TIM-3 may be bound to an insoluble support, e.g., a microtiter plate, magnetic beads, etc. A candidate agent and soluble, labeled TIM-3 ligand are added to the cells, and the unbound components are then washed off. The ability of the candidate agent to compete with the natural ligand for TIM-3 binding may be determined by quantification of bound, labeled ligand.

[0222] In some embodiments, the assay of interest is directed to antibodies that increase the binding of TIM-3 to its ligand. In some embodiments, TIM-3 ligand is LILRA3. The antibody will be substantially unreactive with related molecules to TIM-3, such as CD28, other B7 superfamily members, and/or other members of the immunoglobulin superfamily. Further, the antibody does not activate TIM-3 signaling. In another embodiment, the antibody, does not activate TIM-3 signaling but, in some embodiments, may also bind to one or more other members of the B7 superfamily, including B7.1, B7.2, ICOS Ligand, PD-L1, PD-L2, B7-H3, B7-H5, B7- H6 and/or B7-H7. In an exemplary embodiment, a functional assay detects that an agent blocks the binding of TIM-3 to its ligand, for example, by measuring CD4⁺ T- cell proliferation and/or cell cycle progression, release of IL-12, IL-4, IFN-gamma, TNF-alpha, or other cytokines, expression of CD25 and CD69, or the production/emission of a reporter expressed in a cell line engineered to change the production/emission of the reporter when TIM-3 does not bind its receptor, etc.

[0223] One skilled in the art may measure changes in cell surface marker expression of TIM-3, expression of secreted LILRA3 or cellular changes following TIM-3 or LILRA3 activation/inhibition (including, for example, cell cycle progression, and/or cytokine release) using assays that are well known in the art. These assays include, but are not limited to, flow cytometry (including, for example, fluorescent activating cell sorting (FACS)), indirect immune-fluorescence, solid phase enzyme-linked immunosorbent assay (ELISA), ELISpot assays, western blotting (including in cell western), immunofluorescent staining, microengraving (see Han Q et ah . Lab Chip. 2010;10(11): 1391— 1400), Quant-iT and Qubit protein assay kits, NanoOrange protein quantitation kit, CBQCA protein quantitation kits, EZQ protein quantitation kit, Click-iT reagents, Pro-Q Diamond phosphoprotein stain, Pro-Q glycoprotein stain kits, peptide and protein sequencing, N-terminal amino acid analysis (LifeScience Technologies, Grand Island, NY), chemiluminescence or colorimetric based ELISA cytokine Arrays (Signosis) Intracellular Cytokine Staining (ICS), BD Phosflow™ and BD™ Cytometric Bead Arrays (BD Sciences, San Jose, CA); RT-PCR (RT2 Profiler™ Human Common Cytokine PCR Arrays (Cat # PAHS -021) (SABiosciences/QIAGEN); CyTOF Mass Cytometer (DVS Sciences, Sunnyvale CA); Mass Spectrometry, Microplate capture and detection assay (Thermo Scientific, Rockland, IL), Multiplex Technologies (for example Luminex, Austin, TX); FlowCellect™ T-cell Activation Kit (EMD Millipore); Surface Plasmon Resonance (SPR)-based technologies (for example Biacore, GE Healthcare Life Sciences, Uppsala, Sweden); CD4⁺ Effector Memory T-cell Isolation Kit and CD8⁺CD45RA⁺ Effector T-cell Isolation Kit (Miltenyi Biotec Inc., CA); The EasySep™ Human T- cell Enrichment Kit (StemCells, Inc., Vancouver, Canada); HumanThl/Th2/Thl7 Phenotyping Kit (BD Biosciences, CA); immunofluorescent staining of incorporated bromodeoxyuridine (BrdU) or 7-aminoactinomycin D. See also, Current Protocols in Immunology (2004) sections 3.12.1-3.12.20 by John Wiley & Sons, Inc., or Current Protocols in Immunology (2013) or by John Wiley & Sons, Inc., the contents of which are herein incorporated by reference in their entirety.

Host Cells

[0224] In some embodiments, a polypeptide that modulates the activity of LILRA3 may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. As an example, heavy chains and/or light chains of an antibody that modulates the activity of LILRA3 may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6^® cells (Crucell); and NSO cells. In some embodiments, anti-TIM-3 antibody heavy chains and/or anti-TIM-3 antibody light chains may be expressed in yeast. See, for example, U.S. Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells. [0225] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.

[0226] Host cells comprising any of the polynucleotides or vectors described herein are also provided. In some embodiments, a host cell comprising an antibody that modulates the activity of LILRA3 is provided. Any host cells capable of over- expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis).

[0227] Antibodies of the invention can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the RORl ECD and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify a TIM-3 antibody. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also be suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (for example anion exchange chromatography and/or cation exchange chromatography) may also be suitable for purifying some polypeptides such as antibodies. Mixed-mode chromatography (for example reversed phase/anion exchange, reversed phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.) may also be suitable for purifying some polypeptides such as antibodies. Many methods of purifying polypeptides are known in the art.

[0228] In some embodiments, the antibody is produced in a cell-free system. Nonlimiting exemplary cell-free systems are described, for example, in Sitaraman et al, Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al, Biotechnol. Adv. 21: 695-713 (2003).

[0229] In some embodiments, antibodies prepared by the methods described above are provided. In some embodiments, the antibody is prepared in a host cell. In some embodiments, the antibody is prepared in a cell-free system. In some embodiments, the antibody is purified. In some embodiments, the antibody prepared in a host cell or a cell-free system is a chimeric antibody. In some embodiments, the antibody prepared in a host cell or a cell-free system is a humanized antibody. In some embodiments, the antibody prepared in a host cell or a cell-free system is a human antibody. In some embodiments, a cell culture medium comprising an antibody is provided. In some embodiments, a host cell culture fluid comprising an antibody is provided.

[0230] In some embodiments, compositions comprising antibodies prepared by the methods described above are provided. In some embodiments, the composition comprises an antibody prepared in a host cell. In some embodiments, the composition comprises an antibody prepared in a cell-free system. In some embodiments, the composition comprises a purified antibody. In some embodiments, the composition comprises a chimeric antibody prepared in a host cell or a cell-free system. In some embodiments, the composition comprises a humanized antibody prepared in a host cell or a cell-free system. In some embodiments, the composition comprises a human antibody prepared in a host cell or a cell-free system.

[0231] In some embodiments, a composition comprising a molecule that modulates the activity of LILRA3 at a concentration of more than about any one of 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL, or 250 mg/mL is provided. In some embodiments, the molecule that modulates the activity of LILRA3 is an antibody. In some embodiments, the composition comprises a chimeric antibody prepared in a host cell or a cell-free system. In some embodiments, the composition comprises a humanized antibody prepared in a host cell or a cell-free system. In some embodiments, the composition comprises a human antibody prepared in a host cell or a cell-free system.

[0232] In some embodiments, the molecule {e.g., an antibody) selectively binds to TEVI-3. In some embodiments, the TEVI-3 antibody is a monoclonal human antibody. In some embodiments, the TIM-3 monoclonal human antibody has a K_d of no larger than 10 -^"7 for TIM-3, for example, the numerical value is less than 10 -^"8 , 10 -^"9 , 10 -^"10 , 10 -^"

11 , 10 -^"12 , or lower. In some embodiments, the TIM-3 antibody inhibits or reduces immune modulation or tolerance to tumor cells. In some embodiments, the TIM-3 antibody inhibits or reduces immune modulation or tolerance to tumor cells by inhibiting or reducing the activity or activation of one or more cells selected from: regulatory T-cells (Tregs); myeloid suppressor cells; tumor associated neutrophils (TANs) and tumor associated macrophages (TAMs). In some embodiments, the TIM- 3 antibody enhances or restores the activity or activation of T-cells against tumor cells. In some embodiments, the TIM-3 antibody enhances or restores the activity or activation of one or more cells selected from: T-cells, T helper cells, cytotoxic T-cells, dendritic cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, anti-tumor macrophages and B-cells. In some embodiments, the TIM-3 antibody enhances or restores a T-cell intrinsic signal.

[0233] In some embodiments, TIM-3 activity in the subject is reduced to a level adequate for a therapeutic treatment of the cancer in the subject. In some embodiments, the TIM-3 antibody blocks TIM-3 activity by at least 10%, for example, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% blockade of TIM-3 activity.

