Anti-ILT2 antibodies and uses thereof

Abstract

The present disclosure provides antibodies and polypeptides that specifically bind to Ig-like transcript 2 (ILT2). Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects using these antibodies are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 268,945, filed March 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing The contents of the electronically submitted Sequence Listing XML (Name: 198451_SL; Size: 59,428 bytes; Created: March 1, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to antibodies specific for human Ig-like transcript 2 (ILT2) and methods of use thereof. [Background technology]

[0004] Ig-like transcript 2 (ILT2) is an inhibitory receptor belonging to type I transmembrane glycoproteins, with four extracellular immunoglobulin-like domains (D1-D4), a transmembrane region, and an intracellular tail with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ILT2 is expressed on various immune cells, including T cells, B cells, natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and subpopulations of monocytes / macrophages. Its ligands are both classical (HLA-A, -B, and -C) and nonclassical MHC class I molecules, but the receptor binds to HLA-G with 3-4 times higher affinity than classical MHC class I molecules. ILT2 binding to HLA-G inhibits the function and activity of both innate and adaptive antitumor immune responses, promoting cancer cell evasion from immune surveillance and antitumor immunity. Overexpression of HLA-G in solid tumors is associated with poor prognosis, tumor metastasis, and shortened disease-free survival, suggesting that blocking ILT2 / HLA-G interactions may be effective in cancer treatment.

[0005] Therefore, there is a need for therapeutics that target ILT2. Summary of the Invention

[0006] The present disclosure provides antibodies and polypeptides that specifically bind to ILT2 (e.g., human ILT2). Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects using these antibodies are also provided. The antibodies disclosed herein are particularly advantageous in that they are potent blockers of the ILT2 / HLA-G interaction and exhibit high enhancement of immune cell functional activity in vitro compared to other ILT2 antibodies currently in clinical development. Applicants believe that this will translate into superior efficacy in vivo.

[0007] In one aspect, the disclosure provides an antibody that specifically binds to human ILT2, including an antibody comprising a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO:1, and a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:8.

[0008] In one embodiment, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0009] In one embodiment, the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 15, 16, and 19, respectively.

[0010] In one embodiment, the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 12, 16, and 17; 13, 16, and 17; 14, 16, and 17; 12, 16, and 18; 13, 16, and 18; or 14, 16, and 18, respectively.

[0011] In one embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 9, 10, 11, 12, 16, and 17; 9, 10, 11, 13, 16, and 17; 9, 10, 11, 14, 16, and 17; 9, 10, 11, 12, 16, and 18; 9, 10, 11, 13, 16, and 18; or 9, 10, 11, 14, 16, and 18.

[0012] In one embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 9, 10, 11, 13, 16, and 18, respectively.

[0013] In one embodiment, the antibody comprises the VH amino acid sequence of SEQ ID NO:1.

[0014] In one embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM.

[0015] In one embodiment, the heavy chain constant region is a human IgG4 heavy chain constant region comprising a P at position 228, numbered according to the EU numbering system.

[0016] In one embodiment, the antibody comprises a heavy chain constant region that is a mutant of a wild-type heavy chain constant region, and the mutant heavy chain constant region binds to an FcγR with a lower affinity than the wild-type heavy chain constant region binds to the FcγR.

[0017] In one embodiment, the amino acid sequence of the heavy chain constant region is (a) one or more of the following amino acid mutations according to the EU numbering system: L234A, L235A, L235E, N297A, N297Q, N297G, P329A, P329G, G236D, P238D, S239D, S267E, L328F, and L328E; or (b) According to the EU numbering system, L234A and L235A; L234A and L235E; L234A, L235A, and L329A; or L234A, L235A, and P329G; S267E and L328F; P238D and L328E; P238D, and E233D, G237D, H268D, P271G, and A330R. one or more substitutions selected from the group consisting of: P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and a set of amino acid mutations selected from the group consisting of V264E, S267E, and L328F.

[0018] In one embodiment, the antibody comprises a heavy chain constant region that is a mutant of a wild-type heavy chain constant region, and the mutant heavy chain constant region binds to an FcγR with higher affinity than the wild-type heavy chain constant region binds to the FcγR.

[0019] In one embodiment, the amino acid sequence of the heavy chain constant region is (a) one or more of the following amino acid mutations according to the EU numbering system: G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L; or (b) The set of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L, and I332E; S298A, E333A, and K334A; G236A, S239D, and I332E; and F243L, R292P, Y300L, V305I, and P396L, according to the EU numbering system.

[0020] In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, or 39.

[0021] In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25, or 26.

[0022] In one embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO:8.

[0023] In one embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, or 7.

[0024] In one embodiment, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31, or 32.

[0025] In one embodiment, the VH and VL comprise the amino acid sequences set forth in SEQ ID NOs: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively.

[0026] In one embodiment, the heavy and light chains are selected from the group consisting of SEQ ID NOs: 26 and 30, 26 and 27, 26 and 28, 26 and 29, 26 and 31, 26 and 32, 25 and 27, 25 and 28, 25 and 29, 25 and 30, 25 and 31, 25 and 32, 24 and 27, 24 and 28, 24 and 29, 24 and 30, 24 and 31, 24 and 32, 23 and 27, 23 and 28, 23 and 29, and 29, 23 and 30, 23 and 31, 23 and 32, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 31, 22 and 32, 21 and 27, 21 and 28, 21 and 29, 21 and 30, 21 and 31, 21 and 32, 20 and 27, 20 and 28, 20 and 29, 20 and 30, 20 and 31, or 20 and 32.

[0027] In one embodiment, the antibody blocks binding of ILT2 to HLA-G, HLA-A, HLA-B, and HLA-C, and / or the antibody blocks inhibition of ILT2-mediated FcγR signaling.

[0028] In one aspect, the disclosure provides a polypeptide comprising a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO:1.

[0029] In one embodiment, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0030] In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO:1.

[0031] In one embodiment, the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:20, 21, 22, 23, 24, 25, or 26.

[0032] In one aspect, the disclosure provides a polypeptide comprising a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:8.

[0033] In one embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 15, 16, and 19, respectively.

[0034] In one embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 12, 16, and 17; 13, 16, and 17; 14, 16, and 17; 12, 16, and 18; 13, 16, and 18; or 14, 16, and 18, respectively.

[0035] In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO:8.

[0036] In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7.

[0037] In one embodiment, the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31, or 32.

[0038] In one embodiment, an antibody or polypeptide disclosed herein is conjugated to a cytocidal agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

[0039] In one aspect, the disclosure provides polynucleotides encoding the VH, VL, heavy chains, and / or light chains of an antibody disclosed herein, or of a polypeptide disclosed herein.

[0040] In one aspect, the disclosure provides a vector comprising a polynucleotide disclosed herein.

[0041] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) a polynucleotide disclosed herein; (b) a vector disclosed herein; (c) a first polynucleotide encoding a heavy chain variable region, or heavy chain, of an antibody disclosed herein, and a second polynucleotide encoding a light chain variable region, or light chain, of an antibody disclosed herein; (d) providing a recombinant host cell comprising a first vector comprising a first polynucleotide encoding a heavy chain variable region, or heavy chain, of an antibody disclosed herein, and a second vector comprising a second polynucleotide encoding a light chain variable region, or light chain, of an antibody disclosed herein.

[0042] In one aspect, the disclosure provides a pharmaceutical composition comprising an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, and a pharma- ceutically acceptable carrier or excipient.

[0043] In one aspect, the disclosure provides a method of producing an antibody, the method comprising culturing a host cell disclosed herein under appropriate conditions such that the polynucleotide is expressed and the antibody is produced.

[0044] In one aspect, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0045] In one aspect, the disclosure provides for the use of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein, for the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[0046] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein for use in medicine.

[0047] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein for use in treating cancer in a subject in need thereof. [Brief description of the drawings]

[0048] [Figure 1] 1 is a graph showing binding of anti-ILT2 antibody BA211, and sequence optimized variants BA212, BA216, BA213, BA214, and BA215 to CHO cells expressing relatively low levels of human ILT2. Binding is shown by mean fluorescence intensity (MFI) as a function of antibody concentration in μg / mL. [Diagram 2]1 is a graph showing binding of anti-ILT2 antibody BA211, and sequence optimized variants BA212, BA216, BA213, BA214, and BA215 to Jurkat cells expressing human ILT2. Binding is shown by mean fluorescence intensity (MFI) as a function of antibody concentration in μg / mL. [Diagram 3] 1 is a graph showing binding of sequence-optimized variant anti-ILT2 antibodies formatted with different Fc backbones to CHO cells expressing relatively high levels of human ILT2. Binding is shown by mean fluorescence intensity (MFI) as a function of antibody concentration in μg / mL. [Figure 4A] FIG. 1 is a graph showing binding of sequence optimized variant anti-ILT2 antibody BA252 and reference antibody 15G8 to CHO cells expressing relatively high levels of human ILT2. [Figure 4B] Figure 1 is a graph showing binding of sequence-optimized variant anti-ILT2 antibody BA252 and reference antibody 15G8 to CHO cells expressing relatively low levels of human ILT2. Binding is shown by mean fluorescence intensity (MFI) as a function of antibody concentration in μg / mL. ***p<0.001 and ****p<0.0001 [Diagram 5] Graph showing the ability of BA211 to block the interaction between HLA-G and ILT2-high expressing CHO cells. Blocking is shown as % of maximum mean fluorescence intensity (MFI) of HLA-G-Fc-PE as a function of antibody concentration (μg / mL). [Figure 6] Graph showing the ability of sequence-optimized mutant anti-ILT2 antibody BA252 to block the interaction between HLA-G and ILT2-high expressing CHO cells. Blocking is shown as % of maximum mean fluorescence intensity (MFI) of HLA-G-Fc-PE as a function of antibody concentration (μg / mL). [Figure 7A] FIG. 1 is a graph showing the ability of sequence-optimized mutant anti-ILT2 antibody BA252 to block the interaction between HLA-A and CHO cells highly expressing ILT2. [Figure 7B] FIG. 1 is a graph showing the ability of sequence-optimized mutant anti-ILT2 antibody BA252 to block the interaction between HLA-B and CHO cells highly expressing ILT2. [Figure 7C] Graph showing the ability of sequence-optimized mutant anti-ILT2 antibody BA252 to block the interaction between HLA-C and CHO cells highly expressing ILT2. Blocking is shown as % of maximum mean fluorescence intensity (MFI) of HLA-A*02:01, HLA-B*07:02, and HLA-C*07:02 pentamers, respectively, as a function of antibody concentration (μg / mL). [Figure 8A] FIG. 13 is a graph showing increased NFAT-luciferase signaling in Jurkat reporter cells expressing ILT2 and CD16 co-cultured with Ramos cells expressing HLA-G in the presence of the anti-CD20 antibody rituximab and increasing concentrations of a sequence-optimized variant of an anti-ILT2 IgG1 antibody. [Figure 8B] Figure 1 shows increased NFAT-luciferase signaling in Jurkat reporter cells expressing ILT2 and CD16 co-cultured with Ramos cells expressing HLA-G in the presence of the anti-CD20 antibody rituximab with increasing concentrations of a sequence-optimized variant of an anti-ILT2 IgG4 antibody. Signaling is shown by luciferase activity in relative light units (RLU) as a function of antibody concentration (μg / mL). [Figure 9A] FIG. 13 is a graph comparing the increase in NFAT-luciferase signaling in ILT2- and CD16-expressing Jurkat reporter cells co-cultured with HLA-G-expressing Ramos cells in the presence of the anti-CD20 antibody rituximab with increasing concentrations of BA252 or the reference antibody 15G8. [Figure 9B]Figure 1 is a graph comparing the increase in NFAT-luciferase signaling in ILT2 and CD16 expressing Jurkat reporter cells co-cultured with HLA-G expressing Ramos cells in the presence of the anti-CD20 antibody rituximab with increasing concentrations of BA252 or the reference antibody 15G8. Blockade is shown by luciferase activity in relative light units (RLU) as a function of antibody concentration (μg / mL). The EC50 of BA252 in this experiment was significantly lower than the EC50 of 15G8. ***p<0.001. [Figure 10] 1 is a graph showing binding of sequence-optimized variant anti-ILT2 antibody BA252 and control antibodies to complement component C1q. Binding is indicated by optical density (OD) at 450 nm as a function of antibody concentration (μg / mL). [Figure 11A] FIG. 13 is a graph showing primary immune cell activation in healthy donor PBMCs primed in the presence of BA252, reference ILT2 antibody 15G8, or isotype control, showing that donors were considered "responders" if at least one of the immune subpopulations analyzed responded to BA252 with a ≧20% increase in CD25 surface expression compared to isotype control. [Figure 11B] FIG. 1 is a graph showing primary immune cell activation in healthy donor PBMCs primed in the presence of BA252, reference ILT2 antibody 15G8, or isotype control, showing CD8+ T, NKT, and NK cell activation as a function of CD25% expression for three donors, otherwise the donor was considered a "non-responder." [Figure 12A] FIG. 1 is a graph showing differential surface expression of CD163 on primary human macrophages (Mφ), expressed as a % of the total population of CD33+ myeloid cells. [Figure 12B]Graph showing differential surface expression of CD86 / CD206 on primary human macrophages (Mφ), expressed as % of the total population of CD33+ myeloid cells. Mφ were differentiated from purified human monocytes in the presence of isotype control, BA252 or 15G8 antibodies and left untreated or polarized to M2-like Mφ using conditioned medium from JEG-3 cancer cells expressing HLA-G (cancer cell CM) or an IL-10+TGFβ cocktail. ***p<0.001, *p<0.05, ns=not significant. [Figure 13A] FIG. 13 is a graph showing binding of conjugated anti-ILT2 antibody BA252-APC to CHO cells expressing relatively high levels of human ILT2 in the absence or presence of unconjugated BA252. [Figure 13B] FIG. 13 is a graph showing binding of conjugated anti-ILT2 antibody VMP55-PE to CHO cells expressing relatively high levels of human ILT2 in the absence or presence of unconjugated BA252. [Figure 13C] FIG. 13 is a graph showing binding of conjugated anti-ILT2 antibody 1Q-G2-APC to CHO cells expressing relatively high levels of human ILT2 in the absence or presence of unconjugated BA252. [Figure 13D] FIG. 13 is a graph showing binding of conjugated anti-ILT2 antibody 292305-PE to CHO cells expressing relatively high levels of human ILT2 in the absence or presence of unconjugated BA252. [Figure 13E] FIG. 1 is a graph showing binding of conjugated anti-ILT2 antibody GHI / 75-PE to CHO cells expressing relatively high levels of human ILT2 in the absence or presence of unconjugated BA252. [Figure 13F] 1 is a graph showing binding of conjugated anti-ILT2 antibody 4F9-FITC to CHO cells expressing relatively high levels of human ILT2 in the absence or presence of unconjugated BA252. Binding is shown as median fluorescence intensity (MFI) as a function of the concentration of unconjugated BA252. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present disclosure provides anti-ILT2 antibodies and polypeptides. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of using these antibodies to treat a subject are also provided. The antibodies disclosed herein are particularly useful for treating cancer in a subject.

