Antibodies to ILT4, bispecific anti-ILT4 / PD-L1 antibodies, and methods of use thereof

Abstract

Provided herein are novel ILT4 antibodies, and antigen-binding fragments thereof, as well as bispecific and multispecific constructs that bind ILT4 and PD-L1, comprising such antibodies linked to at least one additional binding agent. Methods of inducing or enhancing an immune response and treating cancer by administering the antibodies (or fragments), bispecific constructs, or compositions are also described. TIFF2024515560000032.tif56128

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 172,997, filed April 9, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] I. Background of the Invention The inhibitory immune checkpoint receptor "immunoglobulin-like transcript 4" (ILT4) is a member of a family of non-catalytic tyrosine phosphorylated receptors expressed on immune cells (e.g., T cells, B cells, NK cells, dendritic cells, macrophages, and mast cells). Like other receptors in this family, ILT4 contains a conserved sequence of amino acids in its cytoplasmic domain called the immunoreceptor tyrosine-based inhibitory motif (ITIM). (Veillette et al. (2002) Annual Review of Immunology 20(1):669-707). Binding to ILT4 by its cognate ligands (HLA-G and HLA class I in myeloid cells) and its activation have immunosuppressive effects through multiple mechanisms. ILT4 is also found in tumor and stromal cells in the tumor microenvironment of various cancers and has been shown to regulate the biological behavior of tumor cells and thus promote their immune escape. (Gao et al. (2018) Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1869(2):278-285.) Thus, expression of ILT4 in several tumor types is associated with poor outcome.

[0003] Programmed death-ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that is associated with suppressing the immune system during certain events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states, such as hepatitis. Normally, the immune system responds to foreign antigens associated with exogenous or endogenous danger signals, which triggers the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. When PD-L1 binds to its receptor, i.e., programmed cell death protein 1 (PD-1), it transmits an inhibitory signal that reduces the proliferation of these T cells, and apoptosis can also be induced, which is further mediated by the downregulation of the gene Bcl-2. In addition to the well-established inhibitory role of PD-1 on T cells, its expression has also been observed on tumor-infiltrating macrophages, where it can negatively regulate antitumor functions such as phagocytosis (Gordon et al. (2017) Nature 545: 495-9 (Non-Patent Document 3)). PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction of PD-1 with PD-L1 results in a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive if the interaction of PD-1 with PD-L2 is also disrupted (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0004] Despite the advances associated with antibody therapy, there is a need in the art for new and improved therapeutic agents for treating conditions or diseases, such as conditions or diseases in which stimulating an immune response is desired. It is therefore an object of the present invention to provide improved methods for treating subjects with such conditions or diseases, such as cancer. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Veillette et al. (2002) Annual Review of Immunology 20(1):669-707 [Non-Patent Document 2] Gao et al. (2018) Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1869(2):278-285 [Non-Patent Document 3] Gordon et al. (2017) Nature 545: 495-9 [Non-Patent Document 4] Dong et al. (2002) Nat. Med. 8:787-9 [Non-Patent Document 5] Dong et al. (2003) J. Mol. Med. 81:281-7 [Non-Patent Document 6] Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314 [Non-Patent Document 7] Konishi et al. (2004) Clin. Cancer Res. 10:5094-100 [Non-Patent Document 8] Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7 [Non-Patent Document 9] Brown et al. (2003) J. Immunol. 170:1257-66 Summary of the Invention

[0006] II. Summary of the Invention Provided herein are novel antibodies and antigen-binding fragments thereof (e.g., fragments such as Fab, Fab', F(ab')2, Fv, or single chain Fv) that bind human ILT4. Also described are bispecific and multispecific constructs comprising such antibodies (or fragments) linked to at least one additional binding agent (e.g., a ligand, receptor / trap sequence, or an antibody or antigen-binding fragment thereof), such as an additional antibody (or fragment) that binds human PD-L1 and / or human PD-1. As further described herein, the ILT4 antibodies (and fragments) and constructs of the invention can be used in methods of inducing or enhancing immune responses and in methods of treating diseases or conditions (e.g., cancer).

[0007] In one embodiment, the ILT4 antibody or antigen-binding fragment thereof has the following sequence: (i) a heavy chain variable region CDR1 amino acid sequence selected from the consensus sequence GYT(I,M)H (SEQ ID NO: 21) or a conservative sequence variant thereof; (ii) the heavy chain variable region CDR2 amino acid sequence shown in SEQ ID NO: 3, or a conservative sequence variant thereof; (iii) a heavy chain variable region CDR3 amino acid sequence selected from the consensus sequence ERPGGSQFIYYY(P,A)(M,L)DY (SEQ ID NO: 22) or a conservative sequence variant thereof; (iv) a light chain variable region CDR1 amino acid sequence selected from the consensus sequence RAS(A,E)NIYSYLA (SEQ ID NO: 23) or a conservative sequence variant thereof; (v) a light chain variable region CDR2 amino acid sequence selected from the consensus sequence NA(I,D)TLAE (SEQ ID NO: 24) or a conservative sequence variant thereof; (vi) the light chain variable region CDR3 amino acid sequence shown in SEQ ID NO: 8 or a conservative sequence variant thereof The heavy and light chain CDR1, CDR2, and CDR3 domains have the following structure:

[0008] An exemplary ILT4 antibody is antibody 7A3 described herein. In one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 7A3. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 7A3 having the sequence shown in SEQ ID NO: 9, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 7A3 having the sequence shown in SEQ ID NO: 10. In another embodiment, the antibody or antigen binding thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen binding thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9. In another embodiment, the antibody or antigen binding thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the antibody or antigen binding thereof comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In another embodiment, the antibody or antigen binding thereof comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 25. In another embodiment, the antibody or antigen binding thereof comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 26. In another embodiment, the antibody comprises heavy and light chains having the amino acid sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0009] Another exemplary ILT4 antibody is antibody 7B1 described herein. In one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 7B1. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 7B1 having the sequence set forth in SEQ ID NO: 19, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 7B1 having the sequence set forth in SEQ ID NO: 20. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:19. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:20. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NO:19 and SEQ ID NO:20, respectively. In another embodiment, the antibody or antigen binding thereof comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 27. In another embodiment, the antibody or antigen binding thereof comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 28. In another embodiment, the antibody comprises heavy and light chains having the amino acid sequences set forth in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0010] In yet another embodiment, the ILT4 antibody (or antigen-binding fragment thereof) of the invention comprises: (a) a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:104, and SEQ ID NO:105, or a sequence which is at least 80% identical to any one of the foregoing sequences; and / or (b) a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108, or a sequence which is at least 80% identical to any one of the aforesaid sequences.

[0011] In another embodiment, the ILT4 antibody of the invention comprises: (a) a heavy chain amino acid sequence as set forth in SEQ ID NO: 25, SEQ ID NO: 27, or a sequence which is at least 80% identical to either sequence; and / or (b) a light chain amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:28, or a sequence which is at least 80% identical to either sequence.

[0012] In another embodiment, the sequence of the ILT4 antibody or antigen-binding fragment thereof (e.g., CDR and / or variable region sequence) can comprise the exact same amino acid sequence as an antibody described herein (e.g., antibody 7A3 or antibody 7B1). In another embodiment, the antibody comprises the sequence of antibody 7A3 or antibody 7B1, which comprises conservative sequence modifications, but still retains the ability to effectively bind to ILT4. Such sequence modifications can include one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid additions, deletions, or substitutions, e.g., conservative sequence modifications.

[0013] In another embodiment, the antibody comprises a sequence having at least 80% sequence identity to the sequence of antibody 7A3 or antibody 7B1. Sequences that are substantially identical to the ILT4 antibodies or antigen-binding fragments described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences) are also encompassed by the present invention. In one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 9, SEQ ID NO: 19, or a sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 10, SEQ ID NO: 20, or a sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10, or a sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 20, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences).

[0014] In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 25, SEQ ID NO: 27, or a sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a light chain comprising SEQ ID NO: 26, SEQ ID NO: 28, or a sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the ILT4 antibody comprises a heavy chain comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO: 26, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the ILT4 antibody comprises a heavy chain comprising SEQ ID NO: 27 and a light chain comprising SEQ ID NO: 28, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0015] Also encompassed by the present invention are ILT4 antibodies or antigen-binding fragments thereof that compete for binding with any of the antibodies (or fragments) described herein or that bind to the same epitope as any of the antibodies (or fragments) described herein. For example, in one embodiment, an ILT4 antibody or antigen-binding fragment thereof competes with antibody 7A3 and / or antibody 7B1 for binding to ILT4.

[0016] In another embodiment, the ILT4 antibody or antigen-binding fragment has the following characteristics: a. Interfering with ILT4 ligands (e.g., HLA-G ligands) binding to human ILT4; b enhancing or increasing the release of cytokines or chemokines by human macrophages; c enhancing the activating effects of LPS and IFNγ on macrophages; d promoting M1 macrophage polarization; e 10 -9 M or less, or alternatively, 10 +9 M -1 binds to human ILT4 with an equilibrium binding constant Ka of or greater; f lack of cross-reactivity with other ILT family members; g Cross-reactivity with cynomolgus ILT4; and / or h Inhibiting ILT4-expressing tumor cells indicates one or more of.

[0017] The present invention also provides bispecific constructs (or multispecific constructs) comprising an ILT4 antibody or antigen-binding fragment thereof linked to at least one additional binding agent (e.g. a ligand, or an antibody or antigen-binding fragment thereof, e.g. a PD-L1 antibody or PD-1 antibody or antigen-binding fragment thereof). In one embodiment, the bispecific construct comprises a PD-L1 antibody or antigen-binding fragment thereof, wherein the PD-L1 antibody or antigen-binding fragment thereof is: (a) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64, respectively; (b) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; (c) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; (d) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; (e) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; and (f) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; The amino acid sequences of the heavy and light chain CDR1, CDR2, and CDR3 are selected from the group consisting of:

[0018] For example, the bispecific construct can be a chemical conjugate, which can be made by chemical conjugation of an ILT4 antibody with a second binding agent, such as a ligand or an antibody or antigen-binding fragment thereof (e.g., a PD-L1 antibody or antigen-binding fragment thereof). In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof further comprises a constant domain of human IgG1. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof is linked to the C-terminus of the heavy chain of the PD-L1 antibody or antigen-binding fragment thereof. In another embodiment, the ILT4 antigen-binding fragment is an scFv, such as an scFv further comprising disulfide stabilizing modifications with Cys substitutions in VH44 and VL100.