Pharmaceutical compositions

[0234] In some embodiments, compositions comprising molecules that modulate the activity of LILRA3 are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^th ed., Lippencott Williams and Wilkins (2004); Kibbe et al, Handbook of Pharmaceutical Excipients, 3 ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. [0235] In some embodiments, a pharmaceutical composition comprising molecules that modulate the activity of LILRA3 is provided. In some embodiments, the pharmaceutical composition comprises a chimeric antibody that modulates the activity of LILRA3. In some embodiments, the pharmaceutical composition comprises a humanized antibody that modulates the activity of LILRA3. In some embodiments, the pharmaceutical composition comprises a human antibody that modulates the activity of LILRA3. In some embodiments, the pharmaceutical composition comprises a molecule that modulates the activity of LILRA3 prepared in a host cell or cell-free system as described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0236] Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated. In general, molecules that modulate the activity of LILRA3 may be administered in an amount in the range of about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, molecules may be administered in an amount in the range of about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, molecules may be administered in an amount in the range of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, molecules may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.

[0237] In some embodiments, molecules that modulate the activity of LILRA3 can be administered in vivo by various routes, including, but not limited to, intravenous, intra-arterial, parenteral, intraperitoneal or subcutaneous. The appropriate formulation and route of administration may be selected according to the intended application.

Combination Therapy

[0238] Molecules that modulate the activity of LILRA3 can be administered alone or with other modes of treatment. They can be provided before, substantially contemporaneous with, or after other modes of treatment, for example, surgery, chemotherapy, radiation therapy, or the administration of a biologic, such as another therapeutic antibody. In some embodiments, a molecule that modulates the activity of LILRA3 is administered in conjunction with another anti-cancer agent.

[0239] In some embodiments, the molecule that modulates the activity of LILRA3 is given concurrently with a second therapeutic agent, for example, a PD-1 therapy. Nonlimiting exemplary PD-1 therapies include nivolumab (anti-PD-1 antibody; BMS- 936558, MDX-1106, ONO-4538; OPDIVO^®; Bristol-Myers Squibb); pidilizumab (anti-PD-1 antibody, CureTech), pembrolizumab (anti-PD-1 antibody; KEYTRUDA^®, MK-3475, lambrolizumab); durvalumab (anti-PD-Ll antibody, MEDI-4736; AstraZeneca/Medlmmune); RG-7446; MSB-0010718C; AMP-224; BMS-936559 (an anti-PD-Ll antibody; Bristol-Myers Squibb); AMP-514; MDX- 1105; ANB-011; anti-LAG-3/PD- 1 ; anti-PD-1 Ab (CoStim); anti-PD-1 Ab (Kadmon Pharm.); anti-PD-1 Ab (Immunovo); anti-TIM-3/PD-l Ab (AnaptysBio); anti-PD-Ll Ab (CoStim/Novartis); atezolizumab (an anti-PD-Ll antibody, Genentech/Roche); avelumab (an anti-PD-Ll antibody, MSB0010718C, Pfizer); KD-033, PD-1 antagonist (Agenus); STI-A1010; STI-A1110; TSR-042; and other antibodies that are directed against programmed death- 1 (PD-1) or programmed death ligand 1 (PD-L1).

[0240] In some embodiments, the two or more therapeutic agents are administered with a time separation of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes. In some embodiments, the antibody is administered sequentially with a second therapeutic agent. For example, administration of the two or more therapeutic agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.

[0241] In some embodiments, the antibody is administered with a second therapeutic method for treatment. Thus, the administration of an antibody can be in combination with another system of treatment.

[0242] In some embodiments, histological samples of tumors are graded using the antibody described herein according to Elston & Ellis, Histopathology, 1991, 19:403- 10, which is hereby incorporated by reference in its entirety. In some embodiments, the antibody described herein is useful in establishing a tumor grade for the purposes of diagnosis or prognosis of a particular cancer. [0243] In some embodiments, the methods described herein are useful for evaluating a subject and/or a specimen from a subject (e.g. a cancer patient). In some embodiments, evaluation is one or more of diagnosis, prognosis, and/or response to treatment.

[0244] In some embodiments, the methods described herein comprise evaluating a presence, absence, or level of a protein. In some embodiments, the methods described herein comprise evaluating a presence, absence, or level of expression of a nucleic acid. The compositions described herein may be used for these measurements. For example, in some embodiments, the methods described herein comprise contacting a specimen of the tumor or cells cultured from the tumor with a therapeutic agent as described herein.

[0245] In some embodiments, the method can include the measurement of a tumor specimen, including biopsy or surgical specimen samples. In some embodiments, the biopsy is a human biopsy. In various embodiments, the biopsy is any one of a frozen tumor tissue specimen, cultured cells, circulating tumor cells, and a formalin-fixed paraffin-embedded tumor tissue specimen. In some embodiments, the tumor specimen may be a biopsy sample, such as a frozen tumor tissue (cryosection) specimen. As is known in the art, a cryosection may employ a cryostat, which comprises a microtome inside a freezer. The surgical specimen is placed on a metal tissue disc which is then secured in a chuck and frozen rapidly to about -20°C to about -30°C. The specimen is embedded in a gel like medium consisting of, for example, poly ethylene glycol and polyvinyl alcohol. The frozen tissue is cut frozen with the microtome portion of the cryostat, and the section is optionally picked up on a glass slide and stained. In some embodiments, the tumor specimen may be a biopsy sample, such as cultured cells. These cells may be processed using the usual cell culture techniques that are known in the art. These cells may be circulating tumor cells. In some embodiments, the tumor specimen may be a biopsy sample, such as a formalin-fixed paraffin-embedded (FFPE) tumor tissue specimen. As is known in the art, a biopsy specimen may be placed in a container with formalin (a mixture of water and formaldehyde) or some other fluid to preserve it. The tissue sample may be placed into a mold with hot paraffin wax. The wax cools to form a solid block that protects the tissue. This paraffin wax block with the embedded tissue is placed on a microtome, which cuts very thin slices of the tissue. In certain embodiments, the tumor specimen contains less than about 100 mg of tissue, or in certain embodiments, contains about 50 mg of tissue or less. The tumor specimen (or biopsy) may contain from about 20 mg to about 50 mgs of tissue, such as about 35 mg of tissue. The tissue may be obtained, for example, as one or more (e.g. , 1, 2, 3, 4, or 5) needle biopsies (e.g. , using a 14-gauge needle or other suitable size). In some embodiments, the biopsy is a fine-needle aspiration in which a long, thin needle is inserted into a suspicious area and a syringe is used to draw out fluid and cells for analysis. In some embodiments, the biopsy is a core needle biopsy in which a large needle with a cutting tip is used during core needle biopsy to draw a column of tissue out of a suspicious area. In some embodiments, the biopsy is a vacuum-assisted biopsy in which a suction device increases the amount of fluid and cells that is extracted through the needle. In some embodiments, the biopsy is an image-guided biopsy in which a needle biopsy is combined with an imaging procedure, such as, for example, X ray, computerized tomography (CT), magnetic resonance imaging (MRI) or ultrasound. In some embodiments, the sample may be obtained via a device such as the MAMMOTOME® biopsy system, which is a laser guided, vacuum-assisted biopsy system for breast biopsy.

[0246] In some embodiments, the evaluation may direct treatment (including treatment with the antibodies described herein). In some embodiments, the evaluation may direct the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also called adjuvant care, is treatment that is given in addition to the primary, main or initial treatment. By way of non-limiting example, adjuvant therapy may be an additional treatment usually given after surgery where all detectable disease has been removed, but where there remains a statistical risk of relapse due to occult disease. In some embodiments, the antibodies are used as an adjuvant therapy in the treatment of a cancer. In some embodiments, the antibodies are used as the sole adjuvant therapy in the treatment of a cancer. In some embodiments, the antibodies described herein are withheld as an adjuvant therapy in the treatment of a cancer. For example, if a patient is unlikely to respond to an antibody described herein or will have a minimal response, treatment may not be administered in the interest of quality of life and to avoid unnecessary toxicity from ineffective chemotherapies. In such cases, palliative care may be used. [0247] In some embodiments the molecules are administered as a neoadjuvant therapy prior to resection. In some embodiments, neoadjuvant therapy refers to therapy to shrink and/or downgrade the tumor prior to any surgery. In some embodiments, neoadjuvant therapy means chemotherapy administered to cancer patients prior to surgery. In some embodiments, neoadjuvant therapy means an antibody is administered to cancer patients prior to surgery. Types of cancers for which neoadjuvant chemotherapy is commonly considered include, for example, breast, colorectal, ovarian, cervical, bladder, and lung. In some embodiments, the antibodies are used as a neoadjuvant therapy in the treatment of a cancer. In some embodiments, the use is prior to resection.

