EA044253B1 - ANTI-ILT4 ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS - Google Patents

Description

Field of invention

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

State of the art

A common strategy used by tumor cells to evade the innate and adaptive immune response is associated with abnormal expression of human leukocyte antigen (HLA)-G (Curigliano et al. Clin Cancer Res. 2013 and Gonzalez et al. Crit Rev Clin Lab Sci. 2012). HLAG can directly inhibit immune cell function through receptor binding and/or trogocytosis and disruption of chemotaxis (Morandi et al. Cytokine Growth Factor Review. 2014 and Lin et al. Mol Med. 2015). Its high expression in various tumor types, including, for example, colorectal, pancreatic, endometrial, lung, breast, ovarian, and gastric cancers, is associated with advanced disease stage, tumor invasiveness, metastatic potential, and poor 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 the expansion of myeloid-derived suppressor cells (MDSC) (Loumange et al. Int J Cancer. 2014, Lin et al. Hum Immunol. 2013. , and Agaugue et al. Blood. 2011). HLA-G binding to ILT4 can directly inhibit the functioning of monocytes, dendritic cells and neutrophils, thereby impairing the innate antitumor immune response. The interaction between HLA-G and monocytes, mediated by ILT4, inhibits the maturation of antigen presenting cells (APCs) of human monocytic origin, resulting in reduced expression of MHC class II antigens and costimulatory molecules as a result of activation of Stat3 (Colonna et al. J Immunol. 1998; Allan et al. 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 and the arrest of maturation/activation of myeloid DCs, and the induction of tolerogenic DCs by HLA-G occurred through disruption of the MHC class II presentation pathway (Ristich et al. Eur J Immunol. 2005).

Patients who do not respond to T-cell therapy but who may benefit from reducing tissue macrophage/MDSC-mediated tumor tolerance (eg, myeloid cell-rich tumors) have an unmet need for treatment. ILT4 blockade could address this need and would differ from existing T cell-targeted antibodies (eg, anti-PD1, anti-TIGIT) by reducing the suppression of tolerogenic myeloid cells in the tumor microenvironment.

The essence of the invention

The present invention relates to antibodies or antigen-binding fragments thereof that bind to human ILT4. In some embodiments, the antibody or antigen binding fragment thereof binds to one or more amino acid residues in a human ILT4 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 some embodiments, the antibody or antigen-binding fragment thereof protects the epitope from deuterium exchange with a deuterium source, such as D ₂ O. In one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the LYREKKSASW epitope (SEQ ID NO: 59) . In another embodiment, the antibody or antigen binding fragment thereof binds to one or more amino acid residues in the TRIRPEL epitope (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 NGQF epitope (SEQ ID NO: 61). In another embodiment, the antibody or antigen binding fragment thereof binds to one or more amino acid residues within the HTGRYGCQ epitope (SEQ ID NO: 62). In 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 LYREKKSASW epitope (SEQ ID NO: 59) and protects the epitope from deuterium exchange with a deuterium source, such as D2O. In another embodiment, the antibody or antigen binding fragment thereof binds to one or more amino acid residues in the TRIRPEL epitope (SEQ ID NO: 60) and protects the epitope from deuterium exchange with a deuterium source, such as D2O. In yet another embodiment, the antibody or antigen binding fragment thereof binds to one or more amino acid residues in the NGQF epitope (SEQ ID NO: 61) and protects the epitope from deuterium exchange

- 1 044253 with a deuterium source such as D2O. In 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 O. In 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), and protects epitopes from deuterium exchange with a deuterium source such as D ₂ O.

The present invention also provides an antibody or antigen binding fragment thereof that binds to the same human ILT4 epitope as any antibody or antigen binding fragment thereof provided herein. In some embodiments, the antibody or antigen binding fragment thereof binds to the same human ILT4 epitope as the 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 11; 10 and 11; 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 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen binding fragment thereof binds to the same human ILT4 epitope as the 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 ILT4 with the antibody or antigen binding fragment thereof provided herein. In some 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 11; 9 and 11; 10 and 11; 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 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen binding fragment thereof competes for binding to human ILT4 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 to human ILT4, comprising: (a) a complementarity determining region-L1 (CDR-L1), a complementarity determining region-L2 (CDR-L2), and a complementarity determining region-L3 (CDR-L3) immunoglobulin light chain variable domain (V _L ) containing the amino acid sequence shown in SEQ ID NO: 3-7, 11, 13, 15 or 45; and/or (b) a complementarity determining region-H1 (CDR-H1), a complementarity determining region-H2 (CDR-H2), and a complementarity determining region-H3 (CDR-H3) of an immunoglobulin heavy chain variable domain ( _VH ) containing an amino acid the sequence given 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO: 17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or a VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GX1X2NRPS; where X1 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 _VH domain containing: CDR-H1: SYAIS (SEQ ID NO: 22), CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23) and CDR-H3: YFX1X2SGWYKGGAFDI; where X1 is D or S, and X ₂ is S or A (SEQ ID NO: 24), and/or a V _L domain containing: CDR-L1: TLRSGINVDTYRIH (SEQ ID NO: 25), CDR-L2: YKSDSDKHQGS ( SEQ ID NO: 26) and CDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a VH domain containing: CDR-H1: SYAMH (SEQ ID NO: 28), CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO: 29) and CDRH3: VGEWIQLWSPFDY (SEQ ID NO: 30), and/or a V domain _L containing: 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 containing: 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 VL domain containing: CDRL1: 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 containing: 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 domain

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Vh containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or V _l domain, containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _l containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a V _h domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _l containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a V _h domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _l containing: 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 domain V _l containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a V _h domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _l containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or V _l domain containing: CDR-L1: TGSSSNI- 3 044253

GAGYDVH (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 VH 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 VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a VH 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 VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS (SEQ ID NO: 56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21 ).

In some embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain has at least 90% amino acid sequence identity with respect to the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 11, 13, 15 or 45, and/or the immunoglobulin heavy chain has at least 90% amino acid sequence identity with respect to the amino acid sequence shown 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 an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain 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 immunoglobulin heavy chain contains a heavy chain variable domain having at least 90% amino acid sequence identity with respect to amino acid sequence shown in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68 or 69.

In other embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 11, 13, 15 or 45; and/or the immunoglobulin heavy chain contains 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 other embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, 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 contains the amino acid sequence set forth in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68, or 69.

In addition, the present invention provides an antibody or an antigen-binding fragment thereof comprising any of the following sets of immunoglobulin heavy chains and immunoglobulin light chains: (1) an immunoglobulin heavy chain containing the amino acid sequence set forth in SEQ ID NO: 1; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (2) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 4; (3) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; (4) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; (5) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; (6) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (7) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 8; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (8) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 9; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (9) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 10; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (10) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 12; light

- 4 044253 immunoglobulin chain containing the amino acid sequence shown in SEQ ID NO: 13; (11) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 14; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 15; (12) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 79; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (13) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 4; (14) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; (15) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; (16) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; (17) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (18) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 82; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (19) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 83; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (20) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 84; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (21) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 85; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 13; or (22) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 86; an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 15.

Furthermore, the present invention provides an antibody or antigen binding fragment thereof comprising any of the following sets of heavy chain variable domains and light chain variable domains: (1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 63 and a variable a light chain domain containing 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 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 containing 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 containing 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 containing 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 containing 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 containing the amino acid sequence set forth in SEQ ID NO: 75; or (11) 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 containing the amino acid sequence set forth in SEQ ID NO: 57; and a V _L domain containing the amino acid sequence set forth in SEQ ID NO: 58.

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In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises: an immunoglobulin heavy chain containing the amino acid sequence set forth in SEQ ID NO: 2; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 7.

In another preferred embodiment, the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 80; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 7.

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

In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain contains the amino acid sequence set forth in SEQ ID NO: 58, and each heavy chain contains 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 contains the amino acid sequence set forth in SEQ ID NO: 7, and each heavy chain contains 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, where 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, for example, with N-linked glycans of engineered yeast or N-linked glycans of Chinese hamster ovary (CHO) cells. In an embodiment of the invention, the antibody or antigen-binding fragment thereof is an antibody.

The present invention also relates to a pharmaceutical composition containing an antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the composition further comprises a therapeutic agent (eg, pembrolizumab). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In other embodiments, the composition further comprises a therapeutic agent (eg, pembrolizumab) and a pharmaceutically acceptable carrier.

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

The present invention further provides a polypeptide (eg, an isolated polypeptide) comprising an immunoglobulin light chain and/or an immunoglobulin heavy chain or a variable domain thereof from any antibody or antigen binding fragment thereof provided herein. For example, in an embodiment of the invention, the polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-39, 44, 45, 47-58, 6377 and 79-86. The present invention also relates to any polynucleotide (eg, DNA or RNA) encoding any polypeptide presented herein. In another aspect, the present invention provides a vector containing a polynucleotide as described herein. The present invention also relates to a host cell (eg, a CHO cell) containing a polynucleotide or vector as described herein.

The present invention relates to a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F, for example, in vitro or in vivo, for example, in the body of an individual in need thereof (for example, a human), comprising: administering to an individual an effective amount of an antibody or antigen binding fragment thereof as provided herein. In some embodiments, the method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprises administering a therapeutic modality (eg, antitumor radiation therapy or surgical tumorectomy) to an individual. In some embodiments, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprises administering a therapeutic agent (eg, pembrolizumab) to the individual. In other embodiments, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B, and/or HLA-F further comprises administering a therapeutic modality (eg, antitumor radiation therapy or surgical tumorectomy) and administering a therapeutic agent (eg, pembrolizumab) to the individual. ).

The present invention also provides a method of treating cancer in an individual (eg, a human), comprising administering to the individual an effective amount of an antibody or antigen binding fragment thereof as provided herein. In some embodiments, the method of treating a cancer further includes administering a therapeutic method (eg, anticancer radiation therapy or surgical tumorectomy) to the individual. In some embodiments, the method of treating cancer further comprises administering to the individual a therapeutic agent (eg, pembrolizumab). In other embodiments, a method of treating a cancer further includes administering a therapeutic method (eg, anticancer radiation therapy or surgical tumorectomy) and administering a therapeutic agent (eg, pembrolizumab) to the individual.

The present invention also provides a method for producing an antibody, or an antigen binding fragment thereof, of the present invention, or an immunoglobulin chain thereof (e.g., its V _H and/or VL), comprising culturing a host cell (e.g., a CHO cell) containing a polynucleotide (e.g., wherein the polynucleotide is in a vector and/or is inserted into one or more chromosomes of a host cell) encoding an antibody, or an antigen binding fragment thereof, or an immunoglobulin chain thereof, for expression of the antibody, or an antigen binding fragment thereof, or an immunoglobulin chain thereof.

The present invention also relates to a method for producing an antibody, or an antigen binding fragment thereof, of the present invention, or an immunoglobulin chain thereof (for example, a V _H and/or V L thereof), comprising: expressing a polynucleotide encoding the antibody, or an antigen binding fragment thereof, or an immunoglobulin chain thereof .

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

A method for detecting the presence of an ILT4 peptide or fragment thereof in a sample is also part of the present invention. The method includes contacting a sample with an antibody or antigen binding fragment of the present invention and detecting the presence of a complex of the antibody or antigen binding fragment and an ILT4 peptide or fragment thereof, wherein detection of the complex indicates the presence of an ILT4 peptide or fragment thereof. In an embodiment of the invention, the method is carried out in vitro, for example, in a biological sample, for example a surgical biopsy or blood sample, of an individual. In another embodiment, the method is carried out in vivo, for example, in the body of an individual. In yet another embodiment, the individual is a human.

Brief description of drawings

Fig. 1. Heat map of deuterium labeling binding of p1E1(G1) to ILT4-His.

In fig. 2A and 2B show the crystal structure of human ILT4. In fig. 2A shows the levels of deuterium labeling mapped onto the human ILT4 structure. In fig. Figure 2B shows the crystal structure of domains 1 and 2 of human ILT4 in complex with HLA-G. ILT4, HLA-G heavy chain, and beta-2 microglobulin are indicated. Human ILT4 epitopes containing residues LYREKKSASW (SEQ ID NO: 59), TRIRPEL (SEQ ID NO: 60), NGQF (SEQ ID NO: 61) and HTGRYGCQ (SEQ ID NO: 62) are indicated.

In fig. 3A and 3B show ILT4 binding to HLA-G and blockade of 1E1 (G4). Mouse 3A9 T cells transfected with human ILT4 were blocked with Fc blockade and then incubated with titrated concentrations of 1E1 (G4) (starting at 27 μg/ml, 1:3 dilution) or isotype control hIgG4 (27 μg/ml). ml).

Fig. 3A. 1E1 (G4) or hIgG4 was determined using fluorochrome-labeled goat anti-human F(ab')2 antibody and flow cytometry.

Fig. 3B. After pretreatment with 1E1 (G4), cells were incubated with 2 μg/ml biotinylated HLA-Fc or control Fc (mVISTA-Fc). Fc binding was determined using RE-conjugated streptavidin and flow cytometry. The graphs shown are from 3 independent experiments. The IC ₅₀ and EC ₅₀ values presented are the average values for these experiments +/- standard deviation.

Fig. 4. Blockade of non-HLA-G MHC class I binding to ILT4 and p1E1 (G1). Mouse 3A9 T cells transfected with human ILT4 were pretreated with titrated concentrations of p1E1 (G1) (starting at 10 μg/ml, 1:3 dilution) or isotype control hIgG1 ( 10

- 7 044253 μg/ml) before incubation with fluorochrome-labeled HLA-F or CD1d tetramers or fluorochrome-labeled HLA*A2:01 or HLA*B7:02 dexamers. Tetramer/dexamer binding was determined by flow cytometry and the average fluorescence intensity was plotted for each. The dilutions/concentrations used for each dexamer/tetramer are as follows: HLA*A2:01-dexamer PE: 1:25; HLA*B7:02-dexamer FITC: 1:25; CD1d tetramer PE: 1:50; HLA-Ftetramer PE: 1 µg/ml.

Fig. 5A-5C. Blockade of ANGPTL binding to ILT4 and p1E1(G1).

Fig. 5A. Biotinylated ANGPTL proteins were preincubated for 20 min. with p1E1 (G1) or human IgG1, the final concentration of each was 20 μg/ml. The solutions were then added to mouse 3A9 T cells transfected with human ILT4 and incubated for an additional 30 min. ANGPTL binding was determined using PE-conjugated streptavidin and analyzed by flow cytometry. PE-labeled HLA-G tetramer was also added as a positive control for ILT4 binding/blocking. ANGPTL proteins were purchased from R&D SYSTEMS and biotinylated

Fig. 5V. Mouse 3A9 T cells transfected with human ILT4 were blocked using Fc blocking, then incubated with 20 μg/ml p1E1(G1) or h1gG1 isotype control. After incubation, p1E1 (G1) or hIgG1 was detected using a fluorochrome-labeled goat anti-human F(ab') ₂ antibody and analyzed by flow cytometry.

Fig. 5C. 3A9 T cells with control vector were used as a negative control for ANGPTL binding. Cells were treated as described in (A), except that antibody treatment was omitted.

Fig. 6A and 6B. ILT family 1E1(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 isotype control or 1E1 (G4) hIgG4 antibody binding at a fixed dose of 10 μg/ml. 3A9 T cells with control vector were used as an additional negative control. Binding of commercially available anti-ILT antibodies is also shown compared to the corresponding isotype control to demonstrate expression of an ILT family member. The data presented correspond to two experiments with similar results. See figure legend.

Fig. 7. Restoration of IL-2 release from 3A9 T cell transfectants with ILT4 using 1E1 (G4). Mouse 3A9 T cells transfected with human ILT4 were treated with plate-bound anti-CD3 antibody in the presence of soluble 1E1 (G4) or isotype control (huIgG4) at a concentration starting at 27 μg/ml and serially diluted 3-fold to 0. 3 µg/ml. After 24 h of incubation, supernatants were removed and mouse IL-2 was measured by ELISA. The presented graph corresponds to 5 independent experiments. The EC ₅₀ value shown is the mean of these experiments +/- standard deviation.

Fig. 8. Restored by p1E1 (G4) and 1E1 (G4), ILT4:HLA-G-induced suppression of mast cell degranulation. Mouse WTMC mast cells were transfected with human ILT4 and pretreated with titrated concentrations of isotype control 1E1 (G4), p1E1 (G4), or hIgG4 (starting at 10 μg/ml, 1:3 dilution) before stimulation with plate-bound anti-CD200Rla antibody (clone DX89; 1 μg/ml) and plate-bound HLA-G tetramer (0.625 μg/ml). After stimulation for 1 h, degranulation was assessed by collecting mast cell supernatants to measure β-hexosaminidase release using a colorimetric enzymatic assay. Data shown are representative of 2 independent experiments with 3 replicates per data point.

Fig. 9A and 9B. Enhanced 1E1 (G4) LPS-induced expression of pro-inflammatory myeloid cytokines. Whole PBMCs from healthy patients were isolated from leukoreduction chambers and treated with 0.25 μg/ml LPS in the presence of hIgG4 (30 μg/ml; open circles) or 1E1 (G4) (labeled 1E1 in the figure) (30 μg/ml to 3 pg /ml; filled circles) for 3 days. After stimulation, supernatants were analyzed for cytokine expression ((Fig. 9A) GM-CSF and (Fig. 9B) TNFa) using the Meso Scale Discovery Multiple Cytokine Assay Kit. Each color represents data for an individual patient. Conditions without any stimulation are also shown (filled triangles).

Fig. 10A and 10B. Enhanced 1E1 (G4) anti-CD3 antibody-induced expression of pro-inflammatory myeloid cytokines. (A-B) Whole PBMCs from healthy patients were isolated from leukoreduction chambers and treated with 0.01 μg/ml anti-CD3 antibody (HIT3a) in the presence of hIgG4 (30 μg/ml; open circles) or 1E1 (G4) (denoted 1E1 in the figure ) (30 μg/mL to 3 pg/mL; filled circles) for 3 days. After stimulation, supernatants were analyzed for cytokine expression ((Fig. 10A) GM-CSF and (Fig. 10B) TNFa) using the Meso Scale Discovery Multiple Cytokine Assay Kit. Each color represents data for an individual patient. Conditions without any stimulation are also shown (filled triangles).

