Cross Reference to Related Applications

This application claims the benefit of priority to U.S. Provisional Application No.

62/483,019, filed on April 7, 2017, the disclosure of which is incorporated herein by its entirety.

Field of the Invention

The present invention relates to antibodies and antigen-binding fragments thereof that bind to immunoglobulin-like transcript 4 (ILT4) as well as methods of making and using such antibodies and antigen-binding fragments, for example, to treat diseases such as cancer. Background of the invention

A common strategy used by tumor ceils to escape innate and adaptive immune response is associated with aberrant expression of human leukocyte antigen (HLA)-G (Curigliano et al. Clin Cancer Res. 2013 and Gonzalez et a!. Crit Rev Clin Lab Sci. 2012). HLA-G can directly inhibit immune cell function through receptor binding and/or through frogocytosis and impairment of chemotaxis (Morandi et a/. Cytokine Growth Factor Review. 2014 and Lin et al. Mol Med. 2015). Its high expression in multiple tumor types, including for example, colorectal, pancreatic, endometrial, lung, breast, ovarian, and gastric cancer, is associated with advanced disease stage, tumor invasiveness, metastatic potential and an unfavorable prognosis (Lin et al Mol Med. 2015. and Loumange et al. Int J Cancer. 2014). Antibody-mediated blockade of HLA-G function in transgenic mouse models has been shown to inhibit tumor development and block expansion of myeloid-derived suppressor ceils (MDSC) (Loumange et a/. Int J Cancer. 2014., Lin et al. Hum Immunol. 2013., and Agaugue et al. Blood. 201 1). HLA-G binding to ILT4 can directly inhibit the function of monocytes, dendritic cells, and neutrophils, thus impairing the innate immune anti-tumor response. The interaction between HLA-G and monocytes due to ILT4 inhibits maturation of human monocyte-derived antigen-presenting ceils (APCs) resulting in a reduced expression of MHC class II antigens and co-stimulatory molecules through Stat3 activation (Colonna et al. J Immunol. 1998; Allan et al. J Exp Med. 1999, and Liang et al. Proc Natl Sci USA. 2008). Using human monocyte-derived dendritic cells (DCs) and ILT4-transgenic mice, HLA-G was shown to induce the development of tolerogenic APCs with arrest maturation/activation of myeloid DCs, and the induction of tolerogenic DCs by HLA-G was through disrupting the MHC class II presentation pathway (Ristich et a!. Eur J Immunol. 2005).

An unmet medical need exists for patients that do not respond to T-ceil therapy but may benefit from relief of tissue associated-macrophage/ MDSC-mediated tumor tolerance (e.g. myeloid "rich" tumors). ILT4 blockade would fill this need and would differentiate from current T-celi-targeted antibodies (e.g. anti-PD1 , anti-TiGIT) by relieving suppression of tolerogenic myeloid cells in the tumor microenvironment. Summary of the Invention

The present invention provides antibodies or antigen-binding fragments thereof that bind to human ILT4. in certain embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in a human 1LT4 epitope selected from the group consisting of LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61 ), and HTGRYGCQ (SEQ ID NO:62). In certain embodiments, the antibody or antigen-binding fragment thereof protects the epitope from deuterium exchange with a deuterium source, such as D ₂ 0. In one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope LYREKKSASW (SEQ ID NO:59). In another embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope TRIRPEL (SEQ ID NO:60). In yet another embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope NGQF (SEQ ID NG:61 ). In still another embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope HTGRYGCQ (SEQ ID NQ:62). In yet still another embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in two, three, or four ILT4 epitopes selected from the group consisting of LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61), and HTGRYGCQ (SEQ ID NO:62). in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope LYREKKSASW (SEQ ID NO:59) and protects the epitope from deuterium exchange with a deuterium source such as D ₂ 0. In another embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope TRIRPEL (SEQ ID NO:60) and protects the epitope from deuterium exchange with a deuterium source such as D ₂ Q. In yet another embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope NGQF (SEQ ID NO:61) and protects the epitope from deuterium exchange with a deuterium source such as D ₂ 0. In still another embodiment, the antibody or antigen- binding fragment thereof binds to one or more amino acid residues in the epitope

HTGRYGCQ (SEQ ID NO:62) and protects the epitope from deuterium exchange with a deuterium source such as D ₂ 0. In yet still another embodiment, the antibody or antigen- binding fragment thereof binds to one or more amino acid residues in two, three, or four ILT4 epitopes selected from the group consisting of LYREKKSASW (SEQ ID NO:59),

TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61), and HTGRYGCQ (SEQ ID NG:62) and protects the epitopes from deuterium exchange with a deuterium source such as D ₂ 0.

The present invention also provides an antibody or antigen-binding fragment thereof that binds to the same epitope of human ILT4 as any antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of human ILT4 as an antibody or antigen-binding fragment thereof comprising the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 1 1 ; 9 and 1 1 ; 10 and 1 1 ; 12 and 13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 1 1 ; 83 and 1 1 ; 84 and 1 1 ; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of human ILT4 as an antibody or antigen-binding fragment thereof comprising the heavy chain variable domain and light chain variable domain amino acid sequences set forth in SEQ ID NOs:63 and 70; 57 and 71 ; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76; respectively.

The present invention further provides an antibody or antigen-binding fragment thereof that competes for binding to human 1LT4 with an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding to human ILT4 with an antibody or fragment comprising the heavy chain and light chain amino acid sequences set forth in SEQ ID

NOs: 1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 1 1 ; 9 and 1 1 ; 10 and 1 1 ; 12 and 13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 1 1 ; 83 and 1 1 ; 84 and 1 1 ; 85 and 13; and 86 and 15; respectively, in some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human 1LT4 with an antibody or fragment comprising the heavy chain variable domain and light chain variable domain amino acid sequences set forth in SEQ ID NOs:63 and 70; 57 and 71 ; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76; respectively.

In addition, the present invention provides an antibody or antigen-binding fragment thereof that binds human ILT4, comprising: (a) the complementarity determining region-L1 (CDR-L1), complementarity determining region-L2 (CDR-L2), and complementarity determining region-L3 (CDR-L3) of a light chain variable (V _L ) domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 3-7, 1 1 , 13, 15, or 45; and/or (b) the complementarity determining region-H1 (CDR-H1), complementarity determining region-H2 (CDR-H2), and complementarity determining region-H3 (CDR-H3) of a heavy chain variable (V _H ) domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8-10, 12, 14, 44, or 79-86.

In an embodiment of the invention, the antibody or antigen-binding fragment thereof comprises: (1) a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHXGSTNYNPSLKS wherein X is S or A (SEQ ID NO: 17); and CDR-H3:

LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 :

TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GX ₁ X ₂ NRPS; wherein X, is S or A and X ₂ is N, Q, E or D (SEQ ID NO: 20); and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ); (2) a V _H domain comprising: CDR-H1 : SYAIS (SEQ ID NO: 22); CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23); and CDR-H3: YFX^aSGWYKGGAFDI; wherein X, is D or S and X ₂ is S or A (SEQ ID NO: 24); and/or, a V _L domain comprising: CDR-L1 : TLRSGINVDTYRIH (SEQ ID NO: 25); CDR-L2: Y SDSDKHQGS (SEQ ID NO: 26); and CDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a V _H domain comprising: CDR-H1 : SYA H (SEQ ID NO: 28); CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO: 29); and CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30); and/or, a V _L domain comprising: CDR-L1 : RASQG!SSWLA (SEQ ID NO: 31); CDR-L2: AASSLQS (SEQ ID NO: 32); and CDR-L3: QQYNSYPPT (SEQ ID NO: 33); and/or (4) a V _H domain comprising: CDR-H1 : ELSMH (SEQ ID NO: 34); CDR-H2:

GFDPEDGETIYAQKFQG (SEQ ID NO: 35); and CDR-H3: AGPLYTIFGWIIPDNWFDP (SEQ ID NO: 36); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 37); CDR-L2: GNSNRPS (SEQ ID NO: 38); and CDR-L3: QSYDSSLSGSGVV (SEQ ID NO: 39).

In one embodiment, the antibody or antigen-binding fragment comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPSiSEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ). In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51 ), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21), in still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPSiSEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ I D NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ). In another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising:CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6 ,7, 1 1 , 13, 15, or 45, and/or the heavy chain immunoglobulin has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8, 9, 10, 12, 14, 44, 79, 80, 81 , 82, 83, 84, 85, or 86.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin comprises a light chain variable domain having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:70, 71 , 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chain

immunoglobulin comprises a heavy chain variable domain having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NQ:63, 57, 64, 65, 66, 67, 68, or 69.

In still other embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6 ,7, 1 1 , 13, 15, or 45; and/or the heavy chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8, 9, 10, 12, 14, 44, 79, 80, 81 , 82, 83, 84, 85, or 86.

In yet still other embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NQ:70, 71 , 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chain variable domain comprise the amino acid sequence set forth in SEQ ID NQ:63, 57, 64, 65, 66, 67, 68, or 69.

Further provided is an antibody or antigen-binding fragment thereof comprising any of the following sets of heavy chain immunoglobulins and light chain immunoglobulins: (1) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 ; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (2) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:4; (3) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:2;a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:5; (4) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:6; (5) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7; (6) a heavy chain immunoglobuiin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (7) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:8; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (8) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:9; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (9) a heavy chain immunoglobuiin comprising the amino acid sequence set forth in SEQ ID NO: 10; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (10) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:12; a light chain immunoglobuiin comprising the amino acid sequence set forth in SEQ ID NO:13; (1 1) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 14; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15; (12) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:79; a light chain immunoglobuiin comprising the amino acid sequence set forth in SEQ ID NO:3; (13) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:4; (14) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80;a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:5; (15) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobuiin comprising the amino acid sequence set forth in SEQ ID NO:6; (16) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7; (17) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (18) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:82; a light chain

immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (19) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:83; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 11 ; (20) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:84; a light chain immunoglobuiin comprising the amino acid sequence set forth in SEQ ID NO:1 1 ; (21) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:85; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 13; or (22) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:86; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15.

in addition, provided herein is an antibody or antigen-binding fragment thereof comprising any of the following sets of heavy chain variable domain and light chain variable domain: (1 ) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:63; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:7Q; (2) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:71 ; (3) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:72; (4) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:73; (5) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:58; (6) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:70; (7) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:64; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (8) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:65; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (9) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:66; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:74; (10) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:67; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:75; or (1 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:68; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:76.

In one preferred embodiment, the antibody or antigen-binding fragment thereof comprises a V _H domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a V _L domain comprising the amino acid sequence set forth in SEQ ID NO:58. In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7.

In yet another preferred embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:7.

In one embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57.

In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57, wherein the light chain further comprises the amino acid sequence set forth in SEQ ID NO:90,

In yet another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57, wherein the heavy chain further comprises the amino acid sequence set forth in SEQ ID NO:89,

In still another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NG:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57, wherein the light chain further comprises the amino acid sequence set forth in SEQ ID NO:90 and the heavy chain further comprises the amino acid sequence set forth in SEQ ID NO:89,

In one embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NO: 7 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:2.

In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence set forth in SEQ ID NO:7 and each heavy chain consists of the amino acid sequence set forth in SEQ ID NO:2.

In an embodiment of the invention, the antibody or antigen-binding fragment thereof is glycosylated, e.g., with engineered yeast N~linked giycans or Chinese hamster ovary (CHO) cell N-linked glycans. In an embodiment of the invention, the antibody or antigen- binding fragment thereof is an antibody.

The present invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the composition further comprises a therapeutic agent (e.g. , pembrolizumab). in some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In other embodiments, the composition further comprises a therapeutic agent (e.g. ,

pembrolizumab) and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition comprises: (i) an antibody that consists of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence set forth in SEQ ID NO:7 and each heavy chain consists of the amino acid sequence set forth in SEQ ID NO:2, and (ii) pembrolizumab.

The present invention further provides a polypeptide (e.g. , an isolated polypeptide) which includes the immunoglobulin light chain and/or immunoglobulin heavy chain or a variable domain thereof of any antibody or antigen-binding fragment thereof disclosed herein. For example, in an embodiment of the invention, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NQs: 1 -39, 44, 45, 47- 58, 63-77, and 79-86. Also provided by the present invention is any polynucleotide (e.g. , DNA or RNA) that encodes any polypeptide disclosed herein. In another aspect, provided is a vector comprising the polynucleotide disclosed herein. The present invention also provides a host cell (e.g., a CHO cell) comprising the polynucleotide or the vector disclosed herein.

The present invention provides a method for blocking binding of ILT4 to HLA-G, HLA-A, HLA-B, and/or HLA-F, e.g., in vitro or in vivo, for example, in the body of a subject (e.g. , a human subject) in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof disclosed herein, in certain embodiments, the method for blocking binding of ILT4 to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprises performing a therapeutic procedure (e.g., anti-cancer radiation therapy or surgical tumorectomy) to the subject. In some embodiments, the method for blocking binding of ILT4 to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprises administering a therapeutic agent (e.g. , pembrolizumab) to the subject. In other embodiments, the method for blocking binding of ILT4 to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprises performing a therapeutic procedure (e.g., anti-cancer radiation therapy or surgical tumorectomy) and administering a therapeutic agent (e.g. ,

pembrolizumab) to the subject. The present invention also provides a method of treating a cancer in a subject (e.g. , a human subject), comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the method of treating a cancer further comprises performing a therapeutic procedure (e.g., anti-cancer radiation therapy or surgical tumorectomy) to the subject. In some

embodiments, the method of treating a cancer further comprises administering a therapeutic agent (e.g. , pembrolizumab) to the subject. In other embodiments, the method of treating a cancer further comprises performing a therapeutic procedure (e.g., anti-cancer radiation therapy or surgical tumorectomy) and administering a therapeutic agent (e.g. ,

pembrolizumab) to the subject.

The present invention also provides a method of producing the antibody or antigen- binding fragment thereof of the present invention or an immunoglobulin chain thereof (e.g., a Vn and/or V _L thereof), comprising culturing a host cell (e.g., a CHO cell) comprising a polynucleotide (e.g., wherein the polynucleotide is in a vector and/or is integrated into one or more chromosomes of the host cell) encoding the antibody or antigen-binding fragment thereof or an immunoglobulin chain thereof to express the antibody or antigen-binding fragment thereof or an immunoglobulin chain thereof.

Also provided is a method of producing the antibody or antigen-binding fragment thereof of the present invention or an immunoglobulin chain thereof (e.g. , a V _H and/or V _L thereof), comprising: expressing a polynucleotide encoding the antibody or antigen-binding fragment thereof or an immunoglobulin chain thereof.

An antibody or antigen-binding fragment thereof that binds human ILT4 or an immunoglobulin chain thereof which is a product of said method is also part of the present invention.

A method for detecting the presence of an ILT4 peptide or a fragment thereof in a sample also forms part of the present invention. The method comprises contacting the sample with an antibody or antigen-binding fragment of the present invention and detecting the presence of a compiex between the antibody or antigen-binding fragment and the ILT4 peptide or fragment thereof, wherein detection of the complex indicates the presence of the 1LT4 peptide or fragment thereof. In an embodiment of the invention, the method is performed in vitro, e.g. , in a biological sample, e.g., surgical section or blood sample, of a subject. In another embodiment, the method is performed in vivo, e.g., in the body of a subject. In yet another embodiment, the subject is a human being. Brief Description of the Figures

Figure 1. Deuterium labeling Heatmap of p1 E1 (G1) binding to 1LT4-His. Figures 2A and 2B show the crystal structure of human ILT4, Figure 2A depicts the deuterium labeling levels mapped onto the structure of human ILT4. Figure 2B shows the crystal structure of domains 1 and 2 of human 1LT4 complexed with HLA-G. ILT4, HLA- G heavy chain, and beta-2-microglobuiin are indicated. The human ILT4 epitopes, having residues LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61), and HTGRYGCQ (SEQ ID NO:62), are indicated.

Figures 3A and 3B show ILT4 HLA-G binding and 1 E1 (G4) blockade. Mouse 3A9 T ceils transfected with human ILT4 were blocked with Fc block, then incubated with titrated concentrations of 1 E1 (G4) (starting at 27 ug/mL, 1 :3 dilutions), or with hlgG4 isotype control (27 ug/mL). Figure 3A. 1 E1 (G4) or hlgG4 was detected with fluorochrome labeled goat anti-human F(ab') ₂ and detected by flow cytometry. Figure 3B. Following 1 E1 (G4) pre-treatment, cells were incubated with 2 ug/mL biotinylated HLA-Fc or control Fc

(mViSTA-Fc). Fc binding was detected with PE conjugated streptavidin and detected by flow cytometry. Plots shown are representative of 3 independent experiments. IC50 and EC50 values shown are the average of these experiments +/- standard deviation.

Figure 4. Non-HLA-G HC class I ligand binding to ILT4 and p1 E1 (G1) blockade. Mouse 3A9 T cells transfected with human ILT4 were pretreaied with titrated concentrations of p1 E1 (G1) (starting at 10 ug/mL, 1 :3 dilutions), or with hlgG1 isotype control (10 ug/mL) before incubation with fluorochrome labeled tetramers of HLA-F or CDI d, or fluorochrome labeled dexamers of HLA*A2:01 or HLA*B7:02. Tetramer/dexamer binding was determined by flow cytometry and the mean fluorescence intensity of each was plotted. The

dilutions/concentrations used for each dexamer/tetramer are as follows: HLA*A2:01 -dex PE: 1 :25; HLA*B7:Q2-dex FITC: 1 :25; CD1 d-tet PE: 1 :50; HLA-F-tet PE: 1 ug/mL

Figures 5A-5C. ANGPTL binding to ILT4 and p1 E1 (G1 ) blockade. Figure 5A.

Biotinylated ANGPTL proteins were preincubated for 20 min. with p1 E1 (G1) or human igG1 , the final concentration of each was 20 ug/mL. Solutions were then added to mouse 3A9 T cells transfected with human ILT4 and were incubated for an additional 30 minutes. ANGPTL binding was detected with PE conjugated streptavidin and analyzed by flow cytometry. PE labeled HLA-G tetramer was also added as a positive ILT4 binding/blocking control. ANGPTL proteins were purchased from R&D SYSTEMS and biotinylated; Figure SB, Mouse 3A9 T cells transfected with human ILT4 were blocked with Fc block, then incubated with 20 ug/mL of p1 E1 (G1) or hlgG1 isotype control. Following incubation, p1 E1 (G1 ) or hlgG1 was detected with fluorochrome labeled goat anti-human F(ab')2 and analyzed by flow cytometry; Figure 5C. Vector control 3A9 T-celis were used as a negative control for ANGPTL binding. Cells were treated as described in (A) except no treatment with antibody was performed. Figures 6A and 6B. ILT family 1 E1 (G4) binding specificity. Mouse 3A9 T cells transfected with human ILT family members derived from consensus sequences published in the Uniprot database were used to test binding of hlgG4 isotype control antibody or 1 E1 (G4) at a fixed dose of 10 ug/mL. Vector control 3A9 T cells were used as an additional negative control. Also shown is binding of commercially available ILT-reactive antibodies compared to their respective isotype control to demonstrate ILT-family member expression. Data shown is representative of two experiments with similar results. See the legend embedded in the figure.