[0248] In some embodiments, the tumor microenvironment contemplated in the methods described herein is one or more of: tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer- associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T-cells; macrophages; neutrophils; and other immune cells located proximal to a tumor.

Kits

[0249] Also provided are articles of manufacture and kits that include any of the molecules that modulate LILRA3 as described herein, and suitable packaging. In some embodiments, the invention includes a kit with (i) a molecule that modulates the activity of LILRA3 and (ii) instructions for using the kit to administer the antibody to an individual. In some embodiments, the molecule that modulates LILRA3 activity is a LILRA3 polypeptide or functional variant thereof, a LILRA3 fusion protein, or an antibody. In some embodiments, the molecule that modulates the activity of LILRA3 is an antibody that binds TIM-3.

[0250] Suitable packaging for compositions described herein are known in the art, and include, for example, vials (e.g. , sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed. Also provided are unit dosage forms comprising the compositions described herein. These unit dosage forms can be stored in a suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g. , a paper sheet included in the kit), but machine-readable instructions (e.g. , instructions carried on a magnetic or optical storage disk) are also acceptable. The instructions relating to the use of the antibodies generally include information as to dosage, dosing schedule, and route of administration for the intended treatment or industrial use. The kit may further comprise a description of selecting an individual suitable for treatment.

[0251] The containers may be unit doses, bulk packages (e.g. , multi-dose packages) or sub-unit doses. For example, kits may also be provided that contain sufficient dosages of molecules disclosed herein to provide effective treatment for an individual for an extended period, such as about any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of molecules and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. In some embodiments, the kit includes a dry (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form generally a stable aqueous suspension of antibody.

EXAMPLES

[0252] The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1: Activated peripheral blood mononuclear cells respond to anti-TIM-3 blockade.

[0253] Whole blood samples activated with Staphylococcal enterotoxin B (SEB) have been shown to respond to an immune checkpoint blockade using an anti-PD- 1 antibody as shown by increases of IL-2 secretion (EP2170959B 1). The SEB assay was adapted to show that SEB-activated PBMCs demonstrate activity in response to TIM-3 blockade using anti-TEVI-3 mAb F38-2E2, either alone or in conjunction with an anti-PD-1 antibody (Fig. 1A). Alone, F38-2E2 addition to the cultures facilitated IL-2 release similar to anti-PD-Ll blockade, but at approximately 50% the activity compared to anti-PD-Ll . When employing the combination of TIM-3 and PD-L1 blockade in the assay, a synergistic increase in IL-2 secretion was seen in comparison to either antibody on its own. The increases were >250% and >600% when comparing the combination to anti-PD-Ll alone or F38-2E2 alone, respectively.

[0254] Anti-human TIM-3 monoclonal antibodies were generated by immunization of mice and hybridoma fusion techniques. Fig. IB shows the respective diverse bins for the mAb clones when arranged according to their ability to cross-block one another in binding plate-bound TIM-3 protein.

Methods

[0255] Generation of a panel of mouse-anti-human TIMS monoclonal antibodies. BALB/c or SJL mice were immunized and boosted with 50 μg each of Human and Mouse TIM-3-FC protein up to 4 times over 3 months. Splenocytes were fused to mouse myeloma cells and selected in HAT medium (containing hypoxanthine, aminopterin, and thymidine). Hybridoma supematants were screened by ELISA for binding to human and mouse TIM-3-FC protein. ELISA positive clones were expanded and screened for binding to human TIM-3 overexpressing 293FT cells. Hybridomas that bound human TIM-3 were subcloned by limiting dilution and confirmed by binding to human TIM-3 expressing ChoKl or 293FT cells by flow cytometry and binding human and TIM-3-FC by ELISA.

[0256] SEB Assay. Fig. 1A shows TIM-3 blockade enhances T cell cytokine secretion and acts synergistically with PD-L1 Blockade. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of fresh donors by Ficoll separation and frozen in 90% fetal bovine serum (FBS), 10% DMSO at -150°C for long term storage. PBMCs were thawed into complete RPMI medium containing 10% FBS, 50 nM 2-Mercaptoethanol, Non-Essential Amino Acids, 1 mM Sodium Pyruvate, 10 mM HEPES. 100,000 cells were plated in each well of a 96-well plate in complete RPMI. Anti-human PD-L1 (Biolegend Clone 29E.2A3) was added at 10-50 μg/ml and anti-human TIM-3 (Biolegend Clone F38-2E2) was added at 50 μg/ml as indicated. Cells and mAbs were incubated at 37°C for 30 minutes and SEB was added at a final concentration of 1 μg/ml. After 4 days of activation, supernatant was collected and frozen at -20°C. Cytokine concentration was measured using multiparameter cytokine bead array (Becton Dickinson & Co.). IL-2 was found to be the cytokine most significantly measured that was influenced by TIM-3 and PD-L1 blockade. Data are representative of at least 4 healthy donors.

[0257] Antibody epitope bins. Monoclonal antibodies were compared in pairwise fashion. One mAb was bound to a plate (Thermo Scientific MaxiSorp) overnight at 4°C (1 μg /ml). Comparison mAbs were individually combined in excess (10 μg/ml) with biotinylated hTIM-3-Fc (10 nM) and incubated at 25°C for 2h, then applied to the antibody coated wells of the plate and incubated for another hour at 25 °C. Amounts of hTIM-3-Fc captured on the plate were measured in a colorimetric assay using Streptavidin-horseradish peroxidase (HRP) with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The TMB substrate was neutralized with H₂S0₄ prior to reading optical density at 450 nm wave-length (OD₄so) utilizing a Biotek plate reader.

Example 2. SEB induction of TIM-3 on macrophages has different kinetics compared to T cells.

[0258] To characterize the SEB-activated PBMC assay more closely, surface expression of PD-1 and TIM-3 proteins were measured over time via flow cytometry while discriminating the most relevant cell types. PD-1 expression was rather uniform among the different cell populations and gradually increased 10-fold over the course of 3 days to reach its peak (Fig. 2A). TIM-3 was expressed more diversely at the start of the assay where it was found at a much higher degree in CD 14+ monocytes/macrophages and CDl lc+ DCs in comparison to T cells (Fig. 2B). Over time, TIM-3 surface expression initially decreased slightly on monocytes/macrophages and DCs reaching its lowest amounts at 24 hours and then all populations increased surface protein until reaching a pinnacle on day 3. From these data, TIM-3 blockade had a greater impact on monocyte/macrophage and DC biology early on in the assay while potentially influencing all cells directly or indirectly (monocytes, DCs, and T cells) as time passed. Methods

[0259] Surface expression levels of TIM-3 and PD-1 were measured during the first 4 days of SEB activation of human PBMCs. 100,000 PBMCs isolated from blood of healthy human donors were plated in each well of a 96 well plate in complete RPMI. SEB was added to a final concentration of 1 μg/ml. At various time points, replicate wells of cells from 2 donors were removed, washed once with PBS containing 2% FBS and 0.05 M sodium azide, stained for 20 minutes on ice with antibodies specific for human CDl lc (Biolegend Clone 3.9), CD14 (Biolegend Clone M5E2), CD8 (Biolegend Clone HIT 8 a), CD4 (Biolegend Clone SK3), PD-1 (Biolegend Clone EH12.2H7) and TIM-3 (Biolegend Clone F38-2E2). Surface protein expression levels are expressed as the average mean fluorescence intensities (MFI) of replicate wells from 2 donors.

Example 3. SEB induction of innate inflammatory cytokines.

[0260] The secretion of several cytokines during the course of SEB activation of PBMC was examined to measure the impact of TIM-3 blockade at the time of the dominant presence of the target on cells of the innate immune system. Increases in TNFa (Fig. 3B) and IL-Ιβ (Fig. 3C) secretion, in addition to IL-2 secretion (Fig. 3 A), occurred by day 2. The secretion of other cytokines is shown in Fig. 3D. These results suggest that TIM-3 blockade leads to activation of the myeloid cells in the assay. These results also suggest that these analytes can serve as readouts for monitoring the effects of anti-TIM-3 mAbs.

Methods

[0261] Cytokine expression was assessed at various time points during SEB activation. 100,000 PBMCs isolated from blood of healthy human donors were plated in each well of a 96 well plate in complete RPMI. Anti-human PD-L1 (Biolegend Clone 29E.2A3) was added at 10 μg/ml and/or anti-human TIM-3 (Biolegend Clone F38-2E2) was added at 50 μg/ml. Cells and mAbs were incubated at 37°C for 30 minutes and SEB was added at a final concentration of 1 μg/ml. After 1, 2, 3 or 4 days, a sample of supernatant was collected and frozen at -20°C. All samples from each time point were measured for cytokine content using multi-parameter cytokine bead array (Becton Dickinson & Co.). Selected cytokines are shown in Figs. 3A-3D, data are representative of PBMCs from 2 healthy donors.