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Fig. 11A-11E. Treatment with p1E1(G4) results in tumor growth inhibition in the SKMEL5 humanized mouse tumor model. Humanized NSG mice transfused with CD34+ umbilical cord blood from 2 different cord blood donors were inoculated subcutaneously with 1x10 ⁶ SKMEL5 tumor cells in the left flank. After inoculation, tumors were allowed to grow and those mice whose tumors reached an average size of 150 mm ³ were randomly assigned to groups of 6 (3 from each stem cell donor, n=6 per group total). Mice were then stimulated with hIgG4 isotype control or p1E1(G4) (20 mg/kg each) every 5 days until the end of the study, while tumors and body weight were measured weekly. Tumor growth in mice treated with isotype control and p1E1(G4) was monitored over time. In fig. 11A shows fresh tumor volume (mm) +/- SD over time for both groups, and FIG. 11B and 11C show tumor volumes (mm ³ ) in individual mice over time for isotype control and p1E1 (G4) treated mice, respectively. In fig. 11D shows tumor burden in individual mice treated with isotype control or p1E1 (G4) over time; in fig. 11E shows weight loss in each treatment group, which was also measured over time. After completion of the study, mice were sacrificed and tumors were collected and weighed.

In fig. 12A-12D show that administration of 1E1(G4) resulted in inhibition of tumor growth in the SK-MEL-5 humanized mouse tumor model. In fig. 12A shows the mean tumor volume (mm ³ ) +/- SD over time in 1E1(G4)-treated mice and IgG4 isotype control-treated mice. In fig. Figure 12B shows the change in body weight over time in both groups. In fig. 12C shows the final tumor weight of individual mice treated with isotype control or 1E1(G4). In fig. 12D shows the final spleen weight of individual mice treated with isotype control or 1E1(G4).

Fig. 13. Linking ILT4 haplotypes. Mouse 3A9 T cells transfected with human ILT4 allelic variants were used to test isotype control or 1E1 (G4) hIgG4 antibody binding at a fixed dose of 10 μg/ml. 3A9 T cells with control vector were used as an additional negative control. Haplotypes are presented in table. 2. The data presented correspond to two experiments with similar results.

Fig. 14A and 14B. ILT4 RNA expression in different tumor types or cell types according to public databases. IN FIG. 14A shows ILT4 RNA expression in various tumor types according to the TCGA database. In fig. 14B shows ILT4 RNA expression in various cell types according to the Blueprint database.

In fig. 15A and 15B show binding of 1E1 (G4) to myeloid cells from renal cell carcinoma (RCC) (FIG. 15A) and colorectal cancer (CRC) histoculture specimens.

Fig. 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-acetylglucosamine (GlcNac); circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).

In fig. 17 shows the antitumor efficacy of various anti-ILT4 antibodies in the SK-MEL-5 humanized mouse tumor model.

Detailed Description of the Invention

For a better understanding of the invention, certain technical and scientific terms are specifically defined below. Unless specifically stated otherwise in the specification, all other technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which the invention belongs.

Throughout the invention, including the claims, terms in the singular include a reference to the corresponding plural unless the context clearly indicates otherwise.

The term affinity refers to the strength of the sum of all non-covalent interactions between a particular binding site of a molecule (eg an antibody) and its binding partner (eg an antigen). Unless otherwise specified, as used herein, the term binding affinity refers to the characteristic binding affinity reflecting the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD). 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, multispecific antibodies (eg, bispecific antibodies), fully human antibodies, and chimeric antibodies.

In the context of the invention, unless otherwise indicated, the term 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, for example, fragments that retain one or more CDR regions. Non-limiting examples of antibody binding fragments include Fab, Fab', F(ab') ₂ , Fv fragments, individual heavy chains or light chains of antibodies, and individual heavy chain or light chain variable regions.

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The Fab fragment consists of one light chain, CH1 and variable regions of one heavy chain.

The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab fragment may be a cleavage product of the antibody by papain.

The Fc'' region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. The two heavy chain units are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

A Fab' fragment contains one light chain and a portion or fragment of one heavy chain containing a VH domain and a CH1 domain, as well as a region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments with the formation of the F(ab') ₂ molecule.

The F(ab') ₂ fragment contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab') ₂ fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The F(ab') ₂ fragment may be a product of the cleavage of the antibody by pepsin.

The Fv region contains variable regions of both heavy and light chains, but lacks constant regions.

The term isolated antibody refers to a purified state and in such a context the term means that 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 denote the complete absence of such material or the absence of water, buffers or salts unless they are present in amounts substantially interfering with the experimental or therapeutic use of the binding compound as provided herein.

As used herein, the term monoclonal antibody refers to a population of substantially homogeneous antibodies, i.e. The antibody molecules that make up the population are identical in amino acid sequence, with the exception of possible natural mutations that may be present in small quantities. In contrast, conventional (polyclonal) antibody preparations typically include many different antibodies having different amino acid sequences of their variable domains, often specific for different epitopes. The designation monoclonal indicates a type of antibody obtained from an essentially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies for use in the present invention can be produced by the hybridoma method first described in Kohler et al. (1975) Nature 256: 495, or by recombinant DNA methods (see, for example, US Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries by methods described, for example, in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

The term fully human antibody refers to an antibody containing only human immunoglobulin protein sequences. A fully human antibody may contain mouse carbohydrate chains if it is produced using a mouse, mouse cells, or a hybridoma derived from mouse cells. Likewise, the term mouse antibody refers to an antibody containing only mouse immunoglobulin sequences. Alternatively, the fully human antibody may contain rat carbohydrate chains if produced using a rat, rat cells, or a rat cell-derived hybridoma. Likewise, the term rat antibody refers to an antibody containing only rat immunoglobulin sequences.

In general, the basic structural unit of an antibody contains a tetramer. In a monospecific antibody, each tetramer comprises two identical pairs of polypeptide chains, and each pair contains one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region or variable domain of approximately 100 to 110 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.

Generally, human light chain constant regions are classified as kappa and lambda light chains. In addition, human heavy chain constant regions are generally classified as mu, delta, gamma, alpha, or epsilon, and they define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Subtypes of these IgG include, for example, IgG1 and IgG4. The present invention relates to anti-ILT4 antibodies and antigen binding fragments containing any of these light and/or heavy chain constant regions.

As used herein, the terms variable region, variable domain, V region or V chain refer to a segment of IgG chains that is variable in sequence among different antibodies. The term antibody variable region refers to the variable region of the light chain of an antibody

- 10 044253 or the variable region of the heavy chain of an antibody, either alone or in combination. The heavy chain variable region may be referred to as VH. The light chain variable region may be referred to as VL. Typically, the variable regions of the heavy and light chains contain three hypervariable regions, also referred to as complementarity determining regions (CDRs), located in relatively conserved framework regions (FRs). CDRs are typically aligned to framework regions and allow binding to a specific epitope. Generally, from N-terminus to C-terminus, the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is generally carried out in accordance with the definitions given in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

The term CDR refers to one of three hypervariable regions (H1, H2, or H3) in the non-framework region of the VH β-sheet framework of an antibody or one of three hypervariable regions (L1, L2, or L3) in the non-framework region of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed with framework region sequences. CDR regions are well known to those skilled in the art and are defined, for example, by Kabat as regions of greatest hypervariability in antibody variable domains. CDR region sequences are also defined structurally by Chothia as residues that are not part of the conserved β-sheet framework, and thus can adapt to a different conformation. Both terminologies are generally accepted in the field. CDR region sequences are also determined using AbM, Contact and IMGT. CDR positions in the canonical variable region of an antibody are determined by comparison of multiple structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues in the hypervariable region varies among antibodies, and additional residues relative to canonical positions are commonly numbered a, b, c, etc. next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is also well known to those skilled in the art. The correspondence between numbering systems, including, for example, Kabat numbering and the unique IMGT numbering system, is well known to one skilled in the art and is presented below in Table 1. 1. In some embodiments, the CDR is determined using the Kabat numbering system. In other embodiments, CDRs are determined by the IMGT numbering system. In other embodiments, CDRs are determined by the AbM numbering system. In other embodiments, CDRs are determined by the Chothia numbering system. In other embodiments, the CDR is determined by the Contact numbering system.

Table 1

Correspondence between CDR numbering systems

Kabat+ Chothia IMGT Kabat AbM Chothia Contact CDRl V _H 26-35 27-38 31-35 26-35 26-32 30-35 CDR2 _VH 50-65 56-65 50-65 50-58 52-56 47-58 CDR3 _VH 95-102 105-117 95-102 95-102 95-102 93-101 CDR1 V _L 24-34 27-38 24-34 24-34 24-34 30-36 CDR2 V _L 50-56 56-65 50-56 50-56 50-56 46-55 CDR3 V _L 89-97 105-117 89-97 89-97 89-97 89-96

The term sequence identity refers to the degree to which amino acids from two polypeptides are the same at equivalent positions in an optimal alignment of the two sequences.

Sequence similarity includes identical residues and non-identical, biochemically related amino acids. Biochemically related amino acids having similar properties that can be used interchangeably are described above.

The terms conservatively modified variants or conservative substitution refer to the replacement 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.) in a manner that can be performed repeatedly changes without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (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. Examples of conservative substitutions are given in table. 2.

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table 2

Examples of conservative amino acid substitutions

Original balance Conservative substitution Ala (A) Gly; Ser Arg(R) Lys; His Asn(N) Gin; His Asp (D) Glu; Asn Cys(C) Ser; Ala Gin (Q) Asn Glu (E) Asp; Gin Gly (G) Ala His(H) Asn; Gin He(I) Leu; Vai Leu (L) He; Vai Lys (K) Arg; His Met(M) Leu; He; Tyr Phe(F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Vai (V) He; Leu

As used herein, the term epitope refers to the region on an antigen to which an antibody or antigen-binding fragment binds. Binding of an antibody or antigen-binding fragment thereof as provided herein to an epitope means that the antibody or antigen-binding fragment thereof binds to one or more amino acid residues in the epitope.

The term isolated nucleic acid molecule or polynucleotide means DNA or RNA, for example, of genomic or synthetic origin, or DNA or RNA derived from mRNA or cDNA or some combination thereof, not associated with part or all of the polynucleotide in which the isolated polynucleotide is found in nature, or associated with a polynucleotide to which it is not associated in nature. For purposes of the present invention, it should be understood that a polynucleotide containing (or the like) a particular nucleotide sequence does not include intact chromosomes. Isolated polynucleotides containing specific nucleic acid sequences may include, in addition to the specific sequences, coding sequences for up to ten or even up to twenty or more other proteins or parts or fragments thereof, or they may include operably linked regulatory sequences that control expression of the coding region specified nucleic acid sequences, and/or may include vector sequences.

The phrase control sequences refers to polynucleotide sequences necessary or useful for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes, for example, include a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation 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 promoter, and/or an intron.

A nucleic acid is operably linked if it is located in a functional relationship with another polynucleotide. For example, DNA for a pre-sequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a precursor protein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence if it is located in such a way as to facilitate translation. Typically, but not necessarily, the term operably linked means that the linked polynucleotide sequences are contiguous and, in the case of a secretory leader sequence, contiguous and in reading frame. However, enhancers may not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If there are no such areas, synthetic oligonucleotide adapters are used.

- 12 044253 ters or linkers in accordance with generally accepted practice.

For the purposes of the invention, the expressions cell, cell line, and cell culture are used interchangeably, and all such designations include their progeny. Thus, the terms transformants and transformed cells include primary cells and cultures derived from them without regard to the number of transfers. It should also be understood that not all offspring will have exactly the same DNA content as a result of intentional or unintentional mutations. The term includes mutant progeny that is screened to have the same function or biological activity as the original transformed cell. Instances where different notations are necessary will be obvious from the context.

Host cells include eukaryotic and prokaryotic host cells, including mammalian cells. Host cells can be used as hosts for the expression of antibodies against ILT4 and antigen binding fragments thereof. Host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells and HEK-293 cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, equine and hamster cells. Other cell lines that can be used are insect cell lines (eg Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast cells and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum , Pichia pijperi, Pichia stiptis, Pichia Memanonica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Ch Rysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica and Neurospora crassa. The present invention relates to any host cell (eg, a CHO cell or a Pichia cell, eg, Pichia pastoris) containing an anti-ILT4 antibody or an antigen-binding fragment thereof, or containing a polynucleotide encoding such an antibody or fragment, or containing a vector containing the polynucleotide.

The terms treat or treat means administering anti-ILT4 antibodies or antigen-binding fragments thereof according to the present invention to an individual having one or more symptoms of a disease for which anti-ILT4 antibodies and antigen-binding fragments are effective, for example, in treating an individual having a cancer or infectious disease or, suspected of having a malignant neoplasm or infectious disease for which the agent has therapeutic activity. Typically, the antibody or fragment is administered in an effective amount or effective dose that will alleviate one or more symptoms (eg, cancer or infectious disease) in the individual or population being treated, inducing regression or elimination of such symptoms, or inhibiting the progression of such symptoms, for example, symptoms of malignancy, such as tumor growth or metastasis, to any clinically significant extent. The effective amount of the antibody or fragment may vary depending on factors such as the stage of the disease, the age and body weight of the patient, and the ability of the drug to produce the desired response in the individual.

As used herein, the term 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 relates to antibodies and antigen-binding fragments thereof, as described herein, that specifically bind to ILT4. An antibody or antigen binding fragment specifically binds to a polypeptide containing the specified sequence (e.g., human ILT4) if it binds to polypeptides containing the sequence with a K _D of approximately 20 nM or higher affinity (e.g., approximately 17 nM, 10 nM, 5 nM , 1 nM, 100 pM or 1 pM), but does not bind to proteins in which the sequence is missing. For example, an antibody or antigen-binding fragment that specifically binds to a polypeptide containing human ILT4 may bind to a FLAG®-tagged form of human ILT4, but does 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:

- 13 044253

MTPIVTVLIC LGLSLGPRTH VQTGTIPKPT LWAEPDSVIT QGSPVTLSCQ GSLEAQEYRL 60 YREKKSASWI TRIRPELVKN GQFHIPSITW EHTGRYGCQY YSRARWSELS DPLVLVMTGA 120 YPKPTLSAQP SPVVTSGGRV TLQCESQVAF GGFILCKEGE EEHPQCLNSQ PHARGSSRAI 180 FSVGPVSPNR RWSHRCYGYD LNSPYVWSSP SDLLELLVPG VSKKPSLSVQ PGPVVAPGES 240

LTLQCVSDVG YDRFVLYKEG ERDLRQLPGR QPQAGLSQAN FTLGPVSRSY GGQYRCYGAH 300

NLSSECSAPS DPLDILITGQ IRGTPFISVQ PGPTVAGEN VTLLCQSWRQ FHTFLLTKAG 360

AADAPLRLRS IHEYPKYQAE FPMSPVTSAH AGTYRCYGSL NSDPYLLSHP SEPLELVVSG 420

PSMGSSPPPT GPISTPAGPE DQPLTPTGSD PQSGLGRHLG VVIGILVAVV LLLLLLLLLF 480

LILRHRRQGK HWTSTQRKAD FQHPAGAVGP EPTDRGLQWR SSPAADAQEE NLYAAVKDTQ 540 PEDGVEMDTR AAASEAPQDV TYAQLHSLTL RRKATEPPPS QEREPPAEPS IYATLAIH 598 (SEQ ID NO: 40; signal sequence underlined). See Uniprot Registration No. Q8N423.

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

QTGTIPKPT LWAEPDSVIT QGSPVTLSCQ GSLEAQEYRL 39

YREKKSASWI TRIRPELVKN GQFHIPSITW EHTGRYGCQY YSRARWSELS DPLVLVMTGA 99

YPKPTLSAQP SPVVTSGGRV TLQCESQVAF GGFILCKEGE EEHPQCLNSQ PHARGSSRAI 159 FSVGPVSPNR RWSHRCYGYD LNSPYVWSSP SDLLELLVPG VSKKPSLSVQ PGPVVAPGES 219 LTLQCVSDVG YDRFVLYKEG ERDLRQLPGR QPQAGLSQAN FTLGPVSRSY GGQYRCYGAH 279 NLSSECSAPS DPLDILITGQ IRGTPFISVQ PGPTVASGEN VTLLCQSWRQ FHTFLLTKAG 339

AADAPLRLRS IHEYPKYQAE FPMSPVTSAH AGTYRCYGSL NSDPYLLSHP SEPLELVVSG 399

PSMGSSPPPT GPISTPAGPE DQPLTPTGSD PQSGLGRHLG VVIGILVAVV LLLLLLLLLF 459

LILRHRRQGK HWTSTQRKAD FQHPAGAVGP EPTDRGLQWR SSPAADAQEE NLYAAVKDTQ 519 PEDGVEMDTR AAASEAPQDV TYAQLHSLTL RRKATEPPPS QEREPPAEPS IYATLAIH 577 (SEQ ID NO: 78).

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

- 14 044253

MTPILMVLIC LGLSLGPRTH VQAGILPKPT LWAEPGSVIS EGSPVTLRCQ GSLQVQEYHL 60

YREKNPASWV RQIRQELVKK GYFAIGFITW EHTGQYRCQY YSHSWWSEPS DPLELVVTGA 120

YSKPTLSALP SPVVASGGNV TLQCDSQVAF DSFTLCKEGE DEHPQRLNCQ SHARGWSWAV 180 FSVGPVSPSR rwsyrcygyi ssapnvwslp sdllellvpg vskkpslsvq PGPVVAPGDK 240

LTLQCGSDAG YDRFALYKEG EGDFLQRPVR QPQAGLSQAN FLLGPVSRSH GGQYRCSGAH 300

NLSSEWSAPS DPLDILIAGQ IRGRPFLSVQ PGPKVVSGEN VTLLCQSSWQ FHAFLLTQAG 360

AADAHLHLRS MYKYPKYQAE FPMSPVTSAH AGTYRCYGSR SSNPYLLSVP SDPLELVVSG 420

PSGGPSSPTT GPTSTCGPED QPLTPTGSAP QSGLGRHLGV VTGVLVAFVL LLFLLLLLFL 480

VLRYRRQGKR WTSAQRKADF QHPAGAVEPE PRDRGLQRRS SPAADTQEEN LYAAVKDTQP 540

EDGVELDSRA AASEDPQDVT YAQLQSLTLR REATEPPPSQ ERAPPVESSI YATLTIH 597 (SEQ ID NO: 43; signal sequence underlined). See NCBI refseq XP_005590753.