[0019] In another embodiment, the ILT4 antibody or antigen-binding fragment thereof further comprises a human IgG1 constant domain. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof is linked to the C-terminus of the heavy chain of the ILT4 antibody or antigen-binding fragment thereof. In another embodiment, the PD-L1 antigen-binding fragment thereof is an scFv.

[0020] In certain embodiments, the bispecific construct comprises a PD-L1 antibody linked to an ILT4 scFv, wherein: (a) the PD-L1 antibody, or antigen-binding fragment thereof, comprises the amino acid sequences of CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, respectively; and (b) the ILT4 scFv comprises: (i) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; or (ii) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0021] In another embodiment, the ILT4 scFv of the bispecific (or multispecific) construct further comprises disulfide stabilizing modifications having Cys substitutions in VH44 and VL100.

[0022] The present invention also provides compositions comprising any of the bispecific (multispecific) constructs, antibodies, or antigen-binding fragments thereof described herein and a pharma- ceutically acceptable carrier, as well as kits comprising any of the bispecific (multispecific) constructs, antibodies, or antigen-binding fragments thereof described herein and instructions for use.

[0023] In a further aspect, isolated nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, and bispecific or multispecific constructs described herein, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors are also provided. In another embodiment, a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof, or bispecific constructs described herein is provided. In another embodiment, the nucleic acid molecule is in the form of an expression vector. In another embodiment, the nucleic acid molecule is in the form of an expression vector that expresses the antibody or antigen-binding fragment thereof, or bispecific construct when administered to a subject in vivo.

[0024] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody heavy chain variable region and / or a light chain variable region, wherein the antibody variable region is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO: 108, or an amino acid sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the foregoing sequences).

[0025] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy and / or light chain of an antibody, wherein the antibody chain comprises an amino acid sequence set forth in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the aforesaid sequences).

[0026] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and light chain variable regions of an antibody, wherein the heavy and light chain variable regions comprise the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:108 or SEQ ID NO:19 and SEQ ID NO:20, respectively, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences).

[0027] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and light chains of an antibody, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:27 and SEQ ID NO:28, respectively, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences).

[0028] In another aspect, a method for inducing or enhancing an immune response (e.g., against an antigen) in a subject comprises administering to the subject any one of the antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein in an amount effective to induce or enhance an immune response (e.g., against an antigen) in the subject.

[0029] In a further aspect, a method is provided for treating a condition or disease (e.g., cancer) in a subject, the method comprising administering to the subject any one of the antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein in an amount effective to treat the condition or disease.

[0030] In another embodiment, a method is provided for treating a tumor in a subject (e.g., a tumor expressing ILT4, HLA-G, HLA class I, angiopoietin-like 2, Nogo, or an ILT4 ligand), the method comprising administering to the subject any one of the antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein in an amount effective to treat the tumor.

[0031] The subject can be, for example, a subject suffering from a condition or disease that is desired to stimulate immune response. In one embodiment, the condition or disease that is desired to stimulate immune response is cancer. The method of inducing or enhancing immune response (e.g., against an antigen) in a subject can further comprise administering the antigen to the subject. The preferred antigen that is administered in combination with the antibody or its antigen-binding fragment, bispecific construct, multispecific construct, or composition described herein is a tumor antigen. [Brief description of the drawings]

[0032] [Figure 1] 1A and 1B are tables showing the antigen binding kinetics of antibody 7A3 and its constructs (FIG. 1A) and antibody 7B1 and its constructs (FIG. 1B). [Diagram 2] Figures 2A, 2B, and 2C are graphs showing representative traces of antigen binding kinetic data for antibody 7A3 (Figure 2A), antibody 7B1 (Figure 2B), and a control antibody (Figure 2C). [Diagram 3]3A and 3B are graphs showing binding of chimeric and humanized monoclonal antibodies to human ILT4 using ELISA; antibody 7A3 (FIG. 3A) and antibody 7B1 (FIG. 3B). [Figure 4] 4A and 4B are graphs showing binding of antibody 7A3 and its constructs (FIG. 4A) and antibody 7B1 and its constructs (FIG. 4B) to HEK293 cells expressing human ILT4. [Diagram 5] 5A and 5B are graphs showing macrophage TNF-α production in the case of antibody 7A3 and constructs thereof (FIG. 5A) and antibody 7B1 and constructs thereof (FIG. 5B). [Figure 6] Figures 6A-6F are graphs showing induction of TNF-α and MIP1-γ production in macrophages by humanized antibodies 7A3 VH6-L17 and 7B1 VH10-L21; untreated (Figures 6A and 6D), with LPS treatment (Figures 6B and 6E), and with IFN-γ treatment (Figures 6C and 6F). [Figure 7] Figures 7A, 7B, and 7C are graphs showing relative gene expression in humanized antibodies 7A3 VH6-L17 and 7B1 VH10-L21; Figure 7A (CD86), Figure 7B (CD54), and Figure 7C (iNOS). [Figure 8] FIG. 8A (antibody 7A3) and FIG. 8B (antibody 7B1) are graphs showing the cross-reactivity of humanized monoclonal antibodies 7A3 VH6-L17 and 7B1 VH10-L21 to cells expressing ILT family members. [Figure 9] FIG. 9A (monocytes), FIG. 9B (macrophages), and FIG. 9C (dendritic cells) are graphs showing binding of humanized monoclonal antibodies 7A3 VH6-L17 and 7B1 VH10-L21 to myeloid cells. [Figure 10] Figure 10A (PD-L1 x ILT4) and Figure 10B (PD-1 x ILT4) are schematics showing the delineation of bispecific constructs. [Figure 11]FIG. 11 is a table showing antigen binding kinetics of bispecific antibody constructs to human ILT4. [Figure 12] Figure 12A (9H9-7A3 HL and 9H9-7A3 LH) and Figure 12B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing the binding profiles of humanized bispecific antibodies to human PD-L1 by ELISA. [Figure 13] Figure 13A (9H9-7A3 HL and 9H9-7A3 LH) and Figure 13B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing the binding profiles of humanized bispecific antibodies to HEK293 cells expressing human PD-L1. [Figure 14] Figure 14A (9H9-7A3 HL and 9H9-7A3 LH) and Figure 14B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing the binding characteristics of humanized bispecific antibodies to HEK293 cells expressing human ILT4. [Figure 15] Figure 15A (9H9-7A3 HL and 9H9-7A3 LH) and Figure 15B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing the bifunctional binding properties of humanized antibodies to HEK293 cells expressing human ILT4 and human PD-L1. [Figure 16] Figure 16A (9H9-7A3 HL and 9H9-7A3 LH) and Figure 16B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing T cell PD-1 / PD-L1 blockade by humanized bispecific antibodies. [Figure 17] FIG. 17A (9H9-7A3 HL and 9H9-7A3 LH) and FIG. 17B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing induction of TNF-α production in macrophages by humanized bispecific antibodies. [Figure 18] Figure 18A (9H9-7A3 HL and 9H9-7A3 LH) and Figure 18B (9H9-7B1 HL and 9H9-7B1 LH) are graphs showing inhibition of HLA-G binding to ILT4 by humanized bispecific antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] IV. DETAILED DESCRIPTION OF THE INVETION In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are provided throughout the detailed description.