[0050] definition The term "ILT2" as used herein refers to immunoglobulin-like transcript 2, also known as leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) or leukocyte immunoglobulin-like receptor 1 (LIR-1). The amino acid sequence of full-length human Ig-like transcript 2 can be found under accession number Q8NHL6 (UniProtKB). ILT2 is an inhibitory receptor for class I MHC antigens that recognizes a wide range of HLA-A, HLA-B, HLA-C, HLA-G, and HLA-F alleles. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species. Thus, the term "ILT2" refers to human ILT2, unless specified as being from a non-human species, e.g., "mouse ILT2", "monkey ILT2", etc. [Table 1] JPEG2025508075000002.jpg212170JPEG2025508075000003.jpg217167JPEG2025508075000004.jpg146167

[0051] As used herein, the terms "antibody" and "antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0052] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens, or to two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies that contain two different antigen-binding sites (excluding the Fc region). Multispecific antibodies include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain and two light chain molecules, antibody light chain monomers, heteroconjugate antibodies, linked single chain antibodies or linked single chain Fvs (scFvs), camelized antibodies, affibodies, linked Fab fragments, F(ab')2 fragments, chemically linked Fvs, and disulfide linked Fvs (sdFvs). Multispecific antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or classes (e.g., human IgG1, IgG2, or IgG4) or subclasses thereof.

[0053] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These particular regions are described, for example, in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety. The definitions herein include overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" refers to the CDRs defined in MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Duebel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to the CDRs defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the heavy and light chain CDRs of an antibody are defined using different conventions. In certain embodiments, the heavy and / or light chain CDRs are defined by performing a structural analysis of the antibody and identifying residues in the variable region(s) that are predicted to contact the epitope region of the target molecule (e.g., human ILT2). CDRH1, CDRH2, and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent the light chain CDRs.

[0054] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region refers to a portion of an antibody, generally a portion of a light or heavy chain, that typically varies widely in sequence between antibodies and is used in the binding and specificity of a particular antibody to its particular antigen, typically about the amino-terminal 110-120 or 110-125 amino acids in mature heavy chains and about 90-115 amino acids in mature light chains. The sequence variability is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable region are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and a primate (e.g., non-human primate) framework region (FR).

[0055] As used herein, the terms "VH" and "VL" refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0056] The term "constant region" as used herein is common in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors).

[0057] As used herein, the term "heavy chain", when used in reference to an antibody, can refer to any of the different types, based on the amino acid sequence of the constant region, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0058] As used herein, the term "light chain" when used in reference to an antibody may refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0059] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," and "immunospecifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immunoconjugate), and such binding as such is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen can generally bind to other peptides or polypeptides with lower affinity, as measured, for example, by immunoassay, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho), or other assays known in the art. In a specific embodiment, a molecule that specifically binds to an antigen has a K that is lower than the K that the molecule would have if it were non-specifically bound to another antigen. AAt least 2 logs (e.g., 10 times), 2.5 logs, 3 logs, 4 logs, or more greater than A binds to the antigen.

[0060] As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, the term "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0061] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0062] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic or prophylactic measures as described herein. A "treatment" method employs administering an antibody to a subject having or prone to having a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate a disease or disorder, or one or more symptoms of a recurrent disease or disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0063] As used herein, in the context of administering a therapy, the term "effective amount" refers to the amount of the therapy that achieves a desired prophylactic or therapeutic effect.

[0064] As used herein, the term "subject" includes any human or non-human animal. In certain embodiments, the subject is a human or non-human mammal. In certain embodiments, the subject is a human.

[0065] As used herein with respect to an antibody or polynucleotide, the term "isolated" refers to an antibody or polynucleotide that is separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) that are present in the natural source of the antibody or polynucleotide. All examples of "isolated antibodies" described herein are further contemplated as antibodies that can be isolated, but do not have to be isolated. All examples of "isolated polynucleotides" described herein are further contemplated as polynucleotides that can be isolated, but do not have to be isolated. All examples of "antibodies" described herein are further contemplated as antibodies that can be isolated, but do not have to be isolated. All examples of "polynucleotides" described herein are further contemplated as polynucleotides that can be isolated, but do not have to be isolated.

[0066] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A specific non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, each of which is incorporated herein by reference in its entirety. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed using, for example, the NBLAST nucleotide program parameters set to score=100, word length=12, etc. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed using XBLAST program parameters set, for example, score 50, word length = 3, etc. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) World Wide Web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0067] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0068] Anti-ILT2 antibody In one aspect, the disclosure provides antibodies that specifically bind to ILT2 (e.g., human ILT2). The amino acid sequences of exemplary antibodies are shown in Table 2 herein. [Table 2] JPEG2025508075000006.jpg249168JPEG2025508075000007.jpg250167JPEG2025508075000008.jpg249167JPEG20255080750 00009.jpg250170JPEG2025508075000010.jpg248169JPEG2025508075000011.jpg254167JPEG2025508075000012.jpg167169

[0069] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a VH domain that comprises one, two, or all three of the CDRs of a VH domain set forth in Table 2. In certain embodiments, the antibody comprises a CDRH1 of a VH domain set forth in Table 2. In certain embodiments, the antibody comprises a CDRH2 of a VH domain set forth in Table 2. In certain embodiments, the antibody comprises a CDRH3 of a VH domain set forth in Table 2.

[0070] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a VL domain that comprises one, two, or all three of the CDRs of a VL domain disclosed in Table 2. In certain embodiments, the antibody comprises a CDRL1 of a VL domain set forth in Table 2. In certain embodiments, the antibody comprises a CDRL2 of a VL domain set forth in Table 2. In certain embodiments, the antibody comprises a CDRL3 of a VL domain set forth in Table 2.

[0071] The individual CDRs of the antibodies disclosed herein may be determined according to any CDR numbering scheme known in the art.

[0072] In certain embodiments, one or more of the CDRs of an antibody disclosed herein can be determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

[0073] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (e.g., human ILT2), which comprise the CDRs of an antibody disclosed in Table 2 herein, as determined by the Kabat numbering scheme.

[0074] In certain embodiments, one or more CDRs of an antibody disclosed herein can be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226, all of which are incorporated by reference in their entirety herein).

[0075] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises the CDRs of an antibody disclosed in Table 2 herein, as determined by the Chothia numbering system.

[0076] In certain embodiments, one or more of the CDRs of the antibody disclosed herein can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Duebel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety.

[0077] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), which comprises the CDRs of an antibody disclosed in Table 2 herein, as determined by the MacCallum numbering system.

[0078] In certain embodiments, the CDRs of the antibodies disclosed herein can be determined according to the IMGT numbering system as described in: Lefranc MP, (1999) The Immunologist 7:132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212, each of which is incorporated by reference in its entirety; and Lefranc MP et al., (2009) Nucleic Acids Res 37:D1006-D1012.

[0079] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises the CDRs of an antibody disclosed in Table 2 herein, as determined by the IMGT numbering system.

[0080] In certain embodiments, the CDRs of the antibodies disclosed herein can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions and represents a compromise between the Kabat CDRs and the Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group Ltd.), which is incorporated herein by reference in its entirety.

[0081] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), which comprises the CDRs of an antibody disclosed in Table 2 herein, as determined by the AbM numbering scheme.

[0082] In certain embodiments, the CDRs of the antibodies disclosed herein can be determined according to the AHo numbering system as described in Honegger and Pluckthun, A., J. Mol. Biol. 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0083] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises the CDRs of an antibody disclosed in Table 2 herein, as determined by the AHo numbering system.

[0084] In certain embodiments, the individual CDRs of the antibodies disclosed herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGt, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, which identifies residues within the variable region(s) that are predicted to contact the epitope region of ILT2.

[0085] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2) comprising a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO:1, and a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:8, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGt, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues within the variable region(s) predicted to contact an epitope region of ILT2 (e.g., human ILT2).

[0086] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences set forth in SEQ ID NOs: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively.

[0087] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a VH comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0088] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 15, 16, and 19, respectively.

[0089] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 12, 16, and 17; 13, 16, and 17; 14, 16, and 17; 12, 16, and 18; 13, 16, and 18; or 14, 16, and 18, respectively.

[0090] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 9, 10, 11, 12, 16, 17; 9, 10, 11, 13, 16, and 17; 9, 10, 11, 14, 16, and 17; 9, 10, 11, 12, 16, and 18; 9, 10, 11, 13, 16, and 18; or 9, 10, 11, 14, 16, and 18, respectively.

[0091] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 9, 10, 11, 13, 16, and 18, respectively.

[0092] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2) comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2) comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0093] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2) comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2) comprising a VL comprising an amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 8.

[0094] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, or 7. In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL comprising an amino acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, or 7. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, or 7.

[0095] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:1, and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:2, 3, 4, 5, 6, or 7. In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 1, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, or 7. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, or 7.

[0096] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), which comprises the VH and VL amino acid sequences set forth in SEQ ID NOs: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively. In certain embodiments, the VH and VL amino acid sequences consist of the amino acid sequences set forth in SEQ ID NOs: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively.

[0097] In certain embodiments, the disclosure provides an antibody that cross-competes with an antibody comprising a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively, for binding to ILT2 (e.g., human ILT2).

[0098] In certain embodiments, the disclosure provides antibodies that bind to the same or overlapping ILT2 epitope (e.g., an epitope of human ILT2) as an antibody described herein, e.g., an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively.

[0099] In certain embodiments, the epitope of an antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be accomplished using any of the methods known in the art (see, e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; Mc Pherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, all of which are incorporated herein by reference in their entirety). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194), and BUSTER (see Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323, all of which are incorporated herein by reference in their entirety). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art.For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al. (1995) and Cunningham BC & Wells JA (1989) ibid. In a specific embodiment, the epitope of the antibody is determined using alanine scanning mutagenesis studies. In addition, antibodies that recognize and bind to the same or overlapping epitopes of ILT2 (e.g., human ILT2) can be identified using conventional techniques such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another antibody to a target antigen (i.e., competitive binding assays). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as ILT2 (e.g., human ILT2). There are many types of competitive binding assays, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15). Solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176:546-52); and direct label RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82) are known, all of which are incorporated herein by reference in their entirety.Typically, such assays involve the use of purified antigen (e.g., ILT2, such as human ILT2) bound to a solid surface or cells containing one of these, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibodies. In a common aspect of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block binding of the labeled antibody to the antigen is then measured using a radioactive or enzymatic label. For further details, see, e.g., Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors, supra, pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0100] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24, 25, or 26. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24, 25, or 26.

[0101] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 27, 28, 29, 30, 31, or 32. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 28, 29, 30, 31, or 32.

[0102] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a heavy chain and a light chain, the heavy chain and the light chain being set forth in SEQ ID NOs: 26 and 30, 26 and 27, 26 and 28, 26 and 29, 26 and 31, 26 and 32, 25 and 27, 25 and 28, 25 and 29, 25 and 30, 25 and 31, 25 and 32, 24 and 27, 24 and 28, 24 and 29, 24 and 30, 24 and 31, 24 and 32, 23 and 27, 23 and 28, 23 and 29, 23 and 30, 23 and 31, 23 and 32, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 31, 22 and 32, 21 and 27, 21 and 28, 21 and 29, 21 and 30, 21 and 31, 21 and 32, 20 and 27, 20 and 28, 20 and 29, 20 and 30, 20 and 31, or 20 and 32.

[0103] In certain embodiments, the amino acid sequences of the heavy and light chains are set forth in SEQ ID NOs: 26 and 30, 26 and 27, 26 and 28, 26 and 29, 26 and 31, 26 and 32, 25 and 27, 25 and 28, 25 and 29, 25 and 30, 25 and 31, 25 and 32, 24 and 27, 24 and 28, 24 and 29, 24 and 30, 24 and 31, 24 and 32, 23 and 27, 23 and 28, 23 and 29, 24 and 31, 24 and 32, 23 and 27, 23 and 28, 23 and 31, 24 and 32, 24 and 27, 24 and 28, 24 ... and 29, 23 and 30, 23 and 31, 23 and 32, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 31, 22 and 32, 21 and 27, 21 and 28, 21 and 29, 21 and 30, 21 and 31, 21 and 32, 20 and 27, 20 and 28, 20 and 29, 20 and 30, 20 and 31, or 20 and 32.

[0104] The anti-ILT2 antigen binding molecules of the present disclosure can be linked or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, thereby generating a bispecific or multispecific antibody with second or additional binding specificities.

[0105] In certain embodiments, the antibodies disclosed herein are conjugated to a cytocidal agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In certain embodiments, a cytocidal agent is capable of inducing the death or destruction of a cell with which it is in contact. In certain embodiments, a cytostatic agent is capable of preventing or substantially reducing the proliferation and / or inhibiting the activity or function of a cell with which it is in contact. In certain embodiments, a cytocidal agent or a cytostatic agent is a chemotherapeutic agent. In certain embodiments, a radionuclide is an isotope 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 Re. In certain embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0106] Any immunoglobulin (Ig) constant region may be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin molecule (e.g., IgG2a and IgG2b).

[0107] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM.

[0108] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a heavy chain constant region that is a mutant of a wild-type heavy chain constant region, and the mutant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR.

[0109] In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, or 39. In certain embodiments, the disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, or 39.

[0110] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced in the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and / or the hinge region (residues 216-230 numbered according to the EU numbering system) of an antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0111] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of an antibody described herein to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, e.g., as described in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region may be altered, for example, to facilitate assembly of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody.

[0112] In a specific embodiment, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably Fc or hinge-Fc fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. See, for example, International Publication No. WO02 / 060919; International Publication No. WO98 / 23289; and International Publication No. WO97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which, for example, mutations that alter (e.g., decrease or increase) the half-life of the antibody in vivo, are incorporated herein by reference in their entirety. In certain embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably the Fc or hinge-Fc fragment) to decrease the half-life of the antibody in vivo. In other embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably the Fc or hinge-Fc fragment) to increase the half-life of the antibody in vivo. In specific embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In a specific embodiment, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Pat. No. 7,658,921, which is incorporated herein by reference in its entirety. This type of mutant IgG, referred to as a "YTE mutant," has been shown to have a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24).(The entire contents of which are incorporated herein by reference.) In certain embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0113] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering system) and / or the hinge region (residues 216-230 numbered according to the EU numbering system) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO02 / 060919; WO98 / 23289; and WO97 / 34631, all of which are incorporated by reference in their entirety.

[0114] In certain embodiments, the antibody comprises a heavy chain constant region that is a mutant of the wild-type heavy chain constant region, and the mutant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In certain embodiments, the mutant heavy chain constant region is a mutant human heavy chain constant region, such as a mutant human IgG1, mutant human IgG2, or mutant human IgG4 heavy chain constant region. In certain embodiments, the mutant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S267E and L328F; P238D and L328E; P238D, and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F, according to the EU numbering system. In certain embodiments, FcγRIIB is expressed on a cell selected from the group consisting of a macrophage, a monocyte, a B cell, a dendritic cell, an endothelial cell, and an activated T cell.

[0115] In further embodiments, one, two or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396 numbered according to the EU numbering system can be replaced with different amino acid residues to retain the antigen binding ability of the parent antibody while altering the affinity for the effector ligand. The effector ligand to which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In certain embodiments, deletion or inactivation of the constant region domain (by point mutation or other means) may decrease Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety, for descriptions of mutations that delete or inactivate the constant region, thereby increasing tumor localization. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, thereby decreasing Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276: 6591-604, incorporated herein by reference in its entirety).In various embodiments, one or more of the following mutations in the constant region of the antibodies described herein may be made: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; C236 deletion; P238A substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution, numbered according to the EU numbering system.

[0116] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, mutations selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody sequence described herein. In certain embodiments, mutations selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody sequence described herein.

[0117] In a specific embodiment, the antibody described herein comprises an IgG1 constant region with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In a particular embodiment, the antibody described herein comprises an IgG1 constant region with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibody described herein comprises an IgG1 constant region with a mutation selected from the group consisting of L234A, L235A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibody described herein comprises an IgG1 constant region with a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, numbered according to the EU numbering system. In a particular embodiment, the amino acid residues at positions in the constant region of the antibody described herein that correspond to positions L234, L235, and D265 of the human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively, numbered according to the EU numbering system.

[0118] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 of the constant region of the antibodies described herein, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238 of the N-terminal region of the CH2 domain of the antibodies described herein, numbered according to the EU numbering system, are altered to alter the ability of the antibody to fix complement. This approach is described further in WO 94 / 29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein is modified by mutating one or more amino acids (e.g., by introducing amino acid substitutions) at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, numbered according to the EU numbering system, to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. , 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, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0119] In certain embodiments, the antibodies described herein comprise a modified constant region of IgG1, where the modification increases the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, 0.1, 1, or 10 μg / mL of the antibody can induce cell death of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of ILT2-expressing cells within 1, 2, or 3 hours, as assessed by the methods described herein and / or methods known to those of skill in the art. In certain embodiments, the modified constant region of IgG1 comprises S239D and I332E substitutions numbered according to the EU numbering system. In certain embodiments, the modified constant region of IgG1 comprises S239D, A330L, and I332E substitutions numbered according to the EU numbering system. In certain embodiments, the modified constant region of IgG1 comprises the following substitutions, numbered according to the EU numbering system: L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the antibody is capable of inducing cell death in effector T cells and Tregs, and the percentage of Tregs undergoing cell death is at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold higher than the percentage of effector T cells undergoing cell death.

[0120] In certain embodiments, the antibodies described herein comprise an IgG2 antibody constant region in which the cysteine ​​at amino acid residue 127 of the heavy chain, numbered according to the EU numbering system, is substituted with a serine.

[0121] In certain embodiments, the antibodies described herein comprise an IgG4 antibody constant region in which the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted with a proline.

[0122] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein that has two heavy chain constant regions.

[0123] Pharmaceutical Compositions Compositions are provided herein that include an anti-ILT2 antibody disclosed herein having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, and the like. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0124] In a specific embodiment, the pharmaceutical composition comprises an anti-ILT2 antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In a specific embodiment, the pharmaceutical composition comprises an anti-ILT2 antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In a particular embodiment, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for reducing or blocking ILT2 (e.g., human ILT2) activity and treating conditions such as cancer. In a particular embodiment, the present disclosure relates to a pharmaceutical composition of the present disclosure comprising an anti-ILT2 antibody of the present disclosure for use as a medicament. In another embodiment, the present disclosure relates to a pharmaceutical composition of the present disclosure for use in a method of treating cancer.

[0125] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, can be added to parenteral formulations packaged in multi-dose containers. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0126] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration, characterized by either subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also include one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0127] Formulations for parenteral administration of antibodies include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle immediately prior to use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0128] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing viscosity enhancing and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0129] Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, douche, spray, suppository, bandage, skin patch, or other formulation suitable for topical administration.

[0130] The anti-ILT2 antibodies disclosed herein can be formulated as aerosols for local application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids useful in the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or in the form of a fine particle powder for inhalation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation will have a diameter of less than 50 microns in certain embodiments, and less than 10 microns in certain embodiments.

[0131] The anti-ILT2 antibodies disclosed herein can be formulated for topical or local application, for example for topical application to mucous membranes such as the skin and in the eye, in the form of gels, creams, and lotions, as well as for application to the eye or intracapsular or intrathecal application. Local administration is also contemplated for transdermal delivery, and administration to the eye or mucous membranes, or inhalation therapy. Intranasal solutions of the antibodies alone or in combination with other pharma- ceutically acceptable excipients can also be administered.

[0132] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entireties.

[0133] In certain embodiments, pharmaceutical compositions comprising the antibodies described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They may also be reconstituted and formulated as solids or gels. Lyophilized powders are prepared by dissolving the antibodies described herein, or pharma- ceutically acceptable derivatives thereof, in a suitable solvent. In certain embodiments, the lyophilized powders are sterile. The solvent may contain excipients or other pharmacological components that improve the stability of the powder or the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art, in certain embodiments at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In certain embodiments, the resulting solution is apportioned into vials for lyophilization. Each vial contains a single or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature. Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. To reconstitute, the lyophilized powder is added to sterile water or other appropriate carrier. Exact amounts will vary with the compound selected. Such amounts can be empirically determined.

[0134] The anti-ILT2 antibodies disclosed herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of the subject being treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the compositions of the invention. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In a specific embodiment, the antibodies described herein target tumors.

[0135] Preparations to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0136] Method and use In another aspect, the present disclosure provides a method of treating a subject using the anti-ILT2 antibody disclosed herein. Any disease or disorder in a subject that would benefit from a reduction in ILT2 (e.g., human ILT2) function can be treated using the anti-ILT2 antibody disclosed herein. In certain embodiments, the disease or disorder is resistant to a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody). In certain embodiments, the disease or disorder recurs after treatment with a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody).

[0137] The anti-ILT2 antibodies disclosed herein are particularly useful for inhibiting immune system tolerance to tumors and can therefore be used as immunotherapy for subjects with cancer. For example, in certain embodiments, the present disclosure provides a method for the inhibition of T cells (e.g., CD8 + Cytotoxic T cells, CD4 + In certain embodiments, the present disclosure provides a method of promoting activation of ILT2 cells (helper T cells, NKT cells, effector T cells, or memory T cells), the method comprising administering to a subject an effective amount of an anti-ILT2 antibody or pharmaceutical composition thereof disclosed herein. In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject an effective amount of an antibody or pharmaceutical composition disclosed herein.

[0138] Cancers that can be treated with the anti-ILT2 antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions. In certain embodiments, the cancer is a solid tumor. Examples of solid tumors include malignant tumors such as sarcomas and carcinomas, including adenocarcinomas affecting the lung, breast, ovary, lymphatic system, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neuronal or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small intestine cancer, and esophageal cancer. The cancer may be early stage, mid-stage, late stage, or metastatic cancer. In certain embodiments, the cancer is resistant to a checkpoint targeting agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1 antibody). In certain embodiments, the cancer recurs after treatment with a checkpoint targeting agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1 antibody).

[0139] In certain embodiments, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two or all of estrogen receptors, progesterone receptors, or Her2 / neu, e.g., triple-negative breast cancer), ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal cancer, thyroid cancer, cervical cancer, epithelial carcinoma, peritoneal cancer, or lymphoproliferative disease (e.g., post-transplant lymphoproliferative disease).

[0140] In certain embodiments, the cancer is a blood cancer, for example, leukemia, lymphoma, or myeloma.In certain embodiments, the cancer is a leukemia, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In certain embodiments, the cancer is a lymphoma, such as B cell lymphoma, diffuse large B cell lymphoma (DLBCL), activated B cell-like (ABC) diffuse large B cell lymphoma, germinal center B cell (GCB) diffuse large B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, relapsed follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In certain embodiments, the cancer is a myeloma, such as multiple myeloma.

[0141] In another embodiment, the cancer is selected from, for example, carcinoma (eg, advanced or metastatic cancer), melanoma, or lung cancer, for example, non-small cell lung cancer.

[0142] In certain embodiments, the cancer is lung cancer, e.g., lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

[0143] In certain embodiments, the cancer is melanoma, for example, advanced melanoma. In certain embodiments, the cancer is advanced or unresectable melanoma that does not respond to other therapies. In other embodiments, the cancer is melanoma with BRAF mutation (e.g., BRAF V600 mutation). In yet other embodiments, the anti-ILT2 antibody or pharmaceutical composition disclosed herein is administered after treatment with anti-CTLA-4 antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

[0144] In another embodiment, the cancer is liver cancer, e.g., advanced liver cancer with or without viral infection, e.g., chronic viral hepatitis.

[0145] In another embodiment, the cancer is prostate cancer, e.g., aggressive prostate cancer.

[0146] In yet another embodiment, the cancer is a myeloma, e.g., multiple myeloma.

[0147] In yet another embodiment, the cancer is a kidney cancer, e.g., a renal cell carcinoma (RCC) (e.g., metastatic RCC, clear cell renal carcinoma (CCRCC) or papillary cell renal carcinoma).

[0148] In yet another embodiment, the cancer is selected from lung cancer, melanoma, renal cancer, breast cancer, colorectal cancer, leukemia, or metastatic lesions of cancer.

[0149] In certain embodiments, the methods further comprise administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent. In certain embodiments, the chemotherapeutic agent is a hypomethylating agent (e.g., azacytidine). In certain embodiments, the chemotherapeutic agent is a DNA damage inducing agent (e.g., gemcitabine). In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-PD-1 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-CD96 antibody, an antagonistic anti-CEACAM1 antibody, an agonist anti-CD137 antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, and an antagonist anti-PD-1 antibody, and wherein an ILT2 (e.g., human ILT2) antibody or pharmaceutical composition disclosed herein synergizes with the checkpoint targeting agent.

[0150] In certain embodiments, the present disclosure relates to an antibody and / or pharmaceutical composition of the present disclosure for use in a method of the present invention, the method further comprising administering an additional therapeutic agent to a subject. In certain embodiments, the present disclosure relates to (a) an antibody and / or pharmaceutical composition of the present disclosure, and (b) an additional therapeutic agent for use as a medicament. In certain embodiments, the present disclosure relates to (a) an antibody and / or pharmaceutical composition of the present disclosure, and (b) an additional therapeutic agent for use in treating cancer. In further embodiments, the present disclosure relates to a pharmaceutical composition, kit, or kit part comprising (a) an antibody and / or pharmaceutical composition of the present disclosure, and (b) an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent.

[0151] In certain embodiments, anti-PD-1 antibodies are used in the methods disclosed herein. In certain embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Mediimmune. In certain embodiments, the anti-PD-1 antibody is PDR001, developed by Novartis Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

[0152] Further non-limiting examples of anti-PD-1 antibodies that may be used in the therapeutic methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated by reference in their entirety for all purposes: U.S. Pat. No. 6,808,710; U.S. Pat. No. 7,332,582; U.S. Pat. No. 7,488,802; U.S. Pat. No. 8,008,449; U.S. Pat. No. 8,114,845; U.S. Pat. No. 8,168,757; U.S. Pat. No. 8,354,509; U.S. Pat. No. 8,686,119; U.S. Pat. No. 8,735,553; U.S. Pat. No. 8,747,847; U.S. Pat. No. 8,779,105; U.S. Pat. No. 8,927,697; U.S. Pat. No. 8,993,731; U.S. Pat. No. 9,102,727; U.S. Pat. No. 9,205,148; U.S. Pat. No. 6,808,710; U.S. Pat. No. 6,332,582; U.S. Pat. No. 7,488,802; U.S. Pat. No. 8,008,449; U.S. Pat. No. 8,114,845; U.S. Pat. No. 8,168,757; U.S. Pat. No. 8,354,509; U.S. Pat. No. 8,686,119; U.S. Pat. No. 8,735,553; U.S. Pat. No. US2013 / 0202623A1; U.S. Publication No. US2013 / 0291136A1; U.S. Publication No. US2014 / 0044738A1; U.S. Publication No. US2014 / 0356363A1; U.S. Publication No. US2016 / 0075783A1; and PCT Publication No. WO2013 / 033091A1; PCT Publication No. WO2015 / 036394A1; PCT Publication No. WO2014 / 179664A2; PCT Publication No. WO2014 / 209804A1; PCT Publication No. WO2014 / 206107A1; PCT Publication No. WO2015 / 058573A1; PCT Publication No. WO2015 / 085847A1; PCT Publication No. WO2015 / 200119A1; PCT Publication No. WO2016 / 015685A1; and PCT Publication No. WO2016 / 020856A1.

[0153] In certain embodiments, anti-PD-L1 antibodies are used in the methods disclosed herein. In certain embodiments, the anti-PD-L1 antibody is atezolizumab, developed by Genentech. In certain embodiments, the anti-PD-L1 antibody is durvalumab, developed by AstraZeneca, Celgene, and Mediimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain embodiments, the anti-PD-L1 antibody is MDX-1105, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

[0154] Non-limiting examples of anti-PD-L1 antibodies that may be used in the therapeutic methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated by reference in their entirety for all purposes: U.S. Patent No. 7,943,743; U.S. Patent No. 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. US2010 / 0203056A1; U.S. Publication No. US2003 / 0232323A1; U.S. Publication No. US2013 / 0323249A1; U.S. Publication No. US2014 / 0341917A1; National Publication No. US2014 / 0044738A1; U.S. Publication No. US2015 / 0203580A1; U.S. Publication No. US2015 / 0225483A1; U.S. Publication No. US2015 / 0346208A1; U.S. Publication No. US2015 / 0355184A1; and PCT Publication No. WO2014 / 100079A1; PCT Publication No. WO201 4 / 022758A1; PCT Publication No. WO2014 / 055897A2; PCT Publication No. WO2015 / 061668A1; PCT Publication No. WO2015 / 109124A1; PCT Publication No. WO2015 / 195163A1; PCT Publication No. WO2016 / 000619A1; and PCT Publication No. WO2016 / 030350A1.

[0155] In certain embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein, hi certain embodiments, the anti-CTLA-4 antibody is ipilimumab, developed by Bristol-Myers Squibb.

[0156] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with a compound that targets an immunomodulatory enzyme(s), such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). Thus, in certain embodiments, the additional therapeutic agent is a compound that targets an immunomodulatory enzyme, such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In certain embodiments, such a compound is selected from the group consisting of epacadostat (IncyteCorp; see, e.g., International Publication No. WO 2010 / 005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In certain embodiments, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In a specific embodiment, the anti-ILT2 antibody disclosed herein is administered to a subject in combination with an IDO inhibitor to treat cancer. The IDO inhibitor described herein for use in treating cancer is present in a solid dosage form of a pharmaceutical composition, such as a tablet, pill, or capsule, the pharmaceutical composition comprising an IDO inhibitor and a pharma- ceutically acceptable excipient. Thus, the antibody described herein and the IDO inhibitor described herein can be administered separately, sequentially, or simultaneously as separate dosage forms. In a specific embodiment, the antibody is administered parenterally and the IDO inhibitor is administered orally. In certain embodiments, the inhibitor is selected from the group consisting of epacadostat (Insight Corporation), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NeuLink Genetics), and NLG919 (NeuLink Genetics). Epacadostat is described in PCT Publication No. WO2010 / 005958, which is incorporated herein by reference in its entirety for all purposes.In certain embodiments, the inhibitor is epacadostat. In another embodiment, the inhibitor is F001287. In another embodiment, the inhibitor is indoximod. In another embodiment, the inhibitor is NLG919.

[0157] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with a vaccine, such as a peptide vaccine, a DNA vaccine, or an RNA vaccine.

[0158] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with an adjuvant. Depending on the treatment context, various adjuvants can be used. Non-limiting examples of suitable adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), Montanide ISA (incomplete Sepic adjuvant), Revia Adjuvant System (RAS), Titormax, muramyl peptides, Syntex Adjuvant Formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvants, Gelb® adjuvant, nitrocellulose-absorbed antigens, encapsulated or entrapped antigens, 3 De-O-acylated monophosphoryl lipid A (3D-MPL), immunostimulatory oligonucleotides, toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, saponins, Quil A, QS-21, QS-7, immunostimulatory conjugates such as ISCOMATRIX. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(U).Combinations of the above adjuvants can also be used.See, for example, U.S. Patent No. 6,645,495; U.S. Patent No. 7,029,678; and U.S. Patent No. 7,858,589, all of which are incorporated herein by reference in their entirety.In certain embodiments, the adjuvant used herein is QS-21 STIMULON.

[0159] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with an additional therapeutic agent comprising a TCR. In certain embodiments, the additional therapeutic agent is a soluble TCR. In certain embodiments, the additional therapeutic agent is a cell expressing a TCR. Thus, in certain embodiments, the present disclosure relates to antibodies and / or pharmaceutical compositions of the present disclosure in combination with an additional therapeutic agent comprising a TCR for use as a medicament and / or for use in a method of treating cancer.

[0160] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with cells expressing a chimeric antigen receptor (CAR). In certain embodiments, the cells are T cells.

[0161] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with a TCR mimetic antibody. In certain embodiments, the TCR mimetic antibody is an antibody that specifically binds to a peptide-MHC conjugate. For non-limiting examples of TCR mimetic antibodies, see, e.g., U.S. Patent No. 9,074,000, U.S. Publication Nos. US2009 / 0304679A1 and US2014 / 0134191A1, all of which are incorporated herein by reference in their entirety.

[0162] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with a bispecific T cell engager (BiTE) (e.g., as described in WO2005061547A2, which is incorporated herein by reference in its entirety) and / or a dual affinity retargeting antibody (DART) (e.g., as described in WO2012162067A2, which is incorporated herein by reference in its entirety). In certain embodiments, the BiTE and / or DART specifically bind to a tumor-associated antigen (e.g., a polypeptide overexpressed in a tumor, a polypeptide from an oncovirus, a polypeptide comprising a tumor-specific post-translational modification, a polypeptide specifically mutated in a tumor), and a molecule on an effector cell (e.g., CD3 or CD16). In certain embodiments, the tumor-associated antigen is EGFR (e.g., human EGFR), and optionally the BiTE and / or DART comprises the VH and VL sequences of cetuximab. In certain embodiments, the tumor-associated antigen is Her2 (e.g., human Her2), and optionally the BiTE and / or DART comprises the VH and VL sequences of trastuzumab. In certain embodiments, the tumor-associated antigen is CD20 (e.g., human CD20).

[0163] The anti-ILT2 antibody and additional therapeutic agent (e.g., chemotherapeutic agent, radiotherapeutic agent, checkpoint targeting agent, IDO inhibitor, vaccine, adjuvant, soluble TCR, cell expressing a TCR, cell expressing a chimeric antigen receptor, and / or TCR mimetic antibody) can be administered separately as separate dosage forms, sequentially, or simultaneously. In certain embodiments, the anti-ILT2 antibody is administered parenterally and the IDO inhibitor is administered orally.

[0164] The antibodies or pharmaceutical compositions described herein may be delivered to a subject by a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intraarterially. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intratumorally. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered to a tumor-draining lymph node.

[0165] The amount of antibody or composition that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0166] The exact dose used in the composition also depends on the route of administration and the severity of the infection or disease caused by it, and should be determined according to the judgment of the practitioner and the circumstances of each subject.For example, the effective dose may also vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight, and health), whether the patient is a human or an animal, other medicines administered, or whether the treatment is preventive or therapeutic.Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated.Therapeutic dosages can be optimally titrated to optimize safety and efficacy.

[0167] The anti-ILT2 antibodies described herein can also be used to assay ILT2 (e.g., human ILT2) protein levels in biological samples using classical immunohistological methods known to those skilled in the art, e.g., immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; iodine ( 125 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 These include radioisotopes such as .Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein, rhodamine, and biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody that recognizes the anti-ILT2 antibodies described herein can be labeled and used in combination with the anti-ILT2 antibodies to detect ILT2 (e.g., human ILT2) protein levels. Thus, in certain embodiments, the present disclosure relates to the use of the anti-ILT2 antibodies of the present disclosure for the in vitro detection of ILT2 (e.g., human ILT2) protein in a biological sample. In further embodiments, the present disclosure relates to the use of the anti-ILT2 antibodies of the present disclosure for the in vitro assay and / or detection of ILT2 (e.g., human ILT2) protein levels in a biological sample. Optionally, the anti-ILT2 antibodies are conjugated to a radionuclide or detectable label and / or carry a label as described herein and / or immunohistological methods are used.

[0168] Assaying for expression levels of ILT2 (e.g., human ILT2) protein is intended to include qualitatively or quantitatively measuring or estimating the level of ILT2 (e.g., human ILT2) protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing with disease-related protein levels in a second biological sample). The ILT2 (e.g., human ILT2) polypeptide expression level in a first biological sample can be measured or estimated and compared to a standard ILT2 (e.g., human ILT2) protein level, for example, taken from a second biological sample obtained from an individual without the disorder or determined by averaging levels from a population of individuals without the disorder. As is understood in the art, once a "standard" ILT2 (e.g., human ILT2) polypeptide level is known, it can be used repeatedly as a standard for comparison. Thus, in a further embodiment, the present disclosure relates to an in vitro method for assaying and / or detecting ILT2 protein levels, e.g. human ILT2, in a biological sample comprising qualitatively or quantitatively measuring or estimating ILT2 protein, e.g. human ILT2 protein levels, in a biological sample by immunohistological methods.

[0169] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cell source that may express ILT2 (e.g., human ILT2). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells (PBMCs).

[0170] The anti-ILT2 antibodies described herein are well known and standard to those skilled in the art and can be used for prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications based on the present description. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and measurement of immune system status and / or immune response may be used to predict, diagnose and monitor patient samples known or suspected to have immune system dysfunction, or assessment of patient samples for expected or desired immune system response, antigen response or vaccine response. Assessment and measurement of immune system status and / or immune response can also help determine whether a patient is suitable for a drug clinical trial or for administration of a particular chemotherapeutic agent, radiotherapeutic agent or antibody (including combinations thereof) over another drug or antibody. This type of prognostic and diagnostic monitoring and evaluation has already been performed using an antibody against the HER2 protein in breast cancer (Dako, HercepTest™), which has also been used to evaluate patients for antibody therapy with Herceptin®. In vivo applications include directed cell therapy, immune system modulation, and radioimaging of immune responses. Thus, in certain embodiments, the present disclosure relates to an anti-ILT2 antibody and / or pharmaceutical composition of the present disclosure for use as a diagnostic agent. In certain embodiments, the present disclosure relates to an anti-ILT2 antibody and / or pharmaceutical composition of the present disclosure for use in a method for predicting, diagnosing, and / or monitoring a subject having or suspected of having an immune system dysfunction, and / or for predicting or desired immune system, antigenic, or vaccine responses. In another embodiment, the present disclosure relates to the use of an anti-ILT2 antibody of the present disclosure for predicting, diagnosing, and / or monitoring a subject having or suspected of having an immune system dysfunction, and / or for predicting or desired immune system, antigenic, or vaccine responses by ex vivo assaying and / or detecting human ILT2 protein levels in a biological sample of the subject.

[0171] In certain embodiments, anti-ILT2 antibodies can be used for immunohistochemistry of biopsy samples. In some embodiments, the method is an ex vivo method. In another embodiment, anti-ILT2 antibodies can be used to detect levels of ILT2 (e.g., human ILT2) or levels of cells with ILT2 (e.g., human ILT2) present on the membrane surface, which levels may be associated with certain disease symptoms. The anti-ILT2 antibodies described herein may carry a detectable or functional label and / or may be conjugated to a radionuclide or detectable label. When fluorescent labels are used, specific binding members can be identified and quantified using currently available microscopy and fluorescence activated cell sorter analysis (FACS), or a combination of both method procedures known in the art. The anti-ILT2 antibodies described herein may carry or be conjugated to a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes, such as aminocoumarins, fluorescein, Texas Red, AlexaFluor dyes, Cy dyes, DyLight dyes, and the like. The anti-ILT2 antibody may carry or be conjugated to a radiolabel or radionuclide, the radiolabel or radionuclide including 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186Isotopes such as Re are included. When a radioactive label is used, currently available counting procedures known in the art can be utilized to identify and quantify the specific binding of the anti-ILT2 antibody to ILT2 (e.g., human ILT2). When the label is an enzyme, detection can be achieved by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gas analytical techniques known in the art. This can be achieved by contacting a sample or a control sample with the anti-ILT2 antibody under conditions that allow the formation of a conjugate between the anti-ILT2 antibody and ILT2 (e.g., human ILT2). The conjugate formed between the anti-ILT2 antibody and ILT2 (e.g., human ILT2) is detected and compared in the sample and the control. Given the specific binding of the anti-ILT2 antibody described herein to ILT2 (e.g., human ILT2), the anti-ILT2 antibody can be used to specifically detect ILT2 (e.g., human ILT2). The anti-ILT2 antibodies described herein can also be used to purify ILT2 (e.g., human ILT2) by immunoaffinity purification. Also included herein are assay systems that can be prepared in the form of test kits, kits, or kit parts, for example, to quantitatively analyze the extent of the presence of ILT2 (e.g., human ILT2) / ILT2 (e.g., human ILT2) ligand conjugates. The systems, test kits, kits, or kit parts may include a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents.

[0172] Polynucleotides, Vectors, and Methods for Producing Antibodies In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody or portion thereof described herein that specifically binds to an ILT2 (e.g., human ILT2) antigen, or fragments thereof (e.g., VL and / or VH, and light and / or heavy chains), as well as vectors, e.g., vectors comprising such polynucleotides for recombinant expression in a host cell (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising a nucleotide sequence encoding the heavy and / or light chain of any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for efficient expression in a host cell, e.g., a mammalian cell.

[0173] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In specific embodiments, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0174] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody that specifically binds to an ILT2 (e.g., human ILT2) polypeptide and comprises an amino acid sequence described herein, as well as antibodies that compete with such antibodies (e.g., in a dose-dependent manner) for binding to an ILT2 (e.g., human ILT2) polypeptide, or that bind to the same epitope as such antibodies.

[0175] In certain aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding a light chain or a heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising the VL FRs and CDRs of an antibody described herein (see, e.g., Table 2), or a nucleotide sequence encoding a heavy chain comprising the VH FRs and CDRs of an antibody described herein (see, e.g., Table 2). In certain embodiments, the polynucleotide encodes a VH, VL, heavy chain, and / or light chain described herein. In another embodiment, the polynucleotide encodes a first VH and a first VL described herein. In another embodiment, the polynucleotide encodes a second VH and a second VL described herein. In another embodiment, the polynucleotide encodes a first heavy chain and a first light chain described herein. In another embodiment, the polynucleotide encodes a second heavy chain and a second light chain described herein. In another embodiment, the polynucleotide encodes a VH and / or VL, or a heavy chain and / or light chain of an antibody described herein.

[0176] Also provided herein are polynucleotides encoding anti-ILT2 antibodies that have been optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and removal of mRNA destabilizing elements. Methods for generating optimized nucleic acids encoding anti-ILT2 antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and / or removing inhibitory regions in the mRNA can be performed by adapting the optimization methods described in, for example, U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, all of which are incorporated by reference in their entirety. For example, potential splice sites or destabilizing elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. This change takes advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In certain embodiments, it may be desirable to make conservative mutations, e.g., changing one or more codons to encode a similar amino acid with similar chemical structure and properties and / or function as the original amino acid. Such methods may increase expression of an anti-ILT2 antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, compared to expression of an anti-ILT2 antibody encoded by a non-optimized polynucleotide.

[0177] In certain embodiments, an optimized polynucleotide sequence encoding an anti-ILT2 antibody or fragment thereof (e.g., VL domain and / or VH domain) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-ILT2 antibody or fragment thereof (e.g., VL domain and / or VH domain) described herein. In specific embodiments, an optimized nucleotide sequence encoding an anti-ILT2 antibody or fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-ILT2 antibody or fragment thereof described herein. In specific embodiments, an optimized nucleotide sequence encoding an anti-ILT2 antibody or fragment thereof described herein hybridizes under high stringency, medium stringency, or low stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-ILT2 antibody or fragment thereof described herein. Information regarding hybridization conditions is provided, for example, in US Patent Application Publication No. US2005 / 0048549 (eg, paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0178] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. The nucleotide sequence encoding the antibodies described herein, such as those described in Table 2, and modified versions of these antibodies, can be determined using methods well known in the art. That is, nucleotide codons known to encode specific amino acids are assembled in such a way as to generate a nucleic acid encoding the antibody. Such polynucleotides encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-6, incorporated herein by reference in its entirety). Briefly, this involves the synthesis of overlapping oligonucleotides that contain portions of the sequence encoding the antibody, annealing and ligating the oligonucleotides, and amplifying the ligated oligonucleotides by PCR.

[0179] Alternatively, polynucleotides encoding the antigen-binding regions described herein or the antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence is performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Using such PCR amplification methods, nucleic acid comprising sequences encoding the light and / or heavy chains of the antibody can be obtained. Using such PCR amplification methods, nucleic acid comprising sequences encoding the variable light and / or variable heavy chain regions of the antibody can be obtained. The amplified nucleic acid can be cloned into a vector for expression in a host cell and further cloning.

[0180] If a clone containing a nucleic acid encoding a particular antigen binding region or antibody is not available, but the sequence of the antigen binding region or antibody molecule is known, nucleic acid encoding an immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses the antibody (such as hybridoma cells selected to express an antibody described herein), or nucleic acid (preferably poly A+ RNA)) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify a cDNA clone from a cDNA library that encodes, for example, the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0181] DNA encoding the anti-ILT2 (e.g., human ILT2) antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an anti-ILT2 (e.g., human ILT2) antibody). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA is placed into an expression vector and transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells (e.g., CHO cells from CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to synthesize the anti-ILT2 antibody in the recombinant host cells.

[0182] To generate a complete antibody or antigen-binding region, the VH or VL sequence within the scFv clone can be amplified using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site. Using cloning techniques known to those of skill in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as human gamma 1 or human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant region, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those of skill in the art to generate a stable or transient cell line expressing full-length antibody, (e.g., IgG).

[0183] The DNA can also be modified, for example, by substituting human heavy and light chain constant region coding sequences for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0184] Also provided are polynucleotides that hybridize under high stringency, medium stringency, or low stringency hybridization conditions to polynucleotides encoding the antibodies described herein. In specific embodiments, the polynucleotides described herein hybridize to polynucleotides encoding the VH and / or VL domains provided herein under high stringency, medium stringency, or low stringency hybridization conditions.

[0185] Hybridization conditions are described in the art and known to those skilled in the art. For example, hybridization under stringent conditions may include hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C. Hybridization under highly stringent conditions may include hybridization to filter-bound nucleic acid in 6xSSC at about 45°C, followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those of skill in the art, see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3, which are incorporated herein by reference in their entirety.

[0186] In certain aspects, provided herein are cells (e.g., host cells) and associated polynucleotides and expression vectors that express (e.g., recombinantly) an antibody described herein that specifically binds to ILT2 (e.g., human ILT2). Provided herein are vectors (e.g., expression vectors) comprising a polynucleotide that comprises a nucleotide sequence encoding an anti-ILT2 antibody or fragment for recombinant expression in a host cell, preferably a mammalian cell (e.g., a CHO cell). Also provided herein are host cells comprising a vector for recombinantly expressing an anti-ILT2 antibody (e.g., a human or humanized antibody) described herein. In certain aspects, provided herein are methods of producing an antibody described herein, comprising expressing the antibody from a host cell.

[0187] Recombinant expression of an antibody described herein (e.g., a full-length antigen-binding region of an antibody described herein, or a heavy and / or light chain of an antibody) that specifically binds to ILT2 (e.g., human ILT2) typically involves the construction of an expression vector comprising a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, the heavy and / or light chain of the antibody, or a fragment thereof (e.g., the heavy and / or light chain variable region) is obtained, vectors for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing a protein by expressing a polynucleotide comprising an antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequence. Methods well known to those skilled in the art can be used to construct expression vectors comprising an antibody or antibody fragment (e.g., light or heavy chain) coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors encoding nucleotide sequences of the antibody molecules described herein, the heavy or light chains of the antibodies, the heavy or light chain variable regions of the antibodies or fragments thereof, or the CDRs of the heavy or light chains operably linked to a promoter. Such vectors can include, for example, nucleotide sequences encoding the constant regions of the antibody molecules (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entirety), and the variable regions of the antibodies can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0188] In certain embodiments, the vector comprises a polynucleotide encoding the VH, VL, heavy chain, and / or light chain of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the heavy and light chains of an antibody described herein.

[0189] The expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques and the resulting cells can be cultured by conventional techniques to produce an antibody or fragment thereof described herein.Thus, provided herein is a host cell containing a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell.

[0190] In certain embodiments, the host cell comprises a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the host cell comprises a vector comprising a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the host cell comprises a first polynucleotide encoding the VH of an antibody described herein and a second polynucleotide encoding the VL of an antibody described herein. In another embodiment, the host cell comprises a first vector comprising a first polynucleotide encoding the VH of an antibody described herein and a second vector comprising a second polynucleotide encoding the VL of an antibody described herein.

[0191] In a specific embodiment, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-ILT2 (e.g., human ILT2) antibody described herein. In certain embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

[0192] In certain embodiments, the vector population provided herein comprises a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-ILT2 (e.g., human ILT2) antibody described herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-ILT2 (e.g., human ILT2) antibody described herein.

[0193] A variety of host-expression vector systems can be utilized to express the antibody molecules described herein (see, e.g., U.S. Patent No. 5,807,715, incorporated herein by reference in its entirety). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells in which the antibody molecules described herein can be expressed when transformed or transfected with the appropriate nucleotide coding sequence. These include bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK) harboring recombinant expression constructs containing promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). 293, NS0, PERC6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells). In a specific embodiment, the cell expressing an antibody described herein is a Chinese Hamster Ovary (CHO) cell, e.g., a CHO cell from CHO GS System™ (Lonza).In certain embodiments, the heavy and / or light chains of the antibodies produced by CHO cells may have an N-terminal glutamine or glutamic acid residue replaced by pyroglutamic acid. In certain embodiments, the cells expressing the antibodies described herein are human cells, e.g., human cell lines. In specific embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as E. coli, or eukaryotic cells (e.g., mammalian cells), particularly for the expression of whole recombinant antibody molecules, are used for the expression of recombinant antibody molecules. For example, mammalian cells such as CHO cells are effective expression systems for antibodies in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus (see Foecking MK & Hofstetter H (1986) Gene 45:101-5; and Cockett MI.et al., (1990) Biotechnology 8(7):662-7, each of which is incorporated herein by reference in its entirety). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In specific embodiments, expression of nucleotide sequences encoding the antibodies described herein that specifically bind to ILT2 (e.g., human ILT2) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0194] In bacterial systems, a number of expression vectors can be advantageously selected depending on the use of the antibody molecule to be expressed. For example, when producing large quantities of such antibodies for the production of pharmaceutical compositions of the antibody molecule, a vector that directs the expression of a high level of fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBOJ 2: 1791-1794), in which the coding sequence is individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); and the like, all of which are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0195] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Coding sequences can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0196] For mammalian host cells, many virus-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control conjugate, e.g., the late promoter and tripartite leader sequence. This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) can result in recombinant viruses that are viable and capable of expressing molecules in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9, incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, to ensure translation of the entire insert, the initiation codon must be in phase with the reading frame of the coding sequence of interest. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, incorporated herein by reference in its entirety).

[0197] In addition, a host cell strain can be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure that the correct modification and processing of the expressed foreign protein is achieved. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PERC6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-ILT2 (e.g., human ILT2) antibodies described herein are produced in mammalian cells, such as CHO cells.

[0198] In a specific embodiment, the antibody described herein has reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibody can be expressed in cells that are defective or lack the ability to fucosylate. In a particular example, a cell line that knocks out both alleles of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. The Potelligent® system (Lonza) is an example of a system that can be used to produce antibodies with reduced fucose content.

[0199] For long-term, high-yield production of recombinant proteins, stable expressing cells can be generated. For example, cell lines can be engineered that stably express the anti-ILT2 (e.g., human ILT2) antibodies described herein. In a specific embodiment, the cells provided herein stably express the light chain / light chain variable region and heavy chain / heavy chain variable region that assemble to form the antigen binding region or antibody described herein.

[0200] In certain embodiments, rather than using an expression vector containing a viral origin of replication, a host cell can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, the engineered cells are grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the anti-ILT2 (e.g., human ILT2) or fragments thereof described herein. Such engineered cell lines can be particularly useful for screening and measuring compositions that interact directly or indirectly with the antibody molecule.

[0201] A number of selection systems can be used, including, but not limited to, the use of herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk-, hgprt-, or aprt- cells, respectively, all of which are incorporated by reference in their entirety. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6)): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56), all of which are incorporated by reference in their entireties.The desired recombinant clones are routinely selected by routine application of methods commonly known in the field of recombinant DNA technology, such as those described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Chapters 12 and 13; Colbere-Garapin Fet al., (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entireties.

[0202] The expression level of an antibody molecule can be increased by vector amplification (for review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), incorporated herein by reference in its entirety). If the marker in the vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is linked to the gene of interest, protein production will also increase (see Crouse GF et al., (1983) Mol Cell Biol 3:257-66, incorporated herein by reference in its entirety).

[0203] A host cell can be co-transfected with two or more expression vectors described herein, a first vector encoding a heavy chain derived polypeptide, and a second vector encoding a light chain derived polypeptide. The two vectors can contain identical selection markers that allow for equal expression of heavy and light chain polypeptides. A host cell can be co-transfected with different amounts of two or more expression vectors. For example, a host cell can be transfected with any one of the following ratios of the first expression vector to the second expression vector: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0204] Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used. In such a situation, the light chain should be placed before the heavy chain to avoid an excess of non-toxic free heavy chain (see Proudfoot NJ (1986) Nature 322:562-565; and Kohler G (1980) PNAS 77:2197-2199, all of which are incorporated herein by reference in their entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. Expression vectors can be monocistronic or multicistronic. Multicistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein): In such an expression vector, transcription of both genes is driven by the promoter, but translation of mRNA from the first gene can be driven by a cap-dependent scanning mechanism and translation of mRNA from the second gene can be driven by a cap-independent mechanism, e.g., an IRES.

[0205] Once the antibody molecules described herein have been produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., by ion exchange, affinity, particularly affinity for a specific antigen following Protein A and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Additionally, to facilitate purification, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or known in the art.

[0206] In specific embodiments, the antibodies described herein are isolated or purified. In certain embodiments, an isolated antibody is an antibody that is substantially free of other antibodies having antigen specificity different from the isolated antibody. For example, in certain embodiments, preparations of antibodies described herein are substantially free of cellular material and / or chemical precursors. The language "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the antibody, e.g., different post-translationally modified forms of the antibody, or other different aspects of the antibody (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In specific embodiments, the antibodies described herein are isolated or purified.

[0207] Anti-ILT2 (e.g., human ILT2) antibodies or fragments thereof can be produced by any method known in the art for the synthesis of proteins or antibodies, for example, chemical synthesis or recombinant expression technology. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature.For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook Jet al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach,IRL Press;Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entirety.

[0208] In specific embodiments, the antibodies described herein are antibodies that are prepared, expressed, created, or isolated by any means involving synthesis, creation through genetic engineering of DNA sequences, etc. In certain embodiments, such antibodies contain sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire in vivo of an animal or mammal (e.g., a human).

[0209] In one aspect, provided herein is a method of making an anti-ILT2 (e.g., human ILT2) antibody, comprising culturing a cell or host cell described herein. In certain embodiments, the method is performed ex vivo. In certain aspects, provided herein is a method of making an anti-ILT2 (e.g., human ILT2) antibody, comprising expressing (e.g., recombinantly expressing) an antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody obtained from the cell or host cell.

[0210] In certain embodiments, the antibody is produced by expressing a polynucleotide encoding the VH and VL of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed and the antibody to be produced. In another embodiment, the antibody is produced by expressing a polynucleotide encoding the heavy and light chains of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed and the antibody to be produced. In certain embodiments, the antibody is produced by expressing a first polynucleotide encoding the VH of the antibody described herein and a second polynucleotide encoding the VL of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed and the antibody to be produced. In certain embodiments, the antibody is produced by expressing a first polynucleotide encoding the heavy chain of the antibody described herein and a second polynucleotide encoding the light chain of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed and the antibody to be produced.

[0211] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, incorporated herein by reference in its entirety).

[0212] Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, which is taught, for example, in Harlow E&Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from a host cell exogenously expressing an antibody or fragment thereof described herein, e.g., the light chain and / or heavy chain of such an antibody.

[0213] In specific embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single cell (e.g., a hybridoma or host cell producing recombinant antibody) that specifically binds to ILT2 (e.g., human ILT2) as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or as shown in the Examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent or polyvalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody is a monospecific or multispecific (e.g., bispecific) antibody. The monoclonal antibodies described herein can be made, for example, by hybridoma methods as described in Kohler G & Milstein C (1975) Nature 256:495, which is incorporated herein by reference in its entirety, or can be isolated from phage libraries, for example, using the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) 5). th Ed., Ausubel FM et al., ibid., see Chapter 11).

[0214] As used herein, an antibody binds multivalently (e.g., bivalently) to an antigen if it contains at least two (e.g., two or more) monovalent binding regions, each of which can bind to an epitope on the antigen. Each monovalent binding region can bind to the same or a different epitope on the antigen.

[0215] Methods for producing and screening specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., ILT2 (e.g., human ILT2)). Alternatively, lymphocytes can be immunized ex vivo. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (see Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). Additionally, animals can be immunized using RIMMS (repeated immunization multiple sites) technology (see Kilpatrick K E et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0216] In certain embodiments, a mouse (or other animal such as a rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., ILT2 (e.g., human ILT2)) and once an immune response is detected, e.g., antibodies specific to the antigen are detected in the mouse serum, the mouse's spleen is harvested and splenocytes are isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, e.g., cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of the immunized mouse are harvested and fused with NS0 myeloma cells.

[0217] The hybridoma cells thus prepared are seeded and grown in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells, For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium will usually contain hypoxanthine, aminopterin, and thymidine (HAT medium), which will prevent the growth of HGPRT-deficient cells.

[0218] Specific embodiments use myeloma cells that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these myeloma cell lines are the NS0 cell line and cell lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, as well as SP-2 or X63-Ag8.653 cells available from the American Collection of Type Cultures, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (see Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel, Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

[0219] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against ILT2 (e.g., human ILT2). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, such as immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0220] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI 1640 medium. Additionally, hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0221] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0222] The antibodies described herein include, for example, antibody fragments that recognize ILT2 (e.g., human ILT2) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to generate Fab fragments) or pepsin (to generate F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of the antibody molecule linked by disulfide bonds in the hinge region.

[0223] Additionally, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or mouse cDNA libraries of diseased tissues). The DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are typically recombinantly fused to either phage gene III or gene VIII. Phage expressing antigen-binding regions that bind to a particular antigen can be selected or identified with the antigen, for example, using labeled antigen, or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies described herein include those disclosed in: Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 1 1236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are incorporated herein in their entireties.

[0224] After phage selection, as described in the above references, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including human antibodies, or other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Techniques for recombinantly producing antibody fragments, such as Fab, Fab', and F(ab')2 fragments, can also be employed using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043, all of which are incorporated herein by reference in their entirety.

[0225] In certain embodiments, to generate a complete antibody, a VH or VL sequence can be amplified from a template, e.g., an scFv clone, using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site. Using cloning techniques known to those of skill in the art, the PCR amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those of skill in the art to generate a stable or transient cell line expressing a full-length antibody, e.g., an IgG.

[0226] A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gillies SD et al., (1989) J Immunol Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0227] A humanized antibody is capable of binding to a given antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can be produced using a variety of techniques known in the art.These techniques include, but are not limited to, CDR grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1997) Prot Engineering 7(6):805-814; and al., (1994) PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895 904; Couto JR et al., (1995) Cancer Res. 55(23 Supp):5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8):1717-22; Sandhu JS (1994) Gene 150(2):409-10; and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73, all of which are incorporated herein by reference in their entirety. See also U.S. Patent Application Publication No. US2005 / 0042664A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0228] Methods for making multispecific antibodies (e.g., bispecific antibodies) have been described, see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713, all of which are incorporated by reference in their entireties.

[0229] Bispecific, bivalent antibodies and methods for their production are described, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, and U.S. Patent Application Publication Nos. 2003 / 020734 and 2002 / 0155537, each of which is incorporated herein by reference in its entirety. Bispecific, tetravalent antibodies and methods for their production are described, for example, in International Patent Application Publication Nos. WO02 / 096948 and WO00 / 44788, the disclosures of both of which are incorporated herein by reference in their entireties. See generally: International Patent Application Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992); each of which is incorporated herein by reference in its entirety.

[0230] The bispecific antibodies described herein can be produced, for example, according to the DuoBody technology platform (Genmab A / S) described in International Publication Nos. WO2011 / 131746, WO2011 / 147986, WO2008 / 119353, and WO2013 / 060867, and in Labrijn AF et al., (2013) PNAS 110(13):5145-5150. The DuoBody technology can be used to combine a first monospecific antibody comprising two heavy chains and two light chains, or half of a first antigen-binding region, with a second monospecific antibody comprising two heavy chains and two light chains, or half of a second antigen-binding region. The resulting heterodimer comprises one heavy chain and one light chain from the first antibody or first antigen-binding region, and one heavy chain and one light chain from the second antibody or second antigen-binding region. When both monospecific antibodies or antigen-binding regions recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0231] The DuoBody technology requires that each of the monospecific antibodies or antigen-binding regions contains a heavy chain constant region with a single point mutation in the CH3 domain. The point mutation allows for a stronger interaction between the CH3 domains of the resulting bispecific antibody than between the CH3 domains of either of the monospecific antibodies or antigen-binding regions. The single point mutation of each monospecific antibody or antigen-binding region is located in the CH3 domain of the heavy chain constant region at residues 366, 368, 370, 399, 405, 407, or 409, numbered according to the EU numbering system, as described, for example, in International Publication No. WO 2011 / 131746. Furthermore, the single point mutation is located at a different residue in one monospecific antibody or antigen-binding region compared to the other monospecific antibody or antigen-binding region. For example, one monospecific antibody or antigen-binding region can contain the mutation F405L (i.e., a phenylalanine to leucine mutation at residue 405), while the other monospecific antibody or antigen-binding region can contain the mutation K409R (i.e., a lysine to arginine mutation at residue 409), numbered according to the EU numbering system. The heavy chain constant regions, or antigen-binding regions, of the monospecific antibodies can be of IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and the bispecific antibodies generated by DuoBody technology can retain Fc-mediated effector functions.

[0232] Another method for generating bispecific antibodies is called the "knobs-into-holes" strategy (see, for example, International Publication No. WO 2006 / 28936). In this technique, mispairing of Ig heavy chains is reduced by mutating selected amino acids that form the interface of the CH3 domain of IgG. At positions in the CH3 domain where the two heavy chains directly interact, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (knob) is introduced at the position of the corresponding interacting residue on the other heavy chain. In some embodiments, the compositions of the present disclosure have immunoglobulin chains whose CH3 domains have been modified by mutating selected amino acids that interact at the interface between the two polypeptides to preferentially form bispecific antibodies. Bispecific antibodies can be composed of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or of different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0233] Bispecific antibodies may optionally include IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chain heterodimers. Such heterodimeric heavy chain antibodies can be routinely engineered to promote the formation of heterodimeric heavy chains, for example, by modifying selected amino acids that form the interface of the CH3 domains of human IgG4 and IgG1 or IgG3.

[0234] In certain embodiments, an antibody described herein that binds to the same epitope of ILT2 (e.g., human ILT2) as an anti-ILT2 (e.g., human ILT2) antibody described herein is a human antibody. In certain embodiments, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) any one of the antibodies described herein from binding to ILT2 (e.g., human ILT2) is a human antibody. Human antibodies can be generated using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring expressing human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of an antigen, e.g., ILT2 (e.g., human ILT2). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. It is incorporated herein by reference in its entirety.For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated by reference herein in their entireties. Examples of mice capable of producing human antibodies include the XenoMouse™ (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Medarex, Inc. / GenPharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex / Kirin), all of which are incorporated by reference herein in their entireties.

[0235] Human antibodies that specifically bind to ILT2 (e.g., human ILT2) can be generated by a variety of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, all of which are incorporated herein by reference in their entirety.

[0236] In certain embodiments, human antibodies can be produced using mouse-human hybridomas. For example, Epstein-Barr Virus (EBV) transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to generate mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., ILT2 (e.g., human ILT2)). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46:19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31, each of which is incorporated herein by reference in its entirety.

[0237] kit Also provided herein is a kit comprising one or more antibodies described herein, or pharmaceutical compositions or conjugates thereof. In a specific embodiment, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, e.g., one or more antibodies described herein. In certain embodiments, the kit comprises the pharmaceutical composition described herein and any prophylactic or therapeutic agent as described herein. In certain embodiments, the kit may comprise, for example, a T cell mitogen, such as phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or a TCR conjugate stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. Optionally, associated with such container(s) may be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for administration to humans.

[0238] Kits that can be used in the above methods are also provided. In certain embodiments, the kits include an antibody, preferably a purified antibody, as described herein in one or more containers. In a specific embodiment, the kits described herein include a substantially isolated ILT2 (e.g., human ILT2) antigen as a control. In another specific embodiment, the kits described herein further include a control antibody that does not react with the ILT2 (e.g., human ILT2) antigen. In another specific embodiment, the kits described herein include one or more elements for detecting the binding of the antibody to the ILT2 (e.g., human ILT2) antigen (e.g., the antibody can be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In a specific embodiment, the kits provided herein can include a recombinantly produced or chemically synthesized ILT2 (e.g., human ILT2) antigen. The ILT2 (e.g., human ILT2) antigen included in the kit can also be bound to a solid support. In a more specific embodiment, the detection means of the kit comprises a solid support to which the ILT2 (e.g., human ILT2) antigen is bound. Such a kit may also comprise an unbound reporter-labeled anti-human antibody or an anti-mouse / rat antibody. In this embodiment, binding of the antibody to the ILT2 (e.g., human ILT2) antigen can be detected by binding of said reporter-labeled antibody. In a particular embodiment, the present disclosure relates to the use of the kit of the present disclosure for in vitro assay and / or detection of ILT2 (e.g., human ILT2) antigen in a biological sample. EXAMPLES

[0239] The following examples are offered by way of illustration and not by way of limitation.

[0240] Example 1: Binding kinetics and optimization of anti-ILT2 antibodies This example describes the characterization of antibodies that specifically bind to human ILT2, the amino acid sequences of which are shown in Table 2 herein.

[0241] An anti-ILT2 antibody generated by mammalian retrocytodisplay was affinity matured by phage display. Variants of this antibody, named BA210, were created to improve properties and optimize the molecule. The BA210 variable domain was formatted using human kappa IgG1, IgG2, or IgG4 constant regions, with substitutions made for heavy chain aglycosylation (N297A), disulfide bond formation and stabilization (C127S, S228P) according to Table 3 below, and a predicted deamidation site at position 34 (G) and an oxidation site at position 105 (M) in the light chain were removed. An additional variant with a C-terminal lysine deletion was created to improve manufacturability. [Table 3]

[0242] Binding to purified human ILT2 protein The binding affinity of anti-ILT2 antibody variants with IgG1 N297A constant region to ECD-His tagged human ILT2 haplotypes PE01, PE02, PE03 (see Table 1) was assessed by surface plasmon resonance (SPR). Antibody BA211 containing identified post-translational modification (PTM) risk was used as a positive isotype control and irrelevant antibody VRC01 was used as a negative isotype control.

[0243] Briefly, SPR experiments were performed using a Biacore T200 instrument to measure binding kinetics (K a ), dissociation rate (K d ), and the dissociation constant (K D) was calculated from each experiment using a 1:1 binding model using the Biacore™ T200 measurement software. Antibody capture from the Human Fab Capture kit (GE Healthcare, 28958325) onto a Series S CM5 sensor chip (GE Healthcare, 29149603) was performed using the immobilization wizard for amine coupling from the Biacore™ control panel, following the manufacturer's recommendations provided with the kit.

[0244] Approximately 3 μg / mL of tested anti-ILT2 antibodies and controls diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured onto individual flow cells of a pre-prepared anti-human Fab CM5 chip, with a single flow cell held as a reference. Antibodies were captured using a 30 second injection at a flow rate of 10 μL / min until approximately 150 resonance units (Rus) were reached. Human ILT2 ECD PE01 (diluted in running buffer at concentrations of 30, 10, 3.3, 1.11, 0.37, 0.12 nM), and human ILT2 ECD PE02 and PE03 (diluted in running buffer at concentrations of 33.3, 11.1, 3.7, 1.2, 0.41, 0.14 nM) were flowed over the chip surface at a flow rate of 30 μL / min with a 3 min association phase and a 10 min dissociation phase. The sensor chip was regenerated with two injections of 10 mM glycine, pH 2.1 at 30 μL / min for 30 s followed by a 60 s stabilization period between cycles. Sensorgrams were measured and fitted to a simple Ran-Muir 1:1 interaction model using the Global Data Analysis option of the BIAevaluation3.1 software. Data quality was visually inspected for deviations and curve fitting and analyzed using R max The binding kinetics were evaluated by evaluating the parameters of , Chi2, and Tc. [Table 4]

[0245] In a separate experiment, the binding affinity of anti-ILT2 antibody variants with light chain substitutions and / or different heavy chain constant regions (BA249, BA250, BA214, BA215, BA217, BA218, BA219, BA220, BA221, and BA222) to ECD-His-tagged human ILT2 haplotype PE01 was assessed by SPR. Antibodies with identified PTM risk (BA210, BA211, BA246, BA247, and BA248) were used as positive isotype controls. Variants of the unrelated antibody VRC01 (with IgG1, IgG1 N297A, IgG4 S228P, IgG2, or IgG2 C127S constant regions) were used as negative isotype controls. Binding kinetics are shown in Table 5 as the average from two independent measurements. [Table 5]

[0246] In a separate experiment, the binding affinity of anti-ILT2 antibody variants BA214, BA221, BA251, and BA252 to ECD-His-tagged human ILT2 haplotypes PE01, PE02, PE03, ECD-His-tagged cynomolgus ILT2 ortholog protein, variant 1, ECD-His-tagged cynomolgus ILT2 ortholog protein, variant 2, ECD-His-tagged cynomolgus ILT2 ortholog protein, variant 3, ECD-His-tagged rhesus ILT2 ortholog protein, variant 1, ECD-His-tagged rhesus ILT2 ortholog protein, variant 2, ECD-His-tagged African green monkey (AGM) ILT2 ortholog protein, and ECD-His-tagged mouse PirB protein (R&D Systems, 2754PB050) was assessed by SPR. An irrelevant antibody, VRC01, with an IgG4 S228P constant region was used as a negative isotype control. Binding kinetics (K a , K d and K D ) or dissociation equilibrium constants (KD) are shown in Tables 6 and 7. [Table 6] [Table 7]

[0247] Specific binding to ILT2 Anti-ILT2 antibodies BA214, BA221, BA251, and BA252 were synthesized using recombinant human LILRA1 / LIR-6 (Sino Biological, 17220-H08H-100), LILRA2 / ILT1 (R&D Systems, 9040-T4-050), LILRA3 / ILT6 (Sino Biological, 13549-H08H-100), LILRA4 / ILT7 (Sino Biological, 16058-H08H-50), LILRA5 / ILT11 (Sino Biological, 16059-H08H-100), LILRA6 / ILT8 (Sino Biological, 29813-H08H-50), and LILRB1 / ILT2. Binding affinities for PE01 (SEQ ID NO: 44), LILRB2 / ILT4 (R&D Systems, 8429-T4-050), LILRB3 / ILT5 (R&D Systems, 9159-T5-050), LILRB4 / ILT3 (Sino Biological, 16742-H08H), and LILRB5 / LIR-8 (Sino Biological, 17221-H08H-100) were assessed by SPR as described above. The irrelevant antibody VRC01 IgG4 S228P was used as a negative isotype control.

[0248] The results showed that it bound to ILT2 with the kinetic parameters shown in Table 8. No physiologically relevant levels of binding were detected for any other LILR family members, indicating specific binding to ILT2. [Table 8]

[0249] Next, binding of BA252 to LILRA and LILRB family members was compared by SPR to commercially available anti-ILT2 antibodies 1Q-G2 (Creative, #DCABH-4494), VMP55 (Novas, #NBP2-50475PE), 4F9 (Bio-Rad, #MCA2515F), 292305 (R&D Systems, #MAB20171), 238145 (Abcam, #EPR22861-6), HP-F1 (Invitrogen, #16-5129-82), and 15G8 (see Table 2). The irrelevant antibody VRC01 IgG1 N297A was used as a negative isotype control.

[0250] The results are shown in Table 9. All anti-ILT2 antibodies bound to ILT2 with measurable affinities ranging from 0.15 nM to 10 nM. Anti-ILT2 antibodies 15G8, 1Q-G2, VMP55, 292305, 4F9 and 238145 also showed measurable binding to LILRA1 and ILT6. Conversely, BA252 did not bind to LILRA1 and showed only a very weak interaction with ILT6.

[0251] None of the anti-ILT2 clones tested bound to LILRA2, LILRA4, LILRA5, LILRA6, LILRB3, LILRB4, or LILRB5. [Table 9]

[0252] Binding to cells expressing human ILT2 The ability of anti-ILT2 antibodies to bind to cells expressing human ILT2 was tested in various cell types.

[0253] Anti-ILT2 antibody variants (BA211, BA212, BA216, BA213, BA214 and BA215) with IgG1 N297A heavy chain constant region were evaluated for their ability to bind human ILT2 expressed on the surface of CHO cells. Briefly, vectors encoding human ILT2 extracellular and transmembrane domains were transfected into CHO cells. Clones stably expressing relatively low levels of ILT2 were cultured in Power CHO-2 medium containing 4 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin and 1×HT-supplement.

[0254] For antibody binding assays, cells were plated in 96-well U-bottom tissue culture plates at 3 × 10 cells per well in 25 μL of PBS supplemented with 0.5% BSA, 1 mM EDTA, and 0.05% sodium azide (FACS buffer). 5 Cells were seeded at a density of 100x1000 ng / mL. Cells were incubated with 25 µL of serial dilutions of anti-ILT2 antibodies or isotype controls at final concentrations ranging from 10 µg / mL to 0.2 ng / mL in FACS buffer for 30 min at 4 °C.

[0255] For antibody staining, cells were washed twice with cold FACS buffer and resuspended in 100 μL of FACS buffer containing R-phycoerythrin goat anti-human IgG antibody (Jackson Immunoresearch / 109-116-098) at a final dilution of 1 / 800. After 30 min incubation at 4 °C in the dark, cells were washed twice with cold FACS buffer and cells were analyzed by flow cytometry (BD LSR Fortessa flow cytometer). CHO cells were identified using a plot of forward scatter area (FSC-A) versus side scatter area (SSC-A) and another plot of FSC-A versus forward scatter height (FSC-H) for the selection of single cells. Samples were analyzed by sequentially gating single cell populations and plotting histograms representing the fluorescence in the PE channel. For each sample, the mean fluorescence intensity (MFI) was calculated and the data were plotted with GraphPad Prism software.

[0256] As shown in Figure 1, BA211 and sequence-optimized variants BA212, BA216, BA213, BA214, and BA215 all bound to CHO cells expressing relatively low levels of human ILT2 in a dose-dependent manner. The calculated geometric mean areas under the curve (AUC) and EC 50 The values ​​are shown in Table 10. [Table 10]

[0257] The same anti-ILT2 antibody variants were tested for their ability to bind to human ILT2 expressed on the surface of Jurkat cells. Briefly, a vector encoding full-length ILT2 was transfected into Jurkat cells and a clone stably expressing ILT2 was selected. This stable cell line also expressed a luciferase reporter gene under the NFAT response element and FcgRIIIa (Promega / G7102) and was cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% non-essential amino acids, 250 μg / mL G418 disulfate solution, 100 μg / mL hygromycin, and 1 mM sodium pyruvate.

[0258] For antibody binding assays, cells were plated in 96-well U-bottom tissue culture plates at 3 × 10 cells per well in 25 μL of PBS supplemented with 0.5% BSA, 1 mM EDTA, and 0.05% sodium azide (FACS buffer). 5 Cells were seeded at a density of 1000 x 1000 cells / well. Cells were incubated with 25 μL of serial dilutions of anti-ILT2 antibodies or isotype control antibodies (VRC01 IgG1) at concentrations ranging from 10 μg / mL to 2.4 ng / mL in FACS buffer for 30 min at 4 °C. Cells were stained and identified as described above.

[0259] As shown in Figure 2, anti-ILT2 antibody BA211 and sequence-optimized variants BA212, BA216, BA213, BA214 and BA215 bound to Jurkat cells expressing human ILT2 in a dose-dependent manner. The results are shown in Table 11. [Table 11]

[0260] Anti-ILT2 sequence-optimized mutant antibodies formatted with different Fc backbones were evaluated for their ability to bind human ILT2, which is highly expressed on the surface of CHO cells. Briefly, vectors encoding human ILT2 extracellular and transmembrane domains were transfected into CHO cells. Clones expressing relatively high levels of ILT2 on the membrane were selected and cultured in Power CHO-2 medium containing 4 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 1xHT-supplement.

[0261] For antibody binding assays, cells were plated in 96-well U-bottom tissue culture plates at 3 × 10 cells per well in 25 μL of PBS supplemented with 0.5% BSA, 1 mM EDTA, and 0.05% sodium azide (FACS buffer). 5 Cells were seeded at a density of 100x1000 ng / mL. Cells were incubated with 25 µL of serial dilutions of anti-ILT2 antibodies or isotype controls at concentrations ranging from 10 µg / mL to 2.4 ng / mL in FACS buffer for 30 min at 4 °C.

[0262] For antibody staining, cells were washed twice with cold FACS buffer and resuspended in FACS buffer with R-phycoerythrin goat anti-human IgG(Fab')2 (Jackson ImmunoResearch / 109-116-097) diluted 1:250. After 30 min incubation at 4°C in the dark, cells were washed twice with cold FACS buffer and analyzed by flow cytometry (BD LSR Fortessa flow cytometer). CHO-ILT2 cells were identified and MFI was calculated as above.

[0263] As shown in Figure 3, all sequence-optimized anti-ILT2 antibodies bound to CHO cells expressing high levels of human ILT2 in a dose-dependent manner. Calculated AUC and EC 50 The values ​​are shown in Table 12. [Table 12]

[0264] The anti-ILT2 antibody variant BA252 was compared to the reference anti-ILT2 antibody 15G8 (see Table 2) for binding to cells expressing different levels of human ILT2.

[0265] CHO cells expressing relatively high and low levels of human ILT2 were plated in 96-well U-bottom tissue culture plates at 2 × 10 cells per well in 50 μL of PBS supplemented with 0.5% BSA, 1 mM EDTA, and 0.05% sodium azide (FACS buffer). 5 Cells were seeded at a density of 100x1000. Cells were incubated with 50 µL of serial dilutions of anti-ILT2 antibodies or isotype control (VRC01 IgG4) at concentrations ranging from 10 µg / mL to 0.2 ng / mL in FACS buffer for 30 min at 4 °C.

[0266] For antibody staining, cells were washed three times with cold FACS buffer and resuspended in FACS buffer containing FITC-conjugated mouse anti-human IgG4 antibody (Southern Biotech / 9190-02) at a final concentration of 1.25 μg / mL. After 30 min incubation at 4°C in the dark, cells were washed three times and analyzed as above. Statistical analysis was performed by comparing the best fit between the BA252 and 15G8 datasets using the "additional sum of squares F-test".

[0267] As shown in Figures 4A and 4B, BA252 and 15G8 bound to CHO cells expressing human ILT2 at relatively high and low levels in a dose-dependent manner, with BA252 showing a significantly higher maximum binding level (Bmax). Calculated AUC and EC 50 The values ​​are shown in Tables 13 and 14. [Table 13] [Table 14]

[0268] Example 2: Anti-ILT2 antibodies block ligand binding to ILT2 Anti-ILT2 antibodies BA211 and BA252 block the binding of the nonclassical major histocompatibility class (MHC) I molecule HLA-G to human ILT2 expressed on CHO cells Anti-ILT2 antibodies were tested for their ability to block binding between ILT2 and the non-classical MHC I ligand, HLA-G.

[0269] For blocking assays, 25 μL of CHO cells expressing relatively high levels of ILT2 were plated into 96-well U-bottom microtiter plates at 2 × 10 cells per well. 5 Cells were dispensed at a final concentration of 10 μg / mL. 25 μL of serial dilutions of anti-ILT2 antibody BA211, commercial anti-ILT2 antibody HP-F1 (Thermofisher / 16-5129-82), or isotype control (VRC01 IgG1) in FACS buffer were added to the wells to obtain final assay concentrations ranging from 10 μg / mL to 2.4 ng / mL. The mixtures were incubated for 30 min at 4 °C. The ILT2 ligand HLA-G-Fc was conjugated with R-phycoerythrin (R-PE) using the LYNX Rapid R-PE antibody conjugation kit (Bio-Rad / LNK022RPE). HLA-G-Fc-PE was resuspended at 1 μg / mL in FACS buffer, and 50 μL of the solution was added to the wells of a 96-well U-bottom tissue culture plate. After 30 min of incubation at 4 °C in the dark, the cells were washed by adding cold FACS buffer. This washing was repeated twice, after which cell fluorescence was analyzed by flow cytometry (BD LSR Fortessa flow cytometer).

[0270] CHO cells were identified using a plot of forward scatter area (FSC-A) versus side scatter area (SSC-A) for single cell selection and another plot of FSC-A versus side scatter height (FSC-H). Samples were analyzed by sequentially gating on single cell populations and plotting histograms representing the fluorescence in the PE channel. For each antibody sample, percent binding was calculated as follows: (MFI(sample) - MFI(HLA-G-PE)バックグラウンド (without antibody) / (MFI(without antibody) 完全結合 )-MFI(HLA-G-PE バックグラウンド None))*100. Data were plotted using GraphPad Prism.

[0271] As shown in Figure 5, BA211 and HP-F1 blocked the binding of HLA-G-Fc to ILT2-expressing cells. The calculated IC 50 The values ​​were 0.159 μg / mL (95% CI 0.099-0.260 μg / mL, N=8) and 0.307 μg / mL (95% CI 0.176-0.530 μg / mL, N=8).

[0272] In a similar experiment, the anti-ILT2 antibody BA252 blocked the binding of HLA-G-Fc to ILT2-expressing CHO cells. Figure 6 shows BA252 compared to the isotype control antibody VRC01 IgG4. The calculated IC of BA252 50 The value was 0.103 μg / mL (95% CI 0.077-0.138 μg / mL, N=3), indicating high inhibitory potency against ligand binding.

[0273] The anti-ILT2 antibody BA252 blocks the binding of classical MHC I molecules HLA-A, HLA-B and HLA-C to CHO cells expressing human ILT2 BA252 was tested for its ability to block binding between ILT2 and classical MHC I molecule ligands.

[0274] Phycoerythrin (PE)-conjugated HLA-A*02:01 pentamers (ProImmune / F008-2A-G), HLA-B*07:02 pentamers (ProImmune / F045-2A-G) and HLA-C*07:02 pentamers (ProImmune / F3269-2A-G) were suspended in FACS buffer at 0.8 μg / mL, 0.4 μg / mL and 0.4 μg / mL, respectively, and 50 μL of each solution was added to three separate wells of a 96-well U-bottom tissue culture plate. 25 μL of serial dilutions of BA252 or isotype control antibody VRC01 IgG4 in FACS buffer were added to the wells to obtain final assay concentrations ranging from 30 μg / mL to 0.5 ng / mL. CHO cells expressing relatively high levels of ILT2 were then cultured at 2 × 10 per well. 5 Cells were added at a final concentration of 25 μL. After 45 min incubation at 4°C in the dark, cells were washed with cold FACS buffer. This wash was repeated twice before analysis of cell fluorescence by flow cytometry (BD LSR Fortessa flow cytometer). CHO cells were identified and samples were analyzed as described above.

[0275] As shown in Figures 7A to 7C, BA252 inhibited the binding of HLA-A*02:01, HLA-B*07:02, and HLA-C*07:02 pentamers to ILT2-expressing cells by IC 50 Blockade values ​​were 0.110 μg / mL (SD + / - 0.020 μg / mL, N=2), 0.136 μg / mL (SD + / - 0.025 μg / mL, N=2), and 0.158 μg / mL (SD + / - 0.022 μg / mL, N=2).

[0276] Example 3: Functionality of anti-ILT2 antibodies A sequence-optimized anti-ILT2 antibody blocks ILT2 and increases FcγRIIIa-NFAT signaling in a human ILT2 T cell reporter assay In this example, anti-ILT2 antibodies BA214, BA221, BA251, and BA252 were tested for their ability to block binding between ILT2-expressing Jurkat reporter cells and HLA-G-expressing Ramos target cells and to enhance FcγRIIIa signaling in Jurkat cells via NFAT. The commercially available anti-ILT2 antibody HP-F1 was used as a positive control and VRC01, which contains IgG1 N297A and IgG4 S228P constant regions, was used as a negative isotype control.

[0277] Jurkat cells (Promega / G7102) expressing FcγRIIIa and NFAT luciferase reporter genes were engineered in-house to express ILT2 and used to model effector immune cells. These effector cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 1% MEM non-essential amino acids, 500 μg / mL G418, 200 μg / mL hygromycin B, and 1 mM sodium pyruvate. For the co-culture reporter assay, Ramos cells engineered in-house to express HLA-G were used as target cells. The medium for target cell growth and co-culture reporter assay was made in RPMI 1640 medium supplemented with 20% fetal bovine serum, 1 mM sodium pyruvate, 10 mM Heps, and 50 μM β-mercaptoethanol.

[0278] Jurkat effector cells were cultured at 1x10 in U-shaped wells of a 96-well microtiter plate. 5 The cells were seeded at 10x100, 1x100, 1x100 and 1x100 cells / well. Rituximab (Genentech / NDC code 50242-053-06), an anti-CD20 antibody, was used as an opsonizing agent to bind CD20 on Ramos target cells and FcγRIIIa on Jurkat effector cells. Rituximab, used at a final assay concentration of 10 μg / mL, was pre-incubated with Jurkat effector cells for 30 minutes at 37°C and 5% CO2. Serial dilutions of anti-ILT2 antibodies ranging from final assay concentrations of 10 μg / mL to 2.4 ng / mL were then added to the relevant wells, followed by addition of a further 5x10 4Ramos target cells were also added. The final assay volume was 100 μL. Plates were incubated overnight at 37 °C and 5% CO2. To measure luciferase reporter gene expression, media from each well was mixed and 60 μL was transferred to a new 96-well flat-bottom microtiter plate. 60 μL of Bio-Glo luciferase substrate (Promega / G7941) was added to each well. Luminescence signal (RLU) was measured using a Tecan Infinite M1000-Pro plate reader. RLU values ​​were plotted against anti-ILT2 antibody concentration using GraphPad Prism software to determine EC 50 values ​​were calculated.

[0279] As shown in Figures 8A-B, all anti-ILT2 antibodies increased FcγRIIIa NFAT signaling compared to isotype control in human ILT2-expressing Jurkat effector cells. Because luminescence signals varied across three independent experiments, the AUC values ​​of BA214, BA221, BA251, and BA252 were normalized to the AUC of the benchmark anti-ILT2 antibody HP-F1. Antibodies BA214, BA251, BA221, and BA251 induced a greater increase in luminescence than the commercially available anti-ILT2 antibody HP-F1. Calculated normalized AUC and geometric mean EC 50 The values ​​are shown in Table 15. [Table 15]

[0280] In a similar experiment, the anti-ILT2 antibody BA252 was compared to the reference antibody 15G8. As shown in Figures 9A-B, BA252 increased FcγRIIIa-NFAT signaling compared to the isotype control and showed superior potency compared to 15G8. The calculated EC 50 The values ​​were 0.07±0.04 μg / mL and 0.43±0.20 μg / mL, respectively.

[0281] Anti-ILT2 antibody BA252 does not bind to C1q The binding of the anti-ILT2 antibody variant BA252 to the complement component 1q (C1q) protein, the essential first step of the classical complement cascade for inducing complement-dependent cytotoxicity (CDC), was assessed.

[0282] C1q binding was measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well NUNC Maxisorp plates (Thermoscientific / 44-2404-21) were coated overnight at 4 °C with 50 μL of serial dilutions (300–0.14 μg / mL) of either BA252 or control antibodies nivolumab (Evidentric GmbH), anti-PD-1 IgG4 antibody, adalimumab (Myonex), anti-TNFα IgG1 antibody, or IgG4 isotype control (CrownBio / C00045-5). Plates were then decanted and blocked for 1 h at room temperature with PBS containing 2% milk powder. Binding assays were then performed by removing the blocking solution and adding 50 μL of a 2 μg / mL solution of the complement component C1q (Sigma / C1740-1 mg). Plates were washed three times with PBS containing 0.05% Tween 20. Bound C1q was detected by adding 50 μL of biotinylated anti-C1q antibody (Invitrogen / MA1-40312) used at 0.2 μg / mL. After 1 h incubation at room temperature, the plates were decanted and washed three times with PBS / 0.05% Tween 20. 50 μL of a 1.5 μg / mL solution of streptavidin-horseradish peroxidase (Jackson ImmunoResearch / 016-030-084) was added to each well and the plates were incubated for 1 h at room temperature. Finally, after washing the plates three times with PBS / 0.05% Tween 20, 50 μL of the chromogen 3,3',5,5'-tetramethylbenzidine (Invitrogen / 00-4201-56) was added to each well. The plate was allowed to develop for approximately 1 min, and then 50 μL of 1 M HCl solution was added to stop the enzymatic reaction. The optical density (OD) of each well was measured at 450 nm using a Tecan Infinite M1000-Pro plate reader.

[0283] As shown in Figure 10, BA252 showed minimal binding to C1q, which was comparable to the control antibody Nivolumab and the human IgG4 isotype control. As a positive control, the IgG1 antibody Adalimumab showed C1q binding. The calculated AUC values ​​are shown in Table 16. [Table 16]

[0284] Anti-ILT2 antibody BA252 enhances activation of primary human T, NK, and NKT cells The potential of BA252 to promote the functional activity of primary immune cells was evaluated in ex vivo experiments using cocultures of peripheral blood mononuclear cells (PBMCs) and tumor cell lines.

[0285] Pharmacodynamic effects were assessed by priming PBMCs from healthy donors overnight with IL-2 and IL-15 in the presence of BA252, 15G8, or isotype control. The following day, JEG-3 expressing HLA-G and HLA-A2 was primed. HLA-A2 Cancer cells were added to primed PBMCs at a tumor cell:PBMC ratio of 1:40 and co-cultured for 72 h. Activation of T, NKT, and NK cells was assessed by profiling CD25 expression on T cells (CD3+CD4+ and CD3+CD8+), NKT (CD3+CD56+), and NK (CD3-CD56+) cells by flow cytometry. PBMC donors were assigned to the "responder" group if the following criteria were met: at least one of the analyzed immune subpopulations (NK, NKT, CD8+T cells) responded to BA252 by a ≥ 20% increase in CD25 surface expression compared to an isotype control antibody. Otherwise, they were classified into the "non-responder" group.

[0286] Results showing that BA252 is superior to 15G8 in promoting the functional activity of primary immune cells are shown in Figures 11A-11B.

[0287] BA252-mediated ILT2 inhibition modulates the phenotype of primary human macrophages The efficacy of BA252 in modulating the functional activity of myeloid cells was assessed in vitro using primary human macrophages (Mφ).

[0288] Mφ were differentiated from purified monocyte fractions of PBMCs in the presence of BA252, 15G8, or IgG4 isotype control. Mφ were polarized in vitro to M2-like Mφ or left unstimulated. To induce the pro-tumorigenic M2 phenotype, Mφ were treated with IL-10+TGFβ cocktail or conditioned medium from HLA-G-expressing JEG-3 cancer cells (cancer cell CM). Mφ phenotype was assessed by profiling the surface expression of CD86, CD163, and CD206 on CD33+ myeloid cells by flow cytometry. Statistical analysis was performed by paired t-test.

[0289] Results demonstrating that BA252 is superior to 15G8 in suppressing the M2-like phenotype and enhancing the antigen-presenting phenotype are shown in Figures 12A-12B.

[0290] Example 4: Competitive Binding BA252 does not compete with commercially available anti-ILT2 antibodies for binding to human ILT2-expressing cells. The ability of BA252 to block binding of the anti-ILT2 antibodies 1Q-G2 (Creative, #DCABH-4494, APC conjugate), VMP55 (Novas, #NBP2-50475PE, PE conjugate), 4F9 (Bio-Rad, #MCA2515F, FITC conjugate), 292305 (R&D Systems, #FAB20171P-100, PE conjugate), and GHI / 75 (Biolegend, #333708, PE conjugate) to human ILT2 expressed on cells was assessed by flow cytometry.

[0291] For epitope competition assays, 8 × 10 CHO cells expressing ILT2 were cultured at 4 °C for 1 h. 6Resuspend in FACS buffer at 2 × 10 cells / mL and add 25 µL to wells of a U-bottom 96-well microtiter plate, with 2 × 10 cells / mL per well. 5 gives the final cell concentration of cells. 25 μL of serial dilutions of unconjugated BA252 in FACS buffer were added to the wells to obtain final assay concentrations ranging from 100 μg / mL to 0.01 μg / mL. After 1 h preincubation on ice, 50 μL of a second anti-ILT2 antibody conjugated to a fluorescent dye was added to the wells. The final assay concentrations of antibodies 1Q-G2, VMP55, 4F9, 292305, and GHI / 75 were 0.2 μg / mL, 1.7 μg / mL, 2.7 μg / mL, 12.5 μg / mL, and 60 μg / mL, respectively, based on previously established titration curves. As a positive control to indicate epitope competition, APC-conjugated BA252 was also included and used at a final concentration of 0.3 μg / mL. After a further 1 h incubation on ice in the dark, cells were centrifuged at 300 g for 5 min and washed by adding 200 μL cold FACS buffer, this wash was repeated once, and then analyzed for cell fluorescence by flow cytometry (BD LSR Fortessa flow cytometer).

[0292] CHO cells were identified using a plot of forward scatter area (FSC-A) versus side scatter area (SSC-A) for single cell selection and another plot of SSC-A versus side scatter height (SSC-H). Samples were analyzed by sequentially gating on single cell populations and plotting histograms representing fluorescence in either the FITC, PE, or APC channel depending on the fluorochrome used for the secondary antibody. Data were plotted using GraphPad Prism.

[0293] As shown in Figure 13A, unconjugated BA252 competed with the binding of APC-conjugated BA252. BA252 did not block the binding of commercial anti-ILT2 clones 1Q-G2, VMP55, 4F9, 292305, and GHI / 75, indicating that BA252 binds to a different epitope on human ILT2 (Figures 13B-F). ***

[0294] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0295] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0296] Other embodiments are within the scope of the following claims.

Claims

1. An antibody that specifically binds to human ILT2, comprising a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 1, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO:

8.

2. The antibody of claim 1, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 9, 10, 11, 13, 16, and 18; 9, 10, 11, 12, 16, and 17; 9, 10, 11, 13, 16, and 17; 9, 10, 11, 14, 16, and 17; 9, 10, 11, 12, 16, and 18; or 9, 10, 11, 14, 16, and 18, respectively.

3. The antibody of claim 1 , wherein the antibody comprises the VH amino acid sequence of SEQ ID NO: 1 and / or the VL amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8.

4. The antibody of claim 3, wherein the VH and VL comprise the amino acid sequences shown in SEQ ID NOs: 1 and 5; 1 and 2; 1 and 3; 1 and 4; 1 and 6; or 1 and 7, respectively.

5. The antibody is a human IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ 2. The antibody of claim 1, comprising a heavy chain constant region selected from the group consisting of IgA1, IgA2, and IgM.

6. The heavy chain constant region comprises: (a) a human IgG 4 heavy chain constant region containing a P at position 228, numbered according to the EU numbering system; (b) a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a lower affinity than the wild-type heavy chain constant region binds to the FcγR; (c) a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a higher affinity than the wild-type heavy chain constant region binds to the FcγR; and / or (d) The antibody of claim 5, comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, or 39.

7. The amino acid sequence of the heavy chain constant region is (b) a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a lower affinity than the wild-type heavy chain constant region binds to the FcγR, and the amino acid sequence of the variant heavy chain constant region is: (i) one or more amino acid mutations among L234A, L235A, L235E, N297A, N297Q, N297G, P329A, P329G, G236D, P238D, S239D, S267E, L328F, and L328E according to the EU numbering system; or (ii) selected from the group consisting of L234A and L235A; L234A and L235E; L234A, L235A, and L329A; or L234A, L235A, and P329G; S267E and L328F; P238D and L328E; P238D, and E233D, G237D, H268D, P271G, and A330R, according to the EU numbering system. and / or a variant of a wild-type heavy chain constant region comprising one or more substitutions; a set of amino acid mutations selected from the group consisting of P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F; and / or (c) a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with higher affinity than the wild-type heavy chain constant region binds to the FcγR, and the amino acid sequence of the variant heavy chain constant region is: (i) one or more amino acid mutations: G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L according to the EU numbering system; or (ii) The antibody of claim 6, which is a mutant of a wild-type heavy chain constant region comprising a set of amino acid mutations selected from the group consisting of S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L, and I332E; S298A, E333A, and K334A; G236A, S239D, and I332E; and F243L, R292P, Y300L, V305I, and P396L according to the EU numbering system.

8. The antibody of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25, or 26 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31, or 32.

9. the heavy and light chains are sequences of SEQ ID NOs: 26 and 30, 26 and 27, 26 and 28, 26 and 29, 26 and 31, 26 and 32, 25 and 27, 25 and 28, 25 and 29, 25 and 30, 25 and 31, 25 and 32, 24 and 27, 24 and 28, 24 and 29, 24 and 30, 24 and 31, 24 and 32, 23 and 27, 23 and 28, 23 and 29, 23 and 3 9. The antibody of claim 8, comprising the amino acid sequence set forth in 0, 23 and 31, 23 and 32, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 31, 22 and 32, 21 and 27, 21 and 28, 21 and 29, 21 and 30, 21 and 31, 21 and 32, 20 and 27, 20 and 28, 20 and 29, 20 and 30, 20 and 31, or 20 and 32.

10. (a) the antibody blocks the binding of ILT2 to HLA-G, HLA-A, HLA-B, and HLA-C; and / or (b) the antibody blocks ILT2-mediated inhibition of FcγR signaling.

11. The antibody of claim 1 , wherein the antibody is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

12. A polynucleotide encoding the VH, VL, heavy chain, and / or light chain of the antibody of claim 1.

13. A vector comprising the polynucleotide of claim 12.

14. (a) the polynucleotide of claim 12; (b) the vector according to claim 13 ; (c) a first polynucleotide encoding the heavy chain variable region or heavy chain of the antibody according to any one of claims 1 to 11, and a second polynucleotide encoding the light chain variable region or light chain of the antibody according to any one of claims 1 to 11; or (d) a recombinant host cell comprising a first vector comprising a first polynucleotide encoding the heavy chain variable region or heavy chain of the antibody of any one of claims 1 to 11, and a second vector comprising a second polynucleotide encoding the light chain variable region or light chain of the antibody of any one of claims 1 to 11.

15. A pharmaceutical composition comprising the antibody of any one of claims 1 to 11, the polynucleotide of claim 12, or the vector of claim 13, and a pharmaceutically acceptable carrier or excipient.

16. 15. A method of producing an antibody, comprising culturing the host cell of claim 14 under suitable conditions such that the polynucleotide is expressed and the antibody is produced.

17. 16. The pharmaceutical composition of claim 15 for use in treating cancer in a subject in need thereof.

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