In an embodiment of the invention, the signal sequence for expression of ILT4 or any other polypeptide provided herein is MTPILMVLICLGLSLGPRTHV (amino acids 1-21 SEQ ID NO: 40), or MTPIVTVLICLGLSLGPRTHV (amino acids 1-21 SEQ ID NO: 43), or MPLLLLLPLLWAGALA (SEQ ID NO: 46).

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

QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPEL VKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTS GGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRC YGYDLNSPYVWSSPSDLLELL VPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFV LYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLD ILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYP KYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLE LVVSGPSMGSSPPPTGPISTP AGPEDQPLTPTGSDPQSGLGRHLGV (amino acids 22-461 SEQ ID NO: 40), or an immunoglobulin fusion protein (e.g., IgG1 or IgG4), or a transmembrane form (TM) expressed on the cell surface:

QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPEL VKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTS GGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRC YGYDLNSPYVWSSPSDLLELL VPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFV LYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLD ILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYP KYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLE LVVSGPSMGSSPPPTGPISTP AGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQGKH (amino acids 22-491 SEQ ID NO: 40). Antibodies and antigen-binding fragments.

The present invention relates to antibodies and antigen binding fragments thereof (eg, fully human antibodies) that bind to ILT4 (herein referred to as anti-ILT4 antibodies), and methods of using the antibodies or antigen binding fragments thereof in the treatment or prevention of disease. In one embodiment, the invention relates to antagonistic antibodies against ILT4 and methods of using the antibodies or antigen-binding fragments thereof in the treatment or prevention of a disease.

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

binds to human ILT4 at one or more amino acid residues in LYREKKSASW (SEQ ID NO: 59), TRIRPEL (SEQ ID NO: 60), NGQF (SEQ ID NO: 61) and/or HTGRYGCQ (SEQ ID NO: 62) and/or 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 exchange (eg, D ₂ O), for example , determined by hydrogen-deuterium exchange mass spectrometry, and/or associated with ILT4 with a heat map substantially such as that presented in FIG. 1;

- 15 044253 binds to human ILT4 in domain 1 (see Wilcox et al. BMC Structural Biology 2:6 (2002));

binds to the extracellular domain of human ILT4 or the TM form of ILT4 expressed on the surface of a cell, such as pre-B cells, Chinese hamster ovary cells, U937 cells, or Jurkat JE6 cells;

calculated pI ~7.29 (e.g. 7.29 or 7.30);

experimentally determined pI ~7.2;

differs in a thermogram having an initial Tm >60°C, Tm1 ~65.2°C and Tm2 ~78.8°C;

binds to human ILT4 with a KD of approximately 1.7 x 10 ^-8 M (eg, as determined by surface plasmon resonance, eg, binding of anti-ILT4 antibody to polyhistidine-labeled human ILT4);

Ka=5.5x10 ⁵ M ^-1 s ^-1 (eg, as determined by surface plasmon resonance binding of anti-ILT4 antibody to polyhistidine-labeled human ILT4);

Kd=9x10 ^-3 s ^-1 (eg, as determined by surface plasmon resonance, eg, binding of anti-ILT4 antibody to polyhistidine-labeled human ILT4);

blocks the binding of HLA-G (eg, Fc fusion HLA-G) to human ILT4 (eg, ILT4 on mouse 3A9 T cells transfected with and expressing ILT4), e.g., with an IC ₅₀ of approximately 0.25 μg/ml (±0.06 μg/ml), for example, as determined by surface plasmon resonance;

blocks the binding of HLA-A, HLA-B (eg, fluorochrome-labeled dexamers of HLA-A, such as HLA*A2:01, or HLA-B, such as HLA*B7:02), and/or HLA-F (eg, fluorochrome-labeled HLA-F tetramers) with ILT4 (eg, ILT4 on mouse 3A9 T cells transfected with and expressing ILT4), for example, as determined by surface plasmon resonance;

blocks the binding of ILT4 (eg, ILT4 on mouse 3A9 T cells transfected with and expressing ILT4) to ANGPTL1, ANGPTL4 and/or ANGPTL7 (eg, biotinylated ANGPTL proteins), for example, as determined by surface plasmon resonance;

does not bind to ILT2, ILT3, ILT5, LILRB5, LILRA1, LILRA2, ILT7, ILT8 and/or ILT11;

reverses ILT4-mediated suppression of IL-2 in ILT4-transfected 3A9 cells, e.g., with an EC _{50 of} 0.43 μg/ml (±0.14 μg/ml);

restores ILT4:HLA-G-induced suppression of mast cell degranulation (e.g., in the presence of a plate-bound HLA-G tetramer), e.g., where mast cells express ILT4 and CD200RLa and are stimulated, e.g., using antibody-mediated cross-linking of CD200RLa ;

increases lipopolysaccharide (LPS)-induced expression of proinflammatory myeloid cytokines, such as GM-CSF and/or TNFa, from peripheral blood mononuclear cells (PBMCs);

increases anti-CD3 antibody-induced expression of proinflammatory myeloid cytokines, such as GM-CSF and/or TNFa, from peripheral blood mononuclear cells (PBMCs);

inhibits tumor growth in humans or, for example, other mammals, such as mice (eg, immunodeficient NSG mice) reconstituted using human hematopoietic stem cells, for example, including human peripheral CD45+ immune cells, for example, where the tumor is a human cutaneous melanoma , for example, from the SKMEL5 cell line;

facilitates macrophage/myeloid-derived suppressor cell (MDSC)-mediated tumor tolerance in an individual (eg, human) with a tumor;

does not bind to cynomolgus ILT4 and/or mouse pirB; and/or it is used to stain human CD14+ monocytes and/or human CD11B+ granulocytes;

binds to one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10) of the human ILT4 haplotypes listed in Table. 7.

Antibody heavy chain 1E1 (Q1E) (IgG4).

Heavy chain

EVOLOQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIROPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNOFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGR GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1; variable domain underlined; CDRs indicated by double underscore).

Heavy chain variable domain

- 16 044253

EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGR GTLVTVSS (SEQ ID NO: 63).

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

Heavy chain

EVQLOOWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIROPPGKGLEWIGEINHA GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVT AARTA VYYCARLPTRWVTTRYFDLWGR GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHA GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARLPTRWVTTRYFDLWGR GTLVTVSS (SEQ ID NO: 57).

Heavy chain of antibody 1E1 (IgG1).

Heavy chain

OVOLQOWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIROPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVT AARTA VYYCARLPTRWVTTRYFDLWGR GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGR GTLVTVSS (SEQ ID NO: 69).

Heavy chain CDR 1E1.

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

CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (for example, EINHSGSTNYNPSLKS EINHAGSTNYNPSLKS) (SEQ ID NO: 17);

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

Antibody light chain 1E1 (Q1E) (lambda).

Light chain or

ESVLTOPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOLPGTAPKLLIYGNSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCOSFDNSLSAYVFGGGTOLTVLGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL (SEQ ID NO: 70).

Antibody light chain 1E1 (Q1E, S54A) (lambda).

Light chain

ESVLTQPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOLPGTAPKLLIYGNAN RPSGVPDRFSVSKSGASASLAITGLOAEDEADYYCOSFDNSLSAYVFGGGTQLTVLGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 4; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNAN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

- 17 044253 (SEQ ID NO: 71).

Antibody light chain 1E1 (Q1E, N53Q) (lambda).

Light chain

ESVLTOPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOLPGTAPKLLIYGQSN RPSGVPDRFSVSKSGASASLAITGLOAEDEADYYCOSFDNSLSAYVFGGGTOLTVLGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS (SEQ ID NO: 5; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGQSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL (SEQ ID NO: 72).

Antibody light chain 1E1 (Q1E, N53E) (lambda).

Light chain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGESN RPSGVPDRFSVSKSGASASLAITGLOAEDEADYYCOSFDNSLSAYVFGGGTQLTVLGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 6; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGESN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL (SEQ ID NO: 73).

Antibody light chain 1E1 (Q1E, N53D) (lambda).

Light chain

ESVLTQPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOLPGTAPKLLIYGDSN RPSGVPDRFSVSKSGASASLAITGLOAEDEADYYCOSFDNSLSAYVFGGGTQLTVLGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 7; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL (SEQ ID NO: 58).

Antibody light chain 1E1 (lambda).

Light chain

OSVLTOPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOLPGTAPKLLIYGNSN RPSGVPDRFSVSKSGASASLAITGLOAEDEADYYCOSFDNSLSAYVFGGGTQLTVLGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 45; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL (SEQ ID NO: 77).

Light chain CDR 1E1.

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

CDR-L2: GX1X ₂ NRPS; where X ₁ is N, Q, E or D, and X ₂ is S or A (for example, GNSNRPS, GNANRPS, GQSNRPS, GESNRPS or GDSNRPS) (SEQ ID NO: 20);

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

Antibodies and antigen binding fragments thereof comprising the 1E1 heavy and light chain CDRs, or 1E1 VH and VL, or 1E1 heavy chain and light chain (or a variant thereof, for example as defined herein), may be referred to as 1E1.

Antibody heavy chain 2A6 (Q1E) (IgG4).

Heavy chain

- 18 044253

EVQLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMGGIIPIF GTANYAOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDSSGWYKGGAFDI WGOGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTCNV DHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIF GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDSSGWYKGGAFDI WGQGTMVTVSS (SEQ ID NO: 64).

Antibody heavy chain 2A6 (Q1E, S102A, M119L) (IgG4).

Heavy chain

EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGOGLEWMGGIIPIF GTANYAOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDASGWYKGGAFDI WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIF GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDASGWYKGGAFDI WGQGTLVTVSS (SEQ ID NO: 65).

Antibody heavy chain 2A6 (Q1E, D101S, M119L) (IgG4).

Heavy chain

EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMGGIIPIF GTANYAOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFSSSGWYKGGAFDI WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIF GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFSSSGWYKGGAFDI WGQGTLVTVSS (SEQ ID NO: 66).

The present invention relates to 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.

Heavy chain CDR 2A6.

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

CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23);

CDR-H3: YFXjX ₂ SGWYKGGAFDI; where X1 is D or S, and X ₂ is S or A (for example, YFDSSGWYKGGAFDI, YFDASGWYKGGAFDI or YFSSSGWYKGGAFDI) (SEQ ID NO: 24).

Antibody light chain 2A6 (lambda).

Light chain

QSVLTQPSSLSASPGASASLTCTLRSGINVDTYRIHWYOOKPGSPPOYLLRYKSDS DKHOGSGVPSRFSGSKDPSANAGILLISGLOSEDEADYYCAIWYSSTWVFGGGTQLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS (SEQ ID NO: 11; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

- 19 044253

QSVLTQPSSLSASPGASASLTCTLRSGINVDTYRIHWYQQKPGSPPQYLLRYKSDS

DKHQGSGVPSRFSGSKDPSANAGILLISGLQSEDEADYYCAIWYSSTWVFGGGTQLTVL (SEQ ID NO: 74).

The present invention relates to antibodies and antigen-binding fragments thereof, where the residue

SEQ ID NO: 11 or 74 is E instead of Q.

Light chain CDR 2A6.

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 comprising 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, for example as defined herein) may be referred to as 2A6.

Antibody heavy chain 3G7 (Q1E) (IgG4).

Heavy chain

EVOLVESGGGVVOPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISY DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGEWIOLWSPFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISY DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGEWIQLWSPFDY WGQGTLVTVSS (SEQ ID NO: 67).

The present invention relates to antibodies and antigen binding fragments thereof, wherein residue 1 of SEQ ID NO: 12 or 67 is Q instead of E. Heavy chain CDR 3G7 CDR-H1: SYAMH (SEQ ID NO: 28) CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO : 29) CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30).

Antibody light chain 3G7 (kappa).

Light chain

DIQMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOQKPGKAPKFLIYAASSLO SGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCOOYNSYPPTFGGGTKVEIKRtVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKFLIYAASSLQ SGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK (SEQ ID NO: 75).

Light chain CDR 3G7.

CDR-L1: RASQGISSWLA (SEQ ID NO: 31);

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

CDR-L3: QQYNSYPPT (SEQ ID NO: 33).

Antibodies and antigen binding fragments thereof comprising the 3G7 heavy and light chain CDRs, or the 3G7 V _H and V _L , or the 3G7 heavy chain and light chain (or a variant thereof, for example as defined herein) may be referred to as 3G7.

Antibody heavy chain 2C1 (Q1E) (IgG4).

Heavy chain

EVQLVOSGAEVKKPGASVKVSCKVSGYTLTELSMHWVROAPGKGLEWMGGFD PEDGETIYAQKFOGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARAGPLYTIFGVVIIP DNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 14; variable domain underlined; CDRs indicated by double underscore). Heavy chain variable domain

- 20 044253

EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFD PEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARAGPLYTIFGVVIIP DNWFDPWGQGTLVTVSS (SEQ ID NO: 68).

The present invention relates to antibodies and antigen binding fragments thereof, wherein residue 1 of SEQ ID NO: 14 or 68 is Q instead of E.

Heavy chain CDR 2C1.

CDR-H1: ELSMH (SEQ ID NO: 34);

CDR-H2: GFDPEDGETIYAQKFQG (SEQ ID NO: 35);

CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQ ID NO: 36).

Antibody light chain 2C1 (Q1E) (lambda).

Light chain

ESVLTOPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOQLPGTAPKLLIYGNSN RPSGVPDRFSGSKSGTSASLAITGLOAEDEADYYCOSYDSSLSGSGVVFGGGTOLIILGOP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS (SEQ ID NO: 15; variable domain is underlined; CDRs are indicated by double underscore). Light chain variable domain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSN RPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGSGVVFGGGTQLIIL (SEQ ID NO: 76).

The present invention relates to antibodies and antigen-binding fragments thereof, where the residue

SEQ ID NO: 15 or 76 is Q instead of E. Light chain CDR 2C1.

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 comprising the 2C1 heavy and light chain CDRs, or the 2C1 VH and V _L , or the 2C1 heavy chain and light chain (or a variant thereof, for example as defined herein), may be referred to as 2C1.

In various embodiments, the antibody or antigen binding fragment thereof lacks the C-terminal lysine of the immunoglobulin heavy chain.

Thus, in some embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO: 3, 4, 5. 6, 7, 11, 13, 15 or 45; and/or the immunoglobulin heavy chain contains 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: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1 or 79; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 3.

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

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

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

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

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

In some embodiments, the antibody or antigen binding fragment thereof comprises:

- 21 044253 immunoglobulin heavy chain containing the amino acid sequence given in

SEQ ID NO: 8 or 82; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 11.

In other embodiments, the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 or 83; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 11.

In other embodiments, the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 84; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 11.

In other embodiments, the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12 or 85; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 13.

In other embodiments, the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 14 or 86; and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 15.

In some other embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, 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 contains the amino acid sequence set forth in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68, or 69.

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: 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 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 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 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: 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 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: 66, 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: 67, and a light chain variable domain containing the amino acid sequence set forth in SEQ ID NO: 75.

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: 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 to ILT4 comprises an immunoglobulin light chain variable domain (V _L ) containing CDR-L1, CDR-L2 and CDR-L3 1E1 (e.g., SEQ ID NO: 19-21), and an immunoglobulin heavy chain variable domain (VH) containing CDR-H1, CDR-H2 and CDR-H3 of 1E1 (eg, SEQ ID NO: 16-18).

In a further embodiment, the antibody or antigen binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable domain (VL) comprising CDR-L1, CDR-L2 and CDR-L3 2A6 (e.g., SEQ ID NO: 25-27), and an immunoglobulin heavy chain variable domain ( _VH ) containing CDR-H1, CDR-H2 and CDR-H3 2A6 (eg, SEQ ID NO: 22-24).

In a further embodiment, the antibody or antigen binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable domain (V _L ) containing CDR-L1, CDR-L2 and CDR-L3 3G7 (e.g., SEQ ID NO: 31-33), and an immunoglobulin heavy chain variable domain ( _VH ) domain containing CDR-H1, CDR-H2 and CDR-H3 of 3G7 (eg, SEQ ID NO: 28-30).

In a further embodiment, the antibody or antigen binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable domain (VL) comprising CDR-L1, CDR-L2 and CDR-L3 2C1 (e.g., SEQ ID NO: 37-39), and an immunoglobulin heavy chain variable domain ( _VH ) containing CDR-H1, CDR-H2 and CDR-H3 2C1 (eg, SEQ ID NO: 34-36).

In one embodiment, the antibody or antigen binding fragment comprises: a V _H domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _L containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _L containing: 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 Vh 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 domain V _L containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO : 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a V _H domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _L containing: 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 Vh 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 domain V _L containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _L containing: 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 Vh 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 domain V _L containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO : 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a V _H domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or V _L domain containing: CDR-L1: TGSSSNI-23 044253

GAGYDVH (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 containing: 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 VL domain containing: 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 VH 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 VL domain containing: 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 VH 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 VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a VH 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 VL domain containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _L containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _L containing: 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 containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL ( SEQ ID NO: 18), and/or domain V _L containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, the antibody or antigen binding fragment thereof comprises: a V _H domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or domain V _L containing: 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 to ILT4 and contains 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 to ILT4 and contains 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 to ILT4 and contains the V _L domain of the 3G7 antibody (eg, SEQ ID NO: 75) and/or the V _H domain of the 3G7 antibody (eg, SEQ ID NO: 67).

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

The present invention further provides an antibody or antigen-binding fragment thereof that binds to ILT4 and contains an immunoglobulin light chain of antibody 1E1 (e.g., SEQ ID NO: 3, 4, 5, 6, 7 or 45) and/or an immunoglobulin heavy chain 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 to ILT4 and contains an immunoglobulin light chain of antibody 2A6 (e.g., SEQ ID NO: 11) and/or an immunoglobulin heavy chain of antibody 2A6 (e.g., SEQ ID NO: 8, 9, 10 , 82, 83 or 84).