[0034] A definition The term "immunoglobulin-like transcript 4" or "ILT4" as used herein refers to a member of the inhibitory immune checkpoint receptor and non-catalytic tyrosine phosphorylated receptor family. ILT4 is also called leukocyte immunoglobulin-like receptor B2 (LILRB2), LIR2, MIR10, and CD85d. ILT4 is expressed on immune cells where it binds to MHC class I molecules on antigen-presenting cells and transmits negative signals that inhibit stimulation of the immune response, for example, by controlling inflammatory responses and cytotoxicity to focus the immune response and limit autoreactivity. Multiple isoforms of human ILT4 have been identified. Isoform 1 (Accession No. Q8N423-1; SEQ ID NO: 89) represents the canonical sequence of 598 amino acid residues. The ILT4 antibody (or antigen-binding fragment thereof) of the present invention may cross-react with ILT4 from species other than human. Alternatively, the ILT4 antibody or antigen-binding fragment thereof may be specific for human ILT4 and may not show any cross-reactivity with other species. ILT4 or any variants and isoforms thereof may be isolated from cells or tissues which naturally express them or may be recombinantly produced using techniques well known in the art and / or described herein.

[0035] Ligands that bind to ILT4 are known in the art and include, inter alia, HLA-G, HLA class I, angiopoietin-like 2, b-amyloid, SEMA4A, CD1c / d, CSP, and myelin inhibitors such as Nogo66, MAG, OMgp.

[0036] The term "human leukocyte antigen G" or "HLA-G" (also known as "histocompatibility antigen, class I, G") refers to the ligand for ILT4. HLA-G belongs to the paralogs of the non-classical class I heavy chains of HLA. This class I molecule is a heterodimer (beta-2 microglobulin) consisting of a heavy chain and a light chain. The heavy chain is membrane anchored. HLA-G is expressed on placental cells of fetal origin. The heavy chain is approximately 45 kDa and its gene contains 8 exons.

[0037] As used herein, the terms "programmed cell death 1 ligand 1," "PD-L1," "PDCD1 ligand 1," "programmed cell death ligand 1," "B7 homolog 1," "B7-H1," and "ILT44" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-L1 that share at least one epitope in common with PD-L1. The complete PD-L1 sequence can be found under GenBank Accession No. NP_001254635 and is set forth in SEQ ID NO: 176.

[0038] Programmed death-ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that is speculated to play a major role in suppressing the immune system during certain events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states, such as hepatitis. Normally, the immune system responds to foreign antigens associated with exogenous or endogenous danger signals, which triggers the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. When PD-1 binds to PD-L1, an inhibitory signal is transmitted that reduces the proliferation of these T cells, and apoptosis can also be induced, which is further mediated by downregulation of the gene Bcl-2. As used herein, the terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1", and "hPD-I" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one epitope in common with PD-1. The complete PD-1 sequence can be found under GenBank accession number NP_005009 and is set forth in SEQ ID NO:175.

[0039] PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8 :787-9). The interaction of PD-1 with PD-L1 results in a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81 :281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54 :307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive if the interaction of PD-1 with PD-L2 is also disrupted (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99 :12293-7; Brown et al. (2003) J. Immunol. 170 :1257-66).