Example 4. TIM-3 is more strongly associated with myeloid cells than T cells in human cancers.

[0262] A set of -8000 human tumors representing 20 different indications was used for this analysis. Immune genes that are known to come from the same cell type show very high levels of correlation. For example, expression of CD3 genes (CD3y, CD35, and CD3s), that are a part of the TCR complex common to T cells, show very high correlation with the expression of both CD4 and CD8 genes. The expression of genes that indicate state of T cell activation, such as PD-1 and ICOS, also show good correlation.

[0263] As TIM-3 was believed to function as a T cell function inhibitor, TIM-3 expression was evaluated for correlation with major T cell markers. However, the correlation of TIM-3 expression and T cell markers was poor to average across multiple tumor types (Fig. 4A). Surprisingly, TIM-3 expression showed a very tight correlation with various established myeloid cell markers, such as CD l ib or CD 11c, across multiple tumor types (Fig. 4B). The strength of these correlations suggests that TIM-3 is predominantly expressed by, and its function is majorly mediated by, tumor- associated monocyte/macrophages and dendritic cells in the human tumor microenvironment.

Methods

[0264] RNA sequencing data from -8000 individual tumors. The sequence data were normalized and processed for expression and mutational analysis by specialized software (OmicSoft, Cary, NC). TIM-3 transcripts levels were correlated to various immune cell type specific genes across all of the available tumor samples using MatLabR2013b software (Mathworks Inc., Natick, MA).

Example 5. Human LILRA3 binds to human TIM-3.

[0265] The macrophage and DC monoculture results related to TIM-3 suggest that a relevant TIM-3 ligand is present on these cells as blockade of TIM-3 leads to functional consequences. Bioinformatic data were used to examine genes whose expression correlates with TIM-3 expression in human tumor samples. The list of expressed proteins was limited to surface receptors that could serve as a ligand for TIM-3. From this list, several candidate proteins were tested for binding to TIM-3. Of these candidates, LILRB2 bound TIM-3 with an affinity of -30 nM (data not shown; see U.S. Patent Application No. 14/987,703). From these results, other members of the LILRB family were investigated. LILRA3 bound to TIM-3 with an affinity of -10 nM (Fig. 5 and Table 2). LILRA3 was not previously reported as a counter receptor for TIM-3.

Table 2. Binding parameters

Methods

[0266] Binding affinity was determined by using the OctetRed 96 System with anti- Human IgG FC capture biosensors (ForteBio) according to the manufacturer's instructions. Human LILRA3-FC Chimera (produced in-house using the sequence from NCBI Ref. Seq. NM_006865.4) was coated to anti-Human IgG FC capture sensors at 10 μg/ml. Saturated sensors were then rinsed in Kinetics Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% azide) and dipped in hTIM-3-HIS protein at 100 nM. Data were analyzed with Octet Data Analysis Software v. 8.0 (ForteBio). Association (K_a) and Dissociation (K_d) rates were determined for each mAb with sensor background subtracted. Equilibrium dissociation constant (K_D) is the ratio K_a/K_d, as determined by the Octet Analysis Software.

Example 6. Activation of macrophages by soluble human LILRA3-Fc.

[0267] To examine the effects of LILRA3 solely on the monocyte/macrophage population, monoculture systems of activated cells were evaluated in isolation. PBMC derived macrophages upon stimulation with LPS were activated to a greater degree when cultured with LILRA3-Fc protein. The addition of LILRA3-Fc protein led to increases in the secretion of effector molecules TNFa, IL-Ιβ, GM-CSF, and IL-6 with a decrease in CCL5. Such changes in these macrophages resemble those of the more inflammatory type population typically referred to as "Ml". These results confirm that LILRA3-Fc protein can impact macrophage biology.

Methods

[0268] PBMCs were isolated by Ficoll separation from 100 ml of fresh whole blood from two donors. CD14 negative selection (Stemcell Technologies EasySep Human Monocyte Enrichment Kit without CD 16 Depletion) was carried out on all cells from each donor according to manufacturer's protocol. 1 million cells per well were added to a 6 well plate in in RPMI with 10% FBS containing M-CSF (50 ng/ml) (Biolegend). Media was renewed on culture days 2, 4 and 6. On culture day 8, macrophages were harvested, pooled, and counted. Macrophages were arrayed in fresh RPMI 10% FBS at 50,000 per well in 100 μΐ in a 96- well round bottom plates as outlined above. 100 μΐ of RPMI containing 200 ng/ml LPS was added to each well, as indicated. LILRA3-Fc protein (produced in-house using the sequence from NCBI Ref. Seq. NM_006865.4 with a human IgGl Fc) was added in a dose titration scheme to achieve final concentrations ranging from 0.1-100 μg/ml. Mouse IgGl isotype antibody was used as a negative control. After 24 hours of LPS activation, supernatant was collected and frozen at -20°C. Supernatant cytokine concentration was measured using multi-parameter cytokine bead array (Becton Dickinson & Co.). The cytokines secreted amounts demonstrated the following approximate maxima and ICso values: GM-CSF max=700 pg/ml, EC₅₀=0.417 μ^πύ; IL-Ιβ 700 pg/ml, EC₅₀=21.74 μg/ml; TNFa 13 ng/ml EC₅₀=0.402 μ^πύ; IL-6 15 ng/ml, EC₅₀=0.403 μg/ml. Notably, mouse IgGl isotype control had baseline values <1% of each cytokine's maximal value in the assay. Conversely, CCL5 decrease reached its minimum of roughly 300 pg/ml from a starting point of 1 ng/ml with an ECso=0.414 μg/ml (Fig. 6).

[0269] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein. SEQUENCES

Human TIM-3 Isoform 1 amino acid sequence

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLR TDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAP TRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQI STLANELRDSRLANDLRDSGATIRIGIYIGA GICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEE PNEYYCYVSSRQQPSQPLGCRFAMP

(SEQ ID NO:l)

Human TIM-3 isoform 1 nucleic acid sequence

agaacactta caggatgtgt gtagtgtggc atgacagaga actttggttt cctttaatgt gactgtagac ctggcagtgt tactataaga atcactggca atcagacacc cgggtgtgct gagctagcac tcagtggggg cggctactgc tcatgtgatt gtggagtaga cagttggaag aagtacccag tccatttgga gagttaaaac tgtgcctaac agaggtgtcc tctgactttt cttctgcaag ctccatgttt tcacatcttc cctttgactg tgtcctgctg ctgctgctgc tactacttac aaggtcctca gaagtggaat acagagcgga ggtcggtcag aatgcctatc tgccctgctt ctacacccca gccgccccag ggaacctcgt gcccgtctgc tggggcaaag gagcctgtcc tgtgtttgaa tgtggcaacg tggtgctcag gactgatgaa agggatgtga attattggac atccagatac tggctaaatg gggatttccg caaaggagat gtgtccctga ccatagagaa tgtgactcta gcagacagtg ggatctactg ctgccggatc caaatcccag gcataatgaa tgatgaaaaa tttaacctga agttggtcat caaaccagcc aaggtcaccc ctgcaccgac tcggcagaga gacttcactg cagcctttcc aaggatgctt accaccaggg gacatggccc agcagagaca cagacactgg ggagcctccc tgatataaat ctaacacaaa tatccacatt ggccaatgag ttacgggact ctagattggc caatgactta cgggactctg gagcaaccat cagaataggc atctacatcg gagcagggat ctgtgctggg ctggctctgg ctcttatctt cggcgcttta attttcaaat ggtattctca tagcaaagag aagatacaga atttaagcct catctctttg gccaacctcc ctccctcagg attggcaaat gcagtagcag agggaattcg ctcagaagaa aacatctata ccattgaaga gaacgtatat gaagtggagg agcccaatga gtattattgc tatgtcagca gcaggcagca accctcacaa cctttgggtt gtcgctttgc aatgccatag atccaaccac cttatttttg agcttggtgt tttgtctttt tcagaaacta tgagctgtgt cacctgactg gttttggagg ttctgtccac tgctatggag cagagttttc ccattttcag aagataatga ctcacatggg aattgaactg ggacctgcac tgaacttaaa caggcatgtc attgcctctg tatttaagcc aacagagtta cccaacccag agactgttaa tcatggatgt tagagctcaa acgggctttt atatacacta ggaattcttg acgtggggtc tctggagctc caggaaattc gggcacatca tatgtccatg aaacttcaga taaactaggg aaaactgggt gctgaggtga aagcataact tttttggcac agaaagtcta aaggggccac tgattttcaa agagatctgt gatccctttt tgttttttgt ttttgagatg gagtcttgct ctgttgccca ggctggagtg caatggcaca atctcggctc actgcaagct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc tgagtggctg ggattacagg catgcaccac catgcccagc taatttgttg tatttttagt agagacaggg tttcaccatg ttggccagtg tggtctcaaa ctcctgacct catgatttgc ctgcctcggc ctcccaaagc actgggatta caggcgtgag ccaccacatc cagccagtga tccttaaaag attaagagat gactggacca ggtctacctt gatcttgaag attcccttgg aatgttgaga tttaggctta tttgagcact gcctgcccaa ctgtcagtgc cagtgcatag cccttctttt gtctccctta tgaagactgc cctgcagggc tgagatgtgg caggagctcc cagggaaaaa cgaagtgcat ttgattggtg tgtattggcc aagttttgct tgttgtgtgc ttgaaagaaa atatctctga ccaacttctg tattcgtgga ccaaactgaa gctatatttt tcacagaaga agaagcagtg acggggacac aaattctgtt gcctggtgga aagaaggcaa aggccttcag caatctatat taccagcgct ggatcctttg acagagagtg gtccctaaac ttaaatttca agacggtata ggcttgatct gtcttgctta ttgttgcccc ctgcgcctag cacaattctg acacacaatt ggaacttact aaaaattttt ttttactgtt aaaaaaaaaa aaaaaaaa