- 24 044253

The present invention further provides an antibody or antigen binding fragment thereof that binds to ILT4 and contains the light chain of the 3G7 antibody immunoglobulin (e.g.

SEQ ID NO: 13) and/or 3G7 antibody immunoglobulin heavy chain (eg, SEQ ID NO: 12 or 85).

The present invention further provides an antibody or antigen binding fragment thereof that binds to ILT4 and comprises an antibody 2C1 immunoglobulin light chain (e.g., SEQ ID NO: 15) and/or an antibody 2C1 immunoglobulin heavy chain (e.g., SEQ ID NO: 14 or 86).

The present invention further relates to an antibody consisting of two heavy chains and two light chains, wherein each light chain contains a V _L or an immunoglobulin light chain of an antibody 1E1, 2A6, 3G7 or 2C1, and each heavy chain contains a VH or an immunoglobulin heavy chain of an antibody 1E1, 2A6, 3G7 or 2C1.

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

In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain contains the amino acid sequence set forth in SEQ ID NO: 58, and each heavy chain contains 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 contains the amino acid sequence set forth in SEQ ID NO: 7, and each heavy chain contains 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, where 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 of the present invention contains a VL (with or without a signal sequence), for example, a VL in any of SEQ ID NO: 58 or 70-77, containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions; and/or VH (with or without a signal sequence), for example, VH in any of SEQ ID NO: 57 or 63-69, containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions, but still binds to ILT4.

The present invention also relates to polypeptides containing the amino acid sequences presented herein, for example SEQ ID NO: 1-39, 44, 45, 47-58, 63-77 or 79-86, as well as polypeptides containing 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.

In some embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain has at least 90% amino acid sequence identity with respect to the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 11, 13, 15 or 45, and/or the immunoglobulin heavy chain has at least 90% amino acid sequence identity with respect to the amino acid sequence shown 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 an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain 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 immunoglobulin heavy chain contains a heavy chain variable domain having at least 90% amino acid sequence identity with respect to amino acid sequence shown in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68 or 69.

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

MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 42).

In an embodiment of the invention, the N-terminal glutamine (Q) of the immunoglobulin chain described herein (eg, heavy and/or light) is replaced with pyroglutamic acid. In one embodiment, the N-terminal Q of the immunoglobulin heavy chain is replaced with pyroglutamic acid. In another embodiment, the N-terminal Q of the immunoglobulin light chain is replaced with pyroglutamic acid. In yet another embodiment, the N-terminal Q of the immunoglobulin heavy chain and the N-terminal Q of the immunoglobulin heavy chain are replaced with pyroglutamic acid.

The present invention also provides antibodies or antigen binding fragments that bind to the same epitope of ILT4 (eg, human ILT4) as any anti-ILT4 antibody or antigen binding fragment thereof provided herein (eg, 1E1, 2A6, 3G7 or 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 another embodiment, the epitope is HTGRYGCQ (SEQ ID NO: 62). In some embodiments, the antibody or antigen binding fragment thereof binds to the same human ILT4 epitope as the 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 11; 10 and 11; 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 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen binding fragment thereof binds to the same human ILT4 epitope as the 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 includes antibodies and antigen binding fragments that cross-block the binding of any anti-ILT4 antibody or antigen binding fragment thereof provided herein (e.g., 1E1, 2A6, 3G7 or 2C1) with ILT4 (e.g., human ILT4), or compete with any antibody against ILT4 or an antigen-binding fragment thereof as provided herein (eg, 1E1, 2A6, 3G7 or 2C1), with ILT4 (eg, human ILT4). Cross-blocking antibodies and antigen-binding fragments thereof provided herein can be identified by their ability to block the binding of any of the antibodies or fragments specifically described herein to ILT4 in binding assays (e.g., biolayer interferometry (BLI; e.g., binding FORTEBIO OCTET; Pall ForteBio Corp; Menlo Park, CA), surface plasmon resonance (SPR), BIACore, ELISA, flow cytometry). For example, in an embodiment of the invention using BLI, the tip of a fiber optic probe is coated with a ligand (e.g., ILT4) and used as a biosensor where binding of an anti-ILT4 antibody or antigen binding fragment to ILT4 changes the interference pattern of white light reflected from the ILT4-bound probe layer. and an internal reference layer. The shift indicates ILT4/anti-ILT4 binding. In an embodiment of the invention, the ILT4-coated tip is immersed in an analyte solution containing an antibody or antigen-binding fragment, for example, in a well of a 96- or 384-well plate. In an embodiment of the invention, the plate is shaken during reading to create orbital flow. For reading analysis, white light is directed along the length of the fiber. As stated above, the interference of light reflected from the reference layer and immobilized surfaces containing ILT4 from the tip creates a characteristic pattern of light returning up the fiber. When molecules bind to an immobilized sensor surface, this pattern changes in proportion to the degree of binding. For example, assays may be used in which an ILT4 protein (eg, human ILT4) is immobilized on a BLI probe or plate, a reference anti-ILT4 antibody or fragment binds to ILT4 (eg, at a saturating concentration), and a test anti-ILT4 antibody or fragment is added. The ability of the test antibody to compete with a reference antibody for binding to ILT4 is then determined. In the BLI format, ILT4 complex interference is monitored to determine whether the antibody competes effectively with a reference antibody, for example, by monitoring a shift in the wavelength of light in nanometers over time, where the shift indicates additional binding of the test antibody and lack of cross-blocking. In the BLI embodiment, cross-blocking is qualitatively considered to occur between antibodies if no additional binding of the test antibody is observed. In an embodiment of the invention, cross-blocking of the reference antibody by itself is confirmed as a control; where it is believed that the analysis was carried out correctly,

- 26 044253 if the reference antibody can cross-block its own binding to ILT4. The ability of a test antibody to inhibit the binding of an anti-ILT4 antibody or fragment 1E1, 2A6, 3G7 or 2C1 to ILT4 (e.g., human ILT4) indicates that the test antibody can cross-block binding of the antibody or fragment to ILT4 (e.g., human ILT4) and thus thus, in some cases, may bind to the same epitope of ILT4 (eg, human ILT4) as 1E1, 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 are also part of the present invention. In an embodiment of the invention, BLI is performed in a sandwich format where an anti-ILT4 reference antibody or antigen-binding fragment is immobilized on a probe and then binds to ILT4. The test anti-ILT4 antibody or antigen binding fragment is then tested for its ability to bind to a reference antibody or fragment.

In some 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 11; 9 and 11; 10 and 11; 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 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15; respectively. In some embodiments, the antibody or antigen binding fragment thereof competes for binding to human ILT4 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.

The present invention relates to anti-ILT4 antibodies and antigen binding fragments thereof containing N-linked glycans typically added to immunoglobulins produced in Chinese hamster ovary cells (N-linked CHO glycans) or engineered yeast cells (N-linked engineered yeast glycans) , such as Pichia pastoris. For example, in an embodiment of the invention, anti-ILT4 antibodies and antigen binding fragments thereof comprise one or more of the engineered yeast N-linked glycans or the CHO N-linked glycans shown in Figures 16 (e.g., G0 and/or G0-F and/or G1, and/or G1-F, and/or G2-F, and/or Man5). In an embodiment of the invention, the anti-ILT4 antibodies and antigen binding fragments thereof comprise engineered yeast N-linked glycans, i.e. G0 and/or G1 and/or G2, optionally further including Man5. In an embodiment of the invention, anti-ILT4 antibodies and antigen-binding fragments thereof contain N-linked CHO glycans, i.e. G0-F, G1-F and G2-F, optionally further including G0 and/or G1 and/or G2 and/or Man5. 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 anti-ILT4 antibodies and antigen binding fragments thereof are engineered yeast N-linked glycans or N-linked glycans CHO. See Nett et al. Yeast. 28(3): 237252 (2011); Hamilton et al. Science. 313(5792): 1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5): 387-392 (2007). For example, in an embodiment of the invention, the engineered yeast cell is GFI5.0, or YGLY8316, or the strains shown in US Pat. No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also International Patent Application Publication No. WO 2013/066765.

Polynucleotides.

The present invention includes polynucleotides (eg, DNA or RNA) encoding immunoglobulin chains from anti-ILT4 antibodies and antigen-binding fragments thereof provided herein. For example, the present invention relates to nucleic acids encoding immunoglobulin heavy and/or light chains from antibodies 1E1, 2A6, 3G7 and 2C1 (for example, SEQ ID NOs: 1-15, 44 and 45 or a variable domain thereof), as provided herein description, as well as nucleic acids that hybridize with them.

The present invention relates to polynucleotides encoding the variable domain (VL) of an antibody light chain containing CDR-L1, CDR-L2 and CDR-L3 1E1 (for example, containing the amino acids set forth in SEQ ID NO: 19-21), and/or variable domain ( _VH ) of the antibody heavy chain containing CDR-H1, CDR-H2 and CDR-H3 of 1E1 (eg, containing the amino acids set forth in SEQ ID NO: 1618).

The present invention relates to polynucleotides encoding the variable domain (V _L ) of an antibody light chain containing CDR-L1, CDR-L2 and CDR-L3 2A6 (for example, containing the amino acids set forth in SEQ ID NO: 25-27), and/or an antibody heavy chain variable domain (VH) comprising CDR-H1, CDR-H2 and CDR-H3 2A6 (eg, containing the amino acids set forth in SEQ ID NO: 2224).

The present invention relates to polynucleotides encoding the variable domain (VL) of an antibody light chain containing CDR-L1, CDR-L2 and CDR-L3 3G7 (for example, containing the amino acids set forth in SEQ ID NO: 31-33), and/or variable domain (VH) of the antibody heavy chain containing CDR-H1, CDR-H2 and CDR-H3 3G7 (for example, containing the amino acids set forth in SEQ ID NO: 28-27 044253

thirty).

The present invention relates to polynucleotides encoding the variable domain (V _L ) of an antibody light chain containing CDR-L1, CDR-L2 and CDR-L3 2C1 (for example, containing the amino acids set forth in SEQ ID NO: 37-39), and/or an antibody heavy chain variable domain (VH) comprising CDR-H1, CDR-H2 and CDR-H3 2C1 (eg, containing the amino acids set forth in SEQ ID NO: 3436).

The present invention provides polynucleotides encoding the V _L domain of antibody 1E1 (for example, SEQ ID NO: 70, 71, 72, 73, 58 or 77) and/or the VH domain of antibody 1E1 (for example, SEQ ID NO: 63, 57 or 69 ).

The present invention provides polynucleotides encoding the V _L domain of antibody 2A6 (eg, SEQ ID NO: 74) and/or the VH domain of antibody 2A6 (eg, SEQ ID NO: 64, 65 or 66).

The present invention provides polynucleotides encoding the V _L domain of antibody 3G7 (eg, SEQ ID NO: 75) and/or the VH domain of antibody 3G7 (eg, SEQ ID NO: 67).

The present invention provides polynucleotides encoding the VL domain of antibody 2C1 (eg, SEQ ID NO: 76) and/or the _VH domain of antibody 2C1 (eg, SEQ ID NO: 68).

The present invention provides polynucleotides encoding the immunoglobulin light chain of antibody 1E1 (e.g., SEQ ID NO: 3, 4, 5, 6, 7, or 45) and/or the immunoglobulin heavy chain of antibody 1E1 (e.g., SeQ ID NO: 1, 2 or 44).

The present invention provides polynucleotides encoding a 2A6 immunoglobulin light chain (eg, SEQ ID NO: 11) and/or a 2A6 immunoglobulin heavy chain (eg, SEQ ID NO: 8, 9 or 10).

The present invention provides polynucleotides encoding a 3G7 antibody immunoglobulin light chain (eg, SEQ ID NO: 13) and/or a 3G7 antibody immunoglobulin heavy chain (eg, SEQ ID NO: 12).

The present invention provides polynucleotides encoding a 2C1 immunoglobulin light chain (eg, SEQ ID NO: 15) and/or a 2C1 immunoglobulin heavy chain (eg, SEQ ID NO: 14).

In a specific embodiment, the polynucleotide contains the nucleotide sequence set forth in SEQ ID NO: 87. In another specific embodiment, the polynucleotide contains the nucleotide sequence set forth in SEQ ID NO: 88. In yet another embodiment, the polynucleotide contains the nucleotide sequence set forth in SEQ ID NO: : 87, and the nucleotide sequence given in SEQ ID NO: 88.

The present invention also provides expression vectors containing isolated nucleic acids of the invention, wherein the nucleic acid is operably linked to one or more control sequences, eg, recognized by a host cell if the host cell is transfected using the vector. The present invention also relates to host cells containing the expression vector of the present invention. In some embodiments, the host cells are CHO cells.

Methods for producing antibodies and their antigen-binding fragments

Antibodies against ILT4 and antigen binding fragments presented herein (eg, 1E1, 2A6, 3G7 and/or 2C1) can be produced recombinantly. In this embodiment, nucleic acids encoding one or more of the antibody immunoglobulin chains and antigen binding fragments of the invention (e.g., any of SEQ ID NOs: 1-15, 44, 45 or 79-86 or containing VH or V _L 1E1, 2A6, 3G7 or 2C1 shown in any of SEQ ID NO: 57, 58, 63-77) can be inserted into a vector and/or host cell chromosome and expressed in a recombinant host cell. There are several methods known in the art by which recombinant antibodies are produced.

Antibodies (eg, 1E1, 2A6, 3G7 or 2C1) can be isolated from the culture medium using standard protein purification methods. In addition, the expression of the immunoglobulin chains of the invention from production cell lines can be increased using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is a common approach to increase expression in some conditions. The present invention relates to vectors containing one or more polynucleotides encoding one or more of these immunoglobulin chains and the glutamine synthetase (GS) gene. In an embodiment of the invention, the vector is located in a host cell that lacks functional glutamine synthetase. In an embodiment of the invention, the host cell is in a culture medium that is substantially free of glutamine. Methods for producing one or more of such immunoglobulin chains, or an anti-ILT4 antibody, or an antigen-binding fragment thereof, comprising culturing such a host cell in a culture medium that is substantially free of glutamine are within the scope of the present invention, as are such antibodies. and fragments obtained in this way.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation profile characteristic of the glycoproteins produced in the line

- 28 044253 cells or transgenic animal. Thus, the particular glycosylation profile of the immunoglobulin chain, or of the antibody, or antigen binding fragment containing the 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 profile including only non-fucosylated N-glycans may be preferred because these antibodies and antigen-binding fragments have been shown to generally exhibit greater potency than their corresponding fucosylated counterparts in vitro and in vivo (see, e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003) ; US patents Nos. 6946292 and 7214775). These anti-ILT4 antibodies and antigen binding fragments (eg 1E1, 2A6, 3G7 or 2C1) with non-fucosylated Nglycans are part of the present invention.

The present invention further includes antibody fragments from the anti-ILT4 antibodies provided herein (eg, 1E1, 2A6, 3G7 or 2C1). Antibody fragments include F(ab) ₂ fragments, which can be produced by enzymatic digestion of IgG, for example with pepsin. Fab fragments can be obtained by, for example, reduction of F(ab) ₂ with dithiothreitol or mercaptoethylamine.

The Fab fragment is a VL-CL chain connected to a VH-CH1 chain via a disulfide bridge. The F(ab) ₂ fragment consists of two Fab fragments, which, in turn, are connected by two disulfide bridges. The Fab parts of F(ab) ₂ molecules include parts of the Fc regions, between which there are disulfide bridges. The Fv fragment is a V _L or V _H region.

The present invention relates to antibodies against ILT4 and their antigen binding fragments (eg, 1E1, 2A6, 3G7 or 2C1), classified as IgA, IgD, IgE, IgG and IgM. In one embodiment, the anti-ILT4 antibody or antigen binding fragment comprises a VH as defined herein and a heavy chain constant region, for example, a human constant region, such as a human γ1, γ2, γ3, or γ4 heavy chain constant region or a variant thereof. In an embodiment of the invention, the antibody or antigen binding fragment comprises a VH as defined herein and a light chain constant region, for example a human light chain constant region such as a human light chain lambda or kappa region or a variant thereof. As a non-limiting example, the human heavy chain constant region may be γ1 or γ4, and the human light chain constant region may be a kappa region. As a non-limiting example, the human heavy chain constant region may be γ1 or γ4, and the human light chain constant region may be a lambda region. In an embodiment of the invention, the Fc region of the antibody is γ4 with the Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38: 1-8, 2001).

In some embodiments, various constant domains can be coupled to the V _L and V _H regions derived from the CDRs provided herein. For example, if the particular intended use of the antibody or antigen binding fragment of the present invention relates to altered effector functions, a heavy chain constant domain other than human IgG1 or a hybrid IgG1/IgG4 may be used. Such anti-ILT4 antibodies and their antigen binding fragments (eg, 1E1, 2A6, 3G7 or 2C1) and hybrids containing IgG1 and IgG4 constant domains are part of the present invention.

In one embodiment, the IgG4 constant domain differs from the native human IgG4 constant domain (Swiss-Prot accession number 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 by Pro to prevent potential formation of an interchain 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), which may interfere with the correct formation of intrachain disulfide bonds. See Angal et al. (1993) Mol. Imunol. 30:105. In other cases, a modified IgG1 constant domain that has been modified to increase half-life or reduce effector function may be used. Such anti-ILT4 antibodies and antigen binding fragments thereof (eg 1E1, 2A6, 3G7 or 2C1) containing a modified IgG4 constant domain are part of the present invention.