Figure 7. Rescue of IL-2 release from 1LT4 3A9 T cell transfectants with 1 E1 (G4). Mouse 3A9 T ceils transfected with human ILT4 were treated with platebound anti-CD3 antibody in the presence of soluble 1 E1 (G4) or isotype control (hulgG4), starting at 27ug/mL and serially diluted 3-fold to 0.3 ug/mL. After 24 hrs of incubation, supernatants are removed and mouse IL-2 is measured by ELISA. Plot shown is representative of 5 independent experiments. EC50 value shown is the average of these experiments +/- standard deviation.

Figure 8. p1 E1 (G4) and 1 E1 (G4) rescued ILT4:HLA-G induced suppression of mast cell degranuiation. Mouse WTMC mast cells were transfected with human 1LT4 and pretreated with titrated concentrations of 1 E1 (G4), p1 E1 (G4), or higG4 isotype control (starting at 10 ug/mL, 1 :3 dilutions) before stimulating with platebound anti~CD200Ria (Clone DX89; 1 ug/mL) and platebound HLA-G fetramer (0.625 ug/mL). Following stimulation for 1 hour, degranuiation was assessed by collecting supernatants from the mast cells to measure the release of β-hexoseaminidase using a coiorimetric enzymatic assay. Data shown is representative of 2 independent experiments with 3 technical replicates per data point.

Figures 9A and 9B. 1 E1 (G4) enhanced LPS-induced expression of proinflammatory myeloid cytokines. Whole PBMCs from healthy patients were isolated from ieukoreduction chambers and treated with 0.25 ug/mL LPS in the presence of either hlgG4 (30 ug/rnL; open circles) or 1 E1 (G4) (marked as "1 E1" in figure) (between 30 ug/mL and 3 pg/mL; closed circles) for 3 days. Following stimulation, supernatants were assayed for cytokine expression ((Figure 9A) GM-CSF and (Figure 9B) TNFa) using a Meso Scale Discovery multi-cytokine assay kit. Each color represents data from an individual patient. Conditions without any stimulation are also shown (closed triangles).

Figures 1QA and 10B. 1 E1 (G4) enhanced anti~CD3-induced expression of proinflammatory myeloid cytokines. (A-B) Whole PBMCs from healthy patients were isolated from ieukoreduction chambers and treated with 0.01 ug/mL anti-CD3 (HIT3a) in the presence of either hlgG4 (30 ug/mL; open circles) or 1 E1 (G4) (marked as "1 E1" in figure) (between 30 ug/mL and 3 pg/mL; closed circles) for 3 days. Following stimulation, supernatants were assayed for cytokine expression ((Figure 10A) GM-CSF and (Figure 10B) TNFa) using a Meso Scale Discovery multi-cytokine assay kit. Each color represents data from an individual patient. Conditions without any stimulation are also shown (closed triangles).

Figures 11 A~11 E. p1 E1 (G4) treatment leads to tumor growth inhibition in a humanized mouse SKMEL5 tumor model. CD34+ Cord blood-engrafted humanized NSG mice, from 2 different cord blood donors, were subcutaneously inoculated with 1x1 Q ⁶ SK EL5 tumor cells in their left flanks. Following inoculation, tumors were allowed to grow and those which reached an average size of 150 mm ³ were randomized into groups of 8 (3 from each stem cell donor, n=6 per group total). Mice were then challenged with either hlgG4 isotype control or p1 E1 (G4) (20 mgs/kg each) every 5 days, with tumors and weights measured weekly, until the end of the study. Tumor growth in the isotype control and p1 E1 (G4) treated mice were tracked ove time. Figure 11 A shows mean tumor volume (mm ³ ) +/- SD over time for both groups, and Figures 11 B and 11C show individual mouse tumor volumes (mm ³ ) over time for isotype treated and p1 E1 (G4), respectively. Figure 11 D shows tumor weight in individual mice treated with isotype control or p1 E1 (G4) as followed over time; Figure 11 E shows weight loss of each treatment group was also measured over time. Following study completion, mice were sacrificed and tumors were harvested and weighed.

Figures 12A-12D demonstrate that 1 E1 (G4) treatment led to tumor growth inhibition in a humanized mouse SK- EL-5 tumor model. Figure 12A shows mean tumor volume (mm ³ ) +/- SD ove time for both 1 E1 (G4)-treated mice and lgG4 isotype control-treated mice. Figure 12B shows body weight change over time for both groups. Figure 12C shows endpoint tumor weight in individual mice treated with isotype control or 1 E1 (G4).

Figure 12D shows endpoint spleen weight in individual mice treated with isotype control or 1 E1 (G4).

Figure 13. ILT4 hapiofype binding. Mouse 3A9 T ceils fransfecfed with human ILT4 allelic variants were used to test binding of hlgG4 isotype control antibody or 1 E1 (G4) at a fixed dose of 10 ug/mL. Vector control 3A9 T ceils were used as an additional negative control. Hapiotypes are explained in Table 2. Data shown is representative of two experiments with similar results.

Figures 14A and 14B. ILT4 RNA expression in different tumor types or ceil types according to public databases. Figure 14A depicts ILT4 RNA expression in various tumor types according to the TCGA database. Figure 14B depicts ILT4 RNA expression in various ceil types according to the Blueprint database. Figures 15A and 15B show 1 E1 (G4) binding to myeloid cells from renal cell carcinoma (RCC) (Figure 15A) and colorectal cancer (CRC) (Figure 15B) tumor histoculture samples.

Figure 16, Predominant N-linked glycans for monoclonal antibodies produced in Chinese hamster ovary cells (CHO N-linked glycans) and in engineered yeast cells (engineered yeast N-linked glycans): squares: N-acetyiglucosamine (GicNac); circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).

Figure 17 shows anti-tumor efficacy of various anti~ILT4 antibodies in a humanized mouse SK-MEL-5 tumor model.

Det ined Description of the Invention

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise.

"Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. , an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. , antibody and 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, including KinExA and Biacore,

As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multsspecsfic antibodies (e.g. , bispecific antibodies), fully human antibodies, and chimeric antibodies.

As used herein, unless otherwise indicated, "antigen-binding fragment" refers to antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv fragments and individual antibody heavy chains or light chains, and individual heavy chain or light chain variable regions. .

A "Fab fragment" is comprised of one light chain and the CH 1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. An "Fab fragment" can be the product of papain cleavage of an antibody.

An "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CHS domains.

A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain that contains the V _H domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') ₂ molecule.

A "F(ab') ₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab') ₂ fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. An "F(ab') ₂ fragment" can be the product of pepsin cleavage of an antibody, The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

"Isolated antibody" refers to the purification status and in such context means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media.

Generally, the term "isolated" is not intended to refer to a complete absence of such material or to an absence of wafer, buffers, or salts, unless they are present in amounts that substantially interfere with experimental or therapeutic use of the binding compound as described herein.

The term "monoclonal antibody", as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. 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 to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g. , U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Ciackson et a!. (1991) Nature 352: 824-628 and Marks ei al. (1991) J. o!. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 1 16:731.

The term "fully human antibody" refers to an antibody that comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, "mouse antibody" refers to an antibody that comprises mouse immunoglobulin sequences only. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat, in a rat cell, or in a hybridoma derived from a rat ceil. Similarly, "rat antibody" refers to an antibody that comprises rat immunoglobulin sequences only.

In general, the basic "antibody" structural unit comprises a tetramer. In an monospecific antibody, each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a "variable region" or "variable domain" of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.

Typically, human constant light chains are classified as kappa and lambda light chains. Furthermore, human constant heavy chains are typically classified as rnu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as Ig , IgD, IgG, IgA, and IgE, respectively. Subtypes of these IgG include, for example, igG1 and lgG4. The present invention includes anti-ILT4 antibodies and antigen-binding fragments comprising any of these light and/or heavy constant chains,

"Variable region," "variable domain, " "V region," or "V chain" as used herein means the segment of IgG chains which is variable in sequence between different antibodies. A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable region of the heavy chain may be referred to as "VH." The variable region of the light chain may be referred to as "V _L ." Typically, the variable regions of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope, in general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1 , CDR1 , FR2, CDR2, FR3, CDRS, and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proieins of immunological interest, Kabat, et a!.; National Institutes of Health, Bethesda, d.; 5th ed.; N! H PubL No. 91 -3242 (1991 ); Kabat (1978) Adv. Prot. Chem. 32: 1 -75; Kabat, et ai. , (1977) J. Biol. Chem. 252:6609-6616; Chothia, et ai., (1987) J ol. Biol. 196:901 -917 or Chothia, et ai,. (1989) Nature 342:878-883.

A "CDR" refers to one of three hypervariable regions (H 1 , H2, or H3) within the non- framework region of the antibody V _H β-sheet framework, or one of three hypervariable regions (L1 , L2, or L3) within the non-framework region of the antibody V _L β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable domains. CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt to different conformation. Both terminologies are well recognized in the art. CDR region sequences have also been defined by Ab , Contact, and !MGT. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Ai-Lazikani ef aL , 1997, J. Mol. Biol. 273:927-48; orea et ai. , 2000, Methods 20:267-79). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme (Ai-Lazikani et a!. , supra). Such nomenclature is similarly well known to those skilled in the art. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art and shown below in Table 1 . In some embodiments, the CDRs are as defined by the Kabat numbering system. In other embodiments, the CDRs are as defined by the IMGT numbering system, in yet other embodiments, the CDRs are as defined by the AbM numbering system, in still other embodiments, the CDRs are as defined by the Chothia numbering system, in yet other embodiments, the CDRs are as defined by the Contact numbering system.

Table 1. Correspondence between the CDR Numbering Systems

V _L CDR1 24-34 27-38 24-34 24-34 24-34 30-36

V _L CDR2 50-56 56-65 50-56 50-56 50-56 46-55

V _L CDR3 89-97 105-1 17 89-97 89-97 89-97 89-96

Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.

Sequence similarity includes identical residues and non-identical, biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable are discussed above.

"Conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar

characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g. , Watson et a!. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)), In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Tab!!e 2.

TABLE 2. Exemplary Conservative Amino Acid Substitutions

The term "epitope," as used herein, refers to an area or region on an antigen to which an antibody or antigen-binding fragment binds. Binding of an antibody or antigen- binding fragment thereof disclosed herein to an epitope means that the antibody or antigen- binding fragment thereof binds to one or more amino acid residues within the epitope.

"Isolated" nucleic acid molecule or polynucleotide means a DNA or RNA, e.g., of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that "a polynucleotide comprising" (or the like) a particular nucleotide sequence does not encompass intact chromosomes, isolated polynucleotides "comprising" specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

The phrase "control sequences" refers to polynucleotide sequences necessary or helpful for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenyiation signals, and enhancers, in an embodiment of the invention, the polynucleotide is operably linked to a promoter such as a viral promoter, a CMV promoter, an SV40 promoter or a non-viral promoter or an elongation factor (EF)~1 promotor; and/or an intron.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another polynucleotide. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

Generally, but not always, "operably linked" means that the polynucleotide sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic

oligonucleotide adaptors or linkers are used in accordance with conventional practice. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words

"transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be dear from the context.

Host cells include eukaryotic and prokaryotic host ceils, including mammalian cells. Host cells may be used as hosts for expression of the anti-ILT4 antibodies and antigen- binding fragments thereof. Host cells include, inter alia, Chinese hamster ovary (CHO) ceils, NSO, SP2 ceils, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. , Hep G2), A549 ceils, 3T3 cells and HEK-293 cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g. , Spodoptera frugiperda or Trichopiusia ni), amphibian ceils, bacterial cells, plant ceils and fungal cells. Fungal ceils include yeast and filamentous fungus cells including, for example, Pichia pasioris, Pichia finiandica, Pichia trehaiophiia, Pichia kociamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotoierans, Pichia saiictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanoiica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenuia polymorphs, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosponum lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrel!a patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenuia

polymorphs, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosponum lucknowense, any Fusarium sp., Yarnowia lipolytics, and Neurospora crassa. The present invention includes any host cell (e.g. , a CHO ceil or Pichia ceil, e.g., Pichia pastoris) containing an anti-ILT4 antibody or antigen-binding fragment thereof or containing a polynucleotide encoding such an antibody or fragment or containing a vector that contains the polynucleotide.

"Treat" or "treating" means to administer anti-ILT4 antibodies or antigen-binding fragments thereof of the present invention, to a subject having one or more symptoms of a disease for which the anti-ILT4 antibodies and antigen-binding fragments are effective, e.g., in the treatment of a subject having cancer or an infectious disease, or being suspected of having cancer or infectious disease, for which the agent has therapeutic activity. Typically, the antibody or fragment is administered in an "effective amount" or "effective dose" which will alleviate one or more symptoms (e.g., of cancer or infectious disease) in the treated subject or population, whether by inducing the regression or elimination of such symptoms or by inhibiting the progression of such symptom(s), e.g . , cancer symptoms such as tumor growth or metastasis, by any clinically measurable degree. The effective amount of the antibody or fragment may vary according to factors such as the disease stage, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject.

As used herein, "an anti-ILT4 antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that binds to human ILT4.

The present invention includes antibodies and antigen-binding fragments thereof set forth herein that bind specifically to ILT4. An antibody or antigen-binding fragment binds "specifically" to a polypeptide comprising a given sequence (e.g. , human ILT4) if it binds to polypeptides comprising the sequence with a K _D of about 20 nM or a higher affinity (e.g., about 17 nM, 10 nM, 5 nM, 1 nM, 100 pM, or 1 pM), but does not bind to proteins lacking the sequence. For example, an antibody or antigen-binding fragment that specifically binds to a polypeptide comprising human ILT4 may bind to a FLAG ^® -tagged form of human ILT4 but will not bind to other FLAG ^® -tagged proteins that lack. ILT4 sequences.

ILT4

In an embodiment of the invention, the amino acid sequence of human ILT4 comprises the amino acid sequence:

MTPIVTVLIC LGLSLGPRTH VQTGTIPKPT LWAEPDSVIT QGSPVTLSCQ GSLEAQEYRL 60

YREKKSASWI TRIRPELVKN GQFHIPSITW EHTGRYGCQY YSRARWSELS DPLVLVMTGA 120

YPKPTLSAQP SPWTSGGRV TLQCESQVAF GGEIIiGKEGE EEHPQCLNSQ FHAKGSSRAI 180

FSVGPVSPNR RWSHRCYGYD LNSPYVWSSP SDLLELLVPG VSKKPSLSVQ PGPWAPGES 240

LTLQCVSDVG YDRFVLYKEG ERDLRQLPGR QPQAGEiSQAN FTLGPVSRSY GGQYRCYGAH 300

NLSSECSAPS DPLDILITGQ IRGXPFISVQ PGPTVASGEN VTLLCQSWRQ FHTFLLTKAG 360

AADAPLRLRS IHEYPKYQAE FPMSPVTSAH AGTYRCIGSIi HSDPYI-IiSHP SEPLELWSG 420

PSMGSSPEPT GPISTPAGPE DQPLTPTGSD PQSGLGRHLG WIGILVAW LLLLLLLLLF 480

LILRHRRQGK H TSTQRKAD FQHPAGAVGP EPTDRGLQWR SSPAADAQEE KLYAAVKDTQ 540

PEDGVE DTR AAASEAPQDV TYAQLHSLTL RRKATEPPPS QEREPPAEPS IYATLAIH 598

(SEQ ID NO:40; signal sequence underscored). See Uniprot accession no. Q8N423.

In another embodiment of the invention, the amino acid sequence of human ILT4 comprises the following amino acid sequence without the signal sequence:

QTGTIP PT LWAEPDSVIT QGSPVTLSCQ GSLEAQEYRL 39

YREKKSASWI TRIRPELVKN GQFHIPSITW EHTGRYGCQY YSRARWSELS DPLVLVMTGA 99

YPKPTLSAQP SPWTSGGRV TLQCESQVAF GGFILCKEGE EEHPQCLNSQ PHARGSSRAI 159

FSVGPVSPNR RWSHRCYGYD LNSPYVWSSP SDLLELLVPG VSKKESLSVQ PGEWAPGES 219 LTLQCVSDVG YDRFVLY EG ERDLRQLPGR QPQAGLSQAN FTLGPVSRSY GGQYRCYGAH 279

NLSSECSA S DPLDILITGQ IRGTPFISVQ PGPTVASGEN VTLLCQSWRQ FHTFLLT AG 339

AADAPLRLRS IHEYPKYQAE FEMSPVTSAH AGTYRCYGSL NSDPYLLSHP SEPLELWSG 399

PSMGSSPPPT GFISTPAGPE DQBLTPT6SD PQSGLGRHLG WTGILVAW LLLLLLLLLF 459

LILRHRRQGK HWTSTQRKAD FQHEAGAVGP EPTDRGLQWR SSPAADAQEE NLYAAVKDTQ 519

PED6VEMDTR AAASEAPQDV TYAQLHSI.TL RR ATEPPPS QEREPPAEPS IYATLAIH 577

(SEQ ID NO:78).

In an embodiment of the invention, the amino acid sequence of cynomolgous monkey IL 1 comprises the amino acid sequence:

MTPIl-MVLIC LGLSLGPRTH VQAGILEKPT LWAEPGSVIS EGSPVTLRCQ GSLQVQEYHL 60

YREKNPASWV RQIRQELVKK GYFAIGFITW EHTGQYRCQY YSHSWWSEPS DPLEL TGA 120

YSKPTLSALP SBWASGGNV TLQCDSQVAF DSFTLCKEGE DEHPQRIiNCQ SHARGWSWAV 180

FSVGPVS SR R SYRCYGYI SSAENVWSLP SDLLELLVPG VSKKPSLSVQ PGPWAPGDK 240

LTLQCGSDAG YDRFALYKEG EGDFLQRPVR QPQAGLSQAN FLLGPVSRSH GGQYRCSGAH 300

NLSSEWSAPS DPLDILIAGQ IRGRPFLSVQ GPKWSGEN VTLLCQSSWQ FEIAFLLTQAG 360

AADAHLHLRS MYKYPKYQAE FPMEPVTSAH AGTYRCYGSR SSNPYLLSVP SDPLEL VSG 420

PSGGPSSPTT GPTSXCGPED QPLTPTGSAP QSGLGRHLGV VTGVLVAFVI, LLFLLLLLFL 480

VLRYRRQGKR TSAQRKADF QHPAGAVEPE PRDRGLQRRS SPAADTQEEN LYAAVKDTQP 540

BDGVELDSRA AASEDPQDVT YAQLQSLTLR REATEPPPSQ BRAPPVHSSI YATIiTIH 597

(SEQ ID NO;43; signal sequence underscored). See NCBI refseq XP_005590753.

In an embodiment of the invention, the signal sequence for expression of ILT4 or any other polypeptide set forth herein is MTPILMVLICLGLSLGPRTHV (amino acids 1-21 of SEQ ID NO:40) or MTPIVTVLICLGLSLGERTHV (amino acids 1-21 of SEQ ID NO;43) or

MPLLLLLPLLWAGALA (SEQ ID NO:45).