(SEQ ID NO: 2) TIM-3 isoform 2 amino acid sequence

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLR TDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPGEWTFAC HLYE

(SEQ ID NO:3)

Human TIM-3 isoform 2 nucleic sequence

actgctcatg tgattgtgga gtagacagtt ggaagaagta cccagtccat ttggagagtt aaaactgtgc ctaacagagg tgtcctctga cttttcttct gcaagctcca tgttttcaca tcttcccttt gactgtgtcc tgctgctgct gctgctacta cttacaaggt cctcagaagt ggaatacaga gcggaggtcg gtcagaatgc ctatctgccc tgcttctaca ccccagccgc cccagggaac ctcgtgcccg tctgctgggg caaaggagcc tgtcctgtgt ttgaatgtgg caacgtggtg ctcaggactg atgaaaggga tgtgaattat tggacatcca gatactggct aaatggggat ttccgcaaag gagatgtgtc cctgaccata gagaatgtga ctctagcaga cagtgggatc tactgctgcc ggatccaaat cccaggcata atgaatgatg aaaaatttaa cctgaagttg gtcatcaaac caggtgagtg gacatttgca tgccatcttt atgaataaga tttatctgtg gatcatatta aaggtactga ttgttctcat ctctgacttc cctaattata gccctggagg agggccacta agacctaaag tttaacaggc cccattggtg atgctcagtg atatttaaca ccttctctct gttttaaaac tcatgggtgt gcctgggcgt ggtggctcgc gcctctggtc ccagcacttt gggaggctga ggccggtgga tcatgaggtc aggaattcga gaccagcctg gccaacatgg taaaaccttg tctccactaa aaatacaaaa aattagccag gcatggttac gggagcctgt aattctagct acttgggggg ctgaagcagg agaatcactt gaacctggaa gtcggaggtt gcggtaagcc aagatctcgc cattgtactc cagcctggct gacaagagtg aaactctgtc ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa

(SEQ ID NO 4)

Murine TIM-3 amino acid sequence

MFSGLTLNCVLLLLQLLLARSLENAYVFEVGKNAYLPCSYTLSTPGALVPMCWGKGFCPW SQCTNELLRTDERNVTYQKSSRYQLKGDLNKGDVSLI IKNVTLDDHGTYCCRIQFPGLMN DKKLELKLDIKAAKVTPAQTAHGDSTTASPRTLTTERNGSETQTLVTLHNNNGTKI STWA DEIKDSGETIRTAIHIGVGVSAGLTLALI IGVLILKWYSCKKKKLSSLSLITLANLPPGG LANAGAVRIRSEENIYTIEENVYEVENSNEYYCYVNSQQPS

(SEQ ID NO:5)

Murine TIM-3 nucleic acid sequence

accattttaa ccgaggagct aaagctatcc ctacacagag ctgtccttgg atttcccctg ccaagtactc atgttttcag gtcttaccct caactgtgtc ctgctgctgc tgcaactact acttgcaagg tcattggaaa atgcttatgt gtttgaggtt ggtaagaatg cctatctgcc ctgcagttac actctatcta cacctggggc acttgtgcct atgtgctggg gcaagggatt ctgtccttgg tcacagtgta ccaacgagtt gctcagaact gatgaaagaa atgtgacata tcagaaatcc agcagatacc agctaaaggg cgatctcaac aaaggagacg tgtctctgat cataaagaat gtgactctgg atgaccatgg gacctactgc tgcaggatac agttccctgg tcttatgaat gataaaaaat tagaactgaa attagacatc aaagcagcca aggtcactcc agctcagact gcccatgggg actctactac agcttctcca agaaccctaa ccacggagag aaatggttca gagacacaga cactggtgac cctccataat aacaatggaa caaaaatttc cacatgggct gatgaaatta aggactctgg agaaacgatc agaactgcta tccacattgg agtgggagtc tctgctgggt tgaccctggc acttatcatt ggtgtcttaa tccttaaatg gtattcctgt aagaaaaaga agttatcgag tttgagcctt attacactgg ccaacttgcc tccaggaggg ttggcaaatg caggagcagt caggattcgc tctgaggaaa atatctacac catcgaggag aacgtatatg aagtggagaa ttcaaatgag tactactgct acgtcaacag ccagcagcca tcctgaccgc ctctggactg ccacttttaa aggctcgcct tcatttctga ctttggtatt tccctttttg aaaactatgt gatatgtcac ttggcaacct cattggaggt tctgaccaca gccactgaga aaagagttcc agttttctgg ggataattaa ctcacaaggg gattcgactg taactcatgc tacattgaaa tgctccattt tatccctgag tttcagggat cggatctccc actccagaga cttcaatcat gcgtgttgaa gctcactcgt gctttcatac attaggaatg gttagtgtga tgtctttgag acatagaggt ttgtggtata tctgcaaagc tcctgaacag gtagggggaa taaagggcta agataggaag gtgaggttct ttgttgatgt tgaaaatcta aagaagttgg tagcttttct agagatttct gaccttgaaa gattaagaaa aagccaggtg gcatatgctt aacactatat aacttgggaa ccttaggcag gagggtgata agttcaaggt cagccagggc tatgctggta agactgtctc aaaatccaaa gacgaaaata aacatagaga cagcaggagg ctggagatga ggctcggaca gtgaggtgca ttttgtacaa gcacgaggaa tctatatttg atcgtagacc ccacatgaaa aagctaggcc tggtagagca tgcttgtaga ctcaagagat ggagaggtaa aggcacaaca gatccccggg gcttgcgtgc agtcagctta gcctaggtgc tgagttccaa gtccacaaga gtccctgtct caaagtaaga tggactgagt atctggcgaa tgtccatggg ggttgtcctc tgctctcaga agagacatgc acatgaacct gcacacacac acacacacac acacacacac acacacacac acacacacac acacacatga aatgaaggtt ctctctgtgc ctgctacctc tctataacat gtatctctac aggactctcc tctgcctctg ttaagacatg agtgggagca tggcagagca gtccagtaat taattccagc actcagaagg ctggagcaga agcgtggaga gttcaggagc actgtgccca acactgccag actcttctta cagaagaaaa aggttacccg caagcagcct gctgtctgta aaaggaaacc ctgcgaaagg caaactttga ctgttgtgtg ctcaagggga actgactcag acaacttctc cattcctgga ggaaactgga gctgtttctg acagaagaac aaccggtgac tgggacatac gaaggcagag ctcttgcagc aatctatata gtcagcaaaa tattctttgg gaggacagtc gtcaccaaat tgatttccaa gccggtggac ctcagtttca tctggcttac agctgcctgc ccagtgccct tgatctgtgc tggctcccat ctataacaga atcaaattaa atagaccccg agtgaaaata ttaagtgagc agaaaggtag ctttgttcaa agattttttt gcattgggga gcaactgtgt acatcagagg acatctgtta gtgaggacac caaaacctgt ggtaccgttt tttcatgtat gaattttgtt gtttaggttg cttctagcta gctgtggagg tcctggcttt cttaggtggg tatggaaggg agaccatcta acaaaatcca ttagagataa cagctctcat gcagaaggga aaactaatct caaatgtttt aaagtaataa aactgtactg gcaaagtact ttgagcatat ttaaa