The present invention relates to recombinant methods for producing an anti-ILT4 antibody, or an antigen binding fragment thereof of the present invention (for example, 1E1, 2A6, 3G7 or 2C1), or an immunoglobulin chain thereof, comprising (i) inserting a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (e.g., containing an amino acid sequence including any one or more of the sequences set forth in SEQ ID NOs: 1-15, 44 and/or 45), for example, where the polynucleotide is in a vector and/or operably linked to a promoter (e.g. viral promoter, CMV promoter or SV40 promoter); (ii) culturing a host cell (e.g., mammalian host cell, fungal host cell, bacterial host cell, Chinese hamster ovary (CHO) cell, NSO cells, SP2 cells, HeLa cells, newborn hamster kidney (BHK) cells, kidney cells monkeys (COS), cages

- 29 044253 human hepatocellular carcinoma (for example, Hep G2), A549 cells, 3T3 cells, HEK293 cells, Pichia cells or Pichia pastoris cells) under conditions favorable for the expression of polynucleotides and, (iii) optionally, isolation of the antibody or fragment, or chains from the host cell and/or the environment in which the host cell is grown. When producing an antibody or antigen-binding fragment containing multiple immunoglobulin chains, for example, an antibody containing two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell results in the binding of the chains, for example, in the cell, or on the surface of the cell, or outside the cell if such chains are secreted in such a way that an antibody or antigen-binding fragment molecule is formed. The methods of the present invention include methods where only the immunoglobulin heavy chain or only the immunoglobulin light chain is expressed (eg, any of the chains presented herein, including mature fragments and/or variable domains thereof). Such chains can be used, for example, as intermediates in the expression of an antibody or antigen-binding fragment comprising such a chain.

Construction of the antibody Fc region.

Anti-ILT4 antibodies and antigen binding fragments of the present invention (eg, 1E1, 2A6, 3G7 and/or 2C1) can also be designed to include modifications in the F region, typically to change one or more functional properties of the antibody, such as half-life in serum, complement fixation, Fc receptor binding, and/or effector function (eg, antigen-dependent cellular cytotoxicity). In addition, the antibodies and fragments provided herein can be modified chemically (eg, one or more chemical compounds can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region corresponds to the EU Kabat index.

Antibodies and antigen binding fragments provided herein (eg, 1E1, 2A6, 3G7 and/or 2C1) also include antibodies with modified (or blocked) F regions to achieve altered effector functions. See, for example, US Pat. No. 5,624,821; WO 2003/086310; WO 2005/120571; WO 2006/0057702. Such modification can be used to enhance or suppress various immune system responses with possible diagnostic and therapeutic benefits. Fc region changes include amino acid changes (substitutions, deletions and/or insertions), glycosylation or deglycosylation, and addition of multiple Fc domains. Changes in Fc can also lead to changes in the half-life of therapeutic antibodies, allowing for less frequent dosing and thus increasing convenience and reducing material utilization. See Presta (2005) J. Allergy Clin. Immunol. 116:731, pp. 734-35.

In one embodiment, the anti-ILT4 antibody or antigen binding fragment (eg, 1E1, 2A6, 3G7 and/or 2C1) is an IgG4 isotype antibody or fragment containing a serine to proline mutation at a position corresponding to position 228 (S228P; EU index) in hinge region heavy chain constant region. This mutation has been shown to eliminate the heterogeneity of disulfide bridges between heavy chains in the hinge region (Angal et al. supra; position 241 follows the Kabat numbering system).

In one embodiment, the CH1 hinge region of an anti-ILT4 antibody or antigen binding fragment thereof of the present invention (e.g., 1E1, 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 in US Pat. No. 5,677,425. The number of cysteine residues in the CH1 hinge region is varied, for example, to facilitate the assembly of 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 (eg, 1E1, 2A6, 3G7 and/or 2C1) is mutagenized to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are included in the surface region of the CH2-CH3 domain of the Fc hinge region such that the antibody has reduced binding to staphylococcal protein A (SpA) relative to the SpA binding of the native Fc hinge domain. This approach is described in detail in US Patent No. 6,165,745.

In another embodiment, the anti-ILT4 antibody or antigen binding fragment (eg, 1E1, 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 inserted: T252L, T254S, T256F, as described in US Pat. No. 6,277,375. Alternatively, to increase the biological half-life, the CH1 or CL region of the antibody can be altered to contain the rescue receptor binding epitope derived of the two loops of the CH2 domain of the IgG Fc region, as described in US Patent Nos. 5,869,046 and 6,121,022.

In other embodiments, the Fc region is modified by replacing at least one amino acid residue with another amino acid residue to alter the effector functions of the anti-ILT4 antibody or antigen binding fragment (eg, 1E1, 2A6, 3G7 and/or 2C1). On

- 30 044253 example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with another amino acid residue such that the antibody has an altered affinity for the effector ligand, but retains the ability to binding of the parent antibody antigen. The effector ligand for which the affinity is changed may be, for example, an Fc receptor or a complement component C1. This approach is described in detail in US 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 another amino acid residue such that the anti-ILT4 antibody or antigen binding fragment (eg, 1E1, 2A6, 3G7 and/or 2C1) has altered C1q binding and/or reduced or eliminated complement-mediated cytotoxicity (CDC). This approach is described in detail in US Patent No. 6194551.

In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered to alter the ability of the antibody to bind complement. This approach is described in PCT publication No. WO 94/29351.

In another example, the Fc region is modified to increase or decrease the ability of an anti-ILT4 antibody or antigen binding fragment (e.g., 1E1, 2A6, 3G7, and/or 2C1) to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or increase or decrease the affinity of the antibody or fragment for the receptor Fcy by modification of 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 one the approach is described in PCT publication No. WO 00/42072. In addition, the binding sites of human IgG1 to FcyR1, FcyRII, FcyRIII 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 have been shown to improve binding to FcyRIII. In addition, the following combination mutants have been shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A.

In one embodiment, the Fc region is modified to reduce the ability of an anti-ILT4 antibody or antigen binding fragment (e.g., 1E1, 2A6, 3G7, and/or 2C1) to mediate effector function and/or enhance anti-inflammatory properties through modification of residues 243 and 264. In one of In embodiments, the Fc region of the antibody is modified by replacing residues at positions 243 and 264 with alanine. In one embodiment, the F region is modified to reduce the ability of the antibody to mediate effector function and/or enhance anti-inflammatory properties by modifying residues 243, 264, 267, and 328.

Post-translational modifications.

In another embodiment, the antibody has a particular glycosylation profile. For example, an aglycosylated anti-ILT4 antibody or antigen binding fragment thereof (eg, 1E1, 2A6, 3G7 and/or 2C1) that lacks glycosylation is part of the present invention. The glycosylation profile of an antibody or fragment can be altered, for example, to increase the affinity or avidity of the antibody for an antigen. Such modifications can be made, for example, by changing one or more glycosylation sites in the sequence of the antibody or fragment. For example, one or more amino acid substitutions may be made to remove one or more of the glycosylation sites of the variable region backbone, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody or fragment for the antigen. See, for example, US Patent Nos. 5,714,350 and 6,350,861.

It is also possible to produce an anti-ILT4 antibody or antigen binding fragment (e.g., 1E1, 2A6, 3G7 and/or 2C1) of the invention, wherein the glycosylation profile includes hypofucosylated or afucosylated glycans, such as hypofucosylated antibodies and antigen binding fragments or afucosylated antibodies and fragments having reduced number of fucosyl residues on the glycan. The antibody or antigen binding fragment may also include glycans having an increased number of GlcNac structures at branch points. These altered glycosylation profiles have been shown to enhance the anti-ADCC potency of antibodies. Such modifications can be accomplished, for example, by expressing an antibody or fragment in a host cell in which the glycosylation pathway is genetically engineered to produce glycoproteins with specific glycosylation profiles. These cells are known in the art and can be used as host cells in which the recombinant antibodies of the invention are expressed to produce an antibody with altered glycosylation. For example, the Ms704, Ms705, and Ms709 cell lines lack the fucosyltransferase gene, FUT8 (a(1,6)-fucosyltransferase), such that the carbohydrate antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose. The present invention relates to anti-ILT4 antibodies and antigen binding fragments in which

- 31 044253 lacking fucose or which are obtained using a host cell that lacks FUT8. FUTS ^-/- cell lines Ms704, Ms705 and Ms709 are generated by targeting the FUT8 gene in CHO/DG44 cells using two replacement vectors (see US Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614 -22). As another example, EP 1176195 describes a cell line with a functionally damaged FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation due to the reduction or elimination of the enzyme acting at the α-1,6 linkage . EP 1176195 describes human cell lines having low or no enzymatic activity for the addition of fucose to N-acetylglucosamine binding to the Fc region of an antibody, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication No. WO 03/035835 describes a variant of the CHO cell line, Lec13 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of antibodies expressed in this 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 No. WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells such as Lemna (US Pat. No. 7,632,983). Methods for producing antibodies in a plant system are described in US Patent Nos. 6,998,267 and 7,388,081. PCT Publication No. WO 99/54342 describes human cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines have an increased number of GlcNac structures at the branch points, resulting in increased antibody ADCC activity (also see Umana et al. (1999) Nat. Biotech. 17: 176-180). The anti-ILT4 antibodies and antigen binding fragments thereof of the present invention produced by such host cells are part of the present invention.

Alternatively, the fucose residues in the anti-ILT4 antibody or antigen binding fragment (eg, 1E1, 2A6, 3G7 and/or 2C1) can be cleaved using the enzyme fucosidase; for example, a-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23). The present invention relates to antibodies and fragments treated with the enzyme fucosidase.

Anti-ILT4 antibody or antigen binding fragments (e.g., 1E1, 2A6, 3G7 and/or 2C1) provided herein further include antibodies and fragments produced in lower eukaryotic host cells, in particular fungal host cells such as yeast and filamentous fungi genetically engineered to produce glycoproteins having glycosylation profiles similar to those of mammals or humans (see, for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton et al. , (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 glycosylation profile of the glycoproteins produced in the cells so that glycoprotein compositions can be produced where a particular N-glycan structure predominates (see, for example, the patent US No. 7,029,872 and US Pat. No. 7,449,308). These genetically modified host cells are used to produce antibodies preferentially having specific N-glycan structures (see, for example, Li et al., (2006) Nat. Biotechnol. 24: 210-215).

Antibody conjugates.

Antibodies against ILT4 and antigen binding fragments presented herein (eg, 1E1, 2A6, 3G7 and/or 2C1) can also be conjugated to a peptide or chemical compound. The chemical compound may be, among other things, a polymer, a radionuclide, or a therapeutic or cytotoxic agent. In specific embodiments of the invention, the chemical compound is a polymer that increases the half-life of the antibody molecule in the body of an individual. Polymers include, but are not limited to, hydrophilic polymers including, but are not limited to, polyethylene glycol (PEG) (e.g., 2, 5, 10, 12, 20, 30, or 40 kDa PEG), dextran, and monomethoxy polyethylene glycol (mPEG) ). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) describe PEG-conjugated single chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) describe the conjugation of antibodies with PEG coupled to a radioactive metal chelator (diethylenetriaminepentaacetic acid (DTPA)).

Antibodies and antigen binding fragments provided herein (eg, 1E1, 2A6, 3G7 and/or 2C1) can also be conjugated to labels, such as radioactive labels. Labels include, as non-limiting examples, ⁹⁹ Tc, ⁹⁰ Y, ⁿ¹ In, ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, 11 C, ¹⁵ O, ¹³ N, ¹⁸ F, ³⁵ S, ⁵¹ Cr, ⁵⁷ To, ²²⁶ Ra, ⁶⁰ Co, ⁵⁹ Fe, ⁵⁷ Se, ¹⁵² Eu, ⁶⁷ CU, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, ²³⁴ Th, and ⁴⁰ K, ¹⁵⁷ Gd, ⁵⁵ Mn, ⁵² Tr and ⁵⁶ Fe.

Antibodies against ILT4 and antibody fragments presented herein (eg,

- 32 044253

1E1, 2A6, 3G7 and/or 2C1) can also be PEGylated (eg using 1 PEG or a 3, 12 or 40 kDa PEG polymer molecule), for example, to increase biological half-life (eg in serum). To PEGylate an antibody or antigen-binding fragment thereof, typically, the antibody or fragment 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 are attached to the antibody or antigen-binding fragment. In specific embodiments, PEGylation is accomplished through an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol is intended to include any of the forms of PEG used to derivatize other proteins, such as mono-(C1C ₁₀ )-alkoxy- or -aryloxy-polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for PEGylation of proteins are known in the art and can be used with the antibodies of the invention. See, for example, EP 0 154 316 and EP 0 401 384.

Anti-ILT4 antibodies and antigen binding fragments provided herein (e.g., 1E1, 2A6, 3G7 and/or 2C1) can also be conjugated to labels, such as fluorescent or chemiluminescent labels, including fluorophores such as rare earth metal chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, ¹⁵² Eu, dansyl, umbelliferone, luciferin, luminol label, isoluminol label, aromatic acridinium ester label, imidazole label, acridinium salt label , oxalate ester label, aequorin label, 2,3dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.

Anti-ILT4 antibodies and antigen binding fragments provided herein (e.g., 1E1, 2A6, 3G7 and/or 2C1) can also be conjugated to a cytotoxic factor such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain , modecin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g. fatty acids), diantine proteins, PAPI, PAPII and PAP-S proteins Phytoiacca americana, Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogellin, restrictocin , fenomycin and enomycin.

Any method known in the art for conjugating antibodies and antigen binding fragments to various compounds can be used, including the methods described in 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 antibody conjugation are conventional and well known in the art.

Pharmaceutical compositions and administration.

Pharmaceutical compositions containing anti-ILT4 antibodies (e.g., fully human antibodies, such as antagonistic fully human antibodies (e.g., 1E1, 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 optional physiologically acceptable carriers, excipients or stabilizers (see, for example, 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 contains an antibody consisting 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 contains: (i) an antibody consisting of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence given in SEQ ID NO: 7, and each heavy chain consists of the amino acid sequence given in SEQ ID NO: 2, and (ii) pembrolizumab.

Therapeutic and diagnostic agent formulations can be prepared by admixture with suitable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, suspensions or aqueous solutions (see, for example, Hardman, et al. (2001) Goodman and Gilman's 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).

The toxicity and therapeutic efficacy of anti-ILT4 antibody or antigen binding fragment compositions (e.g., 1E1, 2A6, 3G7 and/or 2C1), administered alone or in combination with another therapeutic agent, can be determined by standard pharmaceutical methods using cell cultures or experimental animals, e.g. , to determine LD ₅₀

- 33 044253 (dose lethal for 50% of the population) and ED ₅₀ (dose effective for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD ₅₀ /ED ₅₀ ). In certain aspects, antibodies that exhibit high therapeutic indices are desirable. Data obtained from these cell culture assays and animal studies can be used to determine dose ranges for use in humans. The dose of such compounds preferably falls within a range of circulating concentrations including the ED ₅₀ with little or no toxicity. Doses may vary within this range depending on the dosage form used and route of administration.

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

The route of administration may 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 relates to methods of administering an anti-ILT4 antibody or an antigen-binding fragment thereof (eg, 1E1, 2A6, 3G7 and/or 2C1), comprising administering the antibody or fragment to a subject. For example, the method includes puncturing the body of an individual with a syringe needle and injecting the antibody or fragment into the individual's body, for example, into a vein, artery, tumor, muscle tissue or subcutaneous fat of the individual.

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

The present invention also relates to an injection device containing any of the anti-ILT4 antibodies or antigen binding fragments (eg, 1E1, 2A6, 3G7 and/or 2C1) described herein, or a pharmaceutical composition thereof. An injection device is a device by which a substance is introduced into the body of an individual parenterally, for example, intramuscularly, subcutaneously or intravenously. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector), e.g., including a barrel for holding a liquid to be injected (e.g., containing an antibody or fragment, or a pharmaceutical composition thereof), a needle for piercing the skin and/or blood vessels for fluid injection; and a piston for pushing fluid out of the cylinder and through the cavity of the needle. In an embodiment of the invention, the injection device containing the 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 an antibody or fragment, or a pharmaceutical composition thereof, in a cannula or trocar/needle that is connected to a tube that can be connected to a bag or reservoir for retaining a fluid (for example, saline or lactated Ringer's solution containing NaCl, sodium lactate, KCl, CaCl ₂ and optionally including glucose) introduced into the individual's body through a cannula or trocar/needle.

The pharmaceutical compositions provided herein can also be administered using a needle-free subcutaneous injection device, such as those described in US Pat. 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 45 96556. Such needleless devices, containing a pharmaceutical composition are also part of the present invention. The pharmaceutical compositions presented herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include those described in: US Pat. No. 4,487,603, which describes an implantable microinfusion pump for dispensing a drug at a controlled rate; US Patent No. 4,447,233, which describes an infusion pump for administering a drug at a precise infusion rate; US Pat. No. 4,447,224, which describes an implantable variable-flow infusion device for continuous drug administration; US Patent No. 4,439,196, which describes a system for osmotic drug delivery having multi-chamber compartments. Many other such implants, delivery systems and modules are well known to those skilled in the art, and implants, delivery systems and modules containing the pharmaceutical compositions of the present invention are within the scope of the present invention.

Antibodies (e.g. fully human antibodies, such as fully antagonistic

- 34 044253 human antibodies) or their antigen binding fragments presented in the present description (for example, 1E1, 2A6, 3G7 and/or 2C1), can be administered by continuous infusion or by doses administered, for example, daily, 1-7 times per weekly, once a week, once every two weeks, monthly, once every two months, once a quarter, once every six months, annually, etc. Doses may be administered, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinal, 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 to about 30 mg/kg (body weight), for example, for the treatment or prevention of cancer or infectious diseases.

Determination of the appropriate dose is made by the clinician, for example, using parameters or factors that are known or suspected in the art to influence treatment. Typically, when determining a dose, start with an amount slightly less than the optimal dose and increase it in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic parameters include symptom parameters such as inflammation or the level of inflammatory cytokines produced. In general, it is desirable to obtain the biological agent to be used from the same species as the animal to be treated, thereby minimizing any immune response to the reagent. In the case of humans, for example, chimeric and fully human antibodies may be desirable. Guidelines for selecting appropriate doses of anti-ILT4 antibodies or fragments are available (see, for example, 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 et al (2003) New Engl J Med 348:601- 608; Milgrom et al. (1999) New Engl J Med 341:1966-1973; Slamon et al (2001) New Engl J Med 344:783-792; Beniaminovitz et al (2000) New Engl J Med 342:613-619 Ghosh et al (2003) New Engl J Med 348:24-32 Lipsky et al (2000) New Engl J Med 343:1594 1602).