In an embodiment of the invention, an anti-ILT4 antibody or antigen-binding fragment thereof of the present invention binds to the extracellular domain of ILT4:

QTGTIP PTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSI TWEHTGRYGC QYYSRAR SELSDPLVLVMTGAYPKPTLSAQPGPWTSGGRVTJ-QCESQVAFGGFILCKEGEEEHPQC LNSQPHARG SSRAIFSVGPVSPffl^WSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKK^

RFVLYKEGERDLRQLPGRQPQAGLSQAIIFTLGPVSRSYGGQYRCYGAHNISSECSA PSDPLDILITGQIRGTPFISV OPGPWASGElWTr.I.CQSWRQFHTFI,r.TKAGAADAPLRiRsmEYP YQAEFEMSPVTSAHAGTYRCYGSLNSDPYL LSHPSEPLELWSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGV

(amino acids 22-461 of SEQ ID NO:40) or an imrnunoglobulin-fusion thereof (e.g., lgG1 or lgG4) or a cell surface transmembrane (T ) form which is expressed on the surface of a cell:

QTGTIPKPTL AEPDSVirQGSPV LSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGC QYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPWTSGGRVTLQCESQVAFGGFILCKEG EEEHPQCLNSQPHARG SSRAIFSVGPVSPmRWSHRCYGYDLNSPYVWfSGPSDLLEnLVPGVSKKPSLSVQPGPVV APGESLTLQCJVSDVGYD RFVtY EGERDLROLPGRQPQAGLSQAirF LePVSRSYGGQYRCYGAHKLSSECEAPSDPLDXLITGQIRGTP ISV QPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFP SPVTSAHAG YRCYGSLNSDPYL LSHPSEPLELVVSSPSMSSSPPPTSPrSTPAGPEDQPLTPTeSDPQSGLGRHLGVVIGIL VAVVLLLLLLLLLFLIL RHRRQGKH

(amino acids 22-491 of SEQ ID NO:40).

Antibodies and Antigen-Binding Fragments

The present invention provides antibodies and antigen-binding fragments thereof (e.g. , fully human antibodies) that bind to ILT4 (herein referred to as "anti-lLT4") and methods of use of the antibodies or antigen-binding fragments thereof in the treatment or prevention of disease. In one embodiment, the invention provides for antagonistic anti-l LT4 antibodies and methods of use of the antibodies or antigen-binding fragments thereof in the treatment or prevention of disease.

In one aspect, the present application includes anti-ILT4 antibodies and antigen- binding fragments thereof as set forth herein having one or more of the properties set forth below:

» binds human ILT4 at one or more amino acid residues in LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ I D NO:60), NGQF (SEQ ID NO:61), and/or HTGRYGCQ (SEQ ID NO:B2), and/or protects LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61 ). and/or HTGRYGCQ (SEQ ID NO:62) from deuterium (e.g., D2O) exchange, e.g. , as determined by hydrogen-deuterium exchange mass spectrometry and/or binds to ILT4 with a heat map essentially as shown in Figure 1;

• binds human ILT4 at domain 1 (see Wilcox ef al. BMC Structural Biology 2:6 (2002)); e binds human ILT4 extracellular domain or TM form of ILT4 expressed on a cell surface, e.g., a pre-B cell, Chinese hamster ovary cell, U937 cell, or Jurkat JE6 cell.

• calculated pi -7.29 {e.g. , 7.29 or 7.30);

® experimentally determined pi ~ 7.2;

o is characterized by a thermogram having Tm onset > 60°C, Tm1 ~ 65.2°C and Tm2 ~78.8°C;

© binds human ILT4 with a K _D of about 1.7 X 10 ^"8 fvl (e.g., as determined by surface plasmon resonance, e.g., binding of anti-lLT4 to polyhistidine tagged human ILT4); o Ka=5.5 X 10 ⁵ M ^"1 s ^"1 (e.g. , as determined by surface plasmon resonance, e.g. , binding of anti-l LT4 to polyhistidine tagged human ILT4);

© Kd=9X10 ^"3 s ^" (e.g., as determined by surface plasmon resonance, e.g., binding of anti-ILT4 to polyhistidine fagged human ILT4);

« blocks binding of HLA-G (e.g., Fc fused HLA-G) to human ILT4 (e.g., ILT4 on mouse

3A9 T cells transfected with and expressing ILT4), e.g. , with an IC50 of about 0.25 micrograms/mi (+0,06 rnicrograms/rnl), e.g. , as determined by surface piasmon resonance;

» blocks binding of HLA-A, HLA-B (e.g. , fluorochrome labeled dexamers of HLA-A, such as HLA*A2:01 or HLA-B such as HLA*B7:02), and/or HLA-F (e.g. ,

fluorochrome labeled teiramers of HLA-F) to I LT4 (e.g. , I LT4 on mouse 3A9 T cells transfected with and expressing I LT4), e.g. , as determined by surface plasmon resonance;

» blocks I LT4 (e.g. , I LT4 on mouse 3A9 T cells transfected with and expressing I LT4), binding to ANGPTL1 , ANGPTL4, and/or ANGPTL7 (e.g. , biotinylated ANGPTL proteins), e.g. , as determined by surface plasmon resonance;

• does not bind to I LT2, ILT3, ILT5, LILRB5, LI LRA1 , LILRA2, I LT7, I LT8, and/or ILT1 1 ;

• reverses ILT4-mediated suppression of IL2 in I LT4 transfected 3A9 ceils, e.g. , with an EC ₅₀ of 0.43 micrograms/ml (±0.14 micrograms/ml);

» rescues ! LT4:HLA-G induced suppression of mast cell degranulation (e.g. , In the presence of plate-bound HLA-G teframer), for example, wherein the mast cells express I LT4 and CD200RLa and are stimulated, for example, with antibody- mediated cross-linking of CD200RLa;

• enhances lipopolysaccharide (LPS)-induced expression of proinflammatory myeloid cytokines, for example, G -CSF and/or TNFalpha, from a peripheral blood mononuclear cell (PB C);

» enhances anti-CD3-induced expression of pro-inflammatory myeloid cytokines for example, GM-CSF and/or TNFalpha, from a peripheral blood mononuclear cell (PBMC);

· inhibits tumor growth in humans or, for example, in other mammals such as mice (e.g., Immuno-deficient NSG mice) which were reconstituted with human hematopoietic stem cells, for example, which harbor peripheral human CD45+ immune cells, for example, wherein the tumor is a human skin melanoma tumor such as from the cell line SKMEL5;

· relieves macrophage/myeloid-derived suppressor cell (MDSC)-mediated tumor tolerance in the body of a subject (e.g. , human subject) with a tumor;

• does not bind to cynomoigous monkey ILT4 and/or mouse pirB; and/or

• stains CD14+ human monocytes and/or CD1 1 B+ human granulocytes

• binds to one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or all 10) of the human ILT4

hapiotypes set forth in Table 7. Antibody 1E1 (Q1E) heavy chain (IgG4)

Heavy chain

EVQLQQWGAGLLKESETLSkTCAVYGGSFSG ^SWIRQPBGKGLE

FSI.KLSSVT ADTA YYCARI^gWVOTgg^ iWe GTl-VT SSASTKGBSVFPLAPCSRSTSESTAALCCLVKDYF PBPVTVSWMSGALTSGVHTFPAVLQSSGLYSLSSVVXVBSSSLGT TYTCtWDHKPEHTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPP P DTI-MISRTPEVTCVWDVSQEDPEYQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLHGKEYItCKVSH GLPSSIEKTISKA GQPREPQVYTLPPSQEEHTKNQVSLTCLV GFYPSDIAVEWESN GQPENlTOTTPPVLCSDGSFFLYSRLXVD SRWQEGrWFSCSVMHEALHNHYXQKSLSIiSIiGK

(SEQ ID NO;1; variable domain underscored; CDRs double underscored)

Heavy chain variable domain

-SVQnQQWGAGLUCPSETLSnTCAVYGGSFSGYYWS IRQPEG GLEWIGEINHSGSTNYNPSL SRVTISVDTSKNQ FSLKLSSVTAADTAVYYCARLPTR TTRYFDLWGRGTLVTVSS

(SEQ ID NO:63)

Antibody 1E1 (Q1E, S54A) heavy chain (IgG4)

Heavy chain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGr-TTSWIROPPGKGLE^

FSLKLgSVTAADTAVYYCARLJPTgWVrroY

PEPVTVSWNSGALTSGVHTI^AVLQSSGLYSLSSVV VPSSSLGT TYTCNVDHKPSN KVDKRVES YGPPCPPCP APEFLGGPSVFLFPP P I ⁾ TI^ SRTPEVTCVWDVSQEDEEVQFNWWDGVEVHNAKT PREEQFNSTYR7VSVLT VLHQDWLNeKEY C VSNKGLPSSIEKTISKAKGQPREEQVYTLPPSQEEMT NQVSLTCLVKGFYPSDIAVEWESN GQPEM !nTTPPVI-DSDGSFFLYSRLrVDKSRWQEGOT

(SEQ ID NO:2; variable domain underscored; CDRs double underscored)

Heavy chain variable domain

EVQLQQ GAGLLKPSEXLSLTCAVYGGSFSGYY SWIROPPGKGLEWIGEINHAGS imiPSLKSRVTISVDTSKNQ FSLKLSSVTAADTAVYYCARLPTR VTTRYFDLWGRGTLVTVSS

(SEQ ID NO:57)

Antibody 1E1 heavy chain (lgG1)

Heavy chain

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS IRQgPG GI-EWIGEINHSGST Y PSIiKSRVTISVDTSKNQ gSLKLSSVTAADTAVYYPAR^PTRWVTTRYTOt,WGReTLVTVSS¾STKGPSVgBIAPS SKSTSCCTAALGCLVKDYF PEPV VSVraSGALTSGVHTFPAVLQSSGLYSLSSYVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCD THTCP PCPAPELLGGPSVFIiFPPKPKDT^ISRTEEVTGWVDVSHEDPEV FlSn!nn/DGVEVHNA T PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSN ALPAPIEKTISKAKGQPREPQVYTIJPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNy TTPPVLDSDGSFFLYS LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO:44; variable domain underscored; CDRs double underscored) Heavy chain variable domain

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGK^

FSLKLSSVTAADTAVYYCARLPTRWVTTRYFDL G GTLVTVSS

(SEQ ID NO:69)

1E1 heavy chain CDRs

CDR-H1: GYYWS (SEQ ID NO:16)

CDR-H2: El HXGST YN PSLKS wherein X is S or A (e.g. , EINHSGSTNYNPSLKS or EINHAGSTNYNPSLKS) (SEQ ID NO: 17)

CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18)

Antibody 1E1 (Q1 E) light chain (lambda)

Light chain

ESVLTQPPSVSGAPC-gRVTISCTGjiSSN^

ADSSPV AGVETTTPSKQSNNKYAASSYIiSLTPEQ KSHRSYSCQVTHEGSTVEKTVAPTECS

(SEQ ID NO:3; variable domain underscored; CDRs double underscored)

Light chain variable domain

ESVLTQPPSV3GAPGQRVTISCTGSSSNIGAGYDVHMY0QLPGTAPKLI.IYGNSNRPSG VPDRFSVSKSGASASLAI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

(SEQ ID O:70) Antibody 1E1 (Q1 E, S54A) light chain (lambda)

Light chain

.^SSPVKASVETTTESKQSNITi-YAASSTiLSI-^

(SEQ I D NO:4; variable domain underscored; CDRs double underscored)

Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVH YQQLPGTAPKLLIYGNANPSGVPDRFSVS SGASASIAI TGLQAEDEADYYCQSF NSLSAYVFGGGTQLTVL

(SEQ ID NO:71 )

Antibody 1 E1 (Q1 E, W53Q) light chain (lambda)

Light chain ESVLTQggSVSGaPGQRVTISCTGSSSNISAGYjDjre^QQ

TGLQAEDEADYYCOSFPNSLSAYVFGGGTQLTVLGQEKAAPSVTLFPPSSEELDAN ATLVCLISDFYPGAVTVAWK ADSSPVKAGVEXTTPS QSKM YAASSYLSl-TPEQW SHRSYSCQVTHEGSrVEKTVAPTECS

(SEQ I D NO:5; variable domain underscored; CDRs double underscored)

Light chain variable domain

ESVLTQPPSVSGAPGQ VTISCTGSSSNIGAGYDVH YQQLPGrAP LLIYGQSNRPSGVPDRFSVS SGASASJ-AI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

(SEQ ID NO-.72) Antibody 1E1 (Q1 E, N53E) light chain (lambda)

Light chain

ESVLTQPPSVSGAPGQ VTI-SCTGSSSHIGAGTOTOWYQQIiPGTAPKLLIYGESNRPSGVPDRFSVSKSGASASL AI TGLQAEDEADYYCQSFDWSLS^YJgFGGGTQIjTVIiG ?PKAAPSVriiFPPSSEELQAtn ATLVCLlSDFYPGAVTVAWK ADSSPVKAGVETTTPSKQSNWKYAASSYLSLTPEQW SHRSYSCQV HEGSTVEK VAPTECS

(SEQ ID NQ:6; variable domain underscored; CDRs double underscored)

Light chain variable domain

ESVLTQPPSVSGAPGQ VTXSCTGSSSNIGAGYDVHWYQQLPGrAPKLLIYGESNRPSGVPDRFSVS SGASASIAI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

(SEQ ID NO:73)

Antibody 1E1 (Q1 E, IM53D) light chain (lambda)

Light chain

ESVLTQPPSVSGAPGQ VTISCTGSSSNIGAGYDVHWYQOLPaTAPKLLIYGDSHlRPSGVPDRgSVSKSGASASI- AI

ADSSPV AGVETTTESKQSNNKYAASSYLSLXPEQWKSHRSYSCQVTHEGSTVE TVAPTECS

(SEQ ID NO:7; variable domain underscored; CDRs double underscored)

Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGV PDRFSVSKSGASASIAI TGLQAEDEADYYCQEFDNSLSAYVFGGGTQLTVL

(SEQ ID NO:58)

Antibody 1 E1 light chain (lambda)

Light chain

QSVI,TQPPSVSGAPGQRV'JSCTGSSSNIGAGYDVHWYOQLPGTAPKLLIYGMSNRPSG VPDRFSVSKSGASAi5LAI TGLQASDEAITYYCQSFPMSLSAYVFGGGTQLTVk

ADSSPVKAGVETTTPSKQSKNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE TVAPTECS

(SEQ ID NO:45; variable domain underscored; CDRs double underscored)

Light chain variable domain QSVLTQPPSVSGAPGQRV ISCTGSSSNIGAGYDVHWYQQLPGTAP LLIYGNS EPiSGVPDRFSVSKSGASASLAI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

(SEQ ID NO:77) 1 E1 light chain CDRs

CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19)

CDR-L2: GX ₁ X ₂ NRPS; wherein X _! is N,Q,E or D and X ₂ is S or A (e.g. , GNSNRPS, GNANRPS, GQSNRPS, GESNRPS or GDSNRPS) (SEQ ID NO: 20)

CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21)

Antibodies and antigen-binding fragments thereof including the 1 E1 heavy and light chain CDRs or the 1 E1 V _H and V _L or the 1 E1 heavy chain and light chain (or a variant thereof, e.g. , as set forth herein) may be referred to as "1 E1." Antibody 2A6 (Q1E) heavy chain (lgG4)

Heavy chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY^Ajr^WRQAP^GLEmGGIIPIFGTA-^A QKl^RVTITADBSTS TAYMELSSLRSEETAVYYCARYFPSSGWYKgmFDIWGQGT^

DYFPEPVTVSVmSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCWDHKPSH TKVDKRVESKYGPPCP PCPAPKFLGGP SVFLFPBKPKDTJ MI SRTE" EVTCWVDV SQEDPEVQFNVrYVDGVEVHlSlAKTKPREEQFNSTYRVV S VLTVLHQD LKGKEY CKySN GLPSSIEKTIS AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE ESNGQPElTlTCKTTPPVLDSDGSFFLYSRLTVDKSRViQEGlWFSCSVMHEALHNHY QKSLSLSLGK

(SEQ ID NO:8; variable domain underscored; CDRs double underscored)

Heavy chain variable domain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSCTAISOTRQAPGQGLEXraGGIIPIFG TANYAQ PQGRVTITADESTS TAYMELSSLRSEDTAVYYCARYFDSSGWYKGGAFDX GQGTMVTVSS

(SEQ ID NO:64)

Antibody 2A6 (Q1 E, S102A, M119L) heavy chain (I9G4)

Heavy chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISVTVRQAPGQGLE^iMGGIIPIFGTA YAOKFOGRVTITADESTS TAYMELSSLRSEDTAVYYCARYFDASGTmCGGAFDlWGQGTLVTVSSASTKGPSVFPLAP C SRSTSESTAALGCLVK DYFPEPVTVSVraSGALTSGVHTFPAVLQSSGLYSLSSW VPSSSLGT TYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPP P DTIMISRTPEVTCVTVDVSQEDPEYQFNWYVDGVEVTJNAKr PREEQFNSr!fRVVS VLTViaQDWLNGKEYKCKVSlWGLPSSIEKTISK-A eQPREPQVYrLPPSQEE TKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNY TTPPVLDSDGSFFLYiSRLTTOKSR QEGl^FSCSVMHEAtHNHYTQKSLSLSLGK

(SEQ I D NO:9; variable domain underscored; CDRs double underscored)

Heavy chain variable domain EVQLVQSGAEVKKPGSSV VSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIBIFGTANYAQKFQGRVTITADESTS TAYMELSSLRSEBTAVYYCARYFEASGWYKGGAFDIWGQCTLVrVSS

(SEQ ID NO:65) Antibody 2A6 (Q1E, D101S, M119L) heavy chair (lgG4)

Heavy chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQSLEWMGGIIPIFGTA yAQKFQGRVTITADESTS TAYMELSSLRS PTAVYYCARYFSSSGWYKGGAFDI GQGTLVrVSSASTKGPSVFPLAPCSRSTSESTAALGCLYK DYFPEPVTVS^SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG KTYTCNVDHKPSN KVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKEKDTIMISRTPWTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVS VLTVLHQDWLKGKEY C VSIWGLPSSIEKTISKA GQPREPQVYTLPPSQEEMTKNQVSLTCLV GFYPSDIAVE ESNGQPEPnSTfKTTPPVLDSDGSFFLYSRlTVDKSR QEGNVFSGSVIiHEALHNHYTQKSLSLSLGK

(SEQ ID NO:10; variable domain underscored; CDRs double underscored)

Heavy chain variable domain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRgAPGQGLEWMGGIIPIFGT ANYAQ FQGRVTITADESTS TAYMELSSLRSEDTAVYYCARYFSSSGWYKGGAFDIWGQGTLVTVSS

(SEQ ID NO:66)

The present invention includes antibodies and antigen-binding fragments thereof wherein residue 1 of SEQ ID NO:8, 9, 10, 64, 65, or 66 is Q instead of E.

2A6 heavy chain CDRs

CDR-H1: SYAIS (SEQ ID NO:22)

CDR-H2: GII PIFGTANYAQKFQG (SEQ I D NO:23)

CDR-H3: YFXiX ₂ SGWYKGGAFDI; wherein X-, is D or S and X ₂ is S or A (e.g.,

YFDSSGWYKGGAFDl, YFDASGWYKGGAFDI or YFSSSGWYKGGAFDI) (SEQ I D NO;24)

Antibody 2A6 light chain (lambda)

Light chain

QSVLTQPSSLSASPGASASLTCTLRSGI DTYRmWQQKPGSPPQYLLRYKSDSDKHga^GVPSRFSGSKDPaAIJ AGILLISGLQSEPEADYYCAI YSS WVgGGGTOLTVLGOPKAAPSVTLFPPSSEELOAMKATI-VClTRDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNHKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS

(SEQ ID NO:1 1 ; variable domain underscored; CDRs double underscored)

Light chain variable domain

QSVLTQPSSLSASPGASASLTCTLRSGINVDTYRIHWYQQKPGSPPQYLLRY SDSDKHQGSGVPSRFSGSKDPSAN AGILLISGLQSEDEADYYCAIWYSST VFGGGTQLTV!.