(SEQ ID NO: 6)

Human LILRA3-isoform 1 amino acid sequence

MTPILTVLICLGLSLDPRTHVQAGPLPKPTLWAEPGSVITQGSPVTLRCQGSLETQEYHLYREKKTALW ITRIPQELVKKGQFPILSITWEHAGRYCCIYGSHTAGLSESSDPLELWTGAYSKPTLSALPSPWTSG GNVTIQCDSQVAFDGFILCKEGEDEHPQCLNSHSHARGSSRAIFSVGPVSPSRRWSYRCYGYDSRAPYV WSLPSDLLGLLVPGVSKKPSLSVQPGPWAPGEKLTFQCGSDAGYDRFVLYKEWGRDFLQRPGRQPQAG LSQANFTLGPVSRSYGGQYTCSGAYNLSSEWSAPSDPLDILITGQIRARPFLSVRPGPTVASGENVTLL CQSQGGMHTFLLTKEGAADSPLRLKSKRQSHKYQAEFPMSPVTSAHAGTYRCYGSLSSNPYLLTHPSDP LELWSGAAETLSPPQNKSDSKAGE

(SEQ ID NO:7)

Human LILRA3-isoform 1 amino acid sequence

gagcctccaa gtgtccacac cctgtgtgtc ctctgtcctg ccagcaccga gggctcatcc atccacagag cagtgcagtg ggaggagacg ccatgacccc catcctcacg gtcctgatct gtctcgggct gagcctggac cccaggaccc acgtgcaggc agggcccctc cccaagccca ccctctgggc tgagccaggc tctgtgatca cccaagggag tcctgtgacc ctcaggtgtc aggggagcct ggagacgcag gagtaccatc tatatagaga aaagaaaaca gcactctgga ttacacggat cccacaggag cttgtgaaga agggccagtt ccccatccta tccatcacct gggaacatgc agggcggtat tgctgtatct atggcagcca cactgcaggc ctctcagaga gcagtgaccc cctggagctg gtggtgacag gagcctacag caaacccacc ctctcagctc tgcccagccc tgtggtgacc tcaggaggga atgtgaccat ccagtgtgac tcacaggtgg catttgatgg cttcattctg tgtaaggaag gagaagatga acacccacaa tgcctgaact cccattccca tgcccgtggg tcatcccggg ccatcttctc cgtgggcccc gtgagcccaa gtcgcaggtg gtcgtacagg tgctatggtt atgactcgcg cgctccctat gtgtggtctc tacccagtga tctcctgggg ctcctggtcc caggtgtttc taagaagcca tcactctcag tgcagccggg tcctgtcgtg gcccctgggg agaagctgac cttccagtgt ggctctgatg ccggctacga cagatttgtt ctgtacaagg agtggggacg tgacttcctc cagcgccctg gccggcagcc ccaggctggg ctctcccagg ccaacttcac cctgggccct gtgagccgct cctacggggg ccagtacaca tgctccggtg catacaacct ctcctccgag tggtcggccc ccagcgaccc cctggacatc ctgatcacag gacagatccg tgccagaccc ttcctctccg tgcggccggg ccccacagtg gcctcaggag agaacgtgac cctgctgtgt cagtcacagg gagggatgca cactttcctt ttgaccaagg agggggcagc tgattccccg ctgcgtctaa aatcaaagcg ccaatctcat aagtaccagg ctgaattccc catgagtcct gtgacctcgg cccacgcggg gacctacagg tgctacggct cactcagctc caacccctac ctgctgactc accccagtga ccccctggag ctcgtggtct caggagcagc tgagaccctc agcccaccac aaaacaagtc cgactccaag gctggtgagt gaggagatgc ttgccgtgat gacgctgggc acagagggtc aggtcctgtc aagaggagct gggtgtcctg ggtggacatt tgaagaatta tattcattcc aacttgaaga attattcaac acctttaaca atgtatatgt gaagtacttt attctttcat attttaaaaa taaaagataa ttatccatga gaaagcta

(SEQ ID NO: 8)

Human LILRA3-isoform 2 amino acid sequence

MTPILTVLICLGLSLDPRTHVQAGPLPKPTLWAEPGSVITQGSPVTLRCQGSLETQEYHLYREKKTALW ITRIPQELVKKGQFPILSITWEHAGRYCCIYGSHTAGLSESSDPLELWTGAYSKPTLSALPSPWTSG GNVTIQCDSQVAFDGFILCVSKKPSLSVQPGPWAPGEKLTFQCGSDAGYDRFVLYKEWGRDFLQRPGR QPQAGLSQANFTLGPVSRSYGGQYTCSGAYNLSSEWSAPSDPLDILITGQIRARPFLSVRPGPTVASGE NVTLLCQSQGGMHTFLLTKEGAADSPLRLKSKRQSHKYQAEFPMSPVTSAHAGTYRCYGSLSSNPYLLT HPSDPLELWSGAAETLSPPQNKSDSKAGE

(SEQ ID NO: 9)

Human LILRA3-isoform 2 nucleic acid sequence

caccgagggc tcatccatcc acagagcagt gcagtgggag gagacgccat gacccccatc ctcacggtcc tgatctgtct cgggctgagc ctggacccca ggacccacgt gcaggcaggg cccctcccca agcccaccct ctgggctgag ccaggctctg tgatcaccca agggagtcct gtgaccctca ggtgtcaggg gagcctggag acgcaggagt accatctata tagagaaaag aaaacagcac tctggattac acggatccca caggagcttg tgaagaaggg ccagttcccc atcctatcca tcacctggga acatgcaggg cggtattgct gtatctatgg cagccacact gcaggcctct cagagagcag tgaccccctg gagctggtgg tgacaggagc ctacagcaaa cccaccctct cagctctgcc cagccctgtg gtgacctcag gagggaatgt gaccatccag tgtgactcac aggtggcatt tgatggcttc attctgtgtg tttctaagaa gccatcactc tcagtgcagc cgggtcctgt cgtggcccct ggggagaagc tgaccttcca gtgtggctct gatgccggct acgacagatt tgttctgtac aaggagtggg gacgtgactt cctccagcgc cctggccggc agccccaggc tgggctctcc caggccaact tcaccctggg ccctgtgagc cgctcctacg ggggccagta cacatgctcc ggtgcataca acctctcctc cgagtggtcg gcccccagcg accccctgga catcctgatc acaggacaga tccgtgccag acccttcctc tccgtgcggc cgggccccac agtggcctca ggagagaacg tgaccctgct gtgtcagtca cagggaggga tgcacacttt ccttttgacc aaggaggggg cagctgattc cccgctgcgt ctaaaatcaa agcgccaatc tcataagtac caggctgaat tccccatgag tcctgtgacc tcggcccacg cggggaccta caggtgctac ggctcactca gctccaaccc ctacctgctg actcacccca gtgaccccct ggagctcgtg gtctcaggag cagctgagac cctcagccca ccacaaaaca agtccgactc caaggctggt gagtgaggag atgcttgccg tgatgacgct gggcacagag ggtcaggtcc tgtcaagagg agctgggtgt cctgggtgga catttgaaga attatattca ttccaacttg aagaattatt caacaccttt aacaatgtat atgtgaagta ctttattctt tcatatttta aaaataaaag ataattatcc atgagaaagc ta

(SEQ ID NO: 10) LILRB2 variant 1 animo acid sequence

MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASW ITRIRPELVKNGQFHIPS ITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPWTSGG RVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVW SSPSDLLELLVPGVSKKPSLSVQPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGL SQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLC QSWRQFHTFLLTKAGAADAPLRLRS IHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPL ELWSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGWIGILVAWLLLLLLLLLFLIL RHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAA ASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPS IYATLAIH

(SEQ ID NO:ll)