Whether a symptom of a disease has been reduced can be assessed by any clinical measurement typically used by treating physicians or other health care professionals to assess the severity or progression status of that symptom. Although an embodiment of the present invention (e.g., a treatment method or article of manufacture) may not be effective in reducing the symptoms of the target disease in every individual, it should reduce the symptoms of the target disease in a statistically significant number of individuals, as determined by any statistical test known in the art. areas such as Student's t-test, chi-square test, Mann and Whitney U-test, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test and Wilcoxon test.

Therapeutic use of anti-ILT4 antibody.

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

In some embodiments, the malignancy is a solid tumor. In other embodiments, the malignancy is a hematologic malignancy. In some embodiments, the cancer is metastatic. In some embodiments, the malignancy is recurrent. In other embodiments, the malignancy is refractory. In other embodiments, the malignancy is recurrent and refractory.

In some embodiments, the malignancy is an anaplastic astrocytoma, astrocytoma, bladder cancer, bone cancer, brain malignancy, breast cancer (e.g., characterized by a BRCA1 and/or BRCA2 mutation), carcinoid tumor, cervical cancer, chondrosarcoma, papilloma choroid plexus, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, gallbladder cancer, stomach cancer, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, idiopathic myelofibrosis, kidney cancer, leukemia, liver cancer, lung cancer ( For example, with non -coclic lung cancer), lymphoma, medulloblastoma, melanium, meningioma, mercinoma, mesothelioma, multiple myeloma, neuroblastoma, oligodendroglioma, osteosarcoma, ovarian cancer, pancreatic cancer, true polycytemia, and primitive neuro -ethno -ethno -ethno -ethno -ethno -ethno -ethno -ethnoes , prostate cancer, kidney cell cancer, transitional cell kidney cancer, retinoblastoma, rhabdoid tumor of the kidney, rhabdomyosarcoma, salivary gland cancer, sarcoma, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma (eg, squamous cell carcinoma of the skin), synovial sarcoma, thrombocythemia,

- 35 044253 thyroid cancer, uterine cancer, vestibular schwannoma or Wilms tumor. In an embodiment of the invention, the cancer is a metastatic cancer, for example, of the types described above.

In an embodiment of the invention, the malignancy is a myeloid cell-rich tumor (e.g., mesothelioma, kidney cancer, lymphoma, sarcoma, melanoma, head and neck cancer, breast cancer, bladder cancer, gastric cancer, ovarian cancer, or thyroid cancer; see eg Burt et al Cancer 117 (22):5234-44 (2011) Dannenmann et al Oncoimmunology 2(3):e23562 (2013) Steidl et al N Engl J Med 362:875 -885 (2010); Fujiwara et al., Am. J. Pathol. 179(3): 1157-70 (2011); Bronkhorst et al. Invest. Ophthalmol. Vis. Sci. 52(2):643-50 ( 2011); Balermpas et al. Br. J. Cancer 111 (8): 1509-18 (2014); and Zhang et al. PLoS One 7(12):e50946 (2012)). Because ILT4 is expressed primarily by myeloid cells and granulocytes, and infiltration of tumors by myeloid cells is generally associated with a poor prognosis as a result of the immunosuppressive effects of these cells, which may antagonize antitumor T cell responses, treatment with anti-ILT4 antibody or antigen-binding fragment The present invention will benefit individuals with high infiltration of myeloid or immunosuppressive myeloid cells.

Thus, the present invention provides methods for treating cancer in a human in need thereof, comprising administering to the human an effective amount of an antibody or antigen-binding fragment thereof comprising (a) light chain variable domain (VL) CDR-L1, CDR-L2 and CDR-L3 an immunoglobulin chain containing the amino acid sequence shown in SEQ ID NO: 3-7, 11, 13, 15 or 45; and/or (b) CDR-H1, CDR-H2 and CDR-H3 of the variable domain ( _VH ) of the heavy chain of the immunoglobulin chain containing the amino acid sequence shown in SEQ ID NO: 1, 2, 8-10, 12, 14, 44 or 79-86.

In an embodiment of the invention, a method of treating a cancer in a human in need thereof comprises administering to the human an effective amount of an 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, where X is S or A (SEQ ID NO: 17), and complementarity determining region-H3 (CDR-H3): LPTRWVTTRYFDL (SEQ ID NO: 18) , and/or domain V _L containing: complementarity determining region-L1 (CDR-L1): TGSSSNIGAGYDVH (SEQ ID NO: 19), complementarity determining region-L2 (CDR-L2): GX1X ₂ NRPS; where X1 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 VH domain containing: CDR-H1: SYAIS (SEQ ID NO: 22), CDRH2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23) and CDR-H3: YFX1X2SGWYKGGAFDI; where X1 is D or S, and X2 is S or A (SEQ ID NO: 24), and/or a VL domain containing: CDR-L1: TLRSGINVDTYRIH (SEQ ID NO: 25), CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26) and CDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) a VH domain containing: CDR-H1: SYAMH (SEQ ID NO: 28), CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO: 29) and CDR-H3: VGEWIQLWSPFDY (SEQ ID NO: 30), and/or domain V _L containing: 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 containing: 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 containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 37), CDRL2: GNSNRPS (SEQ ID NO: 38) and CDR-L3: QSYDSSLSGSGVV (SEQ ID NO: 39).

In one embodiment, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 VL domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 Vl domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of treating a cancer in a human in need thereof comprises administering to the human an effective amount of an 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 VL domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 VL domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 VL domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 VL domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 V _L domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an antibody or antigen binding fragment thereof comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EIN-37 044253

HAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or a VL domain containing: 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 domain V _L containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS (SEQ ID NO: 56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In other embodiments, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an antibody or an antigen-binding fragment thereof comprising: an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain comprises an amino acid sequence given in SEQ ID NO: 3, 4, 5, 6, 7, 11, 13, 15 or 45; and/or the immunoglobulin heavy chain contains 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 other embodiments, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an antibody or an antigen-binding fragment thereof comprising: an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the light chain variable domain comprises an amino acid the sequence shown in SEQ ID NO: 70, 71, 72, 73, 58, 74, 75, 76 or 77, and/or the heavy chain variable domain contains the amino acid sequence shown in SEQ ID NO: 63, 57, 64, 65 , 66, 67, 68 or 69.

In other embodiments, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an antibody or antigen-binding fragment thereof comprising: any of the following sets of immunoglobulin heavy chains and immunoglobulin light chains: (1) an immunoglobulin heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (2) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 4; (3) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; (4) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; (5) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; (6) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (7) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 8; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (8) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 9; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (9) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 10; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (10) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 12; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 13; (11) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 14; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 15; (12) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 79; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (13) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence given in SEQ ID NO: 4; (14) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; (15) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; (16) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; (17) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (18) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 82; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (19) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 83; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (20) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 84; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (21) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 85; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 13; or (22) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 86; an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 15.

In other embodiments, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an antibody or antigen binding fragment thereof comprising: any of the following sets of heavy chain variable domains and light chain variable domains: (1) a heavy chain variable domain comprising an amino acid the sequence shown in SEQ ID NO: 63, and a light chain variable domain containing the amino acid sequence shown 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 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 containing 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 containing 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 containing 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 containing 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 containing the amino acid sequence set forth in SEQ ID NO: 75; or (11) 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, a method of treating a cancer in a person in need thereof comprises administering to the person an effective amount of an 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 containing amino acid sequence given in SEQ ID NO: 58.

In another preferred embodiment, a method of treating a malignancy in a person in need thereof comprises administering to the person an effective amount of an antibody or antigen binding fragment thereof comprising: an immunoglobulin heavy chain containing the amino acid sequence set forth in SEQ ID NO: 2, and an immunoglobulin light chain containing the amino acid sequence given in SEQ ID NO: 7.

- 39 044253

In another preferred embodiment, a method of treating a malignancy in a person in need thereof comprises administering to the person an effective amount of an antibody or antigen-binding fragment thereof comprising: an immunoglobulin heavy chain containing the amino acid sequence set forth in SEQ ID NO: 80, and an immunoglobulin light chain containing the amino acid sequence given in SEQ ID NO: 7.

The present invention also provides methods for blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a person in need thereof, comprising administering to the person an effective amount of an antibody or antigen binding fragment thereof comprising (a) CDR- L1, CDRL2 and CDR-L3 of the variable domain (VL) of an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3-7, 11, 13, 15 or 45; and/or (b) CDR-H1, CDR-H2 and CDR-H3 of an immunoglobulin heavy chain variable domain ( _VH ) containing 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, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: (1) a V _H domain containing: complementarity determining region-H1 (CDR-H1): GYYWS (SEQ ID NO: 16), complementarity determining region-H2 (CDR-H2): EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO: 17), and a complementarity determining region-H3 (CDR-H3): LPTRWVTTRYFDL (SEQ ID NO: 18), and/or a VL domain containing: a complementarity determining region-L1 (CDR-L1): TGSSSNIGAGYDVH (SEQ ID NO: 19), determining complementarity determining region-L2 (CDR-L2): GX1X ₂ NRPS, where X1 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 VH domain containing: CDR-H1: SYAIS (SEQ ID NO: 22), CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO: 23) and CDR-H3: YFX1X2SGWYKGGAFDI, where X1 is D or S, and X ₂ is S or A (SEQ ID NO: 24), and/or domain V _L containing: CDR-L1: TLRSGINVDTYRIH (SEQ ID NO: 25), CDR-L2: YKSDSDKHQGS (SEQ ID NO: 26) and CDR-L3: AIWYSSTWV (SEQ ID NO: 27); (3) V _H domain containing: 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 VL domain containing: 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 containing: 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 VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 37), CDRL2: GNSNRPS (SEQ ID NO: 38) and CDR-L3: QSYDSSLSGSGVV (SEQ ID NO: 39).

In one embodiment, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 Vl domain containing: 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, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 Vl domain containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: GYYWS (SEQ ID NO : 16), CDRH2: EINHSGSTNYNPSLKS (SEQ ID NO: 47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or a V _L domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 Vl domain containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 V _L domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS (SEQ ID NO: 53) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

- 40 044253

In another embodiment, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 VL domain containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 Vl domain containing: 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, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 Vl domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 V _L domain containing: 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, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 containing: 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, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an 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 V _L domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In another embodiment, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a human in need thereof comprises administering to the human an effective amount

- 41 044253 antibody or antigen-binding fragment thereof, containing: CDR-H1: GYYWS (SEQ ID NO: 16),

CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18), and/or VL domain containing: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2 : GDANRPS (SEQ

ID NO: 56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

In other embodiments, a method of blocking ILT4 binding to HLA-G, HLA-A, HLAB, and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: an immunoglobulin light chain, an immunoglobulin heavy chain, or both an immunoglobulin light and a heavy chain, wherein the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 11, 13, 15, or 45; and/or the immunoglobulin heavy chain contains 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 other embodiments, a method of blocking ILT4 binding to HLA-G, HLA-A, HLAB, and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: an immunoglobulin light chain, an immunoglobulin heavy chain, or both a light and a heavy chain of an immunoglobulin, wherein the light chain variable domain contains 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 contains the amino acid sequence set forth in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68 or 69.

In other embodiments, a method of blocking ILT4 binding to HLA-G, HLA-A, HLAB, and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: any of the following sets of immunoglobulin heavy chains and immunoglobulin light chains: (1) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 1; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (2) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 4; (3) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; (4) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; (5) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; (6) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (7) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 8; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (8) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 9; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (9) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 10; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (10) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 12; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 13; (11) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 14; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 15; (12) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 79; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (13) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 4; (14) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; (15) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; (16) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; (17) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; (18) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 82; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (19) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 83; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (20) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 84; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 11; (21) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 85; an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 13; or (22) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 86; an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 15.

In other embodiments, a method of blocking ILT4 binding to HLA-G, HLA-A, HLAB, and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: any of the following sets of heavy chain variable domains and light chain variable domains: (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 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 containing 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 containing 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 containing 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 containing 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 containing the amino acid sequence set forth in SEQ ID NO: 75; or (11) 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, a method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: a VH domain comprising the amino acid sequence shown in SEQ ID NO: 57, and a VL domain containing the amino acid sequence shown in SEQ ID NO: 58.

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

In another preferred embodiment, a method of blocking ILT4 binding to HLA-G, HLA-A, HLA-B and/or HLA-F in a subject in need thereof comprises administering to the subject an effective amount of an antibody or antigen binding fragment thereof comprising: an immunoglobulin heavy chain, containing the amino acid sequence shown in SEQ ID NO: 80, and an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7.

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

The present invention relates to methods of treating any of the malignancies or infectious diseases described herein by administering a therapeutically effective amount of an anti-ILT4 antibody or antigen binding fragment thereof (e.g., 1E1, 2A6, 3G7 and/or 2C1), optionally together with any of the additional chemotherapeutic agents or therapies provided herein, as well as compositions comprising such antibody or fragment together with such additional chemotherapeutic agent (eg, co-formulated antibody or fragment and additional chemotherapeutic agent).

The individual is a mammal, such as, for example, a human, a dog, a cat, a horse, a cow, a mouse, a rat, a monkey (eg, a cynomolgus macaque, eg Masasa fascicularis or Masasa mulatta) or a rabbit.

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

The term in association with means that the components, an anti-ILT4 antibody (for example, a fully human antibody, such as antagonistic fully human antibodies) or an antigen binding fragment thereof of the present invention (for example, 1E1, 2A6, 3G7 and/or 2C1) together with another agent agents, such as pembrolizumab or nivolumab, can be formulated in a single composition, for example, for simultaneous administration, or formulated separately in two or more compositions (eg, a kit). Each component may be administered to an individual at a different time than the other component administered; for example, each administration may be performed non-simultaneously (eg, separately or sequentially) at intervals over a specified period of time. In addition, the individual components can be administered to an individual by one or different routes (e.g., wherein an anti-ILT4 antibody (e.g., a fully human antibody, such as antagonistic fully human antibodies) or an antigen binding fragment thereof (e.g., 1E1, 2A6, 3G7, and/or 2C1) administered parenterally, and paclitaxel administered orally).

Assays and experimental and diagnostic applications.

The present invention relates to any method of producing a complex between an anti-ILT4 antibody or an antigen-binding fragment thereof of the present invention and ILT4 or a fragment thereof, comprising contacting the ILT4 polypeptide or fragment with an anti-ILT4 antibody or antigen-binding fragment under conditions suitable for binding and complex formation.

Anti-ILT4 antibodies (eg, fully human antibodies, such as antagonistic fully human antibodies) and antigen binding fragments thereof provided herein (eg, 1E1, 2A6, 3G7 and/or 2C1) can be used as affinity purification agents. In this method, anti-ILT4 antibodies and antigen binding fragments thereof are immobilized on a solid phase such as Sephadex, glass or agarose resin, or filter paper by methods well known in the art. The immobilized antibody or fragment is contacted with a sample containing the ILT4 protein (or fragment thereof) to be purified, and the support is then washed with a suitable solvent, which removes material from the sample other than the ILT4 protein associated with the immobilized antibody or fragment. Finally, the support is washed with a solvent that elutes bound ILT4 (eg, protein A). Such immobilized antibodies and fragments are part of the present invention.

The present invention relates to cellular ELISA methods using antibodies against ILT4 and their antigen binding fragments of the present invention (eg, 1E1, 2A6, 3G7 and/or 2C1). In an embodiment of the invention, the method is for determining whether cells contain ILT4, and the method includes the steps of: (i) bringing said cells, immobilized on a solid support (eg, microplate) and tested for the presence of ILT4, into contact with an anti-ILT4 antibody or antigen-binding fragment of the present invention, (ii) optionally, washing the mixture to remove unbound anti-ILT4 antibody or fragment, (iii) bringing the antibody

- 44 044253 against ILT4 or fragment in contact with a labeled secondary antibody or antigen binding fragment thereof, binding to the anti-ILT4 antibody or fragment, (iv) optionally, washing the complex to remove unbound antibodies or fragments and (v) detecting the presence of a label on the secondary antibody, or fragment; where detection of the label indicates that cells containing ILT4 are immobilized on a solid surface.

The present invention relates to ELISA assays (enzyme-linked immunosorbent assays) comprising the use of an anti-ILT4 antibody (eg, fully human antibodies, such as antagonistic fully human antibodies) or an antigen binding fragment thereof as provided herein (eg, 1E1, 2A6, 3G7 and/or or 2C1). For example, such a method for determining whether a sample contains ILT4 or a fragment thereof involves the following steps:

(a) coating a substrate (e.g., the surface of a well of a microtiter plate, e.g., a plastic plate) with an anti-ILT4 antibody (e.g., fully human antibodies, such as antagonistic fully human antibodies) or an antigen binding fragment thereof (e.g., 1E1, 2A6, 3G7, and/or or 2C1);

(b) applying the sample to be tested for the presence of ILT4 to the substrate;

(c) washing the substrate to remove unbound material in the sample;

(d) applying detectably labeled antibodies (eg, enzyme-linked antibodies) also specific for the ILT4 antigen;

(e) washing the substrate to remove unbound labeled antibodies;

(f) detecting labels on antibodies; if the labeled antibodies are bound to an enzyme, applying a chemical that is converted by the enzyme into a detectable, eg fluorescent, signal; and detection of a label, eg, associated with a 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 an ABTS-reactive peroxidase (e.g., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) or 3,3',5,5'-tetramethylbenzidine , to achieve a color change subject to detection. Alternatively, the labeled antibody or fragment is labeled with a detectable radioactive isotope (eg, ³ H), which can be detected using a scintillation counter in the presence of a scintillator.

An anti-ILT4 antibody (eg, fully human antibodies, such as antagonistic fully human antibodies) or an antigen binding fragment thereof of the invention (eg, 1E1, 2A6, 3G7 and/or 2C1) can be used in Western blotting or immunoblotting. Such a method is part of the present invention and includes, for example:

(1) providing a membrane or other solid substrate containing the sample to be tested for the presence of ILT4 or a fragment thereof, for example, optionally including the step of transferring proteins from the sample to be tested for the presence of ILT4 (for example, after electrophoretic separation of proteins in the sample using PAGE or PAGE in the presence of SDS), onto a membrane or other solid substrate (for example, by methods known in the art (for example, semi-dry blotting or tank blotting)); and contacting a membrane or other solid substrate to be tested for bound ILT4 or a fragment thereof with an anti-ILT4 antibody or antigen binding fragment thereof of the invention (eg, 1E1, 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-ILT4 antibody or fragment.