(SEQ ID NO:74) The present invention includes antibodies and antigen-binding fragments thereof wherein residue 1 of SEQ ID NO:11 or 74 is E instead of Q.

2A6 light chain CDRs

CDR-L1 : TLRSGINVDTYRIH (SEQ ID NO:25)

CDR-L2: YKSDSDKHQGS (SEQ ID NO:26)

CDR-L3: AIWYSSTWV (SEQ ID NO:27)

Antibodies and antigen-binding fragments thereof including the 2A6 heavy and light chain CDRs or the 2A6 V _H and V _L or the 2A6 heavy chain and light chain (or a variant thereof, e.g. , as set forth herein) may be referred to as "2A6."

Antibody 3G7 (Q1E) heavy chain (lgG4)

Heavy chain

EVQkVESGGG QPGRS RLSCAASGFTFSSYA HWVRQAPGKGLE AVIj^

TLYiaMHSLRAEDTAVYYCARVGEWIQLWSPFDY GQGTLVTVSSASTKGPSVgPl-AECSRSTSESTAALGCLVK Y FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT TYTCNTOH PSN VD RVESKYGPPCPPC PAPEFLGGPSVFLFPP PKDTI-MISRTPEVTCVVVDVSQEDPEVQFNVri^DGVEVHNA T PREEQFNSTYRWSTL TVLHQD LNG EYKCKVSNKGLPSSIE TIS AKGQPREPQVYTLPPSQEEMTKNQVSLTCLV GFYPSDIAVEWES NGQPENlT!fKTTPPVLDSDGSFFLYSRLTVDKSRWQEGJ^FSCSVMHEAIiHNHYTQ SljSLSIiG

(SEQ ID NO:12; variable domain underscored; CDRs double underscored).

Heavy chain variable domain

EVQLVESG-revyQPGRSLRLSCAASGFTFSSYAMHOTRQAPGKGLEWAVISYDGSNKYY ADSVKGRFTISRDNS H TLYLQMNSLRAEDTAVYYCARVGE IQLWSPFDY GQGTLVTVSS

(SEQ ID NO:67)

The present invention includes antibodies and antigen-binding fragments thereof wherein residue 1 of SEQ ID NO: 12 or 67 is Q instead of E. 3G7 heavy chain CDRs

CDR-H1: SYAMH (SEQ I D NO:28)

CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO:29)

CDR-H3: VGEWIQLWSPFDY (SEQ ID NO:30) Antibody 3G7 light chain (kappa)

Light chain DIQMTQ3PSSVSASVGDRVTITCRASQ^ISSP^WYQ0KBGKAEKFLIYA¾SSLQSGVPS KFSGSGSGTDFTI,TISS LQPEDFATYYCQOYHSYPPTFGGGTKVEI RtVAAESVFIFPPSDEQl_KSGTASWCLLl>rNF^PRE¾CTQWKVDMAL QSGNSQESVTEQDS DSTYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:13; variable domain underscored; CDRs double underscored)

Light chain variable domain

DIQMTQSPSSVSASVGDRYTITCRASQGISSWIAVrYQQ PGKAPKFLIYAASSLQSGVPS FSGSGSGTDFTLTISS LQPEDFA YYCQQY SYPPTFGGGT VEIK

(SEQ I D O:75) 3G7 light chain CDRs

CDR-L1 : RASQG!SSWLA (SEQ ID N0.31)

CDR-L2: AASSLQS (SEQ ID NO:32)

CDR-L3: QQYNSYPPT (SEQ ID IMO:33) Antibodies and antigen-binding fragments thereof including the 3G7 heavy and light chain CDRs or the 3G7 V _H and V _u or the 3G7 heavy chain and light chain (or a variant thereof, e.g., as set forth herein) may be referred to as "3G7."

Antibody 2C1 (Q1 E) heavy chain (lgG4)

Heavy chain

EVQLVQSGAEVTKPGASVKVSCKVSGYTLTELSMHWVROAPGKGIJEWMGGFDPEDGETI YAQKFQGRVTMTEDTSTD TAYMELSSLRSEDTAVYYCARAGPLYTIFGWIIPDNWFDPWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAAL GCLVKDYFPEPVTV£3VWSGALTSGVHTFPAVLQSSGLYSL£5£VVTVPSSSLGT TYTC1WI>H PSOT VDKRVES Y GPPCPPCEAPEFLGGFSWLFPPKPKDTMISRTPEVTCWVDVSQ YRWSVLTVLHQDTONG EYKCKVSH GLPSSIE TIS AKGQPREEQVYTLPPSQEEMTKHQVSI-TCLVKGFYPSD IAVEWESNGQPEN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGWFSCSVMHEALHNHYTQ SLSLSLGK

(SEQ ID NO: 14; variable domain underscored; CDRs double underscored)

Heavy chain variable domain

EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLE MGGFDPEDGETIYAQKFQGRVT TEDTSTD TAYMELSSLRSSDTAVYYCARAGPLYTIFGWIIPDKWFDPWGQGTLVTVSS

(SEQ ID NO:68)

The present invention includes antibodies and antigen-binding fragments thereof wherein residue 1 of SEQ ID NO: 14 or 68 is Q instead of E. 2C1 heavy chain CDRs

CDR-H 1 : ELSMH (SEQ ID NO:34)

CDR-H2: GFDPEDGETiYAQKFQG (SEQ ID NO:35)

CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQ ID NO:36) Antibody 2C1 light chain (Q1E) (lambda)

Light chain

ESVLTQPPSVSGAPSQKViaSCTGSS,SNIGA^^

TGI 3AEDEADYYCqSTOS3I^G^GVyFGCSGTQIilILGOPKAAP SVTLFPF SSEELQ ANKATLVCLI SDFYPGAVTVA WKADSSPV AGVETTTPSKQSHN YAASSYLSIiTPEQWKSHRSYSCQVTHEGSTVK VAPTECS

(SEQ ID NO: 15; variable domain underscored; CDRs double underscored)

Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGASYDVHVreQQLPCTAPKLLIYGNSHH PSCSVPD PSGSKSeTSASLAI TGLQAEDEADYYCOSYDSSLSGSGVVFGGGTQLIIL

(SEQ I D NO:76)

The present invention includes antibodies and antigen-binding fragments thereof wherein residue 1 of SEQ ID NO:15 or 76 is Q instead of E.

2C1 light chain CDRs

CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO:37)

CDR-L2: GNSNRPS (SEQ ID NO:38)

CDR-L3: QSYDSSLSGSGVV (SEQ ID NO:39)

Antibodies and antigen-binding fragments thereof including the 2C1 heavy and light chain CDRs or the 2C1 V _M and V _L or the 2C1 heavy chain and light chain (or a variant thereof, e.g. , as set forth herein) may be referred to as "2C ." In various embodiments of the antibody or antigen-binding fragment thereof, a C- terminal lysine of a heavy chain immunoglobulin is absent.

Thus, in some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin comprises the amino acid sequence set forth in SEQ I D NO:3, 4, 5, 6 ,7, 11 , 13, 15, or 45; and/or the heavy chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8, 9, 10,

12, 14, 44, 79, 80, 81 , 82, 83, 84, 85, or 86.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 or 79; and a light chain immunoglobulin comprising the amino acid sequence set forth in

SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 2 or 80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 4.

In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 2 or 80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 5.

In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 2 or 80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 6.

In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 2 or 80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 7.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 2 or 80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO:

8 or 82; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 .

In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO:

9 or 83; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 .

In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 10 or 84; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 1 1 .

In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 12 or 85; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ I D NO: 13. in yet still embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 14 or 86; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15.

In certain other embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:70, 71 , 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chain variable domain comprise the amino acid sequence set forth in SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69,

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:63; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:70,

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:71.

In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID

NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:72.

In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:73.

In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:58.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:70.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:64; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74.

In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:65; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:74.

In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:66; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74.

In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NG:67; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:75.

In yet still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NG:68; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:76.

In a further embodiment, the antibody or antigen-binding fragment thereof that binds ILT4 comprises an immunoglobulin light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 1 E1 (e.g., SEQ ID NOs: 19-21); and an immunoglobulin heavy chain variable (V _H ) domain comprising a CDR-H1 , CDR-H2 and CDR-H3 of 1 E1 (e.g., SEQ ID NOs: 16-18).

In a further embodiment, the antibody or antigen-binding fragment thereof that binds ILT4 comprises an immunoglobulin light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 2A6 (e.g. , SEQ ID NOs: 25-27); and an immunoglobulin heavy chain variable (V _H ) domain comprising a CDR-H1 , CDR-H2 and CDR-H3 of 2A6 (e.g., SEQ ID NOs: 22-24).

In a further embodiment, the antibody or antigen-binding fragment thereof that binds ILT4 comprises an immunoglobulin light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 3G7 (e.g. , SEQ ID NOs: 31-33); and an immunoglobulin heavy chain variable (V _H ) domain comprising a CDR-H1 , CDR-H2 and CDR-H3 of 3G7 (e.g. , SEQ ID NOs: 28-30).

In a further embodiment, the antibody or antigen-binding fragment thereof that binds ILT4 comprises an immunoglobulin light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 2C1 (e.g. , SEQ ID NOs: 37-39); and an immunoglobulin heavy chain variable (V _H ) domain comprising a CDR-H1 , CDR-H2 and CDR-H3 of 2C1 (e.g. , SEQ ID NOs: 34-36).

In one embodiment, the antibody or antigen-binding fragment comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21),

In another embodiment, the antibody or antigen-binding fragment comprises: a V _H domain comprising: CDR-H1 : GYYVVS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51 ), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

El NHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising:CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51 ), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the antibody or antigen-binding fragment thereof comprises: a V _H domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2:

EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the V _L domain of antibody 1E1 (e.g., SEQ ID NO:70, 71, 72, 73, 58 or 77) and/or the V _H domain of antibody 1E1 (e.g., SEQ ID NO:63, 57 or 69).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the V _L domain of antibody 2A6 (e.g., SEQ ID NO:74) and/or the V _H domain of antibody 2A6 (e.g., SEQ ID NO:64, 65 or 66).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the V _L domain of antibody 3G7 (e.g., SEQ ID NO:75) and/or the V _H domain of antibody 3G7 (e.g., SEQ ID NO:67).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the V _L domain of antibody 2C1 (e.g., SEQ ID NO:76) and/or the V _H domain of antibody 2C1 (e.g., SEQ ID NO:68).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the light chain immunoglobulin of antibody 1 E1 (e.g., SEQ ID NO:3, 4, 5, 6, 7 or 45) and/or the heavy chain immunoglobulin of antibody 1E1 (e.g., SEQ ID NO:1, 2, 44, 79, 80, or 81).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the light chain immunoglobulin of antibody 2A6 (e.g., SEQ ID NO:11) and/or the heavy chain immunoglobulin of antibody 2A6 (e.g., SEQ ID NO:8, 9, 10, 82, 83, or 84).

The present invention further provides an antibody or antigen-binding fragment thereof that binds ILT4 and comprises the light chain immunoglobulin of antibody 3G7 (e.g., SEQ ID NO: 13) and/or the heavy chain immunoglobulin of antibody 3G7 (e.g., SEQ ID NO:12 or 85). The present invention further provides an antibody or antigen-binding fragment thereof that binds I LT4 and comprises the light chain immunoglobulin of antibody 2C1 (e.g. , SEQ I D NO: 15) and/or the heavy chain immunoglobulin of antibody 2C1 (e.g. , SEQ ID NO: 14 or 86).

The present invention further provides an antibody that consists of two heavy chains and two light chains, wherein each light chain comprises the V _L or light chain

immunoglobulin of antibody 1 E1 , 2A6, 3G7, or 2C1 , and each heavy chain comprises the V _H or heavy chain immunoglobulin of antibody 1 E1 , 2A6, 3G7, or 2C1 .

In one embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ I D NO:57.

In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ I D NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57, wherein the light chain further comprises the amino acid sequence set forth in SEQ I D NO:90,

In yet another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ I D NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57, wherein the heavy chain further comprises the amino acid sequence set forth in SEQ ID NO:89.

In still another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ I D NO:58 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57, wherein the light chain further comprises the amino acid sequence set forth in SEQ I D NO:90 and the heavy chain further comprises the amino acid sequence set forth in SEQ I D NO:89,

in one embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence set forth in SEQ I D NO: 7 and each heavy chain comprises the amino acid sequence set forth in SEQ I D NO:2.

In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence set forth in SEQ I D NO:7 and each heavy chain consists of the amino acid sequence set forth in SEQ ID NO:2.

In an embodiment of the invention, the antibody or antigen-binding fragment of the present invention comprises a V _L (with or without signal sequence), e.g. , the V _L in any of SEQ I D NO:58 or 70-77, having up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions; and/or a V _H (with or without signal sequence), e.g. , the V _H in any of SEQ ID NO:57 or 63-69, having up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions, while still binding to ILT4.

The present invention also includes polypeptides comprising the amino acid sequences disclosed herein, e.g. SEQ ID NOs: 1-39, 44, 45, 47-58, 63-77, or 79-86, as well as polypeptides comprising such amino acid sequences with up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative or non-conservative amino acid substitutions therein.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6 ,7, 1 1 , 13, 15, or 45, and/or the heavy chain immunogiobulin has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8, 9, 10, 12, 14, 44, 79, 80, 81 , 82, 83, 84, 85, or 86.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunogiobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunogiobulin comprises a light chain variable domain having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:70, 71 , 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chain

immunoglobulin comprises a heavy chain variable domain having at least 90% amino acid sequence identify to the amino acid sequence set forth in SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In an embodiment of the invention, an immunoglobulin heavy chain of an anti-ILT4 antibody or antigen-binding fragment of the present invention is operably linked to a signal sequence, e.g., comprising the amino acid sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO:41) and/or an immunoglobulin light chain of an anti-ILT4 antibody or antigen-binding fragment of the present invention is operably linked to a signal sequence, e.g., comprising the amino acid sequence MSVPTQVLGLLLLWLTDARC (SEQ ID NO:42).

In an embodiment of the invention, an N-terminal g!utamine (Q) of an

immunoglobulin chain set forth herein (e.g., heavy and/or light) is replaced with a pyrogiutamic acid. In one embodiment, an N-terminai Q of a heavy chain immunogiobulin is replaced with a pyrogiutamic acid. In another embodiment, an N-terminal Q of a light chain immunoglobulin is replaced with a pyrogiutamic acid. In yet another embodiment, an N- terminal Q of a heavy chain immunoglobulin and an N-terminal Q of a heavy chain immunoglobulin are replaced with a pyrogiutamic acid. Further provided herein are antibodies or antigen-binding fragments that bind to the same epitope of ILT4 (e.g. , human ILT4) as any anti-!LT4 antibody or antigen-binding fragment thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 o 2C1 ), In one embodiment, the epitope is LYREKKSASW (SEQ ID NO:59). In another embodiment, the epitope is

TRIRPEL (SEQ ID NO:60). in yet another embodiment, the epitope is NGQF (SEQ ID NO:61). In still another embodiment, the epitope is HTGRYGCQ (SEQ ID NO:62). In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of human ILT4 as an antibody or antigen-binding fragment thereof comprising the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 11 ; 9 and 1 1 ; 10 and 1 1 ; 12 and 13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 1 1 ; 83 and 1 1 ; 84 and 1 1 ; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen- binding fragment thereof binds to the same epitope of human ILT4 as an antibody or antigen-binding fragment thereof comprising the heavy chain variable domain and light chain variable domain amino acid sequences set forth in SEQ ID NOs:63 and 70; 57 and 71 ; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76; respectively.

The present invention include antibodies and antigen-binding fragments that cross- block the binding of any anfi-ILT4 antibody or antigen-binding fragment thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 or 2C1) to ILT4 (e.g., human ILT4) or compete with any anti- ILT4 antibody or antigen-binding fragment thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 or 2C1 ) to ILT4 (e.g. , human ILT4). The cross-blocking antibodies and antigen-binding fragments thereof discussed herein can be identified based on their ability to block any of the antibodies or fragments specifically set forth herein from binding to ILT4, in binding assays (e.g. , bio-layer interferometry (BLI; for example FORTEBIO OCTET binding assay; Pali ForteBio Corp; enlo Park, CA), surface plasmon resonance (SPR), BIACore, ELISA, flow cytometry). For example, in an embodiment of the invention, when using BLI, the tip of a fiber-optic probe is coated with ligand (e.g. , ILT4) and acts as the biosensor wherein binding of anti-lLT4 antibody or antigen-binding fragment to the 1LT4 alters the interference pattern of white light reflected from the probe layer bound to ILT4 and an internal reference layer. The shift is indicative of ILT4/anti-ILT4 binding. In an embodiment of the invention, the ILT4 coated tip is immersed in a solution of analyte containing antibody or antigen- binding fragment, e.g. , in the well of either a 96- or 384-well plate. In an embodiment of the invention, the plate is shaken during reading to create orbital flow. To read the assay, white light is directed down the length of the fiber. As mentioned above, interference between light reflecting off the reference layer and immobilized surfaces containing 1LT4 of the tip creates a distinctive pattern of light returning up the fiber. As molecules bind to the immobilized sensor surface, that pattern changes in proportion to the extent of binding. For example, assays can be used in which a 1 LT4 (e.g. , human I LT4) protein is immobilized on a BLI probe or plate, a reference anti-I LT4 antibody or fragment binds to ILT4 (e.g. , at saturating concentration) and a test anti-! LT4 antibody or fragment is added. The ability of the test antibody to compete with the reference antibody for ILT4 binding is then determined. In the BLI format, light interference of the ILT4 complex is monitored to determine if the test antibody effectively competes with the reference antibody, e.g. , nanometers of light wavelength shift over time is monitored wherein a shift indicates additional binding of the test antibody and a lack of cross-blocking. In an embodiment of the invention, in the BLI format, cross-blocking is qualitatively deemed to have occurred between the antibodies if no additional binding of test antibody is observed, in an embodiment of the invention, as a control, cross-blocking of the reference antibody with itself is confirmed; wherein the assay is determined to be operating correctly if the reference antibody can cross-block itself from I LT4 binding. The ability of a test antibody to inhibit the binding of the anti-I LT4 antibody or fragment 1 E1 , 2A6, 3G7 or 2C1 , to ILT4 (e.g. , human I LT4) demonstrates that the test antibody can cross-block the antibody or fragment for binding to I LT4 (e.g. , human I LT4) and thus, may, in some cases, bind to the same epitope on I LT4 (e.g. , human ILT4) as 1 E1 , 2A6, 3G7 and/or 2C1 . As stated above, antibodies and fragments that bind to the same epitope as any of the anti-ILT4 antibodies or fragments of the present invention also form part of the present invention. In an embodiment of the invention, BLI is conducted in a sandwich format wherein a reference anti- ILT4 antibody or antigen-binding fragment is immobilized to the probe and then bound with I LT4. Test anti- I LT4 antibody or antigen-binding fragment is then tested for the ability to block binding of the references antibody or fragment.