LILRB2 variant 1 nucleic acid sequence

atttggttga aagaaaaccc acaatccagt gtcaagaaag aagtcaactt ttcttcccct acttccctgc atttctcctc tgtgctcact gccacacaca gctcaacctg gacagcacag ccagaggcga gatgcttctc tgctgatctg agtctgcctg cagcatggac ctgggtcttc cctgaagcat ctccagggct ggagggacga ctgccatgca ccgagggctc atccatccgc agagcagggc agtgggagga gacgccatga cccccatcgt cacagtcctg atctgtctcg ggctgagtct gggccccagg acccgcgtgc agacagggac catccccaag cccaccctgt gggctgagcc agactctgtg atcacccagg ggagtcccgt caccctcagt tgtcagggga gccttgaagc ccaggagtac cgtctatata gggagaaaaa atcagcatct tggattacac ggatacgacc agagcttgtg aagaacggcc agttccacat cccatccatc acctgggaac acacagggcg atatggctgt cagtattaca gccgcgctcg gtggtctgag ctcagtgacc ccctggtgct ggtgatgaca ggagcctacc caaaacccac cctctcagcc cagcccagcc ctgtggtgac ctcaggagga agggtgaccc tccagtgtga gtcacaggtg gcatttggcg gcttcattct gtgtaaggaa ggagaagatg aacacccaca atgcctgaac tcccagcccc atgcccgtgg gtcgtcccgc gccatcttct ccgtgggccc cgtgagcccg aatcgcaggt ggtcgcacag gtgctatggt tatgacttga actctcccta tgtgtggtct tcacccagtg atctcctgga gctcctggtc ccaggtgttt ctaagaagcc atcactctca gtgcagccgg gtcctgtcat ggcccctggg gaaagcctga ccctccagtg tgtctctgat gtcggctatg acagatttgt tctgtacaag gagggggaac gtgaccttcg ccagctccct ggccggcagc cccaggctgg gctctcccag gccaacttca ccctgggccc tgtgagccgc tcctacgggg gccagtacag atgctacggt gcacacaacc tctcctctga gtgctcggcc cccagcgacc ccctggacat cctgatcaca ggacagatcc gtggcacacc cttcatctca gtgcagccag gccccacagt ggcctcagga gagaacgtga ccctgctgtg tcagtcatgg cggcagttcc acactttcct tctgaccaag gcgggagcag ctgatgcccc actccgtcta agatcaatac acgaatatcc taagtaccag gctgaattcc ccatgagtcc tgtgacctca gcccacgcgg ggacctacag gtgctacggc tcactcaact ccgaccccta cctgctgtct caccccagtg agcccctgga gctcgtggtc tcaggaccct ccatgggttc cagcccccca cccaccggtc ccatctccac acctgcaggc cctgaggacc agcccctcac ccccactggg tcggatcccc aaagtggtct gggaaggcac ctgggggttg tgatcggcat cttggtggcc gtcgtcctac tgctcctcct cctcctcctc ctcttcctca tcctccgaca tcgacgtcag ggcaaacact ggacatcgac ccagagaaag gctgatttcc aacatcctgc aggggctgtg gggccagagc ccacagacag aggcctgcag tggaggtcca gcccagctgc cgacgcccag gaagaaaacc tctatgctgc cgtgaaggac acacagcctg aagatggggt ggagatggac actcgggctg ctgcatctga agccccccag gatgtgacct acgcccagct gcacagcttg accctcagac ggaaggcaac tgagcctcct ccatcccagg aaagggaacc tccagctgag cccagcatct acgccaccct ggccatccac tagcccggag ggtacgcaga ctccacactc agtagaagga gactcaggac tgctgaaggc acgggagctg cccccagtgg acaccaatga accccagtca gcctggaccc ctaacaaaga ccatgaggag atgctgggaa ctttgggact cacttgattc tgcagtcgaa ataactaata tccctacatt ttttaattaa agcaacagac ttctcaataa tcaatgagtt aaccgagaaa actaaaatca gaagtaagaa tgtgctttaa actgaatcac aatataaata ttacacatca cacaatgaaa ttgaaaaagt acaaaccaca aatgaaaaaa gtagaaacga aaaaaaaaaa ctaggaaatg aatgacgttg gctttcgtat aaggaattta gaaaaagaat aaccaattat tccaaatgaa ggtgtaagaa agggaataag aagaagaaga gttgctcatg aggaaaaacc aaaacttgaa aattcaacaa agccaatgaa gctcattctt gaaaatatta attacagtca taaatcctaa ctacattgag caagagaaag aaagagcagg cacgcatttc catatgggag tgagccagca gacagcccag cagatcctac acacattttc acaaactaac cccagaacag gctgcaaacc tataccaata tactagaaaa tgcagattaa atggatgaaa tattcaaaac tggagtttac ataatgaacg taagagtaat cagagaatct gactcatttt aaatgtgtgt gtatgtgtgt gtatatatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtga aaaacattga ctgtaataaa aatgttccca tcgtaaaaaa aaaaaaaaaa (SEQ ID NO:12)

LILRB2 variant 2 amino acid sequence

MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASW ITRIRPELVKNGQFHIPS ITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPWTSGG RVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVW SSPSDLLELLVPGVSKKPSLSVQPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGL SQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLC QSWRQFHTFLLTKAGAADAPLRLRS IHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPL ELWSGPSMGSSPPPTGPISTPGPEDQPLTPTGSDPQSGLGRHLGWIGILVAWLLLLLLLLLFLILR HRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAAA SEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPS IYATLAIH

(SEQ ID NO:13)

LILRB2 variant 2 nucleic acid sequence

atttggttga aagaaaaccc acaatccagt gtcaagaaag aagtcaactt ttcttcccct acttccctgc atttctcctc tgtgctcact gccacacaca gctcaacctg gacagcacag ccagaggcga gatgcttctc tgctgatctg agtctgcctg cagcatggac ctgggtcttc cctgaagcat ctccagggct ggagggacga ctgccatgca ccgagggctc atccatccgc agagcagggc agtgggagga gacgccatga cccccatcgt cacagtcctg atctgtctcg ggctgagtct gggccccagg acccgcgtgc agacagggac catccccaag cccaccctgt gggctgagcc agactctgtg atcacccagg ggagtcccgt caccctcagt tgtcagggga gccttgaagc ccaggagtac cgtctatata gggagaaaaa atcagcatct tggattacac ggatacgacc agagcttgtg aagaacggcc agttccacat cccatccatc acctgggaac acacagggcg atatggctgt cagtattaca gccgcgctcg gtggtctgag ctcagtgacc ccctggtgct ggtgatgaca ggagcctacc caaaacccac cctctcagcc cagcccagcc ctgtggtgac ctcaggagga agggtgaccc tccagtgtga gtcacaggtg gcatttggcg gcttcattct gtgtaaggaa ggagaagatg aacacccaca atgcctgaac tcccagcccc atgcccgtgg gtcgtcccgc gccatcttct ccgtgggccc cgtgagcccg aatcgcaggt ggtcgcacag gtgctatggt tatgacttga actctcccta tgtgtggtct tcacccagtg atctcctgga gctcctggtc ccaggtgttt ctaagaagcc atcactctca gtgcagccgg gtcctgtcat ggcccctggg gaaagcctga ccctccagtg tgtctctgat gtcggctatg acagatttgt tctgtacaag gagggggaac gtgaccttcg ccagctccct ggccggcagc cccaggctgg gctctcccag gccaacttca ccctgggccc tgtgagccgc tcctacgggg gccagtacag atgctacggt gcacacaacc tctcctctga gtgctcggcc cccagcgacc ccctggacat cctgatcaca ggacagatcc gtggcacacc cttcatctca gtgcagccag gccccacagt ggcctcagga gagaacgtga ccctgctgtg tcagtcatgg cggcagttcc acactttcct tctgaccaag gcgggagcag ctgatgcccc actccgtcta agatcaatac acgaatatcc taagtaccag gctgaattcc ccatgagtcc tgtgacctca gcccacgcgg ggacctacag gtgctacggc tcactcaact ccgaccccta cctgctgtct caccccagtg agcccctgga gctcgtggtc tcaggaccct ccatgggttc cagcccccca cccaccggtc ccatctccac acctggccct gaggaccagc ccctcacccc cactgggtcg gatccccaaa gtggtctggg aaggcacctg ggggttgtga tcggcatctt ggtggccgtc gtcctactgc tcctcctcct cctcctcctc ttcctcatcc tccgacatcg acgtcagggc aaacactgga catcgaccca gagaaaggct gatttccaac atcctgcagg ggctgtgggg ccagagccca cagacagagg cctgcagtgg aggtccagcc cagctgccga cgcccaggaa gaaaacctct atgctgccgt gaaggacaca cagcctgaag atggggtgga gatggacact cgggctgctg catctgaagc cccccaggat gtgacctacg cccagctgca cagcttgacc ctcagacgga aggcaactga gcctcctcca tcccaggaaa gggaacctcc agctgagccc agcatctacg ccaccctggc catccactag cccggagggt acgcagactc cacactcagt agaaggagac tcaggactgc tgaaggcacg ggagctgccc ccagtggaca ccaatgaacc ccagtcagcc tggaccccta acaaagacca tgaggagatg ctgggaactt tgggactcac ttgattctgc agtcgaaata actaatatcc ctacattttt taattaaagc aacagacttc tcaataatca atgagttaac cgagaaaact aaaatcagaa gtaagaatgt gctttaaact gaatcacaat ataaatatta cacatcacac aatgaaattg aaaaagtaca aaccacaaat gaaaaaagta gaaacgaaaa aaaaaaacta ggaaatgaat gacgttggct ttcgtataag gaatttagaa aaagaataac caattattcc aaatgaaggt gtaagaaagg gaataagaag aagaagagtt gctcatgagg aaaaaccaaa acttgaaaat tcaacaaagc caatgaagct cattcttgaa aatattaatt acagtcataa atcctaacta cattgagcaa gagaaagaaa gagcaggcac gcatttccat atgggagtga gccagcagac agcccagcag atcctacaca cattttcaca aactaacccc agaacaggct gcaaacctat accaatatac tagaaaatgc agattaaatg gatgaaatat tcaaaactgg agtttacata atgaacgtaa gagtaatcag agaatctgac tcattttaaa tgtgtgtgta tgtgtgtgta tatatatgtg tgtgtgtgtg tgtgtgtgtg tgtgtgaaaa acattgactg taataaaaat gttcccatcg taaaaaaaaa aaaaaaa (SEQ ID NO 14)