Such a membrane may take the form of, for example, a nitrocellulose or vinyl (eg, polyvinylidene fluoride (PVDF)) membrane onto which proteins to be tested for ILT4 are transferred by non-denaturing PAGE (polyacrylamide gel electrophoresis) or SDS-PAGE (polyacrylamide gel electrophoresis). gel in the presence of sodium dodecyl sulfate) (for example, after electrophoretic separation in a gel). Before contacting the membrane with the anti-ILT4 antibody or fragment, the membrane is optionally blocked, for example, with nonfat dry milk or the like. to bind nonspecific protein binding sites on the membrane.

Detection of bound anti-ILT4 antibody or fragment indicates that ILT4 protein is present on the membrane or substrate and in the sample. Detection of a bound antibody or fragment can be accomplished by binding the antibody or fragment to a detectably labeled secondary antibody (anti-immunoglobulin antibody) and then detecting the presence of the secondary antibody label.

Anti-ILT4 antibodies (eg, fully human antibodies, such as antagonistic fully human antibodies) and antigen binding fragments thereof provided herein (eg, 1E1, 2A6, 3G7 and/or 2C1) can also be used for immunohistochemistry. Such a method is part of the present invention and includes, for example, (1) contacting cells (for example, myeloid lineage cells such as monocytes, macrophages or dendritic cells) to be tested for the presence of ILT4 protein with an anti-ILT4 antibody or an antigen-binding fragment thereof according to the invention (for example, 1E1, 2A6, 3G7 and/or 2C1); And

- 45 044253 (2) detection of an antibody or fragment on or in cells.

If the antibody or fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or fragment can be coupled to a detectably labeled secondary antibody and then the label is detected.

Examples.

These examples are intended to illustrate the present invention and not to limit it. The compositions and methods given in the examples are part of the present invention.

As used herein, the term p1E1 (G1) refers to the fully human anti-ILT4 mAb 1E1 derived from the germline V genes, IGHV4-34*0 and IGLVl-40*01, respectively; containing human IgG1 and human lambda constant domains.

Heavy chain

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGR GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44).

Light chain

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 45).

As used herein, the term p1E1 (G4) refers to the fully human anti-ILT4 mAb 1E1 (containing the Q1E heavy chain and Q1E light chain mutations) derived from the germline V genes, IGHV4-34*0 and IGLV1-40*01, respectively ; containing human IgG4 (S228P) and human lambda constant domains.

Heavy chain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGR GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1).

Human IgG4 constant domain (S228P)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTV (SEQ ID NO: 89).

Light chain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3).

Human lambda constant domain

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 90).

As used herein, the term 1E1 (G4) refers to the fully human anti-ILT4 mAb 1E1 (containing mutations Q1E and N53D in the light chain and Q1E and S54A in the heavy chain) derived from the germline V genes, IGHV4-34*0 and IGLV1-40 *01, respectively; containing human IgG4

- 46 044253 (S228P) and human lambda constant domains.

Heavy chain

EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHA GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGR GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2).

Light chain

ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSN RPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 7).

Example 1: Identification and characterization of anti-ILT4 antibodies.

Production and identification of antibodies.

The parental human anti-ILT4 mAb 1E1 was identified using the RETROCYTE DISPLAY platform (Breous-Nystroma et al., Methods 65(1): 57-67 (2014)).

The RETROCYTE DISPLAY platform uses retroviral transfer of human antibody genes into mammalian pre-B cells to generate stable, high-diversity antibody display libraries. Human umbilical cord blood containing naïve B cells was used as the starting material for the heavy and light chains of the antibodies. Cellular antibody libraries typically expressed >10 ⁸ different full-length human monoclonal antibodies (isotypes hIgG1-4) on the surface of pre-B cells.

Preliminary antibody panels were compiled from antibody hits identified in 3 separate sets of RETROCYTE DISPLAY screens. Candidates were enriched based on FACS detection of recombinant human ILT4 antigen binding to B cell clones. Putative B cell clones were sorted and their antibody sequences were determined. These sequences were then used to generate candidate antibodies and ILT4 binding was confirmed using a CHO cell transfectant binding assay to ILT4 using parental CHO cells as a negative control. Candidates were also screened for ILT4 specificity against closely related ILT family members (FACS of human ILT2, LILRA1, and LILRA2 CHO transfectants) and ILT family members by flow cytometric analysis (LILRA1, LILRA2, LILRA4, LILRA5, LILRA6, ILT2, ILT5, and ILT3 ).

The monoclonal antibodies were further tested for their ability to bind to cynomolgus ILT4 CHO transfectants (predicted sequence from NCBI) by FACS. Candidate mAbs were also screened for their ability to block the binding of recombinant HLA-G-Fc ligand to recombinant ILT4 protein in Luminex-based assays or to ILT4-expressing CHO cells in FACS-based assays. The functional activity of candidate antibodies was analyzed in three ways: 1) restoration of spontaneous suppression of IL-2 in ILT4-transfected mouse 3A9 T cells; 2) restoration of HLA-G-dependent suppression of CD200RLa-stimulated mast cell degranulation in WTMC ILT4 mouse transfectants; and 3) Modulation of cytokines in the reactions of mixed lymphocyte culture with whole PBMCs.

Considering the above screening criteria, eight anti-ILT4 antibodies (1E1, 1G2, 2A6, 2D5, 3E6, 3G7, 2C1 and 5A6) were selected based on their functional and biophysical properties. Antibodies were further analyzed and re-evaluated in a range of biofunctional, biophysical and physicochemical assays. Functional assays assessed antibody-mediated dose-dependent restoration of IL-2 suppression by 3A9 cells and mast cell degranulation described above. The Luminex assay, cellular ligand blocking assay, and ligand competition assay were performed and binding properties and affinities to the ILT4 target antigen, non-target antigens, and PBMC subsets were determined using Biacore assays and flow cytometry, respectively. Biophysical assays assessed the stability (temperature, pH), degradation and aggregation of antibodies. Sequence deficiencies and potential post-translational modification motifs were examined to eliminate potential risks associated with antibody production. Finally, the candidates were tested in an in vivo tumor regression study using SKMEL5 melanoma cell-stimulated humanized mice.

Biophysical properties.

Studies were carried out using the human 1E1 sequence (human IgG4 backbone with

- 47 044253 mutation S228P), transiently expressed in CHO cells. 1E1 had the following physicochemical characteristics: the calculated and experimentally determined isoelectric points (PI) were ~7.29 and ~7.2, respectively, the level of aggregation (HMW molecules) was <5%, initial Tm >60°C, Tm1 ~65 .2°C, Tm2 ~78.8°C, and was stable for at least 5 freeze/thaw cycles. The 1E1 sequence originally contained an N-glycosylation site in the V _H -CDR2, which was successfully corrected using the S54A mutation without negatively affecting binding according to BIACORE and functional analysis. Studies under stress conditions revealed deamidation of the N53 residue in V _L -CDR3 (>13% under high pH conditions). N53 was successfully corrected (N53D) without negatively affecting binding and in a functional assay (restoring IL-2 release from 3A9 T cell transfectants with ILT4 using 1E1). Both N-terminal Q residues in V _H and V _L of 1E1 were mutagenized to E (VH-Q1E and VL-Q1E) to reduce the risk of heterogeneity due to deamidation. In studies under stress conditions carried out on the mutant sequence 1E1 (V _H Q1E1E1, S54A/Q1E V _L , N53D) IgG4 S228P/lambda), ~17% oxidation of the W102 residue in VH-CDR3 was detected under conditions of forced oxidation using AAPH (2 ,2'-azobis(2-amidinopropane)dihydrochloride) after 6 hours with minimal effects on binding and function. AAPH is used to force the oxidation of Trp and Met residues. Under the same conditions, ~8% oxidation of W7 in the _VH framework was determined without affecting binding or function. Peptide mapping determined that approximately 8% of the antibody was afucosylated. This result may be due to the fact that the molecule was expressed in transiently transfected CHO cells.

Epitope mapping.

The epitope on ILT4 for antibody clone p1E1 (G1) was identified by hydrogen-deuterium exchange mass spectrometry. Antibody p1E1 (G1) was premixed with recombinant histidine-tagged extracellular domain of ILT4, then the complex was incubated in deuterium buffer. The degree of deuterium incorporation was measured by mass spectrometry. ILT4 residues LYREKKSASW (39-48 from ILT4 without signal sequence) (SEQ ID NO: 59) and TRIRPEL (50-56 from ILT4 without signal sequence) (SEQ ID NO: 60) and, to a lesser extent, NGQF (59-62 from ILT4 without signal sequence) (SeQ ID NO: 61) and HTGRYGCQ (71-78 from ILT4 without signal sequence) (SEQ ID NO: 62) were identified as showing the largest differences in deuterium labeling compared to the antigen-only sample, indicating that they are likely residues that interact with p1E1 (G1) (Fig. 1). These peptides are located on domain 1 of ILT4. Other peptides per domain 1 and 2 showing smaller differences in deuterium labeling are likely protected due to conformational stability after antibody binding. No significant differences in labeling were observed in domains 3 and 4.

When mapped to the crystal structure of human ILT4, residues protected by p1E1 (G4) form a nonlinear conformational epitope containing three beta strands and a loop (Fig. 2A).

ILT4 uses two binding surfaces to recruit its HLA-G ligand (Shiroishi et al, 2006): beta-2-microglobulin binding site 1, located in ILT4 domain 2, and HLA-G heavy chain binding site 2, located in the 1 ILT4 (Fig. 2B). Region 1 includes ILT4 residues Trp-67, Asp-177, Asn-179 and Val-183 (numbered after Shiroishi et al, 2006). Region 2 includes ILT4 residues Arg-36, Tyr-38, Lys-42, Ile-47 and Thr-48 (numbered after Shiroishi et al, 2006). The HDX-MS data in this description indicate that Tyr-38, Lys-42 and Thr-48 (numbered according to Shiroishi et al, 2006) are part of the p1E1 (G1) epitope on ILT4 domain 1 as residues Tyr-40, Lys -44 and Thr-50 of human ILT4 (Fig. 2A). This suggests that the human ILT4 p1E1 (G1)-bound epitope overlaps with the HLA-G ligand-bound region 2 epitope.

Example 2. Affinity, binding and blocking properties of mAb against ILT4 1E1.

Binding affinities of 1E1(G4) and HLA-G1 to human ILT4 determined by surface plasmon resonance (SPR).

Binding of 1E1 (G4) and HLA-G1-Fc (recombinant extracellular domain of HLA-G (isoform 1) fused to the Fc domain of human IgG1 to produce soluble HLA-G1 protein) to ILT4-His (recombinant extracellular domain of human ILT4 fused with a polyhistidine tag to produce soluble ILT4 protein) was analyzed by Biacore. 1E1(G4) or HLA-G1 Fc was captured on the Biacore chip using Fc clamping. Monomeric ILT4-His was then tested for binding, and the data suggested that ILT4-His bound 1E1 (G4) with 600-fold higher affinity than HLA-Gl-Fc (Table 3).

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Table 3

ILT4-His binds 1E1 (G4) with more than 600-fold higher affinity than HLA-G1-Fc. 1:1 binding kinetics and equilibrium assays indicate 630- and 670-fold differences

η Ligand ka (l/NTV) kd(1/c) _KD (M) Kd ratio 2 1E1 (G4) 5.5E+05 9.0E-03 l,7E-08 1 2 HLA-G1 Fc (Kinetics) 1DE+05 1DE+00 1DE-05 630 HLA-G 1 Fc (SSA) — — 1DE-05 670

Surface plasmon resonance (SPR) analysis using a Biacore T200 device (GE HEALTHCARE) was used to determine the monovalent affinities of anti-human ILT4 IgG4 mAb (1E1(G4)) and HLA-G1-Fc fusion protein (HLA-G1-Fc) to tagged polyhistidine human ILT4 (ILT4-His). The mAb or Fc fusion protein was fixed to a CM5 sensor chip prepared using a human Fc fixation kit (GE HEATHCARE), and a series of titrated concentrations of ILT4-His were injected onto the surface. Biacore T200 software was used to fit each titration series to a 1:1 binding model. The rate constants of association (ka, M ^-1 s ^-1 ) and dissociation (kd, s ^-1 ) for each set of titrated concentrations were determined and used to calculate the dissociation constant, KD (M)=ko _ff /k _on for each titration. As shown in table. 1, the monovalent affinities (KD) of mAb 1E1(G4) and HLA-G1-Fc for His-tagged human ILT4 were 17 nM and 11 μM, respectively, with the KD ratio indicating a 630-fold difference. Due to the high kinetic constants ka and kd, stationary approximation (SSA) was also used to confirm the low affinity of HLA-G1-Fc for ILT4-His.

Blocking HLA-G binding to 3A9 T cell transfectants with ILT4 using 1E1 (G4).

3A9 T cell transfectants with ILT4 were pretreated with 1E1(G4) or isotype control at various doses, followed by secondary detection of 1E1(G4) (Fig. 3A) or by treatment with a fixed concentration of biotinylated chimeric HLA-G1-Fc to assess 1E1(G4) capacity ) block the cognate ligand (Fig. 3B). 1E1(G4) and HLA-G Fc were determined by flow cytometry. Data suggested that 1E1(G4) blocked HLA-G1 Fc binding in a dose-dependent manner.

Luminex and cell-based ligand blocking and ligand competition assays.

Antibodies 1E1, 2A6, 2C1 and 3G7 were tested in the Luminex assay and the cell-based ligand blocking and ligand competition assay for the ability to inhibit the interaction of recombinant dimeric HLA-G with ILT4 antigen coupled to particles or expressed by CHO/ILT4+ cells.

Luminex-based ligand blocking and ligand competition assays used recombinant human ILT4 antigen chemically coupled to Luminex particles. In the blocking assay, particles were preincubated with a dose range (0.5-9000 ng/ml in serial dilutions of 1:3) of hIgG4 anti-ILT4 antibody variants 1E1, 2A6, 2C1, or 3G7. The particle-bound ILT4 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 particles were preincubated with soluble biotinylated HLA-G/Fc fusion protein at a concentration of 50 nM before titration (0.5-9000 ng/ml in 1:3 serial dilutions) of hIgG4 variant anti-ILT4 antibodies 1E1, 2A6, 2C1 or 3G7. In both assays, the ILT4:HLA-G interaction was determined using a PE-conjugated anti-streptavidin antibody and IC ₅₀ values were determined. All antibodies tested demonstrated a dose-dependent blocking of HLA-G binding to Luminex ILT4/Fc coupled particles, and IC ₅₀ values are shown in Table 1. 4. All antibodies tested also demonstrated dose-dependent competition with HLA-G for binding to particle-bound ILT4/Fc (data not shown).

Table 4

Cellular ligand blocking and ligand competition assays followed a similar principle to that described for the Luminex assays and used

- 49 044253 cell line CHO/ILT4. In the blocking assay, cells were preincubated with test 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 μg/ml. In the competitive analysis, the reverse version was used. Cells were preincubated with soluble biotinylated HLA-G/Fc fusion protein at a concentration of 5 μg/ml before adding titrated doses of test antibody ranging from 1-20,000 ng/ml in serial dilutions of 1:3. ILT4:HLA-G interaction was determined using a PE-conjugated anti-streptavidin antibody. All antibodies tested demonstrated dose-dependent blocking of HLA-G binding to CHO-expressed ILT4, and IC ₅₀ values are shown in Table 1. 5. All antibodies tested also demonstrated dose-dependent competition with HLA-G for binding to CHO-expressed ILT4 (data not shown).

Table 5

Blockade of non-HLA-G MHC class I ligand binding to 3A9 T cell transfectants with ILT4 using p1E1 (G1).

3A9 T cell transfectants with ILT4 were pretreated with p1E1 (G1) or hIgG1 isotype control at various doses, followed by a fixed concentration of fluorochrome-tagged HLA-F or CD1d tetramers or HLA-A02:01 or HLA*B7:02 dexamers to assess p1E1 capacity (G1) block non-HLA-G MHC class I ligands. Binding of HLA-A, HLA-B, and HLA-F to ILT4 was inhibited by p1E1(G1) in a dose-dependent manner (Fig. 4), indicating the ability of p1E1(G1) block other known MHC class I ligands.

Blockade of ANGPTL binding to 3A9 T cell transfectants with ILT4 using p1E1 (G1).

Recently, angiopoietin-like (ANGPTL) proteins were found to bind to ILT4 expressed by human hematopoietic stem cells (Zheng et al., Nature. 2012 May 30;485(7400):656-60 and Deng et al. Blood. 2014 Aug 7 ;124(6):924-35). To test whether p1E1(G1) could block ANGPTL family members from binding to ILT4, commercially available ANGPTL proteins or fragments thereof were purchased and tested for binding to p1E1(G1)-pretreated 3A9 T cell transfectants with ILT4. Binding data suggest that ANGPTL1, 4, and potentially 7 can bind to ILT4, but not control vector cells, at the protein concentrations tested. p1E1 (G1) could completely block ANGPTL protein binding at a saturating dose (Fig. 5).

ILT family specificity of 1E1 (G4) when binding to human ILT 3 A9 T cell transfectants.

The specificity of 1E1 (G4) for the ILT family when binding to human ILT family members was analyzed by cell flow cytometry using the 3A9 T cell line transfected to express ILT4, ILT2, ILT3 (two variants), ILT5, LILRB5, LILRA1, LILRA2, Human ILT7, ILT8 or ILT11. 1E1(G4) specifically bound to human ILT4 and did not show cross-reactivity with any other ILT family member tested (Fig. 6A and 6B).

Example 3. Biological activity of mAb against ILT4 1E1 in engineered and primary cells.

Ability of 1E1 (G4), 2A6, 2C1, and 3G7 to reverse interleukin-2 suppression in assays of engineered 3A9 cells with ILT4.