In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding to human I LT4 with an antibody or fragment comprising the heavy chain and light chain amino acid sequences set forth in SEQ I D NOs: 1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 1 1 ; 9 and 1 1 ; 10 and 1 1 ; 12 and 13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 1 1 ; 83 and 1 1 ; 84 and 1 1 ; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human I LT4 with an antibody or fragment comprising the heavy chain variable domain and light chain variable domain amino acid sequences set forth in SEQ I D NOs:63 and 70; 57 and 71 ; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76; respectively. The present invention includes anti-ILT4 antibodies and antigen-binding fragments thereof comprising N-linked glycans that are typically added to immunoglobulins produced in Chinese hamster ovary cells (CHO N-linked glycans) or to engineered yeast cells (engineered yeast N-linked glycans), such as, for example, Pichia pastoris. For example, in an embodiment of the invention, the anti-ILT4 antibodies and antigen-binding fragments thereof comprise one or more of the "engineered yeast N-linked glycans" or "CHO N-linked glycans" that are set forth in Figure 16 (e.g. , GO and/or GO-F and/or G1 and/or G1 -F and/or and/or G2-F and/or Man5). In an embodiment of the invention, the anti-ILT4 antibodies and antigen-binding fragments thereof comprise the engineered yeast N-iinked glycans, i.e. , GO and/or G1 and/or G2, optionally, further including Man5. In an embodiment of the invention, the anti-ILT4 antibodies and antigen-binding fragments thereof comprise the CHO N-linked glycans, i.e., GO-F, G1-F and G2-F, optionally, further including GO and/or G1 and/or G2 and/or an5. in an embodiment of the invention, about 80% to about 95% (e.g. , about 80- 90%, about 85%, about 90% or about 95%) of all N-linked glycans on the anti-ILT4 antibodies and antigen-binding fragments thereof are engineered yeast N-linked glycans or CHO N-iinked glycans. See Nett et as. Yeast. 28(3): 237-252 (201 1 ); Hamilton et a/.

Science. 313(5792): 1441-1443 (2006); Hamilton et a!. Curr Opin Biotechnoi. 18(5): 387- 392 (2007). For example, in an embodiment of the invention, an engineered yeast cell is GFI5.0 or YGLY8316 or strains set forth in U.S. Patent No. 7,795,002 or Zha et ai. Methods Mol Biol. 988:31-43 (2013). See also international patent application publication no.

WO2013/066765.

Polynucleotides

The present invention comprises polynucleotides (e.g., DNA or RNA) encoding the immunoglobulin chains of anti-ILT4 antibodies and antigen-binding fragments thereof disclosed herein. For example, the present invention includes the nucleic acids encoding immunoglobulin heavy and/or light chains of antibodies 1 E1 , 2A6, 3G7 and 2C1 (e.g. , SEQ ID NOs: 1-15, 44 and 45 or a variable domain thereof) as described herein as well as nucleic acids which hybridize thereto.

The present invention includes polynucleotides encoding an antibody light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 1 E1 (e.g. , comprising the amino acids set forth in SEQ ID NOs: 19-21); and/or an antibody heavy chain variable (V _H ) domain comprising a CDR-H1 , CDR-H2 and CDR-H3 of 1 E1 (e.g. , comprising the amino acids set forth in SEQ ID NOs: 16-18).

The present invention includes polynucleotides encoding an antibody light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 2A6 (e.g. , comprising the amino acids set forth in SEQ I D NOs: 25-27); and/or an antibody heavy chain variable (VH) domain comprising a CDR-H 1 , CDR-H2 and CDR-H3 of 2A6 (e.g. , comprising the amino acids set forth in SEQ I D NOs: 22-24),

The present invention includes polynucleotides encoding an antibody light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 3G7 (e.g., comprising the amino acids set forth in SEQ I D NOs: 31 -33); and/or an antibody heavy chain variable (V _H ) domain comprising a CDR-H 1 , CDR-H2 and CDR-H3 of 3G7 (e.g., comprising the amino acids set forth in SEQ I D NOs: 28-30).

The present invention includes polynucleotides encoding an antibody light chain variable (V _L ) domain comprising a CDR-L1 , CDR-L2 and CDR-L3 of 2C1 (e.g., comprising the amino acids set forth in SEQ I D NOs: 37-39); and/or an antibody heavy chain variable (V _H ) domain comprising a CDR-H 1 , CDR-H2 and CDR-H3 of 2C1 (e.g. , comprising the amino acids set forth in SEQ I D NOs: 34-36).

The present invention includes polynucleotides encoding the V _L domain of antibody 1 E1 (e.g. , SEQ I D NO:70, 71 , 72, 73, 58 or 77) and/or the V _H domain of antibody 1 E1 (e.g. , SEQ I D NO:63, 57 or 69).

The present invention includes polynucleotides encoding the V _L domain of antibody 2A6 (e.g., SEQ I D NO:74) and/or the V _H domain of antibody 2A6 (e.g. , SEQ I D NO:64, 65 or 66).

The present invention includes polynucleotides encoding the V _L domain of antibody

3G7 (e.g. , SEQ I D NO:75) and/or the V _H domain of antibody 3G7 (e.g. , SEQ I D NO:67).

The present invention includes polynucleotides encoding the V _L domain of antibody 2C1 (e.g. , SEQ I D NO:76) and/or the V _H domain of antibody 2C1 (e.g. , SEQ ID NO:68).

The present invention includes polynucleotides encoding the light chain

immunoglobulin of antibody 1 E1 (e.g. , SEQ ID NO: 3, 4, 5, 6, 7 or 45) and/or the heavy chain immunoglobulin of antibody 1 E1 (e.g., SEQ I D NO: 1 , 2 or 44).

The present invention includes polynucleotides encoding the light chain

immunoglobulin of antibody 2A6 (e.g. , SEQ ID NO: 1 1 ) and/or the heavy chain

immunoglobulin of antibody 2A6 (e.g. , SEQ I D NO: 8, 9 or 10).

The present invention includes polynucleotides encoding the light chain

immunoglobulin of antibody 3G7 (e.g. , SEQ ID NO: 13) and/or the heavy chain

immunoglobulin of antibody 3G7 (e.g. , SEQ I D NO: 12).

The present invention includes polynucleotides encoding the light chain

immunoglobulin of antibody 2C1 (e.g. , SEQ ID NO: 15) and/or the heavy chain

immunoglobulin of antibody 2C1 (e.g. , SEQ I D NO: 14). In one specific embodiment., the polynucleotide comprises nucleotide sequence set forth in SEQ I D NO:87. In another specific embodiment, the polynucleotide comprises nucleotide sequence set forth in SEQ ID NO:88, In yet another embodiment, the polynucleotide comprises nucleotide sequence set forth in SEQ ID NO:87 and nucleotide sequence set forth in SEQ I D NO:88.

This present invention also provides expression vectors comprising the isolated nucleic acids of the invention, wherein the nucleic acid is operably linked to one or more control sequences, e.g., that are recognized by a host ceil when the host cell is transfected with the vector. Also provided are host ceils comprising an expression vector of the present invention, in certain embodiments, the host ceils are CHO cells.

!Vlethods of Making Antibodies and Antigen-binding Fragments Thereof The anti-I LT4 antibodies and antigen-binding fragments disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) may be produced recombinantly. In this embodiment, nucleic acids encoding one or more of the immunoglobulin chains of the antibodies and antigen-binding fragments of the invention (e.g. , any one of SEQ I D NOs: 1 -15, 44, 45, or 79-86, or comprising a V _H or V _L of 1 E1 , 2A6, 3G7 or 2C1 as set forth in any one of SEQ ID Nos:57, 58, 63-77) may be inserted into a vector and/or into a host cell chromosome and expressed in a recombinant host cell. There are several methods by which to produce recombinant antibodies which are known in the art.

Antibodies (e.g. , 1 E1 , 2A6, 3G7 o 2C1 ) can be recovered from the culture medium using standard protein purification methods. Further, expression of immunoglobulin chains of the invention from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The present invention includes vectors comprising one or more polynucleotides encoding one or more of said immunoglobulin chains and a glutamine synthetase (GS) gene. In an embodiment of the invention, the vector is in a host ceil that lacks functional glutamine synthetase. In an embodiment of the invention, the host ceil is in a culture medium substantially lacking glutamine. Methods for making one or more of such immunoglobulin chains or an anti-I LT4 antibody or antigen-binding fragment thereof comprising culturing such a host cell in culture medium substantially lacking glutamine are within the scope of the preseni invention as well as such chains, antibodies and fragments produced by such a method.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a giycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an immunoglobulin chain or antibody or antigen-binding fragment containing an immunoglobulin chain will depend on the particular cell line or transgenic animal used to produce the antibody.

Antibodies and antigen-binding fragments with a glycosylation pattern comprising only non- fucosylated N-glycans may be advantageous, because these antibodies and antigen- binding fragments have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et aL , J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These anti- ILT4 antibodies and antigen-binding fragments (e.g., 1 E1 , 2A6, 3G7 or 2C1) with non- fucosylated N-giycans are part of the present invention.

The present invention further includes antibody fragments of the anfi-ILT4 antibodies disclosed herein (e.g. , 1 E1 , 2A6, 3G7 or 2C1). The antibody fragments include F(ab) ₂ fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab) ₂ with dithiothreitol or mercaptoethylamine. A Fab fragment is a V _L -CL chain appended to a V _H - CH1 chain by a disulfide bridge. A F(ab) ₂ fragment is two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab) ₂ molecule includes a portion of the Fc region between which disulfide bridges are located. An FV fragment is a V _L or VH region.

The present invention includes anti-ILT4 antibodies and antigen-binding fragments thereof (e.g. , 1 E1 , 2A6, 3G7 or 2C1) which are of the IgA, IgD, igE, IgG and IgM

classification. In one embodiment, the anti-ILT4 antibody or antigen-binding fragment comprises a V _H as set forth herein and a heavy chain constant region, e.g. , a human constant region, such as γ1 , γ2, y-3, or y4 human heavy chain constant region or a variant thereof. In an embodiment of the invention, the antibody or antigen-binding fragment comprises a V _H as set forth herein and a light chain constant region, e.g. , a human light chain constant region, such as lambda or kappa human light chain region or variant thereof. By way of example, and not limitation the human heavy chain constant region can be y1 or γ4 and the human light chain constant region can be kappa. By way of example, and not limitation the human heavy chain constant region can be γ1 or γ4 and the human light chain constant region can be lambda, in an embodiment of the invention, the Fc region of the antibody is y4 with a Ser228Pro mutation (Schuurman, J et a!. , Mol. Immunol. 38: 1 -8, 2001 ).

In some embodiments of the invention, different constant domains may be appended to V _L and V _H regions derived from the CDRs provided herein. For example, if a particular intended use of an antibody or antigen-binding fragment of the present invention were to call for altered effector functions, a heavy chain constant domain other than human igG1 may be used, or hybrid lgG1/lgG4 may be utilized. Such anti-ILT4 antibodies and antigen- binding fragments thereof (e.g. , 1 E1 , 2A6, 3G7 or 2C1 ) and hybrids thereof comprising !gG1 and !gG4 constant domains are part of the present invention.

In one embodiment, the lgG4 constant domain differs from the native human lgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to position 228 in the EU system and position 241 in the KABAT system, where the native Ser108 is replaced with Pro, in order to prevent a potential inter-chain disulfide bond between Cys106 and Cys109 (corresponding to positions Cys 226 and Cys 229 in the EU system and positions Cys 239 and Cys 242 in the KABAT system) that could interfere with proper intra-chain disulfide bond formation. See Angal ei al. (1993) Mol. Imunol. 30:105. In other instances, a modified lgG1 constant domain which has been modified to increase half- life or reduce effector function can be used. Such anti-ILT4 antibodies and antigen-binding fragments thereof (e.g. , 1 E1 , 2A6, 3G7 or 2C1 ), comprising a modified lgG4 constant domain are part of the present invention.

The present invention includes recombinant methods for making an anti-ILT4 antibody or antigen-binding fragment thereof of the present invention (e.g. , 1 E1 , 2A6, 3G7 or 2C1 , or an immunoglobulin chain thereof, comprising (i) introducing a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (e.g. , comprising an amino acid sequence that includes any one or more of the sequences set forth in SEQ ID NOs: 1-15, 44 and/or 45), for example, wherein the polynucleotide is in a vector and/or is operably linked to a promoter (e.g., a viral promoter, a CMV promoter or SV40 promoter); (ii) culturing the host cell (e.g. , a mammalian host cell, a fungal host cell, a bacterial host cell, a Chinese hamster ovary (CHO) cell, an NSO cell, an SP2 cell, a HeLa cell, a baby hamster kidney (BHK) cell, a monkey kidney ceil (COS), a human hepatocellular carcinoma ceil (e.g., Hep G2), a A549 ceil, a 3T3 cell, a HEK-293 cell, a Pichia cell or a Pichia pastoris ceil) under condition favorable to expression of the polynucleotide(s) and, (iii) optionally, isolating the antibody or fragment or chain from the host cell and/or medium in which the host ceil is grown. When making an antibody or antigen-binding fragment comprising more than one immunoglobulin chain, e.g. , an antibody that comprises two heavy immunoglobulin chains and two light immunoglobulin chains, co-expression of the chains in a single host cell leads to association of the chains, e.g. , in the ceil or on the cell surface or outside the ceil if such chains are secreted, so as to form the antibody or antigen-binding fragment molecule. The methods of the present invention include those wherein only a heavy immunoglobuiin chain or only a light immunoglobuiin chain (e.g. , any of those discussed herein including mature fragments and/or variable domains thereof) is expressed. Such chains are useful, for example, as intermediates in the expression of an antibody or antigen-binding fragment that includes such a chain. Antibody Engineering of the Fc region

The anti-I LT4 antibodies and antigen-binding fragments of the present invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) can also be engineered to include modifications within the Fc region, iypically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g. , antigen- dependent cellular cytotoxicity). Furthermore, the antibodies and fragments disclosed herein can be chemically modified (e.g. , one or more chemical moieties can be attached to the antibody) or be modified to alter its giycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

The antibodies and antigen-binding fragments disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g. , U.S. Pat. No. 5,624,821 ; WO2003/086310; WO2005/120571 ; WO2006/0057702. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and/or insertions), giycosylation or deglycosyiation, and adding multiple Fc domains. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. 1 16:731 at 734-35.

In one embodiment of the invention, the anti-I LT4 antibody or antigen-binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) is an lgG4 isotype antibody or fragment comprising a serine to proline mutation at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy chain constant region. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et a!. supra; position 241 is based on the Kabat numbering system).

In one embodiment of the invention, the hinge region of CH 1 of an anti-ILT4 antibody or antigen-binding fragment thereof of the present invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) is modified such that the number of cysteine residues in the hinge region is increased or decreased (e.g. , by +1 , 2 or 3). This approach is described further in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an anti-ILT4 antibody or antigen- binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745.

In another embodiment, the anti-ILT4 antibody or antigen-binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375.

Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and

6, 121 ,022.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the anti- ILT4 antibody or antigen-binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1). For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the anti- ILT4 antibody or antigen-binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) has altered C1 q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6, 194,551.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351.

In yet another example, the Fc region is modified to increase or decrease the ability of the anti-ILT4 antibody or antigen-binding fragment (e.g., 1 E1 , 2A6, 3G7 and/or 2C1) to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase or decrease the affinity of the antibody or fragment for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301 , 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331 , 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072. Moreover, the binding sites on human igG1 for FcyR1 , FcyRII, FcyRill and FcRn have been mapped and variants with improved binding have been described (see Shields et al. (2001 ) J. Biol. Chem. 276:6591 -6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcyRII I. Additionally, the following combination mutants were shown to improve FcyRill binding: T256A S298A, S298A/E333A,

S298A K224A and S298A/E333A/K334A.

In one embodiment, the Fc region is modified to decrease the ability of the anti-ILT4 antibody or antigen-binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase antiinflammatory properties by modifying residues 243, 264, 267 and 328.

Post-Translationa !Vlodificatsons

In still another embodiment, the antibody comprises a particular glycosylation pattern. For example, an aglycosylated anti-ILT4 antibody or antigen-binding fragment thereof (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ), which lacks glycosylation, is part of the present invention. The glycosylation pattern of an antibody or fragment may be altered to, for example, increase the affinity or avidity of the antibody for an antigen. Such modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody or fragment sequence. For example, one or more amino acid substitutions can be made that result removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody or fragment for antigen. See, e.g., U.S. Patent Nos.

5,714,350 and 6,350,861.

An anti-ILT4 antibody or antigen-binding fragment (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) of the invention may also be made in which the glycosylation pattern includes

hypofucosyiated or afucosylated giycans, such as hypofucosyiated antibodies and antigen- binding fragments or afucosylated antibodies and fragments that have reduced amounts of fucosyl residues on the glycan. The antibody or antigen-binding fragment may also include glycans having an increased amount of bisecting GicNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such modifications can be accomplished by, for example, expressing the antibody or fragment in a host cell in which the glycosylation pathway was been genetically engineered to produce glycoproteins with particular glycosylation patterns. These cells have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines s7Q4, Ms7Q5, and s709 lack the fucosyltransferase gene, FUT8 (a (1 ,6)-fucosyltransferase), such that antibodies expressed in the IV1s704, s705, and s709 ceil lines lack fucose on their carbohydrates. The present invention includes anti- 1LT4 antibodies and antigen-binding fragments lacking fucose or produced by a host ceil that lacks FUT8. The s704, s705, and s709 FUT8 ^{_ "} cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 200401 10704 and Yamane-Ohnuki et a!. (2004)

Bioiechno! Bioeng 87:614-22). As another example, EP 1 176195 describes a ceil line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylaiion by reducing or eliminating the a-1 ,6 bond-related enzyme. EP 1 ,176, 195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylaiion of antibodies expressed in thai host cell (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO

06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant ceils, such as Lemna (US Patent 7,632,983). Methods for production of antibodies in a plant system are disclosed in the U.S. Patents 6,998,267 and 7,388,081 . PCT

Publication WO 99/54342 describes ceil lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., P(1 ,4)-N-aceiylglucosaminyliransferase ill (GnTili)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GicNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech, 17: 176-180). Anti-ILT4 antibodies an antigen-binding fragments thereof of the present invention which are produced by such host cells are part of the present invention. Alternatively, the fucose residues of the anti-ILT4 antibody or antigen-binding fragment (e.g., 1 E1 , 2A6, 3G7 and/or 2C1) can be cleaved off using a fucosidase enzyme; e.g. , the fucosidase a-L-fucosidase removes fucosyi residues from antibodies (Tarentino et a/. (1975) Biochem. 14:5516-23). The present invention includes antibodies and fragments which have been treated with a fucosidase enzyme.

Anfi-ILT4 antibody or antigen-binding fragments (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) disclosed herein further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosyiation patterns (See for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton et a/., (2003) Science 301 : 1244-1246; Hamilton et al. , (2006) Science 313: 1441-1443). A particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosyiation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Patent No. 7,029,872 and U.S. Patent No.