[0040] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat subjects with immune disorders. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0041] The term "antibody" as referred to herein means a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region is composed of three domains, namely CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as V L The light chain constant region is composed of one domain, CL. H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions of relative conservation called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0042] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., human ILT4). Such "fragments" are, for example, about 8 to about 1500 amino acids in length, suitably about 8 to about 745 amino acids in length, suitably about 8 to about 300, such as about 8 to about 200 amino acids in length, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) Fab fragments, i.e., V L Domain, V H (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) a V H (iv) an Fd fragment consisting of the V domain and the CH1 domain of one arm of an antibody; L Domains and V H (v) Fv fragment consisting of V domains; H and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. In addition, the two domains of the Fv fragment are L and V HAlthough the V and VL are encoded by separate genes, recombinant methods can be used to link them together with a synthetic linker that allows them to be produced as a single protein chain, in which the V L Area and V H The domains pair to form a monovalent molecule (known as single chain Fv (sFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0043] As used herein, the term "binding domain" refers to a portion of a protein or antibody that contains amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies (e.g., full-length antibodies) and antigen-binding fragments thereof. A binding domain confers specificity and affinity for an antigen to a binding agent. The term also encompasses any protein having a binding domain that is homologous or nearly homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources or may be partially or fully synthetically produced.

[0044] The term "monoclonal antibody" as used herein refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope.Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has a variable region and any constant region derived from human germline immunoglobulin sequence.In one embodiment, human monoclonal antibody is produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome includes a human heavy chain transgene and a human light chain transgene, and the B cell is fused to an immortalized cell.

[0045] The term "recombinant human antibody", as used herein, includes any human antibody prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant human antibody combinatorial library, and (d) antibodies prepared, expressed, created, or isolated by any other means, including the joining of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region comprises an antigen-binding domain that is encoded by different genes that rearrange to form an antibody specific to a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (called somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region further changes in response to the antigen (i.e., isotype switching). Thus, the nucleic acid molecules that have been rearranged and somatically mutated in response to an antigen that encode light and heavy chain immunoglobulin polypeptides may not have sequence identity to the original nucleic acid molecule, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0046] The term "human antibody" includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. Human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (see Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci 764:536-546). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., chimeric and humanized antibodies).

[0047] "Humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced by the corresponding amino acids derived from human immunoglobulin. In one embodiment of a humanized antibody, some, most, or all of the amino acids outside the CDR domain are replaced by amino acids derived from human immunoglobulin, while some, most, or all of the amino acids in one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not abolish the antibody's ability to bind to a specific antigen. A "humanized" antibody retains the same antigen specificity as that of the original antibody.

[0048] An "isolated antibody," as used herein, is intended to mean an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds human ILT4 is substantially free of antibodies that specifically bind to antigens other than human ILT4; an isolated antibody that specifically binds to human PD-L1 is substantially free of antibodies that specifically bind to antigens other than human PD-L1). However, an isolated antibody that specifically binds to an epitope may have cross-reactivity to the same antigen from a different species. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals.

[0049] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained when exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides derived from an antigen (e.g., ILT4 or PD-L1) are tested for reactivity with a given antibody (e.g., ILT4 or PD-L1 antibody). Methods for revealing the spatial conformation of epitopes include techniques in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0050] The term "an antibody that binds to the same epitope" as another antibody is intended to encompass antibodies that interact with, i.e. bind to, the same structural region on human ILT4 as the reference ILT4 antibody. The "same epitope" that the antibody binds may be a linear epitope or a conformational epitope formed by tertiary folding of the antigen.

[0051] The term "competing antibody" refers to an antibody that competes with a reference ILT4 antibody for binding to human ILT4, i.e., competitively inhibits the binding of the reference ILT4 antibody to ILT4. A "competing antibody" may bind to the same epitope on ILT4 as the reference ILT4 antibody, may bind to an overlapping epitope, or may sterically hinder the binding of the reference ILT4 antibody to ILT4.

[0052] Antibodies that recognize the same epitope or compete for binding can be identified using conventional techniques. Such techniques include, for example, immunoassays that show the ability of one antibody to interfere with the binding of another antibody to a target antigen, i.e., competitive binding assays. Competitive binding is measured in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as ILT4. Many types of competitive binding assays are known, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either of these, i.e., an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competing antibody is present in excess, it will inhibit specific binding of the reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

[0053] Other techniques include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of epitopes. Other methods monitor the binding of antibodies to antigen fragments or mutated variants of antigens, where reduced binding due to alteration of amino acid residues in the antigen sequence is often taken as an indication of epitope components. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. These peptides are then taken as leads to determine the epitopes that correspond to the antibodies used to screen the peptide libraries. For epitope mapping, computational algorithms have also been developed that have been shown to map conformationally discontinuous epitopes.

[0054] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to antigen binding to an epitope on a given antigen. Typically, an antibody binds to an epitope of about 10 as measured by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument (e.g., using recombinant human ILT4 as the analyte and an antibody as the ligand). -7 Less than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 M or an even smaller equilibrium dissociation constant (K D ) and binds to the predetermined antigen with an affinity that is at least two times greater than the affinity of the binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The expressions "antibody that recognizes a certain antigen" and "antibody specific for a certain antigen" are used interchangeably herein with the term "antibody that specifically binds to a certain antigen."

[0055] "K DThe term "antibody-antigen dissociation equilibrium constant," as used herein, is intended to mean the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the human antibodies of the invention have a dissociation equilibrium constant of about 10 as measured by surface plasmon resonance (SPR) technology on a BIACORE2000 instrument (e.g., using recombinant human ILT4 as the analyte and an antibody as the ligand). -8 Less than M or smaller, e.g. 10 -9 M or 10 -10 M or an even smaller equilibrium dissociation constant (K D ) binds to ILT4.