An anti-CD3 antibody was used to stimulate control 3A9 mouse T cells, resulting in increased IL-2 release. In contrast, 3A9 T cell transfectants with ILT4 failed to express IL-2 upon CD3 stimulation, likely due to cross-reactivity of ILT4 with mouse MHC class I molecules or unknown xeno-ligands. This interaction appears to result in spontaneous multimerization/activation of the ILT4 receptor, resulting in suppression of anti-CD3 antibody-mediated IL-2 release. Thus, antibodies that functionally bind to ILT4, block the interaction of ILT4 with xeno-ligands, and/or inhibit receptor multimerization should restore IL-2 release.

1E1 (G4), 2A6, 2C1, and 3G7 were tested to mediate IL-2 release by 3A9 mouse T cell transfectants with ILT4. 1E1(G4), 2A6, 2C1, or 3G7 were added to ILT4+ mouse 3A9 T cell transfectants and IL-2 release was measured photometrically by ELISA after 24 hours of anti-CD3 antibody-mediated cell stimulation. A typical dose response curve for 1E1 (G4) is shown in FIG. 7. The _EC50 values of the antibodies tested were determined using dose-response curves and are

- 50 044253 are included in the table. 6.

Table 6

IL-2 repression assay

Mouse WTMC (wild-type mast cell) degranulation assay (qualitative only) p1E1(G4) and 1E1(G4) were tested for restoration of ILT4:HLA-G-dependent mast cell degranulation. Mouse WTMC mast cells were transfected with human ILT4 and stimulated with plate-bound anti-CD200RLa antibody. Antibody-mediated cross-linking of CD200RLa resulted in mast cell degranulation through activation of ITAM motifs found in the intracellular domain of CD220RLa. Degranulation can be measured colorimetrically by analyzing the release of granule contents in the analyzed supernatants. In the presence of plate-bound HLA-G tetramer, CD200RLa-mediated mast cell degranulation in ILT4 transfectants was inhibited. Pretreatment of WTMC transfectants with ILT4 with p1E1(G4) or 1E1(G4) before stimulation with plate-bound anti-CD200RLa antibody and HLA-G tetramer reversed the inhibition of degranulation in a dose-dependent manner (Fig. 8).

Effect of 1E1 (G4) on the secretion of myeloid-derived cytokines by primary human peripheral blood mononuclear cells (PBMCs).

Evaluation of 1E1(G4) in the LPS stimulation assay of primary human PBMCs.

TNFα, a classic proinflammatory cytokine of myeloid origin, is reported to be expressed by monocytes expressing low levels of ILT4 upon stimulation with LPS. Monocytes with high ILT4 expression did not express as much TNFα, and lack of ILT4 expression was found to be a characteristic feature of monocytes isolated from patients with psoriatic arthritis (Bergamini et al., PLoS One. 2014 Mar 27;9(3):e92018). Expression of ILT4 on monocytes may inhibit the effector activity of myeloid cells and antagonize the induction of proinflammatory cytokines (eg, TNF-α) in the presence of proinflammatory stimuli (eg, LPS). Therefore, whole PBMC isolated from healthy human donors were treated with LPS and the ability of ILT4 antagonism to lead to increased expression of proinflammatory myeloid cytokines was assessed using 1E1(G4). In fig. 9A and 9B show data from one of three experiments using 3 donors each, showing that 1E1(G4) increased LPS-dependent expression of GM-CSF and TNFa (myeloid-derived cytokines) in a dose-dependent manner.

Score 1E1 (G4) in anti-CDS antibody stimulation assay of primary human PBMCs.

To evaluate whether 1E1(G4) treatment could increase the expression of T cell or myeloid effector cytokines in the presence of a suboptimal T cell stimulus, whole PBMCs isolated from healthy human donors were treated with anti-CD3 antibody to induce T cell proliferation, and with Using 1E1 (G4), the ability of ILT4 antagonism to modify cytokine expression was assessed. Figures 10A and 10B show data from one of three experiments with 3 donors each, indicating that 1E1(G4) increased anti-CD3 antibody-dependent expression of GMCSF and TNFa in a dose-dependent manner.

Example 4. Antitumor efficacy of anti-ILT4 antibodies in the SK-MEL-5 tumor model in humanized mice.

Antitumor efficacy of 1E1, 2A6, 2C1 and 2D5 in a humanized mouse tumor model.

Antibodies 1E1, 2A6, 2C1 and 2D5 were tested in an in vivo tumor regression assay. Humanized mice (NSG mice reconstituted using human hematopoietic stem cells to mimic the structure of human immune cells) were inoculated with 1 x 10 ⁶ SKMEL5 melanoma cells (HLA class A*02:01) and tumor growth was monitored until an average size of 150 mm ³ was achieved. approximately 35 days. Seven randomized groups of mice, each containing six animals, were injected subcutaneously with isotype control antibody (hIgG1+hIgG4, 20 mg/kg each) or 20 mg/kg of any of the following anti-ILT4 antibodies: 1E1-IgG1, 1E1-IgG1 (N297Q ) (Fc-naive mutant), 1E1-IgG4, 2A6-IgG4, 2C1-IgG4 and 2D5-IgG4. Mice were dosed every seven days (on days 35, 42, and 49; three doses total) and tumor size was measured until day 63. Results indicate reduced tumor growth in mice dosed with 1E1 (IgG1, IgG1(N297Q), IgG4), 2A6-IgG4 and 2D5-IgG4, compared with isotype control animals (Fig. 17). In contrast, mice treated with the isotype control or the anti-ILT4 antibody candidate 2C1 did not experience a reduction in tumor growth.

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Antitumor efficacy of p1E1(G4) in a humanized mouse tumor model.

A humanized mouse tumor model was developed to test the in vivo efficacy of p1E1(G4) in inhibiting tumor growth. Immunodeficient NSG mice were reconstituted using human hematopoietic stem cells. After confirming that mice had peripheral human CD45+ immune cells (>25% PBMC), they were inoculated with SK-MEL-5 tumor cells, a tumor line derived from human cutaneous melanoma. These cells were selected for HLA-G expression. After inoculation, tumors were allowed to grow to a size of approximately 150 mm ³ . Mice were randomly assigned to groups and treated with hIgG4 isotype control or p1E1 (G4).

Mice treated with p1E1(G4) showed inhibition of tumor growth over the course of the study (Fig. 11A). One complete and one partial regression were observed with p1E1 (G4) (Fig. 11C).

Antitumor efficacy of 1E1(G4) in a humanized mouse tumor model.

The antitumor activity of 1E1(G4) was tested in the SK-MEL-5 tumor model in humanized mice. In this model, immunodeficient NSG (NOD.Cg-Prkdc ^scld Il2rg ^{tml Wjl} /SzJ) mice were irradiated and injected with human hematopoietic CD34+ stem cells isolated from umbilical cord blood. After several months of engraftment, human immune cells can be detected in the mice's blood. Mice were then subcutaneously (SC) implanted with the human melanoma-derived cell line SK-MEL-5.

In this study, NSG mice transplanted with human cord blood-derived CD34+ cells from 3 separate donors at 3-4 weeks of age were injected with 1 x 10 ⁶ SK-MEL-5 SC cells at approximately 20 weeks of age. Humanized NSG mice bearing SK-MEL5 tumors were assigned to 2 treatment groups of 6 mice per group (3 mice from each cohort with donor human CD34+ cells per treatment group) when the average tumor size reached approximately 100 mm ³ at 21 days post-inoculation tumors (DO randomization). Tumor-bearing mice were injected with SC 20 mg/kg 1E1 (G4) or isotype control mAb hIgG4 every 7 days for 4 doses. Tumor volumes were monitored every 7 days after initiation of treatment.

Antitumor efficacy in the 1E1 (G4) treatment group was significantly higher than that in the isotype control group (p<0.001 from day 28 to day 49) (Fig. 12A). The final tumor weight in mice treated with 1E1 (G4) was lower than that in mice treated with isotype control (Fig. 12C). Overall, the results indicate significant antitumor efficacy of 1E1(G4) at a dose of 20 mg/kg in the SK-MEL-5 humanized mouse tumor model. No effect was observed on body weight (FIG. 12B) or spleen weight (FIG. 12D) with 1E1 (G4) administration.

Example 5: ILT4 haplotype-specific binding of 1E1 (G4) to human 3A9 T cell transfectants with ILT.

Single nucleotide polymorphism data from publicly available sources (1000 Genomes Project Phase 3) was used to determine ILT4 allele frequencies in African, European, Asian, and South Asian populations. The sequences of haplotypes expressed with a frequency of 5% or more in any population (haplotypes 1, 2, 5, 7, 9 and 10 in Table 7) were determined and expressed in 3A9 T cells. 1E1 (G4) bound all tested haplotypes when using saturating doses of antibody (Fig. 13). Haplotype 2 corresponded to the consensus sequence given in Uniprot. Haplotype 5 was used in functional and ligand assays. Haplotype binding data indicate that 1E1(G4) binds to all major allelic variants tested.

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Table 7

Frequencies of ILT4 haplotypes in human populations

Gap LO type rs3730 32 rs3860 56 rs7247 538 rs7247 451 rsll28 645 Haplotype frequencies by ethnicity p.Glul 61Asp p.Val2 35Met p.His30 OTyr p.Cys3 06Trp p.Arg3 22His c.483 A>T C.703G >A c.898C >T c.918C >G c.965C >G All Afri Kans Euro peoids Azi And you South Asia 1 AT GG WITH D M H C R 0.23 0.09 0.15 0.60 0.15 2 TS GG WITH E V H C R 0.14 0.04 0.22 0.08 0.28 3 AT A.G. WITH D M Y C R 0.03 4 TS G.C. WITH E M H W R 0.02 5 A.C. A.C. T D V Y w H 0.30 0.17 0.58 0.08 0.37 6 A.C. A.C. C D V Y w R 0.02 0.04 0.02 7 A.C. G.C. T D V H w H 0.02 0.06 8 A.C. GG T D V H c H 0.02 9 A.C. G.C. C D V H w R 0.02 0.01 0.11 10 A.C. GG C D V H c R 0.23 0.54 0.20 0.06 f(S NP) 0.80 0.24 0.45 0.46 0.46

*Haplotypes for each population are based on data from Phase 3 of the 1000 Genomes Project and determined using PLINK to analyze non-synonymous SNPs listed in the EXAC database.

Example 6. ILT4 RNA expression in tumor and cell types based on data from the TCGA and Blueprint databases.

ILT4 expression in tumor types and cell populations was determined using publicly available RNA sequencing datasets through Omicsoft (Qiagen, Cary, NC). The TCGA dataset (TCGA_B38_20171002_v4, https://gdc-portal.nci.nih.gov/) consists of 11,292 samples with RNA-seq data. The Blueprint dataset (Blueprint_B38_20170216_v2, http://www.blueprintepigenome.eu/) consists of 258 normal blood samples for 55 cell types with RNA sequencing data.

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Tumor types with the highest expression of ILT4 at the RNA level include LAML (ie AML), DLBC (ie DLBCL), TGCT, MESO, KIRC (Fig. 14A). Cell types with the highest expression of ILT4 at the RNA level include neutrophils, monocytes, osteoclasts, eosinophils, macrophages and dendritic cells (Fig. 14B). In this data set, lymphocytes had low or no ILT4 expression. FPKM 1 (or LOG2(FPKM+0.1) from 0) is the most widely used heuristic fixed threshold, although lower FPKM values may indicate the presence of genes with low expression in the sample.

Example 7. Binding of 1E1 (G4) to myeloid cells from tumor histoculture samples.

Histocultures were obtained from fresh human tumor specimens (surgical resection) and treated with anti-RSV IgG4 (isotype control) or 1E1 (G4) at a dose of 20 μg/ml for 18-24 hours at 37°C. After processing, tumor sections were digested into single cell suspensions and stained for FACS. Dot plots and contour plots represent FACS data for single cell suspensions from histocultures of RCC (Fig. 15A) or CRC (Fig. 15B) tumors. All myeloid cells can be divided into four subpopulations based on the expression of CD66b and/or CD14. These four myeloid cell subsets were simultaneously analyzed for ILT4 expression using a noncompetitive commercial PE-conjugated anti-ILT4 antibody (BioLegend, cat. no. 338706, San Diego, CA) and cell surface-bound 1E1 (G4) using a secondary anti-ILT4 antibody. A good correlation was observed between ILT4+ cells and 1E1(G4)+ cells in histocultures treated with 1E1(G4). The tumor infiltrating lymphocytes in these samples were observed to be ILT4 and 1E1 (G4) cells.

The scope of the present invention is not limited to the specific embodiments presented in the present description. In fact, the scope of the present invention relates to embodiments specifically described in the present description and other embodiments not specifically described in the present description; The embodiments specifically described herein are not intended to be exhaustive. Various modifications of the invention in addition to the modifications presented in the present description will be apparent to those skilled in the art from the above description. Such modifications are intended to be included within the scope of the claims.

Patents, patent applications, publications, product and method descriptions are cited throughout the present invention, the descriptions of which are incorporated herein by reference in their entirety for all purposes.

List of sequences.

The present invention is filed with a copy of the sequence listing in machine readable form (CRF). The CRF is named 24443_PCT_SEQLIST.txt, created March 22, 2018, is 128042 bytes in size, and is incorporated herein by reference in its entirety.

Claims (29)

(1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 63; and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 70; (1) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 1 or 79; and an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 3; 1, 2, 8, 44, 79, 80 or 81. (1) a heavy chain variable domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHXGSTNYNPSLKS where X is S or A (SEQ ID NO: 17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GX1X ₂ NRPS, where X1 is N, Q, E or D, and X ₂ is S or A (SEQ ID NO: 20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21). 1. An antibody or antigen-binding fragment thereof that binds to human immunoglobulin-like transcript 4 (ILT4), containing: (2) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57; and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 71; (2) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2 or 80; and an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 4; 2. An antibody or an antigen-binding fragment thereof according to claim 1, containing: a heavy chain variable domain containing: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO: 48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS (SEQ ID NO: 52) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21). (3) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57; and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 72; (3) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2 or 80; and an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 5; 3. An antibody or its antigen-binding fragment according to claim 1, containing: an immunoglobulin light chain having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7 or 45, and/or an immunoglobulin heavy chain having at least 90% amino acid identity - 54 044253 sequences in relation to the amino acid sequence given in SEQ ID NO: (4) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57; and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 73; (4) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2 or 80; and an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 6; 4. An antibody or its antigen-binding fragment according to claim 1, containing: an immunoglobulin light chain comprising 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 or 77, and/or an immunoglobulin heavy chain, comprising a heavy chain variable domain having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 63, 57 or 69. (5) a heavy chain variable domain containing an amino acid sequence leading - 55 044253 dated in SEQ ID NO: 57; and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 58; or (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. (5) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2 or 80; and an immunoglobulin light chain containing the amino acid sequence shown in SEQ ID NO: 7; or (6) an immunoglobulin heavy chain containing the amino acid sequence set forth in SEQ ID NO: 2 or 80; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 3. 5. Antibody or antigen-binding fragment thereof according to claim 1, containing: an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7 or 45; and/or an immunoglobulin heavy chain containing the amino acid sequence set forth in SEQ ID NO: 1, 2, 44, 79, 80, 81. 6. The antibody or antigen-binding fragment thereof according to claim 1, containing: a light chain variable domain containing the amino acid sequence given in SEQ ID NO: 70, 71, 72, 73, 58 or 77, and/or a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 63, 57 or 69. 7. An antibody or its antigen-binding fragment according to claim 1, containing: 8. Antibody or antigen-binding fragment thereof according to claim 1, containing: 9. Antibody or antigen-binding fragment thereof according to claim 1, containing: a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 57; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 58. 10. Antibody or antigen-binding fragment thereof according to claim 1, containing: an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 7. 11. An antibody or an antigen-binding fragment thereof according to claim 1, containing: an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 80; and an immunoglobulin light chain containing the amino acid sequence set forth in SEQ ID NO: 7. 12. The antibody or antigen binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen binding fragment thereof is glycosylated using engineered yeast N-linked glycans or CHO N-linked glycans. 13. A pharmaceutical composition containing an antibody or an antigen-binding fragment according to any one of claims 1 to 12 and a therapeutic agent. 14. The pharmaceutical composition according to claim 13, further comprising a pharmaceutically acceptable carrier. 15. A host cell containing a polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, or a vector expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. 16. A method of blocking the binding of ILT4 to HLA-G, HLA-A, HLA-B and/or HLA-F in a person in need thereof, comprising administering to the person an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. 17. A method of treating a malignant neoplasm in a person in need thereof, comprising administering to the person an effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. 18. The method according to claim 16 or 17, further comprising carrying out a therapeutic method and/or administering a therapeutic agent to a person. 19. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, comprising culturing a host cell according to claim 15 to express the antibody or an antigen-binding fragment thereof. 20. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, comprising expressing a polynucleotide encoding the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. 21. An antibody or antigen-binding fragment thereof that binds to human ILT4 and is a product of the method according to claim 19 or 20. 22. A method for detecting an ILT4 peptide or a fragment thereof in a sample, comprising bringing the sample into contact with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 and detecting the presence of a complex between the antibody or an antigen-binding fragment thereof and the ILT4 peptide or a fragment thereof; where detection of the complex indicates the presence of the ILT4 peptide or its fragment. 23. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the treatment of a malignant neoplasm. 24. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the treatment of a malignant neoplasm. 25. An antibody consisting of two heavy chains and two light chains, wherein each light chain contains the variable region amino acid sequence set forth in SEQ ID NO: 58, and each heavy chain contains the variable region amino acid sequence set forth in SEQ ID NO: 57. 26. The antibody of claim 25, wherein the light chain further comprises the amino acid sequence set forth in SEQ ID NO: 90, and/or the heavy chain further comprises the amino acid sequence - 56 044253 sequence given in SEQ ID NO: 89. 27. An antibody consisting of two heavy chains and two light chains, wherein each light chain contains the amino acid sequence set forth in SEQ ID NO: 7, and each heavy chain contains the amino acid sequence set forth in SEQ ID NO: 2. 28. An antibody consisting 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. 29. A pharmaceutical composition comprising: (i) an antibody consisting of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence given in SEQ ID NO: 7, and each heavy chain consists of the amino acid sequence given in SEQ ID NO: 2, and (ii) pembrolizumab.