7,449,308). These genetically modified host ceils have been used to produce antibodies that have predominantly particular /V-g!ycan structures (See for example, Li et a/., (2006) Nat. Biotechnoi. 24: 210-215). Antibody Conjugates

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) may also be conjugated to a peptide or chemical moiety. The chemical moiety may be, inter alia, a polymer, a radionuclide or a therapeutic or cytotoxic agent. In particular embodiments of the invention, the chemical moiety is a polymer which increases the half-life of the antibody molecule in the body of a subject. Polymers include, but are not limited to, hydrophilic polymers which include but are not limited to polyethylene glycol (PEG) (e.g. , PEG with a molecular weight of 2kDa, 5 kDa, 10 kDa, 12kDa, 20 kDa, 30kDa or 40kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain antibodies. Wen, et a/. , (2001 ) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) may also be conjugated with labels, such as radiolabeis. Labels include but are not limited to ⁹⁹ Tc, ⁹⁰ Y, ^{1 11} ln, ³² P, ¹⁴ C, ¹²⁵ l, ³ H, ¹³¹ l, ¹¹ C, ^1s O, ¹³ N, ¹⁸ F, ³⁵ S, ^s1 Cr, ⁵⁷ To, ²²⁶ Ra, ⁶⁰ Co, ⁵⁹ Fe, ⁷ Se, ⁵² Eu, ⁶⁷ CU, ^{2 7} Ci, ²¹¹ At, ²¹² Pb, ⁷ Sc, ¹⁰⁹ Pd, ²³ Th, and ⁴⁰ K, ¹⁵⁷ Gd, ⁵⁵ Mn, ^S2 Tr, and ^S6 Fe. The anti-ILT4 antibodies and antibody fragments disclosed herein (e.g., 1 E1 , 2A6, 3G7 and/or 2C1) may also be PEGy!ated (e.g., with 1 PEG or a 3 kDa, 12 kDa or 40 kDa PEG polymer molecule), for example to increase its biological (e.g., serum) half-life. To PEGylate an antibody or antigen-binding fragment thereof, the antibody or fragment typically is reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antigen-binding fragment. In particular embodiments, the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1 -C10) aikoxy- or aryloxy- polyethyiene glycol or polyethylene g!yco!-ma!eimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401 384.

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) may also be conjugated with labels such as fluorescent or chemiliuminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin,

phycocyanin, ailophycocyanin, o-phthaladehyde, fiuorescamine, ¹⁵² Eu, dansyl,

umbeiliferone, iuciferin, luminal label, isoiuminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3- dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.

The anti-ILT4 antibodies and antigen-binding fragments disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, A!eurites fordii proteins and compounds (e.g. , fatty acids), dianthin proteins, Phytolacca amencana proteins PAPi, ΡΑΡΠ, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogeliin, restrictocin, phenomycin, and enomycin.

Any method known in the art for conjugating the antibodies and antigen-binding to the various moieties may be employed, including those methods described by Hunter, et al. , (1962) Nature 144:945; David, et al. , (1974) Biochemistry 13: 1014; Pain, et al, , (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407.

Methods for conjugating antibodies are conventional and very well known in the art. Pharmaceutical Compositions and Administration The pharmaceutical compositions comprising the anti-ILT4 antibodies (e.g. , fully human antibodies such as antagonist fully human antibodies (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) and antigen-binding fragments thereof can be prepared for storage by mixing the antibodies or antigen-binding fragments thereof having the desired degree of purity with optionally physiologically acceptable carriers, excipients, or stabilizers (see, e.g. ,

Remington, Remington's Pharmaceutical Sciences (18 ^th ed. 1980)) in the form of aqueous solutions or lyophilized or other dried forms.

In one embodiment, the pharmaceutical composition comprises an antibody that consists of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence set forth in SEQ ID NO:7 and each heavy chain consists of the amino acid sequence set forth in SEQ ID NO:2.

In another embodiment, the pharmaceutical composition comprises: (i) an antibody that consists of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence set forth in SEQ ID NQ:7 and each heavy chain consists of the amino acid sequence set forth in SEQ ID NO:2, and (ii) pembrolizumab.

Formulations of iherapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g. , Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY;

Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:

Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

Toxicity and therapeutic efficacy of the anti-ILT4 antibody or antigen-binding fragment (e.g., 1 E1 , 2A6, 3G7 and/or 2C1) compositions, administered alone or in combination with another therapeutic agent, can be determined by standard pharmaceutical procedures in ceil cultures or experimental animals, e.g., for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD _S0 / ED _S0 ), in particular aspects, antibodies exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED _S0 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that is administered to a subject in association with an anti-ILT4 antibody (e.g. , fully human antibody such as antagonist fully human antibodies) or antigen-binding fragment thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) is administered to the subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1 , 2002)).

The mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal,

intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial.

The present invention provided methods for administering an anfi-ILT4 antibody or antigen-binding fragment thereof (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) comprising introducing the antibody or fragment into the body of a subject. For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the antibody or fragment into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.

The present invention provides a vessel (e.g. , a plastic or glass vial, e.g. , with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the anti-ILT4 antibodies or antigen-binding fragments (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) set forth herein or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.

The present invention also provides an injection device comprising any of the anti- ILT4 antibodies or antigen-binding fragments (e.g., 1 E1 , 2A6, 3G7 and/or 2C1 ) set forth herein or a pharmaceutical composition thereof. An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g. , intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g. , pre- filied with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g. , comprising the antibody or fragment or a pharmaceutical composition thereof), a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the invention, an injection device that comprises an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device includes the antibody or fragment or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g. , saline; or lactated ringer solution comprising NaCI, sodium lactate, KCI, CaCI ₂ and optionally including glucose) introduced into the body of the subject through the cannula or trocar/needle.

The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399, 163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824 or 4,596,556. Such needleless devices comprising the pharmaceutical composition are also part of the present invention. The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules for

administering the pharmaceutical compositions include those disclosed in: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439, 196, which discloses an osmotic drug delivery system having multi- chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art and those comprising the pharmaceutical compositions of the present invention are within the scope of the present invention.

Antibodies (e.g. , fully human antibodies such as antagonist fully human antibodies) or antigen-binding fragments thereof disclosed herein (e.g., 1 E1 , 2A6, 3G7 and/or 2C1) may be provided by continuous infusion, or by doses administered, e.g. , daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g. , intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. An effective dose of an anti- ILT4 antibody or antigen-binding fragment thereof of the present invention, is from about 0.05 mg/kg (body weight) to about 30 mg/kg (body weight), e.g. , for treatment or prevention of cancer or infectious diseases.

Determination of the appropriate dose is made by the clinician, e.g. , using parameters or factors known or suspected in the art to affect treatment. Generally, in determining the dose, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g. , the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent, in the case of human subjects, for example, chimeric and fully human antibodies are may be desirable. Guidance in selecting appropriate doses of anti-ILT4 antibodies or fragments is available (see, e.g. , Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert ei ai. (2003) New Engl. J. Med. 348:801-608; Milgrom et ai. (1999) New Engl. J. Med. 341 :1966-1973;

Slamon et ai. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et ai. (2000) New Engl. J. Med. 342:613-619; Ghosh et ai (2003) New Engl. J. Med. 348:24-32; Lipsky et ai. (2000) New Engl. J. Med. 343: 1594-1602).

Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. While an embodiment of the present invention (e.g. , a treatment method or article of manufacture) may not be effective in alleviating the target disease symptom(s) in every subject, it should alleviate the target disease symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the chi ² -test, the U-test according to Mann and Whitney, the Kruskai-Wallis test (H-test), Jonckheere-Terpstra-test and the Wiicoxon-test. Therapeutic Uses of Anti-!!LT4 antibodies

The present invention also provides methods for treating or preventing cancer in subjects, such as human subjects, in need of such treatment by administering an effective amount of the anti-ILT4 antibodies or antigen-binding fragments thereof of the present invention which are disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) which may be effective for such treatment or prevention.

In certain embodiments, the cancer is solid tumor. In other embodiments, the cancer is hematologic cancer. In certain embodiments, the cancer is metastatic. In some embodiments, the cancer is relapsed. In other embodiments, the cancer is refractory, in yet other embodiments, the cancer is relapsed and refractory.

In some embodiments, the cancer is anaplastic astrocytoma, astrocytoma, bladder cancer, bone cancer, brain cancer, breast cancer (e.g. , characterized by a mutation in BRCA1 and/or BRCA2), carcinoid cancer, cervical cancer, chondrosarcoma, choroid plexus papilloma, colorectal cancer, endometrial cancer, ependymoma, esophagus cancer, Ewing's sarcoma, gall bladder cancer, gastric cancer, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, idiopathic myelfsbrosis, kidney cancer, leukemia, liver cancer, lung cancer (e.g., non-small ceil lung cancer), lymphoma, meduilobiastoma, melanoma, meningioma, erkel cell cancer, mesothelioma, multiple myeloma,

neuroblastoma, oligodendroglioma, osteosarcoma, ovarian cancer, pancreatic cancer, polycythemia vera, primitive neuroectodermal tumor, prostate cancer, renal ceil cancer, renal transitional cell cancer, retinoblastoma, rhabdoid tumor of the kidney,

rhabdomyosarcoma, salivary gland cancer, sarcoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma (e.g., cutaneous squamous ceil carcinoma), synovial sarcoma, thrombocythemia, thyroid cancer, uterine cancer, vestibular schwannoma, or Wilm's tumor, in an embodiment of the invention, the cancer is metastatic cancer, e.g. , of the varieties described above.

In an embodiment of the invention, the cancer is a mye!oid-rich tumor (e.g., mesothelioma, kidney cancer, lymphoma, sarcoma, melanoma, head & neck cancer, breast cancer, bladder cancer, gastric cancer, ovarian cancer or thyroid cancer; see e.g., Burt ei a/. Cancer. 1 17 (22):5234-44 (201 1 ); Dannenmann et a!. Oncoimmunology 2(3):e23562 (2013); Steidl et al. N, Engl. J. Med. 362:875-885 (2010); Fujiwara ei al., Am, J. Pathol.. 179(3): 1 157-70 (201 1 ); Bronkhorst ei a/. Invest. Ophthalmol. Vis. Sci. 52(2):643-50 (201 1 ); Balermpas et al. Br. J. Cancer 1 1 1 (8): 1509-18 (2014); and Zhang et al. PLoS One

7(12):e50946 (2012)). Since ILT4 is expressed primarily by myeloid cells and granulocytes, and myeloid cell infiltration into tumors is generally associated with poor prognosis due to the immunosuppressive effects of these cells that can antagonize anti-tumor responses by T-celis, treatment with an anti-iLT4 antibody or antigen-binding fragment of the present invention will benefit subjects with a high myeloid or immunosuppressive myeloid cell infiltration.

Thus, provided herein are methods of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising (a) the CDR-L1 , CDR-L2, and CDR-L3 of a light chain variable (V _L ) domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 3-7, 1 1 , 13, 15, or 45; and/or (b) the CDR-H1 , CDR-H2, and CDR-H3 of a heavy chain variable (V _H ) domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8-10, 12, 14, 44, or 79-86.

In an embodiment of the invention, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: (1) a V _H domain comprising: complementarity determining region-H1 (CDR-H1 ): GYYWS (SEQ ID NO: 16),

complementarity determining region-H2 (CDR-H2): EINHXGSTNYNPSLKS wherein X is S or A (SEQ ID NO: 17); and complementarity determining region-H3 (CDR-H3):

LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: complementarity determining region-L1 (CDR-L1): TGSSSN!GAGYDVH (SEQ ID NO: 19), complementarity determining region-L2 (CDR-L2): GX X ₂ NRPS; wherein X-, is S or A and X ₂ is N, Q, E or D (SEQ ID NO: 20); and complementarity determining region-L3 (CDR-L3): QSFDNSLSAYV (SEQ ID NO: 21); (2) a V _H domain comprising: CDR-H1 : SYAIS (SEQ ID NO: 22); CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23); and CDR-H3: YFX X ₂ SGWYKGGAFDI; wherein Xi is D or S and X ₂ is S or A (SEQ ID NO: 24); and/or, a V _L domain comprising: CDR-L1 : TLRSGINVDTYRIH (SEQ ID NO: 25); CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26); and CDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a V _H domain comprising: CDR-H1 : SYA H (SEQ ID NO: 28); CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO: 29); and CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30); and/or, a V _L domain comprising: CDR-L1 :

RASQGISSWLA (SEQ ID NO: 31); CDR-L2: AASSLQS (SEQ ID NO: 32); and CDR-L3: QQYNSYPPT (SEQ ID NO: 33); and/or (4) a V _H domain comprising: CDR-H1 : ELS H (SEQ ID NO: 34); CDR-H2: GFDPEDGETIYAQKFQG (SEQ ID NO: 35); and CDR-H3: AGPLYTIFGWIIPDNWFDP (SEQ ID NO: 36); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 37); CDR-L2: GNSNRPS (SEQ ID NO: 38); and CDR- L3: QSYDSSLSGSGVV (SEQ ID NO: 39).

In one embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: GYYWS (SEQ ID NO: 16), CDR- H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51 ), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In still another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GGSNRPSiSEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPSiSEQ ID NO: 51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of treating a cancer in a human subject in need ihereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR- H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In yet another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

in still another embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

in still other embodiments, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6 ,7, 1 1 , 13, 15, or 45; and/or the heavy chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8, 9, 10, 12, 14, 44, 79, 80, 81 , 82, 83, 84, 85, or 86.

In yet still other embodiments, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:70, 71 , 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chain variable domain comprise the amino acid sequence set forth in SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In more embodiments, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: any of the following sets of heavy chain immunoglobulins and light chain immunoglobulins: (1) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 ; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (2) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:4; (3) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2;a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:5; (4) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:6; (5) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7; (6) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (7) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:8; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:1 1 ; (8) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:9; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (9) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 10; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 11 ; (10) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:12; a light chain

immunoglobulin comprising the amino acid sequence set forth in SEQ ID NG: 13; (1 1) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NG: 14; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15; (12) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:79; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (13) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:SQ; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:4; (14) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:8Q;a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:5; (15) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain

immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:6; (16) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7; (17) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (18) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:82; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (19) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:83; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (20) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:84; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (21) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:85; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 13; or (22) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:86; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15.

In even more embodiments, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: any of the following sets of heavy chain variable domain and light chain variable domain: (1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:63; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:70; (2) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:71 ; (3) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID

NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:72; (4) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:73; (5) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:58; (6) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NG:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:70; (7) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:64; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (8) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:65; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (9) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:66; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (10) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:67; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:75; or (1 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:68; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:76. In one preferred embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a V _H domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a V _L domain comprising the amino acid sequence set forth in SEQ ID NO:58.

In another preferred embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a heavy chain

immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7.

In yet another preferred embodiment, the method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a heavy chain

immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:7.

Further provided herein are methods of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising (a) the CDR-L1 , CDR-L2, and CDR-L3 of a light chain variable (V _L ) domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 3-7, 1 1 , 13, 15, or 45; and/or (b) the CDR-H1 , CDR-H2, and CDR-H3 of a heavy chain variable (V _H ) domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8-10, 12, 14, 44, or 79-86.

In an embodiment of the invention, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: (1) a V _H domain comprising: complementarity determining region-H1 (CDR-H1): GYYWS (SEQ ID NO: 16), complementarity determining region-H2 (CDR-H2): EINHXGSTNYNPSLKS wherein X is S or A (SEQ ID NO: 17); and complementarity determining region-H3 (CDR-H3): LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: complementarity determining region-Li (CDR-L1 ):

TGSSSNIGAGYDVH (SEQ ID NO: 19), complementarity determining region-L2 (CDR-L2): GX ₁ X ₂ NRPS; wherein is S or A and X ₂ is N, Q, E or D (SEQ ID NO: 20); and

complementarity determining region-L3 (CDR-L3): QSFDNSLSAYV (SEQ ID NO: 21); (2) a \/ _H domain comprising: CDR-H1 : SYAIS (SEQ ID NO: 22); CDR-H2:

GIIPIFGTANYAQKFQG (SEQ ID NO: 23); and CDR-H3: YFX ₁ X ₂ SGWYKGGAFDI; wherein X is D or S and X ₂ is S or A (SEQ ID NO: 24); and/or, a V _L domain comprising: CDR-L1 : TLRSGINVDTYRIH (SEQ ID NO: 25); CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26); and CDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a V _H domain comprising: CDR-H1 : SYAMH (SEQ ID NO: 28); CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO: 29); and CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30); and/or, a V _L domain comprising: CDR-L1 :

RASQG!SSWLA (SEQ ID NO: 31); CDR-L2: AASSLQS (SEQ ID NO: 32); and CDR-L3: QQYNSYPPT (SEQ ID NO: 33); and/or (4) a V _H domain comprising: CDR-H1 : ELSMH (SEQ ID NO: 34); CDR-H2: GFDPEDGETIYAQKFQG (SEQ ID NO: 35); and CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQ ID NO: 36); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 37); CDR-L2: GNSNRPS (SEQ ID NO: 38); and CDR- L3: QSYDSSLSGSGVV (SEQ ID NO: 39).

In one embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR- L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS(SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21),

In yet another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA- A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR- L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA- A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subjeci an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21),

In one embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR- L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21),

In another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In yet another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA- A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-

A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In one embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR- L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS(SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPSiSEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In yet another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-

A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS(SEQ ID NO: 51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

in still another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA- A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS(SEQ ID NO: 52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In one embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR- L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS(SEQ ID NO: 53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In another embodiment, the method of blocking binding of 1LT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS(SEQ ID NO: 54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In yet another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA- A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS(SEQ ID NO: 55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

in still another embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-

A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: CDR-H1 : GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and/or, a V _L domain comprising: CDR-L1 : TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS(SEQ ID NO: 56), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In still other embodiments, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6 ,7, 1 1 , 13, 15, or 45; and/or the heavy chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 1 , 2, 8, 9, 10, 12, 14, 44, 79, 80, 81 , 82, 83, 84, 85, or 86.

In yet still other embodiments, the method of blocking binding of ILT4 to HLA-G,

HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light and heavy chain immunoglobulin, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:70, 71 , 72, 73, 58, 74, 75, 76, or 77, and/or the heavy chain variable domain comprise the amino acid sequence set forth in SEQ ID NO:63, 57, 64, 65, 66, 67, 68, or 69.

In more embodiments, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: any of the following sets of heavy chain immunoglobulins and light chain immunoglobulins: (1) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:1 ; a light chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NO:3; (2) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NO:4; (3) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2;a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:5; (4) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:6; (5) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7; (6) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:3; (7) a heavy chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NO:8; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID

NO: 1 1 ; (8) a heavy chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NG:9; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (9) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:10; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:1 1 ; (10) a heavy chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NO: 12; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 13; (1 1) a heavy chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NO: 14; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15; (12) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:79; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (13) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:4; (14) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80;a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:5; (15) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:6; (16) a heavy chain immunogiobuiin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7; (17) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:80; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:3; (18) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:82; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 11 ; (19) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:83; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:1 1 ; (20) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NQ:84; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 1 1 ; (21) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:85; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 13; or (22) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:86; a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15.

In even more embodiments, the method of blocking binding of ILT4 to HLA-G, HLA- A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: any of the following sets of heavy chain variable domain and light chain variable domain: (1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:63; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:70; (2) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:71 ; (3) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:72; (4) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:73; (5) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:58; (6) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:70; (7) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:64; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (8) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NG:65; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:74; (9) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID

NO:66; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:74; (10) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:67; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:75; or (1 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:68; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:76.

In one preferred embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a V _H domain comprising the amino acid sequence set forth in SEQ ID NO:57; and a V _L domain comprising the amino acid sequence set forth in SEQ ID NO:58.

In another preferred embodiment, the method of blocking binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:2; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7,

In yet another preferred embodiment, the method of blocking binding of 1LT4 to HLA- G, HLA-A, HLA-B and/or HLA-F in a human subject in need thereof, comprising

administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof comprising: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:8Q; and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:7.

The present invention also provides methods for treating or preventing an infectious disease in a subject by administering an effective amount of anti-ILT4 antibodies or antigen- binding fragments thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) to the subject which may be effective for such treatment or prevention. In an embodiment of the invention, the infectious disease is viral infection. In an embodiment of the invention, the infectious disease is bacterial infection. In an embodiment of the invention, the infectious disease is parasitic infection, in an embodiment of the invention, the infectious disease is fungal infection.