[0056] The term "kd" as used herein is intended to mean the dissociation rate constant for dissociation of an antibody from the antibody / antigen complex.

[0057] The term "ka," as used herein, is intended to mean the association rate constant for the binding of an antibody to an antigen.

[0058] The term "EC50" as used herein means the concentration of an antibody or antigen-binding fragment thereof that induces 50% of the maximal response, i.e., a response that is halfway between the maximal response and the baseline, in either an in vitro or in vivo assay.

[0059] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. In one embodiment, the human monoclonal antibodies of the invention are of the IgG1 isotype. In another embodiment, the human monoclonal antibodies of the invention are of the IgG2 isotype.

[0060] As used herein, the terms "inhibit" or "interfere" (e.g., with respect to inhibiting / interfering with binding of an HLA-G ligand to ILT4 and / or PD1 binding to a PD-L1 ligand) are used interchangeably and include both partial inhibition / interference and full inhibition / interference. Preferably, inhibition / interference reduces or alters the normal level or type of activity that occurs when binding occurs uninhibited or uninterfered. Inhibition and interference are also intended to include any measurable decrease in the binding affinity of HLA-G when contacted with an ILT4 antibody compared to HLA-G not contacted with an ILT4 antibody, for example, inhibiting HLA-G binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the ILT4 antibody inhibits HLA-G binding by at least about 70%. In another embodiment, the ILT4 antibody inhibits HLA-G binding by at least 80%. Inhibition and interference are also intended to include any measurable decrease in the binding affinity of PD1 when contacted with a PD-L1 antibody compared to PD1 not contacted with the PD-L1 antibody, for example, inhibiting PD1 binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the PD-L1 antibody inhibits PD1 binding by at least about 70%. In another embodiment, the PD-L1 antibody inhibits PD1 binding by at least 80%.

[0061] The term "cross-react" as used herein refers to the ability of an ILT4 antibody or antigen-binding fragment thereof or a PD-L1 antibody or antigen-binding fragment thereof of the present invention to bind to ILT4 or PD-L1, respectively, from different species. For example, an ILT4 antibody or antigen-binding fragment thereof of the present invention that binds to human ILT4 may also bind to ILT4 of another species. Similarly, a PD-L1 antibody or antigen-binding fragment thereof of the present invention that binds to human PD-L1 may also bind to PD-L1 of another species. As used herein, cross-reactivity is assessed based on detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or binding or otherwise functionally interacting with cells that physiologically express ILT4. Methods for measuring cross-reactivity include standard binding assays described herein, for example, by Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry techniques.

[0062] The term "naturally occurring" as used herein when applied to an object means that an object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that exists in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0063] The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0064] A binding domain, antibody, or portion of an antibody that binds ILT4 and / or PD-L1 (e.g., V H , V LThe term "isolated nucleic acid molecule", as used herein in reference to a nucleic acid encoding an antibody, CDR1, CDR2, CDR3, or CDR4, is intended to mean a nucleic acid molecule in which the nucleotide sequence encoding the antibody or a portion of the antibody is free of other nucleotide sequences that may naturally flank the nucleic acid in human genomic DNA encoding an antibody or portion of an antibody that binds to an antigen other than ILT4 and / or PD-L1.

[0065] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" when they have been purified away from other cellular components or other contaminants, such as other nucleic acids or proteins from cells, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0066] The nucleic acid molecules of the present invention, whether derived from cDNA, genome, or mixtures thereof, often exist in native sequence (except for modified restriction sites, etc.), but may be mutated by standard techniques to provide gene sequences. In the case of coding sequences, these mutations may affect the amino acid sequence as desired. In particular, DNA sequences that are substantially identical to or derived from native V, D, J, constant, switch, and other such sequences described herein are contemplated (wherein "derived" indicates that one sequence is identical to or modified from another sequence).

[0067] A nucleic acid is "operably linked" or "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription control sequences, "operably linked" means that the DNA sequences being linked are contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. With respect to switch sequences, "operably linked" indicates that these sequences have the ability to effect switch recombination.

[0068] The present invention also encompasses "conservative sequence modifications" of any of the sequences described herein, i.e., nucleotide and amino acid sequence modifications that do not abolish the binding of the VH and VL sequences encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. Such conservative sequence modifications include conservative substitutions of nucleotides and amino acids, as well as additions and deletions of nucleotides and amino acids. For example, modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-based mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in an ILT4 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0069] In certain embodiments, conservative amino acid sequence modifications refer to at most 1, 2, 3, 4, or 5 conservative amino acid substitutions relative to the CDR sequences described herein. For example, each such CDR may contain up to 5 conservative amino acid substitutions, such as up to 4 (i.e., 4 or less) conservative amino acid substitutions, such as up to 3 (i.e., 3 or less) conservative amino acid substitutions, such as up to 2 (i.e., 2 or less) conservative amino acid substitutions, or 1 or less conservative amino acid substitutions.

[0070] Alternatively, in another embodiment, mutations can be randomly introduced into all or part of the coding sequence for an ILT4 antibody or PD-L1 or PD-1 antibody, or antigen-binding fragment thereof, such as by saturation mutagenesis, and the resulting modified ILT4 antibody or PD-L1 or PD-1 antibody can be screened for binding activity.