Claims

CLAIMS What is claimed is:

1. A molecule that stimulates the secretion of a myeloid-associated cytokine in an individual, wherein the myeloid associated cytokine is one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6.

2. A molecule that modulates biological activity of LILRA3.

3. The molecule of claim 1 or 2 which specifically binds to TIM-3.

4. The molecule of claim 3, wherein the molecule is an anti-TIM-3 antibody.

5. The molecule of claim 4, wherein the antibody is a monoclonal antibody.

6. The molecule of claim 5, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

7. The molecule of any one of claims 3-5, wherein the antibody is an antigen binding fragment of an antibody.

8. The molecule of claim 7, wherein the antibody fragment is a Fab, Fab', Fv, scFv or (Fab')2 fragment.

9. The molecule of any one of claims 3-8, wherein the TIM-3 is a human TIM-3.

10. The molecule of any one of claims 3-9, wherein the amino acid sequence of the TIM-3 comprises the amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:3.

11. The molecule of any one of claims 3-9, wherein the amino acid sequence of the TIM-3 is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:3.

12. The molecule of any one of claims 1-3, wherein the molecule is a LILRA3 or a functional variant of LILRA3.

13. The molecule of any one of claims 1-3 or 12, wherein the molecule is a LILRA3-FC.

14. The molecule of any one of claims 1-3 or 12, wherein the molecule is a fragment of LILRA3.

15. The molecule of any one of claims 1-3 or 12-14, wherein the LILRA3 is a human LILRA3.

16. The molecule of claim 15, wherein the LILRA3 comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.

17. The molecule of claim 15, wherein the amino acid sequence of the LILRA3 is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9.

18. The molecule of claim 2, wherein the molecule modulates the activity of human LILRA3.

19. The molecule of claim 2 or 18, wherein the molecule modulates the activity of a LILRA3 which comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.

20. The molecule of claim 2 or 18, wherein the amino acid sequence of the LILRA3 is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9.

21. The molecule of any one of claims 1-20, wherein the molecule suppresses the secretion of one or more myeloid-associated cytokines in an individual, wherein the one or more myeloid associated cytokines is IL-10, CCL2, CCL3, CCL4 or CCL5.

22. The molecule of claim 21, wherein the myeloid associated cytokine is IL-10, CCL2, or CCL5.

23. The molecule of claim 21, wherein the myeloid associated cytokine is IL-10 or CCL5.

24. A pharmaceutical composition comprising the molecule of any one of claims 1-23 and a pharmaceutically acceptable carrier.

25. A method of modulating the secretion of a myeloid-associated cytokine in an individual, comprising administering to the individual a therapeutically effective amount of the molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24.

26. The method of claim 25, wherein the molecule stimulates the secretion of one or more myeloid-associated cytokines.

27. The method of claim 26, wherein the myeloid-associated cytokine is one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6.

28. The method of any one of claims 25-27, wherein the molecule suppresses the secretion of a myeloid-associated cytokine, wherein the myeloid associated cytokine is IL-10, CCL2, CCL3, CCL4 or CCL5.

29. The method of claim 28, wherein the myeloid associated cytokine is IL-10, CCL2, or CCL5.

30. The method of claim 28, wherein the myeloid associated cytokine is IL-10, CCL2, or CCL5.

31. The method of any one of claims 25-30 , wherein the molecule is for stimulating one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6 and for suppressing one or more of IL-10, CCL2, CCL3, CCL4 or CCL5.

32. The method of any one of claims 25-31, wherein the molecule or

pharmaceutical composition is administered in combination with an additional therapeutic agent.

33. The method of claim 32, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

34. The method of claim 32 or 33 wherein the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-Ll antibody.

35. The method of any one of claims 25-34, wherein the individual has cancer.

36. A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the molecule of any one of claims 1- 23 or the pharmaceutical composition of claim 24.

37. The method of any one of claims 25-36, wherein the individual is a human.

38. An isolated nucleic acid encoding the molecule of any one of claims 1-23.

39. A vector comprising the nucleic acid of claim 38.

40. A host cell comprising the nucleic acid of claim 38 or the vector of claim 39.

41. A host cell that produces the molecule of any one of claims 1-23.

42. Use of a molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24 for stimulating the secretion of a myeloid-associated cytokine in an individual in need thereof.

43. Use of a molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24 in the manufacture of a medicament for modulating the secretion of one or more myeloid-associated cytokines in an individual in need thereof.

44. The use of claim 43, wherein the medicament is for stimulating the secretion of one or more myeloid-associated cytokines.

45. The use of claim 43 or 44, wherein the myeloid-associated cytokine is one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6.

46. The use of any one of claims 42-45, wherein the medicament is for

suppressing the secretion of one or more myeloid-associated cytokines, wherein the one or more myeloid associated cytokines is IL-10, CCL2, CCL3, CCL4 or CCL5.

47. The use of claim 46, wherein the myeloid associated cytokine is IL-10, CCL2, or CCL5.

48. The use of claim 46, wherein the myeloid associated cytokine is IL-10 or CCL5.

49. The use of any one of claims 42-48, wherein the medicament is for stimulating one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6 and for suppressing one or more of IL-10, CCL2, CCL3, CCL4 or CCL5.

50. The use of any one of claims 42-49, wherein the molecule or pharmaceutical composition is administered in combination with an additional therapeutic agent.

51. The use of claim 50, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

52. The use of claim 50 or 51 wherein the additional therapeutic agent is an anti- PD-1 antibody or an anti-PD-Ll antibody.

53. The use of any one of claims 42-52, wherein the individual has cancer.

54. Use of the molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24 for treating cancer in an individual.

55. Use of the molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24 in the manufacture of a medicament for treating cancer in an individual.

56. A pharmaceutical composition for treating cancer in an individual comprising a therapeutically effective amount of a molecule of any one of claims 1-23 and a pharmaceutically acceptable carrier.

57. A kit for modulating a myeloid-associated cytokine in an individual, comprising the molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24.

58. The kit of claim 57, wherein one or more myeloid-associated cytokines is stimulated.

59. The kit of claim 58, wherein the myeloid-associated cytokine is one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6.

60. The kit of any one of claims 57-59, wherein a second myeloid-associated cytokine is suppressed, wherein the second myeloid-associated cytokine is IL-10, CCL2, CCL3, CCL4 or CCL5.

61. The kit of claim 59 or 60, wherein secretion of one or more of IL-12, TNFa, IL-Ιβ, GM-CSF or IL-6 is stimulated and secretion of one or more of IL-10, CCL2, CCL3, CCL4 or CCL5 is suppressed.

62. The kit of any one of claims 57-61, wherein the individual has cancer.

63. A kit for treating cancer in an individual, comprising the molecule of any one of claims 1-23 or the pharmaceutical composition of claim 24.

64. The kit of any one of claims 57-63, wherein the molecule or pharmaceutical composition of the kit is administered in combination with an additional therapeutic agent.

65. The kit of claim 64, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

66. The kit of claim 64 or 65 wherein the additional therapeutic agent is an anti- PD-1 antibody or an anti-PD-Ll antibody.