The present invention includes methods of treating any of the cancers or infectious diseases discussed herein by administering a therapeutically effective amount of an anti- 1LT4 antibody or antigen-binding fragment thereof (e.g. , 1 E1 , 2A8, 3G7 and/or 2C1) optionally in association with any of the further chemotherapeutic agents or therapeutic procedures discussed herein as well as compositions including such an antibody or fragment in association with such a further chemotherapeutic agent (e.g. , co-formulated antibody or fragment and further chemotherapeutic agent).

A "subject" is a mammal such as, for example, a human, dog, cat, horse, cow, mouse, rat, monkey (e.g. , cynomolgous monkey, e.g. , Macaca fascicu!aris or Macaca mu!atta) or rabbit.

in an embodiment of the invention, an anti-ILT4 antibody (e.g. , fully human antibody such as antagonist fully human antibodies) or antigen-binding fragment thereof of the present invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) is in association with a further chemotherapeutic agent such as an antibody or antigen-binding fragment thereof. In an embodiment of the invention, the further chemotherapeutic agent is an antiemetic, erythropoietin, G -CSF, a vaccine, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-PD~L2 antibody or antigen-binding fragment thereof, an anti-PD1 antibody or antigen-binding fragment thereof (e.g. , pembrolizumab or nivolumab), 5-fluorouracil (5- FU), a platinum compound, bevacizumab, daunorubicin, doxorubicin, temozolomide topotecan, irinotecan, paclitaxel, docetaxei, imatinib or rituximab.

The term "in association with" indicates that the components, an anti-ILT4 antibody (e.g. , fully human antibody such as antagonist fully human antibodies) or antigen-binding fragment thereof of the present invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) along with another agent such as pembrolizumab or nivolumab, can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g. , separately or sequentially) at intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route (e.g. , wherein an anti-ILT4 antibody (e.g. , fully human antibody such as antagonist fully human antibodies) or antigen-binding fragment thereof (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 is administered parenterally and paclitaxel is administered orally). Assays and Experimental and Diagnostic Uses

The present invention includes any method for forming a complex between an anti- ILT4 antibody or antigen-binding fragment thereof of the present invention and ILT4 or a fragment thereof comprising contacting the ILT4 polypeptide or fragment with the anti-ILT4 antibody or antigen-binding fragment under conditions suitable for binding and complex formation. The anti-I LT4 antibodies (e.g. , fully human antibodies such as antagonist fully human antibodies) and antigen-binding fragments thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ) may be used as affinity purification agents. In this process, the anti-I LT4 antibodies and antigen-binding fragments thereof are immobilized on a solid phase such a sephadex, glass or agarose resin or filter paper, using methods well known in the art. The immobilized antibody or fragment is contacted with a sample containing the I LT4 protein (or a fragment thereof) to be purified, and, thereafter, the support is washed with a suitable solvent that will remove the material in the sample except the ILT4 protein which is bound to the immobilized antibody or fragment. Finally, the support is washed with a solvent which elufes the bound I LT4 (e.g. , protein A). Such immobilized antibodies and fragments form part of the present invention.

The present invention includes cell-based ELISA methods using the anti-I LT4 antibodies and antigen-binding fragments thereof of the present invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ), In an embodiment of the invention, the method is for determining whether ceils contain I LT4 and the method includes the steps: (i) contacting said ceils immobilized to a solid surface (e.g. , a micropiate), which are to be tested for the presence of ILT4, with an anti-ILT4 antibody or antigen-binding fragment thereof of the present invention, (ii) optionally, washing the mixture to remove unbound anti-ILT4 antibody or fragment, (iii) contacting the anti-ILT4 antibody or fragment with a labeled secondary antibody or antigen- binding fragment thereof that binds to the anti-I LT4 antibody or fragment, (iv) optionally washing the complex to remove unbound antibodies or fragments and (v) detecting the presence of the label on the secondary antibody or fragment; wherein detection of the label indicates that cells containing I LT4 are immobilized to the solid surface.

The present invention includes ELISA assays (enzyme-linked immunosorbent assay) incorporating the use of an anti-ILT4 antibody (e.g. , fully human antibodies such as antagonist fully human antibodies) or antigen-binding fragment thereof disclosed herein (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 ). For example, such a method, for determining if a sample contains ILT4 or a fragment thereof, comprises the following steps:

(a) coating a substrate (e.g. , surface of a microtiter plate well, e.g. , a plastic plate) with anti- 1 LT4 antibody (e.g. , fully human antibodies such as antagonist fully human antibodies) or antigen-binding fragment thereof (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1 );

(b) applying a sample to be tested for the presence of I LT4 to the substrate;

(c) washing the substrate, so that unbound material in the sample is removed;

(d) applying detectably labeled antibodies (e.g. , enzyme-linked antibodies) which are also specific to the I LT4 antigen;

(e) washing the substrate, so that the unbound, labeled antibodies are removed; (f) detecting the label on the antibodies; if the labeled antibodies are enzyme linked, apply a chemical which is converted by the enzyme into a detectable, e.g., fluorescent, signal; and Detection of the label, e.g., associated with the substrate, indicates the presence of the ILT4 protein.

In an embodiment of the invention, the labeled antibody or antigen-binding fragment thereof is labeled with peroxidase which reacts with ABTS (e.g. , 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid)) or 3,3',S,5'-Tetramethylbenzidine to produce a color change which is detectable. Alternatively, the labeled antibody or fragment is labeled with a detectable radioisotope (e.g. , ³ H) which can be detected by scintillation counter in the presence of a scintiiiant.

An anti-ILT4 antibody (e.g., fully human antibodies such as antagonist fully human antibodies) or antigen-binding fragment thereof of the invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1) may be used in a Western blot or immune-protein blot procedure. Such a procedure forms part of the present invention and includes e.g.,:

(1) providing a membrane or other solid substrate comprising a sample to be tested for the presence of ILT4 or fragment thereof, e.g. , optionally including the step of transferring proteins from a sample to be tested for the presence of ILT4 (e.g., from a PAGE or SDS- PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate (e.g. , using methods known in the art (e.g. , semi-dry blotting or tank blotting)); and contacting the membrane or other solid substrate to be tested for the presence of bound ILT4 or a fragment thereof with an anti~ILT4 antibody or antigen-binding fragment thereof of the invention (e.g., 1 E1 , 2A6, 3G7 and/or 2C1 );

(2) washing the membrane one or more times to remove unbound anti-ILT4 antibody or fragment and other unbound substances; and

(3) detecting the bound anti-!LT4 antibody or fragment.

Such a membrane may take the form, for example, of a nitrocellulose or vinyl-based (e.g. , polyvinylidene fluoride (PVDF)) membrane to which the proteins to be tested for the presence of ILT4 in a non-denaturing PAGE (polyacryiamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacryiamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel). Before contacting the membrane with the anti-iLT4 antibody or fragment, the membrane is optionally blocked, e.g. , with non-fat dry milk or the like so as to bind non-specific protein binding sites on the membrane.

Detection of the bound anti-!LT4 antibody or fragment indicates that the ILT4 protein is present on the membrane or substrate and in the sample. Detection of the bound antibody or fragment may be by binding the antibody or fragment with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody label.

The anti-ILT4 antibodies (e.g. , fully human antibodies such as antagonist fully human antibodies) and antigen-binding fragments thereof disclosed herein (e.g., 1 E1 , 2A6, 3G7 and/or 2C1) may also be used for immunohistochemistry. Such a method forms part of the present invention and comprises, e.g.,

(1) contacting cells (e.g., myeloid lineage cells such as monocytes, macrophages or dendtritic cells) to be tested for the presence of ILT4 protein with an anti-ILT4 antibody or antigen-binding fragment thereof of the invention (e.g. , 1 E1 , 2A6, 3G7 and/or 2C1); and (2) detecting the antibody or fragment on or in the cells.

If the antibody or fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or fragment may be bound by a detectably labeled secondary antibody wherein the label is then detected. Examples

These examples are intended to exemplify the present invention and are not a limitation thereof. Compositions and methods set forth in the Examples form part of the present invention.

As used herein the term "p1 E1 (G1)" refers to a fully human anti-ILT4 1 E1 mAb which is derived from the germline V genes, IGHV4-34*0 and IGLV1 -40*01 , respectively; having the human lgG1 and human lambda constant domains.

> heavy chain

QVQLQQWGAGbLKPSETLSLTCAVYGGSFSGYYWS IRQPPGKGLEWIGEIiraSGSTNYlSnPSLKSRVTISVDTSKNQ FSLKLSSVTAADTAVYYCARLPTRWVTTRYFDL GRGTLV VSSASTKGPSVFPLAPSSKSTSGGTAALGCLV DYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT VD RVEP SCDKTHTCP PCPAPELLGGPSVFLFPP P DTMISRTPEVTCWVDVSHEDPEVKF1WYVDGVEVHNA TKPREEQYNSTYRVVS VLTVLHQDWLNG EYKCKVSN ALPAPIE TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV GFYPSDIAVE ESNGQPENNY TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH HYTQKSLSLSPGK

(SEQ ID NO:44)

>light chain

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSVSKSGASASLAI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQAN ATLVCLISDFYPGAVTVAW ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS

(SEQ ID NO:45)

As used herein "p1 E1 (G4)" refers to a fully human anti-lLT4 1 E1 (having the mutations Q1 E in heavy chain and Q1 E in light chain) mAb which is derived from the germline V genes, IGHV4-34*0 and IGLV1 -40*01, respectively; having the human lgG4 (S228P) and human lambda constant domains.

> heavy chain

EVQLQQWSAeLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYN PSLKSRVTISVDTSKNQ FSL LSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSSAST GPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT TYTCWDHKPSNTKVDKRVES YGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN YVDGVEVHNA TKPREEQFNSTYR SVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKXISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVE ESN GQPErajYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHFALHNHYTQKSLSL SLGK

(SEQ IDNO:1)

> human lgG4 (S228P) constant domain

AST GPSVFPLAPCSRSTSESTAALGCLV DYFPEPVTVS NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG XKTYTC VDHKPSNT VDKRVES YGPPCPPCPAPEFLGGPSVFLFPPKP DTLMISRTPEVTCVWDVSQEDPEVQ

F- VDGVEVHNA T PREEQF STYRVVSVLTVLHQDWLNGKEYKCKVSHKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK

(SEQ ID NO:89)

> light chain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSVSKSGASASLAI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATL VCLISDFYPGAVTVAW ADSSPVKAGVETTTPS QSNN YAASSYLSLTPEQW SHRSYSCQVTHEGSTVEKTVAPTECS

(SEQ ID NO:3)

> human lambda constant domain

GQP AAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA KADSSPVKAGVETTTPS QSNN YAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS

(SEQ ID NO:90)

As used herein ΊΕ1 (G4)" refers to a fully human anti-ILT41E1 (having the mutations Q1E and N53D in light chain and Q1E and S54A in heavy chain) mAb which is derived from the germline V genes, IGHV4-34*0 and IGLV -40*01, respectively; having human lgG4 (S228P) and human lambda constant domains.

> heavy chain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE IGEIN-iAGSTiryTTPSLKSRVTISVDTSKNQ FSL LSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSSAST GPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH PSNT VDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKP DTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKT PREEQFNSTYRWSVLT VLHQDWLNG EY CKVSNKGLPSSIEKTIS A GQPREPQVYTLPPSQEEMT NQVSLTCLVKGFYPSDIAVEWESN GQPEITOYKTTPPVLDSDGSFFLYSRLTVDKSR QEGITVFSCSVMHEALH BYTQKSLSLSLGK

(SEQ ID NO: 2) > light chain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGV PDRFSVSKSGASASLAI TGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQP AAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS

(SEQ ID NO: 7)

Example 1 : Anti-ILT4 Antibody Identification and Characterization

Antibody Generation and Identification

The anti-ILT4 parental human mAb 1 E1 was identified using the RETROCYTE

DISPLAY platform (Breous-Nystroma et ai, Methods 65(1): 57-67 (2014)). The

RETROCYTE DISPLAY platform utilizes retroviral gene transfer of human antibody genes into mammalian pre-B cells to generate stable high diversity antibody display libraries.

Human cord blood containing naive B cells were used as the source material for antibody heavy and light chains. The cellular antibody libraries typically expressed >10 ⁸ different full length (hlgG1-4 isotypes) monoclonal human antibodies on the cell surface of the pre-B cell.

Antibody Pre-panels were compiled from antibody hits identified in 3 separate RETROCYTE DISPLAY screening campaigns. Candidates were enriched based on FACS detection of recombinant human ILT4 antigen-binding to B-cell clones. Putative B-cell clones were sorted out and their antibody sequences determined. These sequences were then used to produce antibody candidates and ILT4 binding was confirmed using ILT4 CHO transfectant cell binding assays, with parental CHO cells serving as a negative control. Candidates were also counter-screened for ILT4 specificity against closely related ILT family members (human ILT2, LILRA1 , and LILRA2 CHO transfectant FACS) and against ILT family members by flow cytometric evaluation (LILRA1 , LILRA2, LILRA4, LILRA5, LILRA6, ILT2, ILT5, and ILT3).

Monoclonal antibodies were further tested for their ability to bind cynomolgous monkey ILT4 (predicted sequence from NCBI) CHO transfectants via FACS. Candidate mAbs were also screened for their ability to block recombinant HLA-G Fc ligand binding to recombinant ILT4 protein in Luminex-based assays or to ILT4-expressing CHO cells in FACS-based assays. Candidate antibody functional activity was assessed by three methods: 1) Rescue of spontaneous IL2 suppression in ILT4-transfected mouse 3A9 T- cells; 2) Rescue of HLA-G-dependent suppression of CD200RLa-stimulated mast cell degranulation in mouse WTMC ILT4 transfectants; and 3) Cytokine modulation in whole PBMC mixed lymphocyte reactions. Based on the above screening criteria, eight anti-ILT4 antibodies (1 E1 , 1 G2, 2A8, 2D5, 3E6, 3G7, 2C1 and 5A6) were selected based on their functional and biophysicai properties. Antibodies were further analyzed and re-evaluated in a set of bio-functional, biophysical, and physicochemicai assays. Functional assays assessed antibody-mediated dose-dependent rescue of IL-2 suppression of 3A9 cells and mast cell degranulation described above. Luminex and cell-based ligand blocking and ligand competition assays were performed and binding properties and affinities to the ILT4 target antigen, off-target antigens, and PB C subsets were determined using Biacore and flow cytometry based assays, respectively. Biophysical assays assessed antibody stability (temperature, pH) and degradation and aggregation behavior. Sequence liabilities and potential posf-transiational modification motifs were addressed in order to exclude potential antibody production risks. Finally, candidates were tested in an in vivo, tumor regression study using SKMELS melanoma-challenged humanized mice.

Studies were conducted on human 1 E1 sequence (human lgG4 backbone with S228P mutation) transiently expressed in CHO cells. 1 E1 presented the following physicochemicai characteristics: calculated and experimentally determined isoelectric point (PI) were respectively -7.29 and ~ 7.2, aggregation level (HMW species) was < 5% , Tm onset > 60°C, Tm1 ~ 65.2°C , Tm2 ~78.8°C, and was stable for at least 5 freeze/thaw cycles. The sequence of 1 E1 originally had a N-giycosyiation site in V _H -CDR2 which was successfully corrected with the S54A mutation without negative impact on binding by BIACORE and in functional assay. Stress studies showed deamidation of the N53 residue in V _L -CDR3 (> 13% under high PH conditions). N53 was successfully corrected (N53D) without a negative impact on binding and in a functional assay (rescue of IL-2 release from ILT4 3A9 T ceil fransfectanfs with 1 E1). Both N~termina! Q residues in the 1 E1 V _H and V _L were mutated to E (V _H -Q1 E and V _L -Q1 E) to reduce risk of heterogeneity due to deamidation. Stress studies conducted on the mutated 1 E1 sequence (1 E1 V _H Q1 E, S54A / V _L Q1 E, N53D) igG4 S228P/Lambda) showed - 17% oxidation of the W102 residue in V _H -CDR3 under forced oxidation with AAPH (2,2'-Azobis(2~amidinopropane) dihydrochloride) at 6 hours with minimal impact on binding and function. AAPH is used to force oxidation of Trp and Met residues. Under the same condition, -8% oxidation of W7 in V _H framework was detected with no impact on binding and function. Around 8% afucosylated antibody was detected by peptide mapping. This finding might be associated with the molecule being expressed in transiently transfected CHO cells. Epitope Mapping

The epitope on ILT4 of the antibody done, p1 E1 (G1), was identified by Hydrogen- Deuterium Exchange Mass Spectrometry. The antibody p1 E1 (G1) was premixed with the recombinant histidine tagged, extrace!iuiar domain of ILT4, then the complex was incubated in deuterium buffer. The amount of deuterium incorporation was measured by mass spectrometry. ILT4 residues LYREKKSASW (39 - 48 of ILT4 without signal sequence) (SEQ ID NO:59) and TRXRPEL (50 - 56 of ILT4 without signal sequence) (SEQ ID NO:60) and to less extent NGQF (59-62 of ILT4 without signal sequence) (SEQ ID NO:61) and HTGRYGCQ (71-78 of ILT4 without signal sequence) (SEQ ID NQ:62) were identified as showing the largest difference in deuterium labeling compared to an antigen-only sample, indicating they are likely the residues interacting with p1 E1 (G1) (Figure 1). These peptides are on domain

1 of ILT4. Other peptides on domain 1 and 2 that showed less deuterium labeling differences are likely protected due to conformational stability upon antibody binding. No significant differences in labeling were seen in domains 3 and 4.

When mapped onto the crystal structure of human ILT4, the residues protected by p1 E1 (G4) are forming non-linear conformational epitope comprising three beta-strands and a loop (Figure 2A).

ILT4 uses two binding interfaces to engage its ligand HLA-G (Shiroishi et al, 2006): site 1 for beta-2-microglobuiin binding, located in domain 2 of ILT4, and site 2 for HLA-G heavy chain binding, located in domain 1 of ILT4 (Figure 2B). Site 1 includes ILT4 residues Trp-67, Asp-177, Asn-179, and Val-183 (numbering according to Shiroishi ef al, 2006). Site

2 includes ILT4 residues Arg-36, Tyr~38, Lys~42, lle-47, and Thr~48 (numbering according to Shiroishi et al, 2006). The HDX-MS data of this application show that Tyr-38, Lys-42, and Thr~48 (numbering according to Shiroishi et al, 2006) are part of the p1 E1 (G1) epitope on ILT4 domain 1 as residues Tyr-40, Lys-44, and Thr-50 of human ILT4 (Figure 2A). This indicates that the human ILT4 epitope bound by p1 E1 (G1) overlaps with the site 2 epitope bound by the HLA-G ligand.

Example 2: Affinity, Binding and Blocking Properties of AniHLT4 mAb 1 E1 Binding Affinities of 1 E1 (G4) and HLA-G1 to Human 1LT4 Determined by Surface Piasmon Resonance (SPR)

Binding of 1 E1 (G4) and HLA-G 1 Fc (recombinant extracellular domain of HLA-G (isoform 1) fused to the human lgG1 Fc domain to make soluble HLA-G1 protein) to ILT4- His (recombinant extracellular domain of human ILT4 fused to a poiy-Histidine tag to make soluble ILT4 protein) was assessed via Biacore. Either 1 E1 (G4) or HLA-G 1 Fc was captured on a Biacore chip via Fc capture. IVlonomeric ILT4~His was then tested for binding and data indicated !LT4-His bound 1 E1 (G4) with a greater than 600-fold higher affinity than HLA-G1 Fc (Table 3). Table 3. !LT4-His binds 1 E1 (G4) with a greater than 6QG~fo!d tighter affinity than HLA~ G1 Fc. 1 :1 binding kinetics and steady-state analyses indicate 630- and 670-foid differences.