[0071] In the case of nucleic acids, the term "substantial homology" indicates that two nucleic acids or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80%, usually at least about 90%-95%, and more preferably at least about 98%-99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0072] In the case of amino acids, the term "substantial homology" indicates that two amino acid sequences or designated sequences, when optimally aligned and compared, are identical in at least about 80% of the amino acids, usually at least about 90%-95%, and more preferably at least about 98%-99% or 99.5% of the amino acids, with appropriate amino acid insertions or deletions.

[0073] The percent identity between two sequences is a function of the number of identical positions common to the two sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0074] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0075] The nucleic acid and protein sequences of the present invention can also be used as a "query sequence" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain identical nucleotide sequences to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain identical amino acid sequences to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Please see http: / / www.ncbi.nlm.nih.gov.

[0076] B ILT4 antibodies and antigen-binding fragments thereof Provided herein are novel ILT4 antibodies (or antigen-binding fragments thereof), e.g., humanized antibodies, characterized by particular functional features or properties. For example, the antibodies (or fragments) of the invention have the following properties: a. Interfering with ILT4 ligands (e.g., HLA-G ligands) binding to human ILT4; b enhancing or increasing the release of cytokines or chemokines by human macrophages; c enhancing the activating effects of LPS and IFNγ on macrophages; d promoting M1 macrophage polarization; e 10 -9M or less, or alternatively, 10 +9 M -1 binds to human ILT4 with an equilibrium binding constant Ka of or greater; f lack of cross-reactivity with other ILT family members; g Cross-reactivity with cynomolgus ILT4; and / or h Inhibiting ILT4-expressing tumor cells indicates one or more of.

[0077] An exemplary ILT4 antibody is antibody 7A3 described herein. In one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 7A3. In another embodiment, the antibody or antigen-binding fragment comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 7A3 having the sequence shown in SEQ ID NO: 9, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 7A3 having the sequence shown in SEQ ID NO: 10. In another embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, respectively, or conservative sequence variants thereof, and the light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, or conservative sequence variants thereof. Alternatively, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9. Alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:97, SEQ ID NO:98, or SEQ ID NO:99. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:10.Alternatively, the antibody or antigen-binding fragment comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 102. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99; and (b) the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:100, SEQ ID NO:101, and SEQ ID NO:102.

[0078] For example, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region having the amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:10.

[0079] Another exemplary ILT4 antibody is antibody 7B1 described herein. In one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 7B1. In another embodiment, the antibody or antigen-binding fragment comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 7B1 having the sequence set forth in SEQ ID NO: 19, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 7B1 having the sequence set forth in SEQ ID NO: 20. In another embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively, or conservative sequence variants thereof, and the light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, or conservative sequence variants thereof. Alternatively, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19. Alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20.Alternatively, the antibody or antigen-binding fragment comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 108. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105; and (b) the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108.

[0080] For example, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region having the amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:20.

[0081] The antibody sequence can also be a consensus sequence of several antibodies. For example, in one embodiment, an ILT4 antibody or antigen-binding fragment comprises a heavy chain variable region CDR1 comprising an amino acid sequence selected from the consensus sequence GYT(I,M)H (SEQ ID NO: 21). In another embodiment, an ILT4 antibody or antigen-binding fragment comprises a heavy chain variable region CDR2 comprising SEQ ID NO: 3. In another embodiment, an ILT4 antibody or antigen-binding fragment comprises a heavy chain variable region CDR3 comprising an amino acid sequence selected from the consensus sequence ERPGGSQFIYYY(P,A)(M,L)DY (SEQ ID NO: 22). In another embodiment, an ILT4 antigen-binding fragment comprises a light chain variable region CDR1 comprising an amino acid sequence selected from the consensus sequence RAS(A,E)NIYSYLA (SEQ ID NO: 23). In another embodiment, the ILT4 antibody or antigen-binding fragment comprises a light chain variable region CDR2 comprising an amino acid sequence selected from the consensus sequence NA(I,D)TLAE (SEQ ID NO: 24). In another embodiment, the ILT4 antibody or antigen-binding fragment comprises a light chain variable region CDR3 comprising SEQ ID NO: 8.

[0082] If each of the described antibodies is capable of binding to human ILT4, it is considered to be an antibody that binds to the V H Array and V L Sequences can be "mixed and matched" to create a variety of ILT4 antibodies or antigen-binding fragments thereof. Binding of such "mixed and matched" antibodies to human ILT4 can be tested using binding assays known in the art and described in the Examples (e.g., ELISA). For example, the ILT4 antibodies and antigen-binding fragments thereof of the present invention comprise combinations of heavy and light chain variable region sequences shown in Table 1.

[0083] Table 1: Combinations of VH and VL sequences (SEQ ID NO.) TIFF2024515560000002.tif65150

[0084] Sequences that are substantially identical to the ILT4 antibodies and antigen-binding fragments thereof described herein (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences) are also provided. For example, in one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:9, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:19, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences). In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 10, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 20, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences).In another embodiment, the ILT4 antibody (or antigen-binding fragment thereof) comprises (a) a heavy chain variable region comprising SEQ ID NO:9, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:19, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences), and (b) a heavy chain variable region comprising SEQ ID NO:10, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO: 20, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 108, or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). For example, ILT4 antibodies or antigen-binding fragments thereof include SEQ ID NO: 9 or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto), and SEQ ID NO: 19 or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).Alternatively, the ILT4 antibody or antigen-binding fragment thereof includes SEQ ID NO: 10 or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto), and SEQ ID NO: 20 or a sequence at least 80% identical thereto (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0085] Other exemplary antibodies include ILT4 antibodies and antigen-binding fragments thereof that compete for binding with any of the ILT4 antibodies or fragments thereof described herein or bind to the same epitope as any of the ILT4 antibodies or fragments thereof described herein. For example, in one embodiment, the ILT4 antibody or antigen-binding fragment thereof competes with antibody 7A3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7A3) for binding to ILT4. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof competes with antibody 7B1 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7B1) for binding to ILT4. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof binds to the same epitope on ILT4 as antibody 7A3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7A3). In another embodiment, the ILT4 antibody or antigen-binding fragment thereof binds to the same epitope on ILT4 as antibody 7B1 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7B1).

[0086] In one embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Alternatively, the ILT4 antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 19, or a sequence at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the foregoing sequences.

[0087] In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. Alternatively, the ILT4 antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:15, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 10 and a light chain variable region comprising SEQ ID NO: 20, or a sequence that is at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the foregoing sequences.

[0088] C PD-L1 antibodies and antigen-binding fragments thereof Provided herein are PD-L1 antibodies and antigen-binding fragments thereof for use together with the ILT4 antibodies or antigen-binding fragments of the invention, for example in bispecific and multispecific constructs and methods of treatment.

[0089] An exemplary PD-L1 antibody is antibody 7H7 described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 7H7. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 7H7 having the sequence set forth in SEQ ID NO:77, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 7H7 having the sequence set forth in SEQ ID NO:78. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:77. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:77. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NO:77 and SEQ ID NO:78, respectively.

[0090] Another exemplary PD-L1 antibody is antibody 1B3 described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 1B3. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 1B3 having the sequence set forth in SEQ ID NO:79, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 1B3 having the sequence set forth in SEQ ID NO:80. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:79. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:80. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NO:79 and SEQ ID NO:80, respectively.

[0091] Another exemplary PD-L1 antibody is antibody 3B6 described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 3B6. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 3B6 having the sequence set forth in SEQ ID NO:81, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 3B6 having the sequence set forth in SEQ ID NO:82. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:81. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:82. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain and light chain variable regions having the amino acid sequences set forth in SEQ ID NO:81 and SEQ ID NO:82, respectively.

[0092] Another exemplary PD-L1 antibody is antibody 8B1 described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 8B1. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 8B1 having the sequence set forth in SEQ ID NO:83, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 8B1 having the sequence set forth in SEQ ID NO:84. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:83. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:84. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain and light chain variable regions having the amino acid sequences set forth in SEQ ID NO:83 and SEQ ID NO:84, respectively.

[0093] Another exemplary PD-L1 antibody is antibody 4A3 described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 4A3. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 4A3 having the sequence set forth in SEQ ID NO:85, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 4A3 having the sequence set forth in SEQ ID NO:86. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:85. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:86. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NO:85 and SEQ ID NO:86, respectively.

[0094] Another exemplary PD-L1 antibody is antibody 9H9 described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs or variable regions of antibody 9H9. In another embodiment, the antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of antibody 9H9 having the sequence set forth in SEQ ID NO:87, and the CDR1, CDR2, and CDR3 domains of the light chain variable region of antibody 9H9 having the sequence set forth in SEQ ID NO:88. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, or conservative sequence variants thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, respectively, or conservative sequence variants thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:87. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:88. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain and light chain variable regions having the amino acid sequences set forth in SEQ ID NO:87 and SEQ ID NO:88, respectively.

[0095] The antibody sequence can also be a consensus sequence of several antibodies. For example, in one embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region CDR1 comprising an amino acid sequence selected from the consensus sequence (T,S)(S,Y,H)WMS (SEQ ID NO: 167). In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region CDR2 comprising SEQ ID NO: 168. In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 169. In another embodiment, the PD-L1 antigen-binding fragment comprises a light chain variable region CDR1 comprising SEQ ID NO: 170. In another embodiment, the PD-L1 antigen-binding fragment comprises a light chain variable region CDR2 comprising SEQ ID NO: 171. In another embodiment, the PD-L1 antigen-binding fragment comprises a light chain variable region CDR3 comprising SEQ ID NO: 172.

[0096] Sequences that are substantially identical to the PD-L1 antibodies and antigen-binding fragments thereof described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences) are also encompassed by the present invention. In one embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, or a sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the PD-L1 antigen-binding fragment comprises a light chain variable region comprising SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences). In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO:77 and a light chain variable region comprising SEQ ID NO:78, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences). In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO: 79 and a light chain variable region comprising SEQ ID NO: 80, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences).In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO:81 and a light chain variable region comprising SEQ ID NO:82, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO:83 and a light chain variable region comprising SEQ ID NO:84, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO: 85 and a light chain variable region comprising SEQ ID NO: 86, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the PD-L1 antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO: 87 and a light chain variable region comprising SEQ ID NO: 88, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences).

[0097] Other exemplary antibodies include PD-L1 antibodies and antigen-binding fragments thereof that compete for binding with