Anti-ILT2 antibodies and uses thereof

Abstract

The present disclosure provides antibodies and polypeptides that specifically bind to Ig-like transcript 2 (ILT2). The disclosure also provides 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.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 268,945, filed on March 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] The contents of the electronically submitted Sequence Listing XML (Name: 198451_SL; Size: 59,428 bytes; Created on: March 1, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to antibodies specific for human Ig-like transcript 2 (ILT2) and methods of using the same. Background Art

[0006] Ig-like transcript 2 (ILT2) is an inhibitory receptor belonging to type I transmembrane glycoprotein, which has 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 a variety of immune cells, such as T cells, B cells, natural killer (NK) cells, myeloid-derived suppressor cells (MDSC), dendritic cells (DC) and subpopulations of monocytes / macrophages. Its ligands are both classical (HLA-A, -B and -C) and non-classical MHC class I molecules, but the affinity of the receptor combined with HLA-G is three to four times the affinity combined with classical MHC class I molecules. The combination of ILT2 and HLA-G can inhibit the function and activity of innate and adaptive anti-tumor immune responses, and promote cancer cells to escape immune surveillance and anti-tumor immunity. Overexpression of HLA-G in solid tumors is associated with poor prognosis, tumor metastasis, and shorter disease-free survival, suggesting that blockade of ILT2 / HLA-G interaction may be effective in cancer therapy.

[0007] Therefore, there is a need for therapies targeting ILT2. Summary of the invention

[0008] The disclosure provides antibodies and polypeptides that specifically bind to ILT2 (e.g., human ILT2). The disclosure also provides pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods for treating subjects using these antibodies. The antibodies disclosed herein are particularly advantageous because they are effective blockers of ILT2 / HLA-G interactions and, compared to other ILT2 antibodies currently in clinical development, exhibit high in vitro enhancement of immune cell functional activity. The applicant believes that this will translate into excellent in vivo efficacy.

[0009] In one aspect, the present invention provides an antibody that specifically binds to human ILT2, comprising: a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 1; and a VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 8.

[0010] In embodiments, the antibody comprises the CDRH1, CDRH2 and CDRH3 amino acid sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0011] In embodiments, the antibody comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences set forth in SEQ ID NOs: 15, 16 and 19, respectively.

[0012] In embodiments, 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.

[0013] In an embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NO: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, respectively.

[0014] In embodiments, 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.

[0015] In an embodiment, the antibody comprises the VH amino acid sequence of SEQ ID NO:1.

[0016] In an embodiment, the antibody comprises a heavy chain constant region, which is optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgM.

[0017] In an embodiment, the heavy chain constant region is a human IgG4 heavy chain constant region comprising P at position 228 according to the EU numbering system.

[0018] In embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds an FcγR with a lower affinity than the wild-type heavy chain constant region binds an FcγR.

[0019] In an embodiment, the amino acid sequence of the heavy chain constant region comprises:

[0020] (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

[0021] (b) a set of amino acid mutations selected from the group consisting of the following 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 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.

[0022] In embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds an FcγR with a greater affinity than the wild-type heavy chain constant region binds an FcγR.

[0023] In an embodiment, the amino acid sequence of the heavy chain constant region comprises:

[0024] (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

[0025] (b) a set of amino acid mutations selected from the group consisting of the following according to the EU numbering system: 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.

[0026] In an embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:33, 34, 35, 36, 37, 38 or 39.

[0027] In an embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26.

[0028] In an embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO:8.

[0029] In embodiments, the antibody comprises the VL amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7.

[0030] In an embodiment, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31 or 32.

[0031] In an embodiment, 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.

[0032] In an embodiment, the heavy chain and the light chain comprise the sequences in SEQ ID NO: 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 The amino acid sequence shown in 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.

[0033] In embodiments, the antibody blocks binding of ILT2 to HLA-G, HLA-A, HLA-B, and HLA-C; and / or the antibody blocks ILT2-mediated inhibition of FcγR signaling.

[0034] In one aspect, the present disclosure provides a polypeptide comprising a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO:1.

[0035] In embodiments, the polypeptide comprises the CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs: 9, 10 and 11, respectively.

[0036] In an embodiment, VH comprises the amino acid sequence of SEQ ID NO:1.

[0037] In an embodiment, the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26.

[0038] In one aspect, the present disclosure provides a polypeptide comprising a VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO:8.

[0039] In an embodiment, the VL comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NOs: 15, 16 and 19, respectively.

[0040] In embodiments, 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.

[0041] In an embodiment, VL comprises the amino acid sequence of SEQ ID NO:8.

[0042] In an embodiment, VL comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7.

[0043] In an embodiment, the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31 or 32.

[0044] In embodiments, an antibody or polypeptide disclosed herein is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

[0045] In one aspect, the present disclosure provides a polynucleotide encoding: the VH, VL, heavy chain and / or light chain of an antibody disclosed herein; or a polypeptide disclosed herein.

[0046] In one aspect, the present disclosure provides a vector comprising a polynucleotide disclosed herein.

[0047] In one aspect, the present disclosure provides a recombinant host cell comprising:

[0048] (a) a polynucleotide disclosed herein;

[0049] (b) a vector disclosed herein;

[0050] (c) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody disclosed herein and a second polynucleotide encoding a light chain variable region or a light chain of an antibody disclosed herein;

[0051] (d) a first vector comprising a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody disclosed herein, and a second vector comprising a second polynucleotide encoding a light chain variable region or a light chain of an antibody disclosed herein.

[0052] In one aspect, the present 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 pharmaceutically acceptable carrier or excipient.

[0053] In one aspect, the present disclosure provides a method for producing an antibody, the method comprising culturing the host cell disclosed herein under suitable conditions so that the polynucleotide is expressed and the antibody is produced.

[0054] In one aspect, the present 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.

[0055] In one aspect, the present disclosure provides 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 in the manufacture of a medicament for treating cancer in a subject in need thereof.

[0056] 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.

[0057] 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

[0058] Figure 1 Is a graph showing the 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.

[0059] Figure 2 Is a graph showing the 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.

[0060] Figure 3 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.

[0061] FIG. 4A to FIG. 4B It is shown that the sequence-optimized variant anti-ILT2 antibody BA252 and the reference antibody 15G8 have relatively high expression ( Figure 4A ) and low ( Figure 4B ) levels of human ILT2 in CHO cells. Binding is shown by mean fluorescence intensity (MFI) as a function of antibody concentration in μg / mL. ***p<0.001 and ****p<0.0001

[0062] Figure 5 : is a graph showing the ability of BA211 to block the interaction between HLA-G and CHO cells that highly express ILT2. The percentage of blocking the maximum mean fluorescence intensity (MFI) by HLA-G-Fc-PE is shown as a function of antibody concentration (μg / mL).

[0063] Figure 6 : is a graph showing the ability of sequence-optimized variant anti-ILT2 antibody BA252 to block the interaction between HLA-G and CHO cells that highly express ILT2. The percentage of maximum mean fluorescence intensity (MFI) blocked by HLA-G-Fc-PE is shown as a function of antibody concentration (μg / mL).

[0064] 7A to 7C It is shown that the sequence-optimized variant anti-ILT2 antibody BA252 blocks HLA-A ( Fig. 7A )、HLA-B( Figure 7B ) or HLA-C( Figure 7C ) and CHO cells that highly express ILT2. The percentage of blocking the maximum mean fluorescence intensity (MFI) of HLA-A*02:01, HLA-B*07:02 and HLA-C*07:02 pentamers, respectively, is shown as a function of antibody concentration (μg / mL).

[0065] FIG. 8A to FIG. 8B The results showed that NFAT-luciferase signaling was increased in Jurkat reporter cells expressing ILT2 and CD16 co-incubated with HLA-G expressing Ramos cells in the presence of the anti-CD20 antibody rituximab, as well as anti-ILT2 IgG1 ( Fig. 8A ) and IgG4( Figure 8B ) Plot of increasing concentrations of sequence optimized variants of the antibody. Signaling is shown by luciferase activity in relative luminescence units (RLU) as a function of antibody concentration (μg / mL).

[0066] FIG. 9A to FIG. 9BThe increase in NFAT-luciferase signaling in Jurkat reporter cells expressing ILT2 and CD16 co-incubated with HLA-G expressing Ramos cells in the presence of the anti-CD20 antibody rituximab, as well as BA252 or the reference antibody 15G8 ( Fig. 9A ) concentrations. Blockade is shown as relative luminescence units (RLU) of luciferase activity as a function of antibody concentration (μg / mL). The EC50 of BA252 in this experiment was significantly lower than the EC50 of 15G8 ( Fig. 9B ). ***p<0.001

[0067] Fig.10 is a graph showing binding of sequence optimized variant anti-ILT2 antibody BA252 and control antibody to complement component C1q. Binding is shown by optical density (OD) at 450 nm as a function of antibody concentration (μg / mL).

[0068] FIG. 11A to FIG. 11B Figure 2 is a graph showing the activation of primary immune cells in healthy donor PBMCs primed in the presence of BA252, reference ILT2 antibody 15G8, or isotype control. Activation of CD8+T, NKT, and NK cells is shown as a function of % CD25 expression for 3 donors. Donors were considered "responders" ( ) if at least one of the analyzed immune subsets responded to BA252 by increasing CD25 surface expression by ≥20% compared to isotype control. Fig.11A ), otherwise they are considered “non-responders” ( Fig. 11B ).

[0069] FIG. 12A to FIG. 12B Primary human macrophages CD163( Fig. 12A ) or CD86 / CD206( Fig. 12B ) and expressed as % of the total CD33+ bone marrow cell population. The cells were not treated or polarized to M2-like cells using conditioned medium from HLA-G expressing JEG-3 cancer cells (cancer cell CM) or IL-10+TGFβ mixture. ***p<0.001, *p<0.05, ns=not significant.

[0070] FIG. 13A to FIG. 13F Figure 2 shows the effect of conjugated anti-ILT2 antibody BA252-APC ( Fig.13A )、VMP55-PE( Fig. 13B )、1Q-G2-APC( Fig. 13C)、292305-PE( Fig.13D )、GHI / 75-PE( Fig.13E ) and 4F9-FITC( Fig.13F ) is a series of graphs showing binding of BA252 to CHO cells expressing relatively high levels of human ILT2. Binding is shown as median fluorescence intensity (MFI) as a function of the concentration of unconjugated BA252. DETAILED DESCRIPTION

[0071] The present disclosure provides anti-ILT2 antibodies and polypeptides. The present disclosure also provides pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods of using these antibodies to treat subjects. The antibodies disclosed herein are particularly useful for treating cancer in subjects.

[0072] definition

[0073] As used herein, the expression "ILT2" refers to immunoglobulin-like transcript 2, which is also referred to as leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) or leukocyte immunoglobulin-like receptor 1 (LIR-1). The amino acid sequence of the full-length human Ig-like transcript 2 can be found in accession number Q8NHL6 (UniProtKB). ILT2 is an inhibitory receptor for class I MHC antigens that can recognize a wide spectrum of HLA-A, HLA-B, HLA-C, HLA-G and HLA-F alleles. Unless explicitly specified as being from a non-human species, all references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide or protein fragment. Therefore, the expression "ILT2" means human ILT2, unless specified as being from a non-human species, for example, "mouse ILT2", "monkey ILT2" etc.

[0074] Table 1. Exemplary ILT-2 amino acid sequences.

[0075]

[0076]

[0077]

[0078]

[0079] As used herein, the term "antibody" includes 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 chain 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, heterologous conjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), 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 colonies. Antibodies can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b). In certain embodiments, antibodies described herein are IgG antibodies or their categories (e.g., human IgG1 or IgG4) or subclasses. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0080] "Multispecific antibodies" are antibodies that specifically bind to two or more different antigens or two or more different regions of the same antigen (e.g., bispecific antibodies). Multispecific antibodies include bispecific antibodies containing two different antigen binding sites (excluding Fc regions). Multispecific antibodies may include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, surface reconstructed antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, heterologous conjugated antibodies, connected single-chain antibodies or connected single-chain Fv (scFv), camelized antibodies, affibodies, connected Fab fragments, F (ab') 2 fragments, chemically connected Fv and disulfide bond-connected Fv (sdFv). Multispecific antibodies can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b). In certain embodiments, the multispecific antibodies described herein are IgG antibodies or a class (eg, human IgG1, IgG2, or IgG4) or subclass thereof.

[0081] As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. For example, Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196: 901-917 (1987) and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) have described these specific regions, all of which are incorporated herein by reference in their entirety, wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" is a CDR as defined by 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 Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, different conventions are used to define the heavy chain CDR and light chain CDR of an antibody. In certain embodiments, the heavy chain CDR and / or light chain CDR are defined by performing structural analysis of the antibody and identifying residues in the variable region predicted to contact the epitope region of the target molecule (e.g., human ILT2). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0082] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminal in a mature heavy chain, and about 90 to 115 amino acids in a mature light chain, which are very different in sequence between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in those regions known as complementary determining regions (CDRs), while the more highly conserved regions in the variable region are known as framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies to antigens. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse 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 regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0083] As used herein, the terms "VH" and "VL" refer to the antibody heavy chain variable region and light chain variable region, 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.

[0084] As used herein, the term "constant region" is common in the art. The constant region is an antibody portion, e.g., the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in binding of the antibody to an antigen, but which may exhibit various effector functions, such as interaction with an Fc receptor (e.g., an Fcγ receptor).

[0085] 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 the subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0086] As used herein, the term "light chain" when used to refer to an antibody can refer to any of the different types based on the amino acid sequence of the constant region, for example, kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0087] As used herein, the terms "specific binding", "specific recognition", "immunospecific binding", and "immunospecific recognition" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex), such binding being understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, immunoassays, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In specific embodiments, a molecule that specifically binds to an antigen has a K of at least 2 logs (e.g., a factor of 10), 2.5 logs, 3 logs, 4 logs, or more. A or greater than the K when the molecule binds nonspecifically to another antigen A Binds to antigen.

[0088] As used herein, the term "affinity" refers to the total strength 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, "binding affinity" refers to intrinsic binding affinity, which 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.

[0089] As used herein, the term "EU numbering system" refers to the EU numbering convention for the constant region of antibodies, 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 Service. s , 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0090] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic or preventive measures as described herein. The method of "treatment" employs administering an antibody to a subject having a disease or disorder or susceptible to such a disease or disorder to prevent, cure, delay, lessen the severity of the disease or disorder or a recurring disease or disorder, or to ameliorate one or more symptoms thereof, or to prolong the survival of the subject beyond the expected survival in the absence of such treatment.

[0091] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to that amount of the therapy that achieves the desired prophylactic or therapeutic effect.

[0092] 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.

[0093] As used herein, with respect to antibodies or polynucleotides, the term "isolated" refers to antibodies or polynucleotides that are separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) present in the natural source of the antibodies or polynucleotides. All instances of "isolated antibodies" described herein are further contemplated as antibodies that may, but need not, be isolated. All instances of "isolated polynucleotides" described herein are further contemplated as polynucleotides that may, but need not, be isolated. All instances of "antibodies" described herein are further contemplated as antibodies that may, but need not, be isolated. All instances of "polynucleotides" described herein are further contemplated as polynucleotides that may, but need not, be isolated.

[0094] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin S and Altschul SF (1990) PNAS 87:2264-2268, as modified in Karlin S and Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set (e.g., for score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameter set (e.g., with a score of 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gap alignments for comparison purposes, gap BLAST as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402 can be utilized, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search (supra) that detects distance relationships between molecules. When utilizing BLAST, gap BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. This 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 may be used.

[0095] 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.

[0096] Anti-ILT2 Antibodies

[0097] In one aspect, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2). The amino acid sequences of exemplary antibodies are listed in Table 2.

[0098] Table 2. Amino acid sequences of exemplary anti-ILT2 antibodies.

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a VH domain comprising one, two, or all three CDRs of the VH domain listed in Table 2. In certain embodiments, the antibody comprises a CDRH1 of a VH domain listed in Table 2. In certain embodiments, the antibody comprises a CDRH2 of a VH domain listed in Table 2. In certain embodiments, the antibody comprises a CDRH3 of a VH domain listed in Table 2.

[0112] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL domain comprising one, two, or all three CDRs of a VL domain disclosed in Table 2. In certain embodiments, the antibody comprises CDRL1 of a VL domain listed in Table 2. In certain embodiments, the antibody comprises CDRL2 of a VL domain listed in Table 2. In certain embodiments, the antibody comprises CDRL3 of a VL domain listed in Table 2.

[0113] The individual CDRs of the antibodies disclosed herein can be identified according to any CDR numbering scheme known in the art.

[0114] In certain embodiments, one or more of the CDRs of the antibodies disclosed herein can be determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

[0115] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2) and comprise the CDRs of the antibodies disclosed in Table 2 herein as determined by the Kabat numbering scheme.

[0116] In certain embodiments, one or more of the CDRs of the antibodies disclosed herein can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and 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. Pat. No. 7,709,226, all of which are incorporated herein by reference in their entirety).

[0117] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2) and comprise the CDRs of the antibodies disclosed in Table 2 herein as determined by the Chothia numbering system.

[0118] In certain embodiments, one or more of the CDRs of the antibodies 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, e.g., Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety.

[0119] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2) and comprise the CDRs of the antibodies disclosed in Table 2 herein as determined by the MacCallum numbering system.

[0120] In certain embodiments, the CDRs of the antibodies disclosed herein can be identified 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 herein by reference in its entirety; and Lefranc M-P et al., (2009) Nucleic Acids Res 37: D1006-D1012.

[0121] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2) and comprise the CDRs of the antibodies disclosed in Table 2 herein as determined by the IMGT numbering system.

[0122] In certain embodiments, the CDRs of the antibodies disclosed herein can be identified according to the AbM numbering scheme, which refers to the AbM hypervariable regions, which represent a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0123] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2) and comprise the CDRs of the antibodies disclosed in Table 2 herein as determined by the AbM numbering scheme.

[0124] In certain embodiments, the CDRs of the antibodies disclosed herein can be identified according to the AHo numbering system as described in Honegger and Plückthun, A., J. Mol. Biol. 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0125] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (eg, human ILT2) and comprise the CDRs of the antibodies disclosed in Table 2 herein as determined by the AHo numbering system.

[0126] In certain embodiments, each CDR of an antibody disclosed herein is each independently determined according to one of the Kabat, Chothia, MacCallum, IMGt, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the variable region predicted to contact an epitope region of ILT2.

[0127] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VH and a VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO:1, and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown 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 multispecific molecules, wherein the structural analysis identifies residues in the variable region predicted to contact an epitope region of ILT2 (e.g., human ILT2).

[0128] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (e.g., human ILT2), wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6, or 1 and 7, respectively.

[0129] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (e.g., human ILT2), wherein the antibody comprises a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0130] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (e.g., human ILT2), wherein the antibody comprises a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NOs: 15, 16, and 19, respectively.

[0131] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a VL, the VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NO: 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.

[0132] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises a VH and a VL, the VH comprises CDRH1, CDRH2 and CDRH3 regions, and the VL comprises CDRL1, CDRL2 and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NO: 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, respectively.

[0133] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), wherein the antibody comprises VH and VL, VH comprises CDRH1, CDRH2 and CDRH3 regions, and VL comprises CDRL1, CDRL2 and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NOs: 9, 10, 11, 13, 16 and 18, respectively.

[0134] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VH, which comprises 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 shown in SEQ ID NO: 1. In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VH, which comprises the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 1.

[0135] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL that comprises 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 shown in SEQ ID NO: 8. In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL that comprises the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 8.

[0136] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL that comprises 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 shown in SEQ ID NO: 2, 3, 4, 5, 6, or 7. In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VL that comprises an amino acid sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, or 7. In certain embodiments, the amino acid sequence of VL consists of an amino acid sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, or 7.

[0137] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a VH and a VL, wherein the VH comprises 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 shown in SEQ ID NO: 1, and the VL comprises 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 shown in SEQ ID NO: 1. NO:2, 3, 4, 5, 6 or 7 shown in the amino acid sequence 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 amino acid sequence. In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising VH and VL, VH comprising the amino acid sequence of SEQ ID NO:1, and VL comprising the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6 or 7. In certain embodiments, the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO:1; and the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO:2, 3, 4, 5, 6 or 7.

[0138] In certain embodiments, the present disclosure provides antibodies that specifically bind to ILT2 (e.g., human ILT2), comprising the VH and VL 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. In certain embodiments, the amino acid sequences of VH and VL consist of 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.

[0139] In certain embodiments, the present disclosure provides antibodies that cross-compete for binding to ILT2 (e.g., human ILT2) with an antibody comprising the VH and VL 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.

[0140] In certain embodiments, the present disclosure provides antibodies that bind to the same or overlapping epitope of ILT2 (e.g., an epitope of human ILT2) as the antibodies described herein (e.g., antibodies comprising the VH and VL 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).

[0141] In certain embodiments, the epitope of the antibody can be identified by, for example, NMR spectroscopy, surface plasmon resonance X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping) are used to determine. For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson 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 can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, Wyckoff HW et al., eds.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, Carter CW, ed.; 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 completed using any method known to those skilled in the art. See, for example, Champe M et al., (1995) supra and Cunningham BC and Wells JA (1989) supra description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In a specific embodiment, alanine scanning mutagenesis studies are used to determine the epitope of the antibody. In addition, or, conventional techniques such as immunoassays can be used, such as by showing that one antibody blocks the ability of another antibody to bind to the target antigen, i.e., competitive binding assays, to identify antibodies that recognize and bind to the same or overlapping epitopes of ILT2 (e.g., human ILT2). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificities to epitopes.Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen such as ILT2 (eg, human ILT2). Many types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (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 labeling assay, solid phase direct labeling sandwich assay (see Harlow E and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (see Cheung et al., (1986) J Immunol 137:3614-9); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow E and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (see Cheung et al., (1986) J Immunol 137:3614-9). RC et al., (1990) Virology 176:546-52); and directly labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigens (e.g., ILT2, such as human ILT2) bound to a solid surface or a cell with any of an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by measuring the amount of labeling bound to a solid surface or a cell in the presence of a test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when the competing antibody is present in excess, it will inhibit the specific binding of the reference or antibody to the common antigen by at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75% or more. Labeled antigens or labeled antibodies can be used to configure competitive binding assays in a large number of different formats. In a common version of this assay, the antigen is immobilized on a 96-well plate. A radioactive or enzymatic label is then used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen.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 and Lane D, eds., supra, pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0142] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a heavy chain comprising the amino acid sequence shown 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 shown in SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26.

[0143] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a light chain comprising the amino acid sequence shown 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.

[0144] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a heavy chain and a light chain, wherein the heavy chain and the light chain comprise 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, 2 3 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, The amino acid sequences of 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.

[0145] In certain embodiments, the amino acid sequences of the heavy and light chains consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 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, 9, 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.

[0146] The anti-ILT2 antigen binding molecules of the present disclosure can be connected or co-expressed with another functional molecule such as another peptide or protein. For example, an antibody or its fragment can be functionally connected (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0147] In certain embodiments, the antibodies disclosed herein are conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In certain embodiments, a cytotoxic agent is capable of inducing death or destruction of cells in contact with it. In certain embodiments, a cytostatic agent is capable of preventing or significantly reducing proliferation and / or inhibiting the activity or function of cells in contact with it. In certain embodiments, a cytotoxic agent or a cytostatic agent is a chemotherapeutic agent. In certain embodiments, a radionuclide is selected from an isotope 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 186 In certain embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0148] Any immunoglobulin (Ig) constant region can be used in the antibodies disclosed herein. In certain embodiments, the Ig region is human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0149] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (eg, human ILT2), the antibody comprising a heavy chain constant region, which is optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM.

[0150] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), the antibody comprising a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR.

[0151] In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38 or 39. In certain embodiments, the present disclosure provides an antibody that specifically binds to ILT2 (e.g., human ILT2), comprising a heavy chain constant region consisting of an amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38 or 39.

[0152] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1)) and / or CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering system) and / or 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.

[0153] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of an antibody described herein such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, e.g., in U.S. Pat. No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region can be altered, e.g., to facilitate assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0154] In a specific embodiment, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into an IgG constant region or an FcRn binding fragment thereof (preferably an Fc or hinge-Fc fragment) to change (e.g., reduce or increase) the in vivo half-life of the antibody. See, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are incorporated herein by reference in their entirety as examples of mutations that will change (e.g., reduce or increase) the in vivo half-life of an antibody. In certain embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into an IgG constant region or an FcRn binding fragment thereof (preferably an Fc or hinge-Fc fragment) to reduce the in vivo half-life of the antibody. In other embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to increase the in vivo half-life of the antibody. In a specific embodiment, 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 constant region of the IgG1 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. Patent 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, which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436 numbered according to the EU numbering system.

[0155] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1)) and / or CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering system) and / or 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 on the surface of an effector cell (e.g., an activated Fc receptor). 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 that can be made in an antibody Fc receptor to alter the affinity of the antibody for the Fc receptor are described, e.g., in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entirety).

[0156] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant 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 variant heavy chain constant region is a variant human heavy chain constant region, for example, a variant human IgG1, a variant human IgG2, or a variant human IgG4 heavy chain constant region. In certain embodiments, the variant 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 according to the EU numbering system; 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. In certain embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0157] In a further embodiment, one, two or more amino acid substitutions are introduced into the IgG constant region Fc region to change the effector function 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 by different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, the C1 component of Fc receptors or complement. This method is described in more 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, the deletion or inactivation of the constant region domain (by point mutation or otherwise) can reduce the Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, e.g., U.S. Pat. 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 constant regions and thereby increase 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, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations, numbered according to the EU numbering system, can be made in the constant region of an antibody described herein: 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.

[0158] In certain embodiments, the mutation selected from the group consisting of D265A, P329A and combinations thereof numbered according to the EU numbering system can be carried out in the constant region of an antibody as described herein. In certain embodiments, the mutation selected from the group consisting of L235A, L237A and combinations thereof numbered according to the EU numbering system can be carried out in the constant region of an antibody as described herein. In certain embodiments, the mutation selected from the group consisting of S267E, L328F and combinations thereof numbered according to the EU numbering system can be carried out in the constant region of an antibody as described herein. In certain embodiments, the mutation selected from the group consisting of S239D, I332E, optionally A330L and combinations thereof numbered according to the EU numbering system can be carried out in the constant region of an antibody as described herein. In certain embodiments, the mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L and combinations thereof numbered according to the EU numbering system can be carried out in the constant region of an antibody as 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.

[0159] In a specific embodiment, the antibody described herein comprises a constant region of an IgG1 having an N297Q or N297A amino acid substitution according to the EU numbering system. In certain embodiments, the antibody described herein comprises a constant region of an IgG1 having 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 a constant region of an IgG1 having 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 a constant region of an IgG1 having a mutation selected from the group consisting of L234F, L235F, N297A and combinations thereof numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the positions corresponding to the positions L234, L235 and D265 (numbered according to the EU numbering system) in the constant region of the antibody described herein are not L, L and D respectively. This method is described in detail in International Publication No. WO 14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain (numbered according to the EU numbering system) are F, E, and A; or A, A, and A, respectively.

[0160] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 (numbered according to the EU numbering system) in the constant region of an antibody described herein 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 more 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 to 238 (numbered according to the EU numbering system) in the N-terminal region of the CH2 domain of an antibody described herein are altered to alter the ability of the antibody to fix complement. This approach is further described in International Publication No. 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 to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcγ receptor by mutating one or more amino acids (e.g., introducing an amino acid substitution) at the following positions numbered according to the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286 , 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 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 method is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0161] In certain embodiments, antibodies as described herein include a modified constant region of IgG1, wherein 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 methods described herein and / or known to those skilled in the art. In certain embodiments, the modified constant region of IgG1 includes S239D and I332E numbered according to the EU numbering system. In certain embodiments, the modified constant region of IgG1 includes S239D, A330L and I332E numbered according to the EU numbering system. In certain embodiments, the modified constant region of IgG1 includes L235V, F243L, R292P, Y300L and P396L numbered according to the EU numbering system. In certain embodiments, the antibody is capable of inducing cell death in effector T cells and Tregs, wherein the percentage of Treg cells 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 greater than the percentage of effector T cells undergoing cell death.

[0162] In certain embodiments, the antibodies described herein comprise the constant region of an IgG2 antibody with a cysteine ​​substituted for serine at amino acid residue 127 of the heavy chain numbered according to the EU numbering system.

[0163] In certain embodiments, the antibodies described herein comprise the constant region of an IgG4 antibody and a proline is substituted with serine at amino acid residue 228 of the heavy chain numbered according to the EU numbering system.

[0164] 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 having two heavy chain constant regions.

[0165] Pharmaceutical composition

[0166] Provided herein are compositions comprising an anti-ILT2 antibody disclosed herein in a physiologically acceptable carrier, excipient or stabilizer having a desired degree of purity (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; 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; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0167] In specific embodiments, the pharmaceutical composition comprises an anti-ILT2 antibody disclosed herein in a pharmaceutically acceptable carrier and optionally one or more additional prophylactic or therapeutic agents. In specific embodiments, the pharmaceutical composition comprises an anti-ILT2 antibody disclosed herein in a pharmaceutically acceptable carrier and optionally one or more additional prophylactic or therapeutic agents. In certain embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to reduce or block ILT2 (e.g., human ILT2) activity and treat conditions such as cancer. In certain embodiments, the present disclosure relates to a pharmaceutical composition of the present disclosure comprising an anti-ILT2 antibody of the present disclosure, which is used as a medicament. In another embodiment, the present disclosure relates to a pharmaceutical composition of the present disclosure, which is used in a method for treating cancer.

[0168] Pharmaceutically acceptable carriers for parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, emulsifiers, multivalent sequestrants or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, glucose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils, cottonseed oil, corn oil, sesame oil and peanut oil of plant origin. Antimicrobial agents in antibacterial or antifungal concentrations can be added to parenteral preparations packaged in multidose containers, and these parenteral preparations include phenol or cresol, mercurial, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and glucose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 ( 80). Metal ion sequestrants or chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0169] The pharmaceutical composition can 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 subcutaneous, intramuscular or intravenous injection is also contemplated herein. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. Injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, glucose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

[0170] Preparations of antibodies for parenteral administration include sterile solutions ready for injection, sterile dry soluble products ready for combination with solvents before use (such as lyophilized powders, including subcutaneous tablets), sterile suspensions ready for injection, sterile dry insoluble products ready for combination with vehicles before use, and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0171] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0172] Topical mixtures containing antibodies are prepared as described for topical and systemic administration. The resulting mixture can be a solution, suspension, emulsion, etc., and can be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0173] The anti-ILT2 antibodies disclosed herein can be formulated as aerosols for topical administration, 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 for treating 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 as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation will have a diameter of less than 50 microns in certain embodiments, less than 10 microns in certain embodiments.

[0174] The anti-ILT2 antibodies disclosed herein can be formulated for topical or local application, such as topical application to the skin and mucous membranes, such as in the eyes, in the form of gels, creams and lotions, and application to the eyes or for intracisternal or intraspinal applications. Topical administration is contemplated for transdermal delivery and may also be used for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of the antibodies alone or in combination with other pharmaceutically acceptable excipients may also be administered.

[0175] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled 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 entirety.

[0176] In certain embodiments, the pharmaceutical composition comprising the antibodies described herein is a lyophilized powder that can be reconstituted to be administered as a solution, emulsion, and other mixtures. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibodies described herein or their pharmaceutically acceptable derivatives in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate or potassium phosphate or other such buffers known to those skilled in the art, in certain embodiments, about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to provide the desired formulation. In certain embodiments, the resulting solution will be dispensed into vials for freeze-drying. Each vial will contain a single dose 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 parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. This amount can be determined empirically.

[0177] 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 subject's body to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the present invention. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. 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 specific embodiments, the antibodies described herein are targeted to tumors.

[0178] Compositions to be used for in vivo administration may be sterile. This can be easily achieved, for example, by filtration through sterile filtration membranes.

[0179] Usage and Purpose

[0180] On the other hand, the present disclosure provides a method for treating a subject using an anti-ILT2 antibody disclosed herein. Any disease or illness that will benefit from the reduction of ILT2 (e.g., human ILT2) function in a subject can be treated with an anti-ILT2 antibody disclosed herein. In certain embodiments, the disease or illness is tolerant 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 disease or illness 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).

[0181] The anti-ILT2 antibodies disclosed herein are particularly useful for suppressing the immune system's tolerance to tumors and are therefore useful as immunotherapy for subjects with cancer. For example, in certain embodiments, the disclosure provides a method for increasing the number of T cells (e.g., CD8 + Cytotoxic T cells, CD4 + A method for activating helper T cells, NKT cells, effector T cells or memory T cells) comprising administering to the subject an effective amount of an anti-ILT2 antibody or a pharmaceutical composition thereof as disclosed herein. In certain embodiments, the disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein.

[0182] Cancers that can be treated with 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, cancer is a solid tumor. Examples of solid tumors include malignancies, such as sarcomas and cancers, such as adenocarcinomas of various organ systems, such as those affecting the lungs, breasts, ovaries, lymphs, gastrointestinal (e.g., colon), anus, genitalia and urogenital tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, nerves or glial cells), head and neck, skin (e.g., melanoma) and pancreas, and malignancies including colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small intestinal cancer and esophageal cancer. Cancer can be in early, mid-term, late 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 relapses 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).

[0183] In certain embodiments, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC or NSCLC adenocarcinoma with squamous and / or non-squamous cell carcinoma histology)), 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 receptor, progesterone receptor 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 cancer, peritoneal cancer, or lymphoproliferative disease (e.g., post-transplant lymphoproliferative disease).

[0184] In certain embodiments, the cancer is a blood cancer, such as a leukemia, lymphoma, or myeloma. In certain embodiments, the cancer is a leukemia, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In certain embodiments, cancer is 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, cancer is myeloma, such as multiple myeloma.

[0185] In another embodiment, the cancer is selected from carcinoma (eg, advanced or metastatic cancer), melanoma, or lung cancer, such as non-small cell lung cancer.

[0186] In certain embodiments, the cancer is lung cancer, such as lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

[0187] In certain embodiments, cancer is melanoma, such as advanced melanoma. In certain embodiments, cancer is advanced or unresectable melanoma that is unresponsive to other therapies. In other embodiments, cancer is a melanoma with a BRAF mutation (e.g., a BRAF V600 mutation). In yet other embodiments, the anti-ILT2 antibody or pharmaceutical composition disclosed herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab), with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

[0188] In another embodiment, the cancer is liver cancer, such as advanced liver cancer, with or without viral infection, such as chronic viral hepatitis.

[0189] In another embodiment, the cancer is prostate cancer, such as advanced prostate cancer.

[0190] In yet another embodiment, the cancer is myeloma, such as multiple myeloma.

[0191] In yet another embodiment, the cancer is renal cancer, such as renal cell carcinoma (RCC) (eg, metastatic RCC, clear cell renal cell carcinoma (CCRCC), or renal papillary cell carcinoma).

[0192] In yet another embodiment, the cancer is selected from lung cancer, melanoma, renal cancer, breast cancer, colorectal cancer, leukemia, or a metastatic lesion of cancer.

[0193] In certain embodiments, these methods further include administering another therapeutic agent to the subject. In certain embodiments, another therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent. In certain embodiments, a chemotherapeutic agent is a hypomethylating agent (e.g., azacitidine). In certain embodiments, a chemotherapeutic agent is a DNA damage inducing agent (e.g., gemcitabine). In certain embodiments, a checkpoint targeting agent is selected from the group consisting of the following: antagonist anti-CTLA-4 antibodies, antagonist anti-PD-L1 antibodies, antagonist anti-PD-L2 antibodies, antagonist anti-PD-1 antibodies, antagonist anti-TIM-3 antibodies, antagonist anti-LAG-3 antibodies, antagonist anti-VISTA antibodies, antagonist anti-CD96 antibodies, antagonist anti-CEACAM1 antibodies, agonist anti-CD137 antibodies, agonist anti-GITR antibodies, and agonist anti-OX40 antibodies. 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, wherein the ILT2 (e.g., human ILT2) antibody or pharmaceutical composition disclosed herein acts synergistically with the checkpoint targeting agent.

[0194] In certain embodiments, the present disclosure relates to antibodies and / or pharmaceutical compositions of the present disclosure, which are used in the methods of the present disclosure, wherein the method further comprises administering another therapeutic agent to a subject. In certain embodiments, the present disclosure relates to (a) antibodies and / or pharmaceutical compositions of the present disclosure and (b) another therapeutic agent, which is used as a medicine. In certain embodiments, the present disclosure relates to (a) antibodies and / or pharmaceutical compositions of the present disclosure and (b) another therapeutic agent, which is used in a method for treating cancer. In a further embodiment, the present disclosure relates to a pharmaceutical composition, a kit or a multi-component kit, which comprises (a) antibodies and / or pharmaceutical compositions of the present disclosure and (b) another therapeutic agent. In certain embodiments, another therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent or a checkpoint targeting agent.

[0195] In certain embodiments, anti-PD-1 antibodies are used in the methods disclosed herein. In certain embodiments, anti-PD-1 antibodies are nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, anti-PD-1 antibodies are pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, anti-PD-1 antibodies are pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, anti-PD-1 antibodies are MEDI0680, also known as AMP-514, developed by Medimmune. In certain embodiments, anti-PD-1 antibodies are PDR001 developed by Novartis Pharmaceuticals. In certain embodiments, anti-PD-1 antibodies are 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.

[0196] Further non-limiting examples of anti-PD-1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No. 7,488,802; U.S. Patent No. 8,008,449; U.S. Patent No. 8,114,845; U.S. Patent No. 8,168,757; U.S. Patent No. 8,354,509; U.S. Patent No. 8,686,119; U.S. Patent No. 8,735,553; U.S. Patent No. 8,747,847; U.S. Patent No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No. 9,102,727; U.S. Patent No. 9,205,148; U.S. Publication No. US2013 / 0202623 A1; U.S. Publication No. US2013 / 0291136 A1; U.S. Publication No. US 2014 / 0044738 A1; U.S. Publication No. US2014 / 0356363 A1; U.S. Publication No. US2016 / 0075783 A1; and PCT Publication No. WO 2013 / 033091 A1; PCT Publication No. WO 2015 / 036394 A1; PCT Publication No. WO 2014 / 179664 A2; PCT Publication No. WO 2014 / 209804 A1; PCT Publication No. WO 2014 / 206107 A1; PCT Publication No. WO 2015 / 058573 A1; PCT Publication No. WO 2015 / 085847 A1; PCT Publication No. WO 2015 / 200119 A1; PCT Publication No. WO 2016 / 015685A1; and PCT Publication No. WO 2016 / 020856 A1.

[0197] In certain embodiments, an anti-PD-L1 antibody is 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 Medimmune. 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.

[0198] Non-limiting examples of anti-PD-L1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein 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. US 2010 / 0203056 A1; U.S. Publication No. US2003 / 0232323 A1; U.S. Publication No. US2013 / 0323249 A1; U.S. Publication No. US2014 / 0341917 A1; U.S. Publication No. US2014 / 0044738 A1; U.S. Publication No. US 2015 / 0203580A1; U.S. Publication No. US2015 / 0225483 A1; U.S. Publication No. US2015 / 0346208 A1; U.S. Publication No. US2015 / 0355184 A1; and PCT Publication No. WO 2014 / 100079 A1; PCT Publication No. WO 2014 / 022758 A1; PCT Publication No. WO 2014 / 055897 A2; PCT Publication No. WO 2015 / 061668 A1; PCT Publication No. WO 2015 / 109124 A1; PCT Publication No. WO 2015 / 195163 A1; PCT Publication No. WO 2016 / 000619 A1; and PCT Publication No. WO 2016 / 030350 A1.

[0199] In certain embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In certain embodiments, the anti-CTLA-4 antibody is Ipilimumab developed by Bristol-Myers Squibb.

[0200] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with a compound targeting an immunomodulatory enzyme such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). Therefore, in certain embodiments, the additional therapeutic agent is a compound targeting an immunomodulatory enzyme, such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In certain embodiments, such compounds are selected from the group consisting of: epacadostat (Incyte Corp; see, e.g., 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 for treating cancer as described herein is present in a solid dosage form of a pharmaceutical composition, such as a tablet, a pill or a capsule, wherein the pharmaceutical composition comprises an IDO inhibitor and a pharmaceutically acceptable excipient. Therefore, the antibody as described herein and the IDO inhibitor as described herein can be administered separately, sequentially or simultaneously as a separate dosage form. In certain embodiments, the antibody is administered parenterally, and the IDO inhibitor is administered orally. In certain embodiments, the inhibitor is selected from the group consisting of ikadolistat (Incyte Corporation), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indolemod (NewLink Genetics) and NLG919 (NewLink Genetics). Ikadolistat has been described in PCT Publication No. WO 2010 / 005958, which is incorporated herein by reference in its entirety for all purposes. In certain embodiments, the inhibitor is ikadolistat. In another embodiment, the inhibitor is F001287. In another embodiment, the inhibitor is indomod. In another embodiment, the inhibitor is NLG919.

[0201] In certain embodiments, an anti-ILT2 antibody disclosed herein is administered to a subject in combination with a vaccine. The vaccine can be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine.

[0202] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with an adjuvant. Various adjuvants can be used depending on the treatment situation. Non-limiting examples of suitable adjuvants include, but are not limited to, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), montanide ISA (Seppic incomplete adjuvant), Ribi adjuvant system (RAS), Titer Max, muramyl peptide, Syntex adjuvant formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvant, Adjuvant, nitrocellulose adsorbed antigen, encapsulated or embedded antigen, 3De-O-acylated monophosphoryl lipid A (3D-MPL), immunostimulatory oligonucleotides, toll-like receptor (TLR) ligands, mannan binding lectin (MBL) ligands, STING agonists, immunostimulatory complexes such as saponin, Quil A, QS-21, QS-7, ISCOMATRIX, etc. 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 Nos. 6,645,495; 7,029,678; and 7,858,589, all of which are incorporated herein by reference in their entirety. In certain embodiments, the adjuvant used herein is QS-21STIMULON.

[0203] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with another therapeutic agent comprising a TCR. In certain embodiments, the other therapeutic agent is a soluble TCR. In certain embodiments, the other therapeutic agent is a cell expressing a TCR. Therefore, in certain embodiments, the disclosure relates to an antibody and / or pharmaceutical composition disclosed herein in combination with another therapeutic agent comprising a TCR, which is used as a drug and / or in a method for treating cancer.

[0204] In certain embodiments, an anti-ILT2 antibody disclosed herein is administered to a subject in combination with a cell expressing a chimeric antigen receptor (CAR). In certain embodiments, the cell is a T cell.

[0205] In certain embodiments, the anti-ILT2 antibodies disclosed herein are administered to a subject in combination with a TCR mimetic antibody. In certain embodiments, a TCR mimetic antibody is an antibody that specifically binds to a peptide-MHC complex. For non-limiting examples of TCR mimetic antibodies, see, e.g., U.S. Patent No. 9,074,000 and U.S. Publication Nos. US2009 / 0304679A1 and US2014 / 0134191 A1, all of which are incorporated herein by reference in their entirety.

[0206] 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, BiTE and / or DART specifically bind to tumor-associated antigens (e.g., polypeptides overexpressed in tumors, polypeptides derived from tumor viruses, polypeptides containing post-translational modifications specific to tumors, polypeptides specifically mutated in tumors) and molecules on effector cells (e.g., CD3 or CD16). In certain embodiments, the tumor-associated antigen is EGFR (e.g., human EGFR), optionally wherein the BiTE and / or DART comprise the VH and VL sequences of cetuximab. In certain embodiments, the tumor-associated antigen is Her2 (eg, human Her2), optionally wherein the BiTE and / or DART comprises the VH and VL sequences of trastuzumab. In certain embodiments, the tumor-associated antigen is CD20 (eg, human CD20).

[0207] Anti-ILT2 antibodies and other therapeutic agents (e.g., chemotherapeutic agents, radiotherapeutic agents, checkpoint targeting agents, IDO inhibitors, vaccines, adjuvants, soluble TCRs, cells expressing TCRs, cells expressing chimeric antigen receptors, and / or TCR mimic antibodies) can be administered separately, sequentially, or simultaneously as separate dosage forms. In certain embodiments, anti-ILT2 antibodies are administered parenterally, and IDO inhibitors are administered orally.

[0208] The antibodies or pharmaceutical compositions described herein can be delivered to the subject by a variety of routes. These 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 a formulation with an atomizer to be used 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 the tumor draining lymph nodes.

[0209] The amount of the antibody or composition effective in the treatment and / or prevention of a disorder will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0210] The precise dose employed in the composition will also depend on the route of administration and the severity of the infection or disease caused thereby, and should be determined according to the practitioner's judgment and the circumstances of each subject. For example, the effective dose may also vary depending on the mode of administration, the target site, the patient's physiological state (including age, weight and health), whether the patient is human or animal, and whether the other drugs or treatments administered are preventive or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, may also be treated. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0211] The anti-ILT2 antibodies described herein can also be used to determine the level of ILT2 (e.g., human ILT2) protein in a biological sample using classical immunohistological methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, or Western blots. Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioactive isotopes such as iodine ( 125 I. 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and 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. Therefore, in certain embodiments, the present disclosure relates to the use of the anti-ILT2 antibodies of the present disclosure for in vitro detection of ILT2 (e.g., human ILT2) proteins in biological samples. In a further embodiment, the present disclosure relates to the use of the anti-ILT2 antibodies of the present disclosure for in vitro determination and / or detection of ILT2 (e.g., human ILT2) protein levels in biological samples, optionally wherein the anti-ILT2 antibodies are conjugated to a radionuclide or a detectable label, and / or carry a label described herein, and / or wherein an immunohistological method is used.

[0212] Determining the expression level of an ILT2 (e.g., human ILT2) protein is intended to include qualitatively or quantitatively measuring or estimating the level of an ILT2 (e.g., human ILT2) protein in a first biological sample directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing with disease-associated protein levels in a second biological sample). The expression level of an ILT2 (e.g., human ILT2) polypeptide in a first biological sample can be measured or estimated and compared with a standard ILT2 (e.g., human ILT2) protein level, the standard being, for example, a second biological sample obtained from an individual not suffering from the disease, or determined by an average level from a population of individuals not suffering from the disease. As will be understood in the art, once a "standard" ILT2 (e.g., human ILT2) polypeptide level is known, it can be repeatedly used as a standard for comparison. Therefore, in a further embodiment, the present disclosure relates to an in vitro method for determining and / or detecting the level of ILT2 protein, such as the level of human ILT2 protein, in a biological sample, which comprises qualitatively or quantitatively measuring or estimating the level of ILT2 protein, such as human ILT2 protein, in the biological sample by an immunohistological method.

[0213] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue or other cell source that potentially expresses 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).

[0214] Anti-ILT2 antibodies as described herein can be used for prognosis, diagnosis, monitoring and screening applications, including in vitro and in vivo applications well known to those skilled in the art and standard and based on this specification. Prognosis, diagnosis, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response can be used for prediction, diagnosis and monitoring to evaluate patient samples, including samples known to suffer from or suspected of suffering from immune system dysfunction, or samples of expected or desired immune system response, antigenic response or vaccine response. Assessment and evaluation of immune system status and / or immune response can also be used to determine the suitability of the patient for the clinical trial of a drug or for the administration of a specific chemotherapeutic agent, radiotherapeutic agent or antibody (including combinations thereof) relative to different medicaments or antibodies. This type of prognosis and diagnostic monitoring and evaluation have been practiced using antibodies (HercepTest) for HER2 protein in breast cancer. TM , Dako), where the assay was also used to evaluate the use of Patients undergoing antibody therapy. In vivo applications include directed cell therapy and immune system modulation and radiographic imaging of immune responses. Therefore, in certain embodiments, the present disclosure relates to anti-ILT2 antibodies and / or pharmaceutical compositions of the present disclosure, which are used as diagnostic agents. In certain embodiments, the present disclosure relates to anti-ILT2 antibodies and / or pharmaceutical compositions of the present disclosure, which are used in methods for predicting, diagnosing and / or monitoring subjects suffering from or suspected of having immune system dysfunction and / or regarding an expected or desired immune system response, antigen response or vaccine response. In another embodiment, the present disclosure relates to the use of anti-ILT2 antibodies of the present disclosure for predicting, diagnosing and / or monitoring subjects suffering from or suspected of having immune system dysfunction and / or regarding an expected or desired immune system response, antigen response or vaccine response by in vitro determination and / or detection of human ILT2 protein levels in a biological sample of the subject.

[0215] In certain embodiments, anti-ILT2 antibodies can be used for immunohistochemistry of biopsy samples. In certain embodiments, the method is an in vitro method. In another embodiment, anti-ILT2 antibodies can be used to detect the level of ILT2 (e.g., human ILT2), or the level of cells containing ILT2 (e.g., human ILT2) on its membrane surface, and its level can then be associated with certain disease symptoms. The anti-ILT2 antibodies described herein can carry detectable or functional markers and / or can be conjugated to radionuclides or detectable markers. When fluorescent markers are used, currently available microscopy and fluorescence activated cell sorting analysis (FACS) or a combination of two method procedures known in the art can be used to identify and quantify specific binding members. The anti-ILT2 antibodies described herein can carry or can be conjugated to fluorescent markers. Exemplary fluorescent markers include, for example, reactive and conjugated probes, such as aminocoumarins, fluorescein and Texas Red, Alexa Fluor dyes, Cy dyes and DyLight dyes. Anti-ILT2 antibodies can carry or can be conjugated to radioactive markers or radionuclides, such as isotopes 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 186 Re. When radioactive labels are used, the specific binding of anti-ILT2 antibodies to ILT2 (e.g., human ILT2) can be identified and quantified using currently available counting procedures known in the art. In the case where the label is an enzyme, detection can be accomplished by any of the currently used colorimetric, spectrophotometric, fluorescence spectrophotometric, current analysis, or gas measurement techniques known in the art. This can be achieved by contacting a sample or a control sample with an anti-ILT2 antibody under conditions that allow a complex to form between the anti-ILT2 antibody and ILT2 (e.g., human ILT2). Any complex formed between the anti-ILT2 antibody and ILT2 (e.g., human ILT2) is detected and compared in the sample and the control. In view of the specific binding of the anti-ILT2 antibodies described herein to ILT2 (e.g., human ILT2), anti-ILT2 antibodies 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) via immunoaffinity purification. Also included herein is a determination system that can be prepared in the form of a test kit, a kit or a multi-component kit for quantitative analysis of, for example, the presence of an ILT2 (e.g., human ILT2) / ILT2 (e.g., human ILT2) ligand complex. The system, test kit, kit or multi-component kit can include a labeled component, such as a labeled antibody and one or more additional immunochemical reagents.

[0216] Polynucleotides, vectors and methods for producing antibodies

[0217] On the other hand, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies described herein or portions thereof or fragments thereof (e.g., VL and / or VH; and light and / or heavy chains), which specifically bind to ILT2 (e.g., human ILT2) antigens, and vectors, e.g., vectors comprising such polynucleotides, for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding the heavy and / or light chains of any antibody provided herein, and vectors comprising such polynucleotide sequences, e.g., expression vectors that are effectively expressed in host cells (e.g., mammalian cells).

[0218] As used herein, "isolated" polynucleotides or nucleic acid molecules are polynucleotides or nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., in mice or humans). In addition, "isolated" nucleic acid molecules, such as cDNA molecules, can be substantially free of other cellular materials or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the wording "substantially free" includes preparations of polynucleotides or nucleic acid molecules with less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (particularly less than about 10%) of other substances (e.g., cellular materials, culture media, other nucleic acid molecules, chemical precursors and / or other chemicals). In a specific embodiment, nucleic acid molecules encoding antibodies described herein are isolated or purified.

[0219] In a specific aspect, 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 as described herein, as well as antibodies that compete with such antibodies for binding to an ILT2 (e.g., human ILT2) polypeptide (e.g., in a dose-dependent manner), or antibodies that bind to the same epitope as such antibodies.

[0220] In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding light chains or heavy chains of antibodies described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising VL FR and CDR of an antibody described herein (see, e.g., Table 2) or a nucleotide sequence encoding a heavy chain comprising VH FR and CDR of an antibody described herein (see, e.g., Table 2). In certain embodiments, the polynucleotide encodes VH, VL, heavy chains and / or light chains described herein. In another embodiment, the polynucleotide encodes the first VH and the first VL described herein. In another embodiment, the polynucleotide encodes the second VH and the second VL described herein. In another embodiment, the polynucleotide encodes the first heavy chain and the first light chain described herein. In another embodiment, the polynucleotide encodes the second heavy chain and the second light chain described herein. In another embodiment, the polynucleotide encodes the VH and / or VL, or the heavy chain and / or light chain of an antibody described herein.

[0221] Also provided herein are polynucleotides encoding anti-ILT2 antibodies, which are optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA unstable elements. The optimized nucleic acid encoding anti-ILT2 antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) is generated for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA. The method can be performed by adjusting accordingly, for example, the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splicing sites and unstable elements (e.g., elements rich in A / T or A / U) within RNA can be mutated without changing the amino acid encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These changes utilize the degeneracy of the genetic code, for example, using alternative codons for the same amino acid. In certain embodiments, it may be necessary to change one or more codons to encode conservative mutations, e.g., similar amino acids having similar chemical structures and properties and / or functions as the original amino acids. Such methods may increase the expression of anti-ILT2 antibodies or fragments thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more relative to the expression of anti-ILT2 antibodies encoded by non-optimized polynucleotides.

[0222] In certain embodiments, the optimized polynucleotide sequence encoding the anti-ILT2 antibody or fragment thereof described herein (e.g., VL domain and / or VH domain) can be hybridized with the antisense (e.g., complementary) polynucleotide of the non-optimized polynucleotide sequence encoding the anti-ILT2 antibody or fragment thereof described herein (e.g., VL domain and / or VH domain). In a specific embodiment, the optimized nucleotide sequence encoding the anti-ILT2 antibody or fragment described herein is hybridized with the antisense polynucleotide of the non-optimized polynucleotide sequence encoding the anti-ILT2 antibody or fragment thereof described herein under high stringency conditions. In a specific embodiment, the optimized nucleotide sequence encoding the anti-ILT2 antibody or fragment thereof described herein is hybridized with the antisense polynucleotide of the non-optimized nucleotide sequence encoding the anti-ILT2 antibody or fragment thereof described herein under high stringency, medium or low stringency hybridization conditions. Information about hybridization conditions has been described, see, for example, U.S. Patent Application Publication No. US2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0223] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of polynucleotides is determined. The nucleotide sequence encoding antibodies described herein (e.g., antibodies described in Table 2) and modified forms of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in a manner to generate nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled by chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, which is incorporated herein by reference in its entirety), in short, it relates to the synthesis of overlapping oligonucleotides containing the sequence portion encoding the antibody, the annealing and connection of these oligonucleotides, and then the oligonucleotides connected by PCR amplification.

[0224] Alternatively, polynucleotides encoding antigen binding regions described herein or antibodies described herein can be generated from nucleic acids from suitable sources (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that can hybridize with the 3' and 5' ends of known sequences can be performed using genomic DNA obtained from hybridoma cells that produce antibodies of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding light chains and / or heavy chains of antibodies. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding variable light chain regions and / or variable heavy chain regions of antibodies. Amplified nucleic acids can be cloned into vectors to be expressed in host cells and used for further cloning.

[0225] 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, then nucleic acids encoding immunoglobulins can be obtained by chemical synthesis or by cDNA library generated from a suitable source (e.g., an antibody cDNA library or nucleic acid (preferably poly A+RNA) isolated from any tissue or cell expressing the antibody (such as a hybridoma cell selected to express an antibody described herein) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, cDNA clones from a cDNA library encoding an 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.

[0226] DNA encoding the anti-ILT2 (e.g., human ILT2) antibodies described herein can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of anti-ILT2 (e.g., human ILT2) antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell (e.g., from a CHO GS System TM (Lonza) CHO cells) or myeloma cells that do not otherwise produce immunoglobulins to obtain synthesis of anti-ILT2 antibodies in recombinant host cells.

[0227] In order to generate a complete antibody or antigen binding region, a PCR primer including a VH or VL nucleotide sequence, a restriction site, and a flanking sequence protecting the restriction site can be used to amplify the VH or VL sequence in the scFv clone. Using cloning techniques known to those skilled in the art, the VH domain amplified by PCR can be cloned into a vector expressing a heavy chain constant region (e.g., a human γ1 or human γ4 constant region), and the VL domain amplified by PCR can be cloned into a vector expressing a light chain constant region (e.g., a human κ or λ constant region). In certain embodiments, the vector for expressing the VH or VL domain comprises 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 a vector expressing the necessary constant region. Then, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0228] The DNA also may be modified, for example, by substituting coding sequences for human heavy and light chain constant regions in place of the murine sequences, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0229] Also provided are polynucleotides that hybridize to polynucleotides encoding antibodies described herein under high stringency, medium or lower stringency hybridization conditions. In specific embodiments, polynucleotides described herein hybridize to polynucleotides encoding VH domains and / or VL domains provided herein under high stringency, medium or lower stringency hybridization conditions.

[0230] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization of DNA bound to a filter 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 involve hybridization of nucleic acids bound to a filter 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 skilled in the art and has been described, 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, pp. 6.3.1-6.3.6 and 2.10.3, which are incorporated herein by reference in their entirety.

[0231] In certain aspects, cells (e.g., host cells) expressing (e.g., recombinantly) antibodies described herein that specifically bind to ILT2 (e.g., human ILT2), as well as related polynucleotides and expression vectors are provided herein. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-ILT2 antibodies or fragments for recombinant expression in host cells, preferably in mammalian cells (e.g., CHO cells). Also provided herein are host cells comprising such vectors for recombinantly expressing anti-ILT2 antibodies described herein (e.g., human or humanized antibodies). In specific aspects, provided herein are methods for producing antibodies described herein, the methods comprising expressing antibodies from host cells.

[0232] The recombinant expression of antibodies (e.g., full-length antigen binding regions or antibodies or heavy and / or light chains of antibodies described herein) that specifically bind to ILT2 (e.g., human ILT2) described herein generally involves constructing an expression vector containing a polynucleotide encoding an antibody. Once a polynucleotide encoding an antibody molecule, a heavy chain and / or light chain of an antibody, or a fragment thereof (e.g., a heavy chain and / or light chain variable region) as described herein is obtained, the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., a light chain or heavy chain) encoding nucleotide sequence is described herein. Expression vectors containing antibody or antibody fragment (e.g., light chain or heavy chain) encoding sequences and appropriate transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. A replicable vector comprising a nucleotide sequence is also provided, the nucleotide sequence encoding comprising an antibody molecule, an antibody heavy chain or light chain, an antibody heavy chain or light chain variable region or a fragment thereof, or a heavy chain or light chain CDR, which is operably connected to a promoter. Such a vector may, for example, include a nucleotide sequence encoding the constant region of an antibody molecule (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464, which are incorporated herein by reference in their entireties), and the variable region of an antibody may be cloned into this vector for expression of an entire heavy chain, an entire light chain, or both an entire heavy chain and a light chain.

[0233] In certain embodiments, the vector comprises polynucleotides encoding the VH, VL, heavy chain and / or light chain of an antibody described herein. In another embodiment, the vector comprises polynucleotides encoding the VH and VL of an antibody described herein. In another embodiment, the vector comprises polynucleotides encoding the heavy chain and light chain of an antibody described herein.

[0234] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce an antibody or fragment thereof as described herein. Therefore, provided herein is a host cell containing a polynucleotide encoding an antibody or fragment thereof as described herein, or a heavy chain or light chain thereof, or a fragment thereof, or a single-chain antibody as described herein, the polynucleotide being operably linked to a promoter for expressing such sequences in a host cell.

[0235] In certain embodiments, the host cell comprises polynucleotides encoding the VH and VL of an antibody described herein. In another embodiment, the host cell comprises a vector comprising polynucleotides 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 and a second vector, the first vector comprising a first polynucleotide encoding the VH of an antibody described herein, and the second vector comprising a second polynucleotide encoding the VL of an antibody described herein.

[0236] In specific embodiments, 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 host cell population comprising such a first host cell and such a second host cell.

[0237] In certain embodiments, provided herein is a vector population comprising 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.

[0238] A variety of host-expression vector systems can be used to express the antibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems represent vehicles that can produce and subsequently purify the coding sequences of interest, but also represent cells that can in situ express the antibody molecules described herein when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces cerevisiae and Pichia pastoris) with, for example, recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with, for example, recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with, for example, recombinant virus expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or with, for example, recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells), including, for example, recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In specific embodiments, cells used to express antibodies described herein are Chinese hamster ovary (CHO) cells, such as those from CHO GS System TM In certain embodiments, the heavy and / or light chains of antibodies produced by CHO cells may have an N-terminal glutamine or glutamic acid residue substituted with pyroglutamic acid. In certain embodiments, the cells used to express the antibodies described herein are human cells, such as human cell lines. In specific embodiments, the mammalian expression vector is pOptiVEC TMOr pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells), particularly cells for expressing complete recombinant antibody molecules, are used to express recombinant antibody molecules. For example, mammalian cells such as CHO cells, combined with vectors such as major intermediate early gene promoter elements from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK and 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, antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of the nucleotide sequence encoding the antibody specifically binding to ILT2 (e.g., human ILT2) described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0239] In bacterial systems, many expression vectors can be advantageously selected according to the intended use of the antibody molecule to be expressed. For example, when a large number of such antibodies are to be produced, in order to produce the pharmaceutical composition of the antibody molecule, it may be necessary to instruct the high-level expression of the fusion protein product that is easily purified. Such vectors include but are not limited to the Escherichia coli expression vector pUR278 (Ruether U and Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the coding sequence can be connected to the vector in the framework with the lac Z coding region alone, so as to produce fusion protein; pIN vector (Inouye S and Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G and Schuster SM (1989) J Biol Chem 24: 5503-5509); etc., all of these documents are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, 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 include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0240] For example, in insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned individually into a non-essential region of the virus (e.g., a polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

[0241] In mammalian host cells, many virus-based expression systems can be utilized. In the case of using adenovirus as an expression vector, the coding sequence of interest can be connected to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., E1 region or E3 region) will produce a recombinant virus that is viable and can express the molecule in an infected host (e.g., referring to Logan J and Shenk T (1984) PNAS 81 (12): 3655-9, which is incorporated herein by reference in its entirety). The effective translation of the inserted coding sequence may also require a specific start signal. These signals include an ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can have a variety of sources, including natural and synthetic. Expression efficiency may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety).

[0242] In addition, host cell strains can be selected that regulate the expression of inserted sequences or modify and process gene products in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign proteins. For this reason, eukaryotic host cells with cellular machinery for appropriate processing of primary transcripts, glycosylation and phosphorylation of gene products 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 murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL703O, COS (e.g., COS1 or COS), PER.C6, 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.

[0243] In specific embodiments, the antibodies described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells lacking or lacking fucosylation ability. In a specific example, a cell line with two alleles of α1,6-fucosyltransferase knockout can be used to produce antibodies with reduced fucose content. The ANTIBODYNAMIC® system (Lonza) is an example of such a system that can be used to generate antibodies with reduced fucose content.

[0244] For long-term, high-yield production of recombinant proteins, stable expression cells can be produced. For example, a cell line stably expressing an anti-ILT2 (e.g., human ILT2) antibody as described herein can be engineered. In a specific embodiment, the cells provided herein stably express light chains / light chain variable regions and heavy chains / heavy chain variable regions, which associate to form antigen binding regions or antibodies as described herein.

[0245] In some aspects, instead of using an expression vector containing a viral origin of replication, a host cell can be transformed with a DNA and a selectable marker controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.). After introducing foreign DNA / polynucleotide, the cell of through engineering approaches can be allowed to grow for 1-2 days in an enrichment medium, and then switch to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and allows cells to stably integrate plasmids into their chromosomes and grow to form a focus, which can then be cloned and expanded into a cell line. The method can be advantageously used for through engineering approaches to express anti-ILT2 as described herein (e.g., human ILT2) or a cell line of its fragment. The cell line of this type of through engineering approaches can be particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0246] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH and Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk cells, hgprt cells, or aprt cells, respectively, all of which are incorporated herein by reference in their entirety. In addition, antimetabolite resistance can be used as the basis for selection for 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 and Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY and 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 and Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ and 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 herein by reference in their entirety.Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are 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); and in Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entirety.

[0247] The expression level of the antibody molecule can be increased by vector amplification (for review, see Bebbington CR and Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When the marker in the vector system is amplifiable, the increase in the inhibitor level present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is related to the gene of interest, the yield of the protein will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, which is incorporated herein by reference in its entirety).

[0248] Host cells can be co-transfected with two or more expression vectors as described herein, the first vector encoding a heavy chain derived polypeptide, and the second vector encoding a light chain derived polypeptide. The two vectors may contain the same selection marker, which enables the heavy chain and light chain polypeptides to be equally expressed. Host cells can be co-transfected with two or more expression vectors in different amounts. For example, host cells can be transfected with a first expression vector and a second expression vector in any one of the following ratios: 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.

[0249] Alternatively, a single vector encoding and capable of expressing both heavy and light chain polypeptides can be used. In this case, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and G (1980) PNAS 77: 2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences of the heavy chain and the light chain may comprise cDNA or genomic DNA. The expression vector may be a monocistronic or polycistronic. The polycistronic nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or within the range of 2-5, 5-10 or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise a promoter, a first gene (e.g., a heavy chain of an antibody described herein) and a second gene (e.g., a light chain of an antibody described herein) in the following order. In this expression vector, transcription of the two genes may be driven by a promoter, and translation of the mRNA from the first gene may be performed by a cap-dependent scanning mechanism, and translation of the mRNA from the second gene may be performed by a cap-independent mechanism, e.g., by an IRES.

[0250] Once the antibody molecules described herein are produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen after protein A, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0251] In a specific embodiment, the antibodies described herein are separated or purified. In certain embodiments, the separated antibodies are antibodies that are substantially free of other antibodies with antigenic specificity different from the separated antibodies. For example, in certain embodiments, the antibody preparations described herein are substantially free of cellular material and / or chemical precursors. The wording "substantially free of cellular material" includes antibody preparations, wherein the antibody is separated from the cellular components of the cells that separate or recombinantly produce the antibody. Therefore, antibodies substantially free of cellular material include antibody preparations with 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 antibody variants (e.g., different post-translational modified forms of antibodies or other different versions of antibodies (e.g., antibody fragments)). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5% or 0.1% of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such antibody preparations have less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

[0252] Anti-ILT2 (e.g., human ILT2) antibodies or fragments thereof can be produced by any method known in the art for synthesizing proteins or antibodies, such as by chemical synthesis or by recombinant expression technology. Unless otherwise indicated, the methods described herein employ 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 technical scope of the art. For example, these techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., 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., 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 et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); 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.

[0253] In specific embodiments, the antibodies described herein are prepared, expressed, produced or isolated by any means involving production, e.g., via synthesis of DNA sequences, genetic engineering. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in the antibody germline repertoire in an animal or mammal (e.g., human).

[0254] In one aspect, a method for preparing an anti-ILT2 (e.g., human ILT2) antibody is provided herein, the method comprising culturing a cell or host cell as described herein. In certain embodiments, the method is performed in vitro. In a certain aspect, a method for preparing an anti-ILT2 (e.g., human ILT2) antibody is provided herein, the method comprising expressing (e.g., recombinantly expressing) an antibody using a cell or host cell as described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody as 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 a step of purifying the antibody obtained from the cell or host cell.

[0255] In certain embodiments, antibodies are produced by expressing polynucleotides encoding the VH and VL of antibodies described herein in cells under suitable conditions, thereby expressing polynucleotides and producing antibodies. In another embodiment, antibodies are produced by expressing polynucleotides encoding the heavy and light chains of antibodies described herein in cells under suitable conditions, thereby expressing polynucleotides and producing antibodies. In certain embodiments, antibodies are produced by expressing a first polynucleotide encoding the VH of antibodies described herein and a second polynucleotide encoding the VL of antibodies described herein in cells under suitable conditions, thereby expressing polynucleotides and producing antibodies. In certain embodiments, antibodies are produced by expressing a first polynucleotide encoding the heavy chain of antibodies described herein and a second polynucleotide encoding the light chain of antibodies described herein in cells under suitable conditions, thereby expressing polynucleotides and producing antibodies.

[0256] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel FM et al., eds., John Wiley and Sons, New York, which is incorporated herein by reference in its entirety).

[0257] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and, for example, Harlow E and Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd Edition 1988); Hammerling GJ et al., In: 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 host cells that exogenously express antibodies described herein or fragments thereof (e.g., light chains and / or heavy chains of such antibodies).

[0258] In a specific embodiment, as used herein, "monoclonal antibody" is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody 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 in the examples provided herein. In certain embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein can be prepared, for example, by the hybridoma method as described in Kohler G and Milstein C (1975) Nature 256:495, which is incorporated herein by reference in its entirety, or can be separated from a phage library, for example, using techniques as described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th ed., Ausubel FM et al., supra).

[0259] As used herein, when an antibody comprises at least two (e.g., two or more) monovalent binding regions, the antibody is multivalently bound to an antigen (e.g., bivalent), and each monovalent binding region is capable of binding an epitope on the antigen. Each monovalent binding region can bind to the same or different epitopes on the antigen.

[0260] The method of using hybridoma technology to produce and screen specific antibodies is conventional and well known in the art. For example, in the hybridoma method, immune mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters or macaques, are used to induce lymphocytes that produce or can produce antibodies, and the antibodies will specifically bind to proteins (e.g., ILT2 (e.g., human ILT2)) for immunity. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using suitable fusogens (such as polyethylene glycol) to form hybridoma cells (Goding JW (editor), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). In addition, RIMMS (repeated immune multisite) technology can be used for immune animals (Kilpatrick KE et al., (1997) Hybridoma 16: 381-9, which is incorporated herein by reference in its entirety).

[0261] In certain embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., ILT2 (e.g., human ILT2)), and once an immune response is detected, such as antigen-specific antibodies are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then mixed with any suitable myeloma cells (e.g., from the American Type Culture Collection) by well-known techniques. (Manassas, VA) cell line SP20 cells) to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of immunized mice are harvested and fused with NS0 myeloma cells.

[0262] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0263] Specific embodiments use myeloma cells, which effectively fuse, support stable high-level antibody production of selected antibody-producing cells, and are sensitive to culture media such as HAT culture media. Among these myeloma cell lines are mouse myeloma cell lines, such as NS0 cell lines or cell lines derived from MOPC-21 and MPC-11 mouse tumors, available from Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells, available from American Type Culture Collection (Rockville, MD, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (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).

[0264] The production of monoclonal antibodies against ILT2 (e.g., human ILT2) is determined in the culture medium in which the hybridoma cells are grown. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, such as immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0265] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 culture media. In addition, hybridoma cells can be grown as ascites tumors in animals.

[0266] 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.

[0267] Antibodies described herein include, for example, antibody fragments that recognize ILT2 (e.g., human ILT2), and can be produced by any technique known to those skilled in the art. For example, Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Fab fragments correspond to one of the two identical arms of an antibody molecule and contain a complete light chain paired with the VH and CH1 domains of a heavy chain. F(ab')2 fragments contain two antigen-binding arms of an antibody molecule connected by a disulfide bond in the hinge region.

[0268] In addition, the antibodies described herein can also be produced using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of phage particles carrying 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 affected tissues). DNA encoding VH and VL domains are recombined with scFv linkers by PCR and cloned into phagemid vectors. The vector is electroporated in E. coli and E. coli is infected with helper phage. The phage used in these methods is generally filamentous phage, including fd and M13, and VH and VL domains are generally recombinantly fused with phage gene III or gene VIII. Phages expressing antigen binding regions that bind to specific antigens can be selected or identified with antigens, for example, using labeled antigens or antigens that are bound or captured to solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies described herein include 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 and Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401 and WO 97 / 13844; and those disclosed in 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 by reference in their entirety.

[0269] As described in the above references, after phage selection, the antibody coding region from the phage can be isolated and used to generate complete antibodies (including human antibodies or any 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. Recombinant production of antibody fragments such as Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art, such as PCT Publication No. WO 92 / 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.

[0270] In certain embodiments, to produce a complete antibody, a PCR primer comprising a VH or VL nucleotide sequence, a restriction site, and a flanking sequence protecting the restriction site can be used to amplify a VH or VL sequence from a template (e.g., scFv clone). Using cloning techniques known to those skilled 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 a 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 skilled in the art to generate a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0271] Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may 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, e.g., Morrison SL (1985) Science 229: 1202-7; Oi VT and Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. 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.

[0272] Humanized antibodies can bind to predetermined antigens, and they include framework regions substantially having the amino acid sequence of human immunoglobulins and CDRs substantially having the amino acid sequence of non-human immunoglobulins (e.g., mouse immunoglobulins). In certain embodiments, humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. Antibodies can also include CH1, hinge, CH2, CH3, and CH4 regions of heavy chains. Humanized antibodies can be selected from any class of immunoglobulins, 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, including, but not limited to, CDR grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneer 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., (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 Suppl):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. Application Publication No. US2005 / 0042664 Al (February 24, 2005), which is incorporated herein by reference in its entirety.

[0273] 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 herein by reference in their entirety.

[0274] Bispecific, bivalent antibodies and methods for their preparation are described in, for example, U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333 and U.S. Application Publication Nos. 2003 / 020734 and 2002 / 0155537; each of these documents is incorporated herein by reference in its entirety. Bispecific tetravalent antibodies and methods for their preparation are described in, for example, International Application Publication Nos. WO 02 / 096948 and WO 00 / 44788, the disclosures of which are incorporated herein by reference in their entirety. See generally, International 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. Pat. 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.

[0275] Bispecific antibodies as described herein can be produced according to the DuoBody technology platform (Genmab A / S) as described in, for example, International Publication Nos. WO 2011 / 131746, WO 2011 / 147986, WO 2008 / 119353, and WO 2013 / 060867 and Labrijn AF et al., (2013) PNAS 110(13):5145-5150. DuoBody technology can be used to combine a first monospecific antibody or half of a first antigen-binding region containing two heavy chains and two light chains with a second monospecific antibody or half of a second antigen-binding region containing two heavy chains and two light chains. The resulting heterodimer contains one heavy chain and one light chain from the first antibody or first antigen-binding region, paired with one heavy chain and one light chain from the second antibody or second antigen-binding region. When the two monospecific antibodies or antigen-binding regions recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0276] DuoBody technology requires that each monospecific antibody or antigen-binding region includes a heavy chain constant region with a single point mutation in the CH3 domain. The point mutation allows the interaction between the CH3 domains in the resulting bispecific antibody to be stronger than the interaction between the CH3 domains in the monospecific antibody or antigen-binding region. The single point mutation in each monospecific antibody or antigen-binding region is located at residues 366, 368, 370, 399, 405, 407 or 409 numbered according to the EU numbering system in the CH3 domain of the heavy chain constant region, as described in, for example, International Publication No. WO 2011 / 131746. In addition, the single point mutation is located at a different residue in one monospecific antibody or antigen-binding region compared to another monospecific antibody or antigen-binding region. For example, one monospecific antibody or antigen-binding region may comprise a mutation F405L (i.e., a mutation from phenylalanine to leucine at residue 405), while another monospecific antibody or antigen-binding region may comprise a mutation K409R (i.e., a mutation from lysine to arginine at residue 409), numbered according to the EU numbering system. The heavy chain constant region or antigen-binding region of the monospecific antibodies may be of the IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and the bispecific antibodies produced by DuoBody technology may retain Fc-mediated effector functions.

[0277] Another method for generating bispecific antibodies is called the "knob-in-hole" strategy (see, e.g., International Publication WO2006 / 028936). In this technique, mispairing of Ig heavy chains is reduced by mutating selected amino acids that form the interface of the CH3 domain in IgG. At the position where the two heavy chains directly interact within the CH3 domain, amino acids with small side chains (holes) are introduced into the sequence of one heavy chain, and amino acids with large side chains (knobs) are introduced into the corresponding interacting residue positions on the other heavy chain. In some embodiments, the compositions of the present disclosure have immunoglobulin chains in which the CH3 domain has been modified by mutating selected amino acids that interact at the interface between the two polypeptides so as to preferentially form bispecific antibodies. Bispecific antibodies can be composed of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0278] In some cases, the bispecific antibody can contain IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG3 or IgG1 and IgG3 chain heterodimers. Such heterodimeric heavy chain antibodies can be routinely engineered by, for example, modifying selected amino acids forming the interface of the CH3 domains in human IgG4 and IgG1 or IgG3 to facilitate heterodimeric heavy chain formation.

[0279] In certain embodiments, the antibodies described herein that bind to the same epitope of ILT2 (e.g., human ILT2) as the anti-ILT2 (e.g., human ILT2) antibodies described herein are human antibodies. In certain embodiments, the competitive blocking described herein (e.g., in a dose-dependent manner) of any of the antibodies described herein and ILT2 (e.g., human ILT2) binding antibodies are human antibodies. Human antibodies can be produced using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be rendered inoperative alone or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, J HHomozygous deletion in the region prevents endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then cultivated to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a normal manner with selected antigens, such as all or part of an antigen (e.g., ILT2 (e.g., human ILT2)). Monoclonal antibodies against antigens can be obtained from immune transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene carried by transgenic mice is rearranged during B cell differentiation, and then undergoes class switching and somatic mutation. Therefore, using this technology, useful IgG, IgA, IgM and IgE antibodies for treatment can be produced. For an overview of this technology for producing human antibodies, see Lonberg N and Huszar D (1995) Int Rev Immunol 13: 65-93, which is incorporated by reference in its entirety. For a detailed discussion of such techniques 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. Pat. 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 herein by reference in their entirety. Examples of mice capable of producing human antibodies include the XenoMouse. TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse TM (Medarex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse TM (Kirin) and KMMouse TM (Medarex / Kirin), all of which are incorporated herein by reference in their entirety.

[0280] Human antibodies that specifically bind to ILT2 (e.g., human ILT2) can be prepared by a variety of methods known in the art, including the above-mentioned phage display methods 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. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, all of which are incorporated herein by reference in their entirety.

[0281] In certain embodiments, mouse-human hybridomas can be used to produce human antibodies. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine the hybridomas that secrete human monoclonal antibodies that specifically bind to target antigens (e.g., ILT2 (e.g., human ILT2)). Such methods are known and described in the art, see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of these documents is incorporated herein by reference in its entirety.

[0282] Reagent test kit

[0283] Also provided is a kit comprising one or more antibodies described herein or its pharmaceutical composition or conjugate.In a specific embodiment, a pharmaceutical package or kit is provided herein, which comprises one or more containers, and the one or more containers are filled with one or more components of a pharmaceutical composition as described herein, such as one or more antibodies provided herein.In certain embodiments, the kit contains a pharmaceutical composition as described herein and any preventive or therapeutic agent, such as those described herein.In certain embodiments, the kit may contain a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA) or TCR complex stimulating antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies.Optionally, associated with such containers may be a notice in the form of a government agency regulation of the manufacture, use or sale of regulatory drugs or biological products, which reflects the approval obtained by the manufacture, use or sale agency for human administration.

[0284] Also provided is a kit that can be used for the above method. In certain embodiments, the kit comprises an antibody described herein in one or more containers, preferably a purified antibody. In a specific embodiment, the kit described herein contains a substantially separated ILT2 (e.g., human ILT2) antigen as a control. In another specific embodiment, the kit described herein further comprises a control antibody that does not react with the ILT2 (e.g., human ILT2) antigen. In another specific embodiment, the kit described herein contains one or more elements for detecting the binding of an antibody to an ILT2 (e.g., human ILT2) antigen (e.g., an antibody can be conjugated with 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 with a detectable substrate). In a specific embodiment, the kit provided herein may include a recombinantly produced or chemically synthesized ILT2 (e.g., human ILT2) antigen. The ILT2 (e.g., human ILT2) antigen provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection tool of the above kit includes a solid support to which the ILT2 (e.g., human ILT2) antigen is attached. This kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse / rat antibody. In this embodiment, the binding of the antibody to an ILT2 (e.g., human ILT2) antigen can be detected by the binding of the reporter-labeled antibody. In certain embodiments, the present disclosure relates to the use of the kit of the present disclosure for in vitro determination and / or detection of an ILT2 (e.g., human ILT2) antigen in a biological sample.

[0285] Examples

[0286] The following examples are offered by way of illustration and not limitation.

[0287] Example 1: Binding kinetics and optimization of anti-ILT2 antibodies

[0288] This example describes the characteristics of antibodies that specifically bind to human ILT2. The amino acid sequences of these antibodies are listed in Table 2 herein.

[0289] Anti-ILT2 antibodies produced by mammalian reverse transcription cell display were affinity matured by phage display. This variant of the antibody was named BA210, aiming to improve the properties and optimize the molecule. The BA210 variable domain was formatted with human κ IgG1, IgG2 or IgG4 constant regions, replacing the heavy chain aglycosylation (N297A), disulfide bond formation and stabilization (C127S, S228P), and removing the predicted deamidation site at position 34 (G) and the oxidation site at position 105 (M) in the light chain according to Table 3 below. Other variants with C-terminal lysine deletions were prepared to improve manufacturability.

[0290] Table 3. ILT2 antibody variants

[0291] Antibody describe Antibody describe BA210 IgG1 BA220 G34Y / M105L IgG2 C127S BA211 IgG1 N297A BA221 G34T / M105L IgG4 S228P BA212 G34T IgG1 N297A BA222 G34Y / M105L IgG4 S228P BA213 M105L IgG1 N297A BA246 IgG2 BA214 G34T / M105L IgG1 N297A BA247 IgG2 C127S BA215 G34Y / M105L IgG1 N297A BA248 IgG4 S228P BA216 G34Y IgG1 N297A BA249 G34T / M105L IgG1 BA217 G34T / M105L IgG2 BA250 G34Y / M105L IgG1 BA218 G34Y / M105L IgG2 BA251 G34T / M105L IgG1 N297A-C-terminal K BA219 G34T / M105L IgG2 C127S BA252 G34T / M105L IgG4 S228P-C-terminal K

[0292] Binds to purified human ILT2 protein

[0293] 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, while an irrelevant antibody VRC01 was used as a negative isotype control.

[0294] Briefly, SPR experiments were performed using a Biacore T200 instrument and were performed by Biacore using a 1:1 binding model. TM The T200 evaluation software calculates the association rate (K) from each experiment. a ), dissociation rate (K d ) and dissociation constant (K D The antibodies were captured from the Human Fab Capture Kit (28958325, GE Healthcare) onto S-series CM5 sensor chips (29149603, GE Healthcare) using the Biacore TM Open the Amine Coupling Immobilization Wizard on the control panel and follow the manufacturer's recommendations provided with the kit.

[0295] Approximately 3 μg / mL of test anti-ILT2 antibodies and controls diluted in electrophoresis buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured in individual flow cells of a previously prepared anti-human Fab CM5 chip, leaving a single flow cell as a reference. Antibodies were captured using a 30 second injection at a flow rate of 10 μL / min to achieve approximately 150 resonance units (Rus). Human ILT2 ECD PE01 (diluted at 30, 10, 3.3, 1.11, 0.37, 0.12 nM in electrophoresis buffer), and human ILT2 ECD PE02 and PE03 (diluted at 33.3, 11.1, 3.7, 1.2, 0.41, 0.14 nM in electrophoresis buffer) were flowed over the chip surface at a flow rate of 30 μL / min for a 3 min association phase and a 10 min dissociation phase. The sensor chip was regenerated between cycles with two 30 s injections of 10 mM glycine pH 2.1 at 30 μL / min followed by a 60 s stabilization period. Sensorgrams were evaluated using the global data analysis option of BIAevaluation 3.1 software and fitted to a simple Langmuir 1:1 interaction model. The bias and curve fit were visually inspected and the R max The parameters of , Chi2 and Tc were used to verify the data quality. The binding kinetics are shown in Table 4.

[0296] Table 4. Kinetic parameters of anti-ILT2 antibody variants binding to human ILT2 haplotypes PE01, PE02, PE03.

[0297] Antibody ILT2 haplotype <![CDATA[K a (1 / Ms)]]> <![CDATA[K d (1 / s)]]> <![CDATA[K D (pM)]]> BA211 PE01 3.4E+05 3.4E-05 99 BA212 PE01 3.7E+04 3.3E-06 89 BA216 PE01 3.6E+05 6.7E-05 185 BA213 PE01 3.2E+05 1.1E-05 33 BA214 PE01 3.3E+05 2.1E-05 64 BA215 PE01 2.8E+05 9.6E-07 3 BA211 PE02 2.6E+05 1.7E-04 671 BA212 PE02 2.7E+05 2.1E-04 783 BA216 PE02 2.3E+05 1.4E-04 617 BA213 PE02 2.3E+05 1.5E-04 630 BA214 PE02 2.6E+05 1.3E-04 513 BA215 PE02 2.3E+05 1.2E-04 541 BA211 PE03 2.7E+05 2.5E-04 929 BA212 PE03 4.6E+05 4.0E-04 875 BA216 PE03 2.7E+05 8.5E-05 317 BA213 PE03 5.0E+05 7.5E-05 149 BA214 PE03 5.4E+05 1.4E-04 257 BA215 PE03 4.9E+05 1.1E-04 216

[0298] In another experiment, the binding affinity of anti-ILT2 antibody variants with light chain substitution and / or different heavy chain constant regions (BA249, BA250, BA214, BA215, BA217, BA218, BA219, BA220, BA221 and BA222) to the human ILT2 haplotype PE01 labeled with ECD-His was assessed by SPR. Antibodies with identified PTM risks (BA210, BA211, BA246, BA247 and BA248) were used as positive isotype controls. Variants of unrelated antibody VRC01 (with IgG1, IgG1N297A, IgG4 S228P, IgG2 or IgG2 C127S constant regions) were used as negative isotype controls. Binding kinetics are shown in Table 5 as the mean values ​​of two independent measurements.

[0299] Table 5. Kinetic parameters for binding of anti-ILT2 antibody Fc isotypes to human ILT2

[0300]

[0301] In another experiment, the binding affinity of anti-ILT2 antibody variants BA214, BA221, BA251 and BA252 to ECD-His-tagged human ILT2 haplotypes PE01, PE02 and PE03, as well as ECD-His-tagged cynomolgus monkey ILT2 orthologous protein variant 1, ECD-His-tagged cynomolgus monkey ILT2 orthologous protein variant 2, ECD-His-tagged cynomolgus monkey ILT2 orthologous protein variant 3, ECD-His-tagged rhesus monkey ILT2 orthologous protein variant 1, ECD-His-tagged rhesus monkey ILT2 orthologous protein variant 2, ECD-His-tagged African green monkey (AGM) ILT2 orthologous 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 constant (KD) are shown in Tables 6 and 7.

[0302] Table 6. Kinetic parameters for binding of anti-ILT2 antibodies to human ILT2 haplotypes

[0303]

[0304]

[0305] NBD = No binding detected up to 100 nM

[0306] Table 7. Binding of anti-ILT2 antibodies to non-human primate ILT2 orthologs and mouse PirB

[0307]

[0308] NBD = No binding detected (up to 10 RU at 5 μM receptor concentration)

[0309] Specific binding to ILT2

[0310] The anti-ILT2 antibodies BA214, BA221, BA251 and BA252 were evaluated by SPR as described above against 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), LILRB1 / ILT2 PE01 (SEQ ID NO: 44), LILRB2 / ILT4 (R&D Systems The binding affinity of LILRB4 / ILT3 (SinoBiological 16742-H08H) and LILRB5 / LIR-8 (Sino Biological 17221-H08H-100) was measured. An irrelevant antibody VRC01 IgG4 S228P was used as a negative isotype control.

[0311] The results showed that the binding kinetic parameters to ILT2 are shown in Table 8. No binding at physiologically relevant levels was detected for any other LILR family members, indicating specific binding to ILT2.

[0312] Table 8. Kinetic parameters of binding of anti-ILT2 antibodies to ILT2

[0313]

[0314] The binding of BA252 to LILRA and LILRB family members was then compared by SPR with commercially available anti-ILT2 antibodies 1Q-G2 (Creative, #DCABH-4494), VMP55 (Novus, #NBP2-50475PE), 4F9 (Biorad, #MCA2515F), 292305 (R&D Systems, #MAB20171), 238145 (Abcam, #EPR22861-6), HP-F1 (Invitrogen, #16-5129-82) and 15G8 (see Table 2). An unrelated antibody VRC01 IgG1N297A was used as a negative isotype control.

[0315] The results are shown in Table 9. All anti-ILT2 antibodies bind 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 show measurable binding to LILRA1 and ILT6. In contrast, BA252 does not bind to LILRA1 and shows only a very weak interaction with ILT6.

[0316] None of the anti-ILT2 clones tested bound to LILRA2, LILRA4, LILRA5, LILRA6, LILRB3, LILRB4 or LILRB5.

[0317] Table 9. Binding affinity of anti-ILT2 antibodies to LILRA and LILRB family members.

[0318]

[0319]

[0320] NBD = No binding detected

[0321] Binding to cells expressing human ILT2

[0322] The ability of anti-ILT2 antibodies to bind to cells expressing human ILT2 was tested in a variety of cell types.

[0323] The ability of anti-ILT2 antibody variants (BA211, BA212, BA216, BA213, BA214 and BA215) with IgG1 N297A heavy chain constant regions to bind to human ILT2 expressed on the surface of CHO cells was assessed. In brief, CHO cells were transfected with vectors encoding human ILT2 extracellular and transmembrane domains. Clones that stably expressed relatively low levels of ILT2 were cultured in Power CHO-2 medium containing 4mM L-glutamine, 100U / mL penicillin, 100μg / mL streptomycin, 1x HT-supplement.

[0324] For antibody binding assays, cells were plated at 3×10 cells / 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 100 cells / mL in a 96-well U-bottom tissue culture plate. Cells were incubated at 4°C for 30 min with 25 μL of serially diluted anti-ILT2 antibody or isotype control in FACS buffer with final concentrations ranging from 10 μg / mL to 0.2 ng / mL.

[0325] For antibody staining, cells were washed twice with cold FACS buffer and resuspended in 100 μL FACS buffer containing R-phycoerythrin goat anti-human IgG antibody (Jackson Immunoresearch / 109-116-098) at a final dilution of 1 / 800. After incubation at 4°C in the dark for 30 minutes, cells were washed twice with cold FACS buffer and analyzed by flow cytometry (BD LSR Fortessa flow cytometer). CHO cells were identified using a forward scatter area (FSC-A) versus side scatter area (SSC-A) and another FSC-A versus forward scatter height (FSC-H) graph to select single cells. Samples were analyzed by sequentially gating single cell populations and plotting histograms representing fluorescence in the PE channel. For each sample, the mean fluorescence intensity (MFI) was calculated and the data were plotted by GraphPad Prism software.

[0326] like Figure 1 As shown, BA211 and the sequence-optimized variants BA212, BA216, BA213, BA214, and BA215 all bind to CHO cells expressing relatively low levels of human ILT2 in a dose-dependent manner. The geometric area under the curve (AUC) and EC were calculated. 50 The values ​​are listed in Table 10.

[0327] Table 10. Binding of anti-ILT2 antibodies to CHO cells expressing ILT2.

[0328]

[0329]

[0330] The ability of the same anti-ILT2 antibody variant to bind to the human ILT2 expressed on the surface of Jurkat cells was tested. In brief, Jurkat cells were transfected with a vector encoding full-length ILT2, and clones of stably expressed ILT2 were selected. The stable cell line also expresses luciferase reporter gene under NFAT response element and FcgRIIIa (Promega / G7102), and is cultivated in the RPMI-1640 culture medium supplemented with 10% heat-inactivated FBS, 1% non-essential amino acids, 250 μg / mL G418 disulfate solution, 100 μg / mL hygromycin and 1mM sodium pyruvate.

[0331] For antibody binding assays, cells were plated at 3×10 cells / well in 25 μL of PBS supplemented with 0.5% BSA, 1 mM EDTA, and 0.05% sodium azide (FACS buffer). 5The cells were seeded at a density of 10 cells / mL in a 96-well U-bottom tissue culture plate. The cells were incubated with 25 μL of serially diluted anti-ILT2 antibody or isotype control antibody (VRC01IgG1) in FACS buffer for 30 minutes at 4°C, ranging from 10 μg / mL to 2.4 ng / mL. The cells were stained and identified as described above.

[0332] like Figure 2 As shown, anti-ILT2 antibody BA211 and sequence optimized variants BA212, BA216, BA213, BA214 and BA215 dose-dependently bind to Jurkat cells expressing human ILT2. The results are listed in Table 11.

[0333] Table 11. Binding of anti-ILT2 antibodies to Jurkat cells expressing ILT2.

[0334] Antibody AUC (Area Under the Curve) <![CDATA[EC 50 (μg / ml)]]> BA211 225681 0.059 BA212 222439 0.057 BA216 198758 0.070 BA213 189474 0.110 BA214 208116 0.055 BA215 172820 0.096

[0335] The ability of variant antibodies optimized for anti-ILT2 sequences formatted with different Fc backbones to bind to human ILT2 highly expressed on the surface of CHO cells was assessed. In brief, CHO cells were transfected with vectors encoding the extracellular and transmembrane domains of human ILT2. Clones expressing relatively high levels of ILT2 on the membrane were selected and cultured in Power CHO-2 medium containing 4mM L-glutamine, 100U / mL penicillin, 100μg / mL streptomycin, 1x HT-supplement.

[0336] For antibody binding assays, cells were plated at 3×10 cells / 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 100 cells / mL in a 96-well U-bottom tissue culture plate. Cells were incubated at 4°C for 30 min with 25 μL of serially diluted anti-ILT2 antibody or isotype control in FACS buffer at concentrations ranging from 10 μg / mL to 2.4 ng / mL.

[0337] For antibody staining, cells were washed twice with cold FACS buffer and resuspended in FACS buffer containing R-phycoerythrin goat anti-human IgG (Fab') 2 (Jackson ImmunoResearch / 109-116-097) at a dilution of 1:250. After incubation at 4°C in the dark for 30 minutes, cells were washed twice with cold FACS buffer and analyzed by flow cytometry (BD LSRFortessa flow cytometer). CHO-ILT2 cells were identified as described above and MFI was calculated.

[0338] like Figure 3As shown, 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.

[0339] Table 12. Binding of anti-ILT2 antibodies to CHO cells expressing ILT2.

[0340] Antibody AUC (Area Under the Curve) 95% CI <![CDATA[EC 50 (μg / mL)]]> 95%CI (μg / mL) BA214 379740 200212-720249 0.104 0.005–2.105 BA215 392229 227748-675500 0.082 0.014–0.478 BA217 264082 200916-347107 0.248 0.094–0.655 BA218 294751 209658-414382 0.227 0.084–0.613 BA219 307176 251396-375333 0.183 0.077–0.432 BA220 304607 209658-414382 0.282 0.045–1.776 BA221 360571 244693-531325 0.141 0.027–0.722 BA222 372656 245051-566709 0.115 0.018–0.728 BA249 349259 184767-660192 0.117 0.0009–1.457 BA250 383800 245703-599514 0.087 0.016–0.490

[0341] The anti-ILT2 antibody variant BA252 was compared with the reference anti-ILT2 antibody 15G8 (see Table 2) for binding to cells expressing different levels of human ILT2.

[0342] CHO cells expressing relatively high and low levels of human ILT2 were cultured at 2×10 cells / 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 100 cells / mL in a 96-well U-bottom tissue culture plate. Cells were incubated at 4°C for 30 min with 50 μL of serially diluted anti-ILT2 antibody or isotype control (VRC01 IgG4) in FACS buffer at concentrations ranging from 10 μg / mL to 0.2 ng / mL.

[0343] 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 incubation at 4°C in the dark for 30 minutes, cells were washed three times and analyzed as described above. Statistical analysis was performed by comparing the best fit values ​​between the BA252 and 15G8 data sets using the "Extra Sum of Squares F Test".

[0344] like Figure 4A and 4B As shown, BA252 and 15G8 bound to CHO cells expressing human ILT2 in a dose-dependent manner at relatively high and low levels, with BA252 showing a significantly higher maximum binding level (Bmax). Calculated AUC and EC 50 The values ​​are listed in Tables 13 and 14.

[0345] Table 13. Binding of anti-ILT2 antibodies to ILT2 high-expressing CHO cells.

[0346]

[0347] Table 14. Binding of anti-ILT2 antibodies to ILT2 low expressing CHO cells.

[0348]

[0349] Example 2: Anti-ILT2 antibody blocks the binding of ligand to ILT2

[0350] 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

[0351] The ability of anti-ILT2 antibodies to block the binding between ILT2 and the non-classical MHC I ligand HLA-G was tested.

[0352] For the blocking assay, 25 μL of CHO cells expressing ILT2 at relatively high levels were plated at 2 × 10 cells per well. 5 The final concentration of 10 cells was distributed into a 96-well U-bottom microtiter plate. 25 μL of anti-ILT2 antibody BA211, commercial anti-ILT2 antibody HP-F1 (Thermofisher / 16-5129-82) or isotype control (VRC01 IgG1) serial dilutions in FACS buffer were added to the wells to obtain a final assay concentration range of 10 μg / mL to 2.4 ng / mL. The mixture was incubated at 4°C for 30 minutes. The ILT2 ligand HLA-G-Fc was conjugated to R-phycoerythrin (R-PE) using the LYNX Rapid R-PE Antibody Conjugation Kit (Bio-Rad / LNK022RPE). HLA-G-Fc-PE was resuspended in FACS buffer at 1 μg / mL, and 50 μL of the solution was added to the wells of a 96-well U-bottom tissue culture plate. After incubation at 4°C in the dark for 30 minutes, the cells were washed by adding cold FACS buffer. This wash was repeated twice before analyzing cell fluorescence by flow cytometry (BD LSR Fortessa flow cytometer).

[0353] CHO cells were identified using a forward scattering area (FSC-A) versus side scattering area (SSC-A) and another FSC-A versus side scattering height (FSC-H) graph to select single cells. Samples were analyzed by gating single cell populations in sequence and drawing a histogram representing the fluorescence in the PE channel. For each antibody sample, the binding percentage was calculated as follows: (MFI (sample) - MFI (without HLA-G-PE 背景 )) / (MFI(without antibody 完全结合 )-MFI(without HLA-G-PE 背景 ))*100. Data were plotted using GraphPad Prism.

[0354] like Figure 5As shown, BA211 and HP-F1 blocked the binding of HLA-G-Fc to cells expressing ILT2. In this assay, the 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), respectively.

[0355] In similar experiments, the anti-ILT2 antibody BA252 blocked the binding of HLA-G-Fc to CHO cells expressing ILT2. Figure 6 Comparison of BA252 with isotype control antibody VRC01 IgG4 is shown. Calculated IC of BA252 50 The value was 0.103 μg / mL (95% CI 0.077-0.138 μg / mL, N=3), demonstrating efficient inhibition of ligand binding. 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

[0356] The ability of BA252 to block the binding between ILT2 and classical MHC I molecule ligands was tested.

[0357] Phycoerythrin (PE)-bound HLA-A*02:01 pentamer (ProImmune / F008-2A-G), HLA-B*07:02 pentamer (ProImmune / F045-2A-G), and HLA-C*07:02 pentamer (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 separate wells of three 96-well U-bottom tissue culture plates. 25 μL of serial dilutions of BA252 or isotype control antibody VRC01 IgG4 in FACS buffer were added to the wells to give a final assay concentration ranging from 30 μg / mL to 0.5 ng / mL. Then 25 μL of CHO cells expressing relatively high levels of ILT2 were added to a final concentration of 2×10 per well. 5 After incubation at 4°C in the dark for 45 minutes, the cells were washed by adding cold FACS buffer. This wash was repeated twice before analyzing the cell fluorescence by flow cytometry (BD LSR Fortessa flow cytometer). CHO cells were identified and samples were analyzed as described above.

[0358] like Figures 7A to 7CAs shown, BA252 has an IC value of 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) respectively. 50 The values ​​blocked the binding of HLA-A*02:01, HLA-B*07:02, and HLA-C*07:02 pentamers to cells expressing ILT2.

[0359] Example 3: Function of anti-ILT2 antibodies

[0360] Sequence-optimized anti-ILT2 antibodies block ILT2 and increase FcγRIIIa-NFAT signaling in a human ILT2 T cell reporter assay

[0361] In this example, the ability of anti-ILT2 antibodies BA214, BA221, BA251, and BA252 to block binding between Jurkat reporter cells expressing ILT2 and Ramos target cells expressing HLA-G and to enhance FcγRIIIa signaling in Jurkat cells through NFAT was tested. The commercial anti-ILT2 antibody HP-F1 was used as a positive control, and VRC01 with IgG1N297A and IgG4 S228P constant regions was used as a negative isotype control.

[0362] The Jurkat cells (Promega / G7102) expressing FcγRIIIa and NFAT-luciferase reporter genes are internally engineered to express ILT2, and used to simulate effector immune cells. These effector cells are expanded in the RPMI 1640 culture medium containing 10% fetal bovine serum, 1%MEM non-essential amino acids, 500μg / mL G418, 200μg / mL hygromycin B and 1mM sodium pyruvate. In the co-culture reporter gene assay, the target cell is composed of the Ramos cells expressing HLA-G by internal engineering. The culture medium for amplifying target cells and for the co-culture reporter gene assay is made up of the RPMI 1640 culture medium supplemented with 20% fetal bovine serum, 1mM sodium pyruvate, 10mM Hepes and 50μM β-mercaptoethanol.

[0363] Jurkat effector cells were cultured at 1 x 10 5The cells were seeded in U-shaped wells of 96-well microtiter plates. The anti-CD20 antibody rituximab (Genentech / NDC code 50242-053-06) was used as a conditioning agent to bind to 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 at 37°C and 5% CO2 for 30 minutes. Serial dilutions of anti-ILT2 antibodies ranging from 10 μg / mL to 2.4 ng / mL and 5 x 10 4 Ramos target cells were added to the relevant wells. The final assay volume was 100 μL. The plates were incubated overnight at 37°C and 5% CO2. To measure luciferase reporter gene expression, the culture medium in 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. The luminescent 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 and EC was calculated. 50 value.

[0364] like Figures 8A to 8B As shown, all anti-ILT2 antibodies increased FcγRIIIa NFAT signaling relative to isotype control in Jurkat effector cells expressing human ILT2. As the luminescence signal varied in 3 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 listed in Table 15.

[0365] Table 15. Effects of anti-ILT2 antibodies on FcγRIIIa-NFAT signaling.

[0366] Antibody Average AUC SD <![CDATA[EC 50 (mg / mL)]]> 95%CI(mg / mL) BA214 145.22 17.77 0.0340 0.0005–0.245 BA251 159.24 10.77 0.0212 0.004–0.108 BA221 143.09 20.17 0.0328 0.0076–0.142 BA252 158.58 27.18 0.0153 0.0003–0.763 HP-F1 100 0 0.0328 0.0191–0.057

[0367] In a similar experiment, the anti-ILT2 antibody BA252 was compared with the reference antibody 15G8. Figures 9A to 9B As shown in , BA252 increased FcγRIIIa-NFAT signaling relative to isotype control, showing superior potency compared to 15G8. The calculated EC values ​​for BA252 and 15G8 were 50The values ​​were 0.07±0.04μg / mL and 0.43±0.20μg / mL, respectively.

[0368] Anti-ILT2 antibody BA252 does not bind C1q

[0369] The anti-ILT2 antibody variant BA252 was assessed for binding to the complement component 1q (C1q) protein, the essential first step in the classical complement cascade that induces complement-dependent cytotoxicity (CDC).

[0370] C1q binding was determined using an enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well NUNC Maxisorp plate (Thermoscientific / 44-2404-21) was coated overnight with 50 μL of BA252 or a control antibody, nivolumab (Evidentric GmbH), an anti-PD-1 IgG4 antibody, adalimumab (Myonex), an anti-TNFα IgG1 antibody, or a serial dilution (300-0.14 μg / mL) of an IgG4 isotype control (CrownBio / C00045-5) at 4°C. The plate was then decanted and blocked with PBS containing 2% milk powder for 1 hour at room temperature. The binding assay was then performed by removing the blocking solution and adding 50 μL of a 2 μg / mL complement component C1q (Sigma / C1740-1 mg) solution. The plate was washed 3 times in 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 incubation at room temperature for 1 hour, the plate was decanted and washed 3 times in 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 plate was incubated for 1 hour at room temperature. Finally, after washing the plate 3 times in 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 about 1 minute, and then the enzymatic reaction was stopped by adding 50 μL of a 1M HCl solution. The optical density (OD) of each well was measured at 450 nm using a Tecan Infinite M1000-Pro plate reader.

[0371] like Fig.10As shown, BA252 exhibited minimal binding to C1q, which was also comparable to the control antibody nivolumab and the human IgG4 isotype control. As a positive control, adalimumab (an IgG1 antibody) exhibited C1q binding. The calculated AUC values ​​are listed in Table 16.

[0372] Table 16. AUC values ​​of anti-ILT2 antibodies.

[0373] Antibody Average AUC SD BA252 0.55 0.09 Nivolumab 0.52 0.18 IgG4 Isotype Control 0.50 0.21 Adalimumab 6.54 0.79

[0374] Anti-ILT2 antibody BA252 enhances activation of primary human T, NK, and NKT cells

[0375] The potential of BA252 to promote the functional activity of primary immune cells was assessed in ex vivo experiments using co-cultures of peripheral blood mononuclear cells (PBMCs) and tumor cell lines.

[0376] Pharmacodynamic effects were assessed by priming PBMCs from healthy donors with IL-2 and IL-15 overnight in the presence of BA252, 15G8 or isotype control. HLA-A2 Cancer cells were added to the induced PBMCs with a ratio of 1:40 tumor cells to PBMCs and co-cultured for 72 hours. The activation of T, NKT and NK cells was assessed by flow cytometry analysis of CD25 expression on T cells (CD3+CD4+ and CD3+CD8+), NKT (CD3+CD56+) and NK (CD3-CD56+) cells. If the following criteria are met, PBMC donors are assigned to the "responder" group: at least one of the analyzed immune subgroups (NK, NKT, CD8+T cells) reacts to BA252, and compared with isotype control antibodies, CD25 surface expression increases by ≥20%. Otherwise, they will be classified as "non-responders" group.

[0377] The results showed that BA252 was superior to 15G8 in promoting the functional activity of primary immune cells. Figures 11A to 11B shown.

[0378] BA252-mediated ILT2 blockade modulates the phenotype of primary human macrophages

[0379] Use of primary human macrophages The potency of BA252 in modulating the functional activity of myeloid cells was assessed in vitro.

[0380] Differentiation of the purified mononuclear cell fraction from PBMCs in the presence of BA252, 15G8 or IgG4 isotype control Polarized to M2-like To induce the tumorigenic M2 phenotype, cells were treated with IL-10+TGFβ mixture or conditioned medium from JEG-3 cancer cells expressing HLA-G (cancer cell CM). Surface expression of CD86, CD163, and CD206 on CD33+ bone marrow cells was assessed by flow cytometry Phenotype. Statistical analysis was performed by paired t-test.

[0381] The results demonstrating that BA252 is superior to 15G8 in suppressing M2-like and enhancing antigen presentation phenotypes are as follows Figures 12A to 12B shown.

[0382] Example 4: Cross-competitive binding

[0383] BA252 does not compete with commercially available anti-ILT2 antibodies for binding to human ILT2-expressing cells

[0384] The ability of BA252 to block the binding of anti-ILT2 antibodies 1Q-G2 (Creative, #DCABH-4494, APC-conjugated), VMP55 (Novus, #NBP2-50475PE, PE-conjugated), 4F9 (Biorad, #MCA2515F, FITC-conjugated), 292305 (R&D Systems, #FAB20171P-100, PE-conjugated), and GHI / 75 (Biolegend, #333708, PE-conjugated) to human ILT2 expressed on cells was evaluated by flow cytometry.

[0385] For epitope competition assays, CHO cells expressing ILT2 were cultured at 8 × 10 6 The cells were resuspended in FACS buffer and 25 μL were added to the wells of a U-bottom 96-well microtiter plate to obtain 2×10 cells / mL per well. 5The final cell concentration of cells was 100 μg / mL. 25 μL of serial dilutions of unconjugated BA252 in FACS buffer were added to the wells to obtain a final assay concentration in the range of 100 μg / mL to 0.01 μg / mL. After pre-incubation on ice for 1 hour, 50 μL of a second anti-ILT2 antibody conjugated with a fluorescent dye was added to the wells. Based on the pre-established titration curve, 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. As a positive control to demonstrate epitope competition, APC-conjugated BA252 was also included and used at a final concentration of 0.3 μg / mL. After incubation on ice for another 1 hour in the dark, the cells were washed by centrifugation at 300 g for 5 minutes and adding 200 μL of cold FACS buffer. This wash was repeated once before analysis of cell fluorescence by flow cytometry (BD LSR Fortessa flow cytometer).

[0386] CHO cells were identified using a plot of forward scatter area (FSC-A) versus side scatter area (SSC-A) and another plot of SSC-A versus side scatter height (SSC-H) to select single cells. Samples were analyzed by sequentially gating the single cell population and plotting a histogram representing the fluorescence in the FITC, PE, or APC channel (depending on the fluorochrome used for the secondary antibody). Data were plotted using GraphPad Prism.

[0387] like Fig.13A As shown, unconjugated BA252 competes with APC-conjugated BA252 for binding. BA252 does not block the binding of commercially available anti-ILT2 clones 1Q-G2, VMP55, 4F9, 292305, and GHI / 75, indicating that it binds to different epitopes on human ILT2 ( Figures 13B to 13F ).

[0388] ***

[0389] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described, will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0390] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and 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.

[0391] Other embodiments are within the scope of the following claims.

Claims

1. An antibody that specifically binds to human ILT2, the antibody comprising: a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 1; and a VL comprising the 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 and CDRH3 amino acid sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

3. The antibody according to any one of the preceding claims, wherein the antibody comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NOs: 15, 16 and 19, respectively.

4. The antibody of any one of the preceding claims, wherein the antibody comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences shown 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.

5. An antibody according to any of the preceding claims, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NO: 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, respectively.

6. The antibody according to any one of the preceding claims, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NOs: 9, 10, 11, 13, 16 and 18, respectively.

7. The antibody according to any one of the preceding claims, wherein the antibody comprises the VH amino acid sequence of SEQ ID NO:

1.

8. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain constant region, which is optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgM.

9. The antibody of claim 8, wherein the heavy chain constant region is a human IgG4 heavy chain constant region comprising P at position 228 numbered according to the EU numbering system.

10. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain constant region that is 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.

11. The antibody according to claim 10, wherein the amino acid sequence of the heavy chain constant region comprises: (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) a set of amino acid mutations selected from the group consisting of L234A and L235A; L234A and L235E; L234A, L235A and L329A; or L234A, L235A and P329G according to the EU numbering system; 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.

12. The antibody of any one of claims 1 to 9, wherein the antibody comprises a heavy chain constant region that is 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.

13. The antibody according to claim 12, wherein the amino acid sequence of the heavy chain constant region comprises: (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) a set of amino acid mutations selected from the group consisting of according to the EU numbering system: 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.

14. The antibody of any one of the preceding claims, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38 or 39.

15. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26.

16. The antibody of any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO:

8.

17. The antibody of any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7.

18. The antibody of any one of the preceding claims, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31 or 32.

19. The antibody of any one of the preceding claims, wherein the VH and the VL comprise the amino acid sequences shown in SEQ ID NO: 1 and 5, 1 and 2, 1 and 3, 1 and 4, 1 and 6 or 1 and 7, respectively.

20. The antibody of claim 18 or 19, wherein the heavy chain and the light chain comprise the residues 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, 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 The amino acid sequence shown in 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.

21. The antibody of any one of the preceding claims, wherein: (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.

22. A polypeptide comprising a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO:

1.

23. The polypeptide of claim 22, wherein the VH comprises the CDRH1, CDRH2 and CDRH3 amino acid sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

24. The polypeptide of claim 22 or 23, wherein the VH comprises the amino acid sequence of SEQ ID NO:

1.

25. The polypeptide of any one of claims 22 to 24, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26.

26. A polypeptide comprising a VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO:

8.

27. The polypeptide of claim 26, wherein the VL comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NOs: 15, 16 and 19, respectively.

28. The polypeptide of claim 26 or 27, wherein the VL comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NO: 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.

29. The polypeptide of any one of claims 26 to 28, wherein the VL comprises the amino acid sequence of SEQ ID NO:

8.

30. The polypeptide of any one of claims 26 to 29, wherein the VL comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7.

31. A polypeptide according to any one of claims 26 to 30, wherein the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31 or 32.

32. The antibody or polypeptide of any one of the preceding claims, wherein the antibody or polypeptide is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

33. A polynucleotide encoding: the VH, VL, heavy chain and / or light chain of the antibody according to any one of claims 1 to 21; or the polypeptide according to any one of claims 22 to 32.

34. A vector comprising the polynucleotide according to claim 33.

35. A recombinant host cell comprising: (a) the polynucleotide according to claim 33; (b) the vector according to claim 34; (c) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 21 and a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of claims 1 to 21; (d) a first vector comprising a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 21, and a second vector comprising a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of claims 1 to 21.

36. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 21 or 32, a polypeptide according to any one of claims 22 to 32, a polynucleotide according to claim 33, a vector according to claim 34 or a host cell according to claim 35, and a pharmaceutically acceptable carrier or excipient.

37. A method for producing an antibody, the method comprising culturing the host cell according to claim 35 under suitable conditions so that the polynucleotide is expressed and the antibody is produced.

38. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 21 or 32, a polypeptide according to any one of claims 22 to 32, a polynucleotide according to claim 33, a vector according to claim 34, a host cell according to claim 35, or a pharmaceutical composition according to claim 36.

39. Use of an antibody according to any one of claims 1 to 21 or 32, a polypeptide according to any one of claims 22 to 32, a polynucleotide according to claim 33, a vector according to claim 34, a host cell according to claim 35 or a pharmaceutical composition according to claim 36 for the manufacture of a medicament for treating cancer in a subject in need thereof.

40. An antibody according to any one of claims 1 to 21 or 32, a polypeptide according to any one of claims 22 to 32, a polynucleotide according to claim 33, a vector according to claim 34, a host cell according to claim 35 or a pharmaceutical composition according to claim 36 for use in medicine.

41. An antibody according to any one of claims 1 to 21 or 32, a polypeptide according to any one of claims 22 to 32, a polynucleotide according to claim 33, a vector according to claim 34, a host cell according to claim 35 or a pharmaceutical composition according to claim 36 for use in treating cancer in a subject in need thereof.

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