A surface plasmon resonance (SPR) assay on a Biacore T200 (GE HEALTHCARE) instrument was used to determine the monovalent affinities of anti-human ILT4 lgG4 mAb (1 E1 (G4)) and HLA-G1 Fc fusion (HLA-G1-Fc) against polyhistidsne-tagged human ILT4 (ILT4-His). Either mAb or Fc fusion protein was captured on a CM5 sensor chip prepared using a Human Fc Capture kit (GE HEATHCARE) and a titrating concentration series of ILT4~His was injected over this surface. Biacore T200 Evaluation Software was used to fit each titration series to a 1 : 1 binding model. The association (ka, M ^"1 s ^"1 ) and dissociation (kd, s ^"1 ) rate constants were determined for each set of titrations and used to calculate the dissociation constant, K _D ( ) = koff/kon, for each titration. As shown in Table 1 , the monovalent affinities (K _D ) of 1 E1 (G4) mAb and HLA-G1-Fc against human ILT4-His were 17 n and 1 1 uM, respectively, with a 630-fold difference indicated by the K _D ratio.

Because of the fast ka and kd kinetics constants, steady-state approximation (SSA) was also used to confirm the low affinity of HLA-G1-Fc for iLT4-His.

Blocking of HLA-G Binding to 1LT4 3A9 T cell Transfectants with 1 EKG4)

ILT4 3A9 T ceil transfectants were pre-treated with 1 E1 (G4) or isotype control at various doses, followed by secondary detection of 1 E1 (G4) (Figure 3A) or by treatment with a fixed concentration of biotinyiated HLA-G1 Fc chimera to assess the ability of 1 E1 (G4) to block cognate ligand (Figure 3B). 1 E1 (G4) and HLA-G Fc was detected via flow cytometry. The data show 1 E1 (G4) blocked HLA-G 1 Fc binding in a dose-dependent manner. Luminex- and Cell-based Ligand Blocking and Competition Assays

Antibodies 1 E1 , 2A6, 2C1 , and 3G7 were tested in Luminex- and cell-based ligand blocking and ligand competition assays for their potential of inhibiting the interaction of recombinant dimeric HLA-G with 1LT4 antigen coupled to beads or expressed by

CHO/ILT4+ cells.

The Luminex based ligand blocking and competition assays used recombinant human ILT4 antigen chemically coupled to Luminex beads, in the blocking assay, the beads were pre-incubated with a dose range (0.5-9,000 ng/mL in 1 :3 serial dilutions) of higG4 variants of the anti-ILT4 antibody 1 E1 , 2A6, 2C1 , or 3G7. Bead-bound 1LT4 antigen was then tested for binding to soluble biotinylated HLA-G/Fc fusion protein at a concentration of 50 nM. In the Luminex ligand competition assay, antigen-coupled Luminex beads were pre-incubated with soluble biotinylated HLA-G/Fc fusion protein at a concentration of 50 nM before dose-titrations (0.5-9,000 ng/mL in 1 :3 serial dilutions) of higG4 variants of the anti~ILT4 antibody 1 E1 , 2A8, 2C1 , or 3G7. in both assay setups, ILT4:HLA-G interaction was detected with an anti-streptavidin-PE antibody and iC ₅₀ values were determined. AH tested antibodies showed dose-dependent blocking of HLA-G binding to Luminex bead- coupled i LT4/Fc and I C ₅₀ values are summarized in Table 4. All tested antibodies also showed dose-dependent competition with HLA-G for binding to Luminex bead- coupled iLT4/Fc (data not shown).

Table 4 Luminex Ligand Blocking Assay

The cell-based ligand blocking and ligand competition assays followed a similar principle as described for the Luminex-based assays and used a CHO/ILT4 ceil line for surface expression of the antigen. In the blocking assay, cells were pre-incubated with the tested antibody using a dose range of 1-20,000 ng/mL in 1 :3 serial dilutions. ILT4 antigens were then tested for binding to soluble biotinylated HLA-G/Fc fusion protein at a

concentration of 5 pg/m!. The competition assay used a reverse setup. Cells were pre- incubated with soluble biotinylated HLA-G/Fc fusion protein at a concentration of 5 pg/ml before dose titrations of the tested antibody were added in a range of 1 -20,000 ng/mL in 1 :3 serial dilutions. ILT4:HLA-G interaction was detected with an anti-streptavidin-PE antibody. All tested antibodies showed dose-dependent blocking of HLA-G binding to CHO-expressed ILT4 and IC5 ₀ values are summarized in Table 5, All tested antibodies also showed dose-dependent competition with HLA-G for binding to CHO-expressed ILT4 (data not shown).

Tab e 5 Ceil-based Ligand Stocking Assay

Blockade of Non-HLA-G MHC Class I Ligand Binding to 1LT4 3A9 T Cell Transfectants with

ILT4 3A9 T cell transfectants were pre-treated with p1 E1 (G1) or hlgG1 isotype control at various doses, followed by treatment with a fixed concentration of fluorochrome labeled HLA-F or CD1 d tetramers, or HLA-A02:01 or HLA*B7:Q2 dexamers to assess the ability of p1 E1 (G1) to block non-HLA-G MHC class I ligands. HLA-A, HLA-B, and HLA-F binding to ILT4 was inhibited by p1 E1 (G1 ) in a dose titratable fashion (Figure 4), indicating the ability of p1 E1 (G1) to block other reported MHC class I ligands.

Blockade of ANGPTL Binding to 1LT4 3A9 T Cell Transfectants with p1 E1 (G1 )

Angiopoietin-like (ANGPTL) proteins were recently reported to bind to ILT4 expressed by human hematopoietic stem cells (Zheng et a!., Nature. 2012 May

3Q;485(7400):656-60 and Deng et al. Blood. 2014 Aug 7; 124(6):924-35). To test whether p1 E1 (G1 ) could block ANGPTL family member binding to 1LT4, commercially available

ANGPTL proteins or protein fragments were purchased and tested for binding to ILT4 3A9 T ceil transfectants that were pre-treated with p1 E1 (G1). Binding data indicate that

ANGPTL1 , 4, and potentially 7 could bind to ILT4 and not vector control cells at the concentration of protein tested. p1 E1 (G1) was able to fully block ANGPTL protein binding at a saturating dose (Figure 5).

ILT Family Specificity Binding of 1 EKG4) to Human ILT 3A9 T Cell Transfectants

ILT family specificity binding of 1 E1 (G4) to human ILT family members was assessed by cell-based flow cytometry using 3A9 T cell lines transfected to express human ILT4, ILT2, 1LT3 (two variants), ILT5, LILRB5, LILRA1 , LILRA2, 1LT7, ILT8, or ILT1 1. 1 E1 (G4) specifically bound human I LT4 and did not have cross-reaciiviiy to any other I LT family member tested (Figures 8A and 6B).

Example 3: Bioaciivity of Anti-iLT4 mAb 1 E1 In Engineered and Primary Ceils

Ability of 1 E1 (G4), 2A6, 2C1 , and 3G7 to Reverse Interleukin 2 Suppression in Engineered I LT4 3A9 Cell-based Assays

An anti-CD3 antibody was used to stimulate control mouse 3A9 T-cells resulting in an increase of I L-2 release, in contrast, ILT4 3A9 T-ceil transfectants could not express I L-2 in the presence of CDS stimulation, possibly due to cross-reactivity with I LT4 with mouse MHC class I molecules or through an unknown xeno-ligand(s). This interaction appeared to lead to spontaneous multimerization/activation of the 1LT4 receptor, resulting in suppression of the anti-CD3 mediated I L-2 release. Accordingly, antibodies that functionally bind to I LT4 and block the interaction of ILT4 with the xeno-ligand(s) and/or inhibit receptor multimerization should restore I L-release.

1 E1 (G4), 2A6, 2C1 , and 3G7 were tested for mediating IL-2 release of ILT4 mouse 3A9 T-cell transfectants. 1 E1 (G4), 2A6, 2C1 , or 3G7 was added to ILT4+ mouse 3A9 T-ceil transfectants and IL-2 release was measured photometrically by ELISA following 24 hours of anti~CD3 mediated cell stimulation. The representative dose response curve of 1 E1 (G4) is shown in Figure 7. EC ₅₀ values of the tested antibodies were determined from the dose response curves and shown in Table 8.

Table 8 !L-2 Repression Assay

WTMC (wild-type mast ceil) Mouse Cell Degranulation Assay (Qualitative Only)

p1 E1 (G4) and 1 E1 (G4) were tested for rescue of ILT4: HLA-G dependent mast cell degranulation. Mouse WTMC mast cells were transfected with human I LT4 and stimulated with plate-bound antibody raised against CD2Q0RLa. Antibody-mediated cross-linking of CD200RLa led to mast ceil degranulation by activation of ITAM motifs found in the intracellular domain of CD220RLa. Degranulation can be measured coiorimetricaily by assaying granule content release in assay supernatants. in the presence of plate-bound HLA-G tetramer, CD2QQRLa-mediated mast cell degranulation was inhibited in I LT4 iransfectanis. Preireatrneni of ILT4 WTMC transfectants with p1 E1 (G4) or 1 E1 (G4) before stimulation with platebound anti-CD200RLa and HLA-G teframer reversed degranuiation inhibition in a dose titratable manner (Figure 8). Effect of 1 EKG4) on Myeloid Derived Cytokine Secretion by Primary Human Peripheral Blood Mononuclear Cells (PB C)

Assessment of 1E1(G4) in human primary PBMC LPS stimulation assays

Expression of TNFa, a prototypical myeloid derived proinflammatory cytokine, was reported to be expressed by monocytes expressing low levels of ILT4 when stimulated with LPS. Monocytes with high expression of ILT4 did not express as much TNFa, and the lack of ILT4 expression was found to be a hallmark of monocytes isolated from patients with psoriatic arthritis (Bergamini et al., PLoS One. 2014 Mar 27;9(3):e92Q18), ILT4 expression on monocytes could inhibit myeloid cell effector activity and antagonize proinflammatory cytokine induction (e.g. , TNFa) in the presence of proinflammatory stimuli (e.g. , LPS). As such, whole PBMCs isolated from healthy human donors were treated with LPS and the ability of ILT4 antagonism to enhance proinflammatory myeloid cyiokine expression was evaluated with 1 E1 (G4). Figures 9A and 9B show data from one of three experiments, with 3 donors each, demonstrating that 1 E1 (G4) enhanced LPS-dependent expression of both GM-CSF and TNFa (both myeloid-derived cytokines) in a dose titratable fashion.

Assessment of 1E1(G4) in human primary PBMC anti-CD3 stimulation assays

To assess whether 1 E1 (G4) treatment could enhance T-cell or myeloid effector cyiokine expression in the presence of a sub-optimal T cell stimulus, whole PBMCs isolated from healthy human donors were treated with anti-CD3 to induce T-cell proliferation, and the ability of ILT4-antagonism to modify cytokine expression was evaluated with 1 E1 (G4). Figures 10A and 10B show data from one of three experiments, with 3 donors each, demonstrating that 1 E1 (G4) enhanced anti-CD3 dependent expression of GM-CSF and TNFa in a dose titratable fashion.

Example 4: Anti-tumor Efficacy of anti-iLT4 antibodies in the Humanized IVIouse S ~i¥iEL-5 Tumor IVlodel

Anti-tumor Efficacy of 1 E1 , 2A6, 2C1 , and 2D5 in the Humanized Mouse Tumor Model

Antibodies 1 E1 , 2A6, 2C1 and 2D5 were tested in an in vivo tumor regression assay. Humanized mice (NSG mice reconstituted with human hematopoeitic stem cells to establish human immune eel! constitution) were inoculated with 1x10° SKMEL5 melanoma cells (HLA class A*02:Q1) and tumor growth was monitored until an average size of 150 mm after approximately 35 days was observed. Seven randomized groups of mice, each containing six animals, were subcutaneously dosed with isotype control antibody (hlgG1 + hlgG4, 20 mg/kg of each), or 20 mg/kg of of either of the following anti-ILT4 antibodies: 1 E1-igG1 , 1 E1-lgG1 (N297Q) (Fc null mutant), 1 E1 -lgG4, 2A6-lgG4, 2C1-lgG4, and 2D5- lgG4, Mice were dosed every seven days (day 35, 42, and 49; total of three doses) and tumor size was measured until day 63. Results indicated impaired tumor growth in mice treated with 1 E1 (lgG1 , igG1-(N297Q), lgG4), 2A6-lgG4, and 2D5-lgG4 compared to animals dosed with the isotype control (Figure 17). in contrast, mice treated with either isotype control or anti~ILT4 candidate 2C1 failed to demonstrate impaired tumor growth.

Anti-tumor Efficacy of p1 E1 (G4) in the Humanized Mouse Tumor Model

A humanized mouse tumor model was developed to test in vivo efficacy of p1 E1 (G4) for tumor growth inhibition. Immuno-deficient NSG mice were reconstituted with human hematopoietic stem ceils. After mice were confirmed to harbor peripheral human CD45+ immune cells (>25% of PBMCs), they were inoculated with SK-MEL-5 tumor cells, a human skin melanoma derived tumor line. These ceils were selected for their genetic expression of HLA-G. Following inoculation, tumors were allowed to grow to an approximate size of 150 mm ³ . Mice were randomized into groups and challenged with either higG4 isotype control or p1 E1 (G4).

Mice treated with p1 E1 (G4) displayed tumor growth inhibition over the course of the study (Figure 11 A). One complete and one partial regression were observed with p1 E1 (G4) (Figure 11C),

Anti-tumor Efficacy of 1 EKG4) in the Humanized Mouse Tumor Model

The anti-tumor activity of 1 E1 (G4) was tested in the humanized mouse SK-MEL-5 tumor model. In this model, immunodeficient NSG {HOD.Cg~Prkdc ^{s d} ii'2rg ^{tr! Wj} ''iSzS) mice are irradiated and injected with human CD34+ hematopoietic stem cells isolated from umbilical cord blood. After several months of engraftment, human immune cells can be detected in mouse blood. The mice were then implanted subcutaneously (SC) with the human melanoma-derived SK-MEL-5 ceil line.

For this study, NSG mice transplanted at 3 to 4 weeks of age with human cord blood-derived CD34÷ cells from 3 separate donors were injected SC with 1 ^χ 10 ⁶ SK-MEL-5 cells at approximately 20 weeks of age. Humanized NSG mice bearing SK-MEL-5 tumors were assigned to 2 treatment groups at 6 mice per group (3 mice from each human CD34+ donor cohort per treatment group) when mean tumor size was approximateiyl 00 mm ³ , 21 days following tumor inoculation (DO tumor randomization). Tumor-bearing mice were injected SC with 20 mg/kg 1 E1 (G4) or a hlgG4 isotype control mAb every 7 days for

4 doses. Tumor volumes were monitored every 7 days following the initiation of treatment.

Anti-tumor efficacy in the 1 E1 (G4) treatment group was significantly greater than the isotype control group (p < 0.001 from Day 28 through Day 49) (Figures 12A). The endpoint tumor weight in 1 E1 (G4)-freated mice was lower than that in isotype-treated mice (Figure 12C). Overall, the results revealed significant anti-tumor efficacy of 1 E1 (G4) at 20 mg/kg in the humanized mouse SK-MEL-5 tumor model. No effect was observed on body weight (Figure 12B) and splenic weight (Figure 12D) with 1 E1 (G4) treatment. Example 5: ILT4 haplotype binding of 1 E1 (G4) to human ILT 3A9 T cell transfectants

Single nucleotide polymorphism data from publicly available sources (1 K Genome Project Phase 3) was used to determine ILT4 allelic frequencies for African, European, Asian, and South Asian populations. Sequences for haplotypes that were expressed with a 5% or greater prevalence in any population (haplotypes 1 , 2, 5, 7, 9 and 10 in Tabfe 7) were determined and expressed in 3A9 T cells. 1 E1 (G4) bound all haplotypes tested using saturating doses of the antibody (Figure 13). Haplotype 2 corresponded to the consensus sequence reported in Uniprot. Haplotype 5 was used in functional and ligand-based assays. Haplotype binding data indicates that 1 E1 (G4) binds all major allelic variants tested.

Table 7. HapHotype frequencies for ILT4 across human populations.

*Hapiotypes for each population were based on the phase 3 data from the 1000 genome project and were determined using PLINK to analyze non-synonymous SNPs listed in the EXAC database.

Example 6: RNA Expression of ILT4 in Tumor and Cell Types Based on TCGA and Blueprint Databases Expression of ILT4 across tumor types and cell populations was determined using publicly available RNAseq datasets, through Omicsoft (Qiagen, Gary, NC). The TCGA dataset (TCGA_B38_20171002_v4, https://gdc-portal.nci.nih.gov ) is comprised of 1 1 ,292 samples with RNA-Seq data. The Blueprint dataset (BlueprintJ338 _m 20170216__v2, http://www.blueprint-epigenome.eu/) is comprised of 258 normal blood samples from 55 cell types with RNA-Seq data. The tumor types with highest expression of 1LT4, at the RNA level, include LA L (i.e. , AML), DLBC (i.e. , DLBCL), TGCT, MESO, KIRC (Figure 14A). The cell types with highest expression of ILT4, at the RNA level, include neutrophils, monocytes, osteoclasts, eosinophils, macrophages, and dendritic cells (Figure 14B).

Lymphocytes had low to no expression of ILT4, in this dataset. FPK of 1 (or

LOG2(FPK ÷0.1) of 0) is the most widely accepted heuristic fixed threshold, although lower FPK values could report on "low expressed" genes within a sample.

Example 7: Binding of 1 E1(G4) to Myeloid Ceils from Tumor Histoculture Samples

Histocultures were prepared from fresh human tumor samples (surgical resections), and were treated with either anti-RSV igG4 (ssotype control) or 1 E1 (G4) at 20 g/mL for 18-24 hours at 37°C. After treatment, tumor slices were digested into single ceil suspensions and stained for FACS. Dot plots and contour plots represent FACS data from either RCC (Figure 15A) or CRC (Figure 15B) tumor histoculture single cell suspensions. Total myeloid ceils can be subdivided into four subsets based on the expression of CD68b and/or CD14. These four myeloid subsets were simultaneously analyzed for ILT4 expression, using a non-competing commercial anti-ILT4-PE (BioLegend, cat# 338706, San Diego, CA), and ceil surface-bound 1 E1 (G4), using an anti-lgG4 secondary antibody. Good correlation between ILT4+ cells and 1 E1 (G4)+ cells was observed in 1 E1 (G4)-treated histocultures. Tumor-infiltrating lymphocytes were observed to be ILT4- and 1 E1 (G4)- in these samples.

The present invention is not to be limited in scope by the specific embodiments described herein, indeed, the scope of the present invention includes embodiments specifically set forth herein and other embodiments not specifically set forth herein; the embodiments specifically set forth herein are not necessarily intended to be exhaustive. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the claims. Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. Sequence Listing

The present specification is being filed with a computer readable form (CRF) copy of the Sequence Listing, The CRF entitled 24443__PCT__SEQLlST.txt, which was created on March 22, 2018 and is 128,042 bytes in size, is incorporated herein by reference in its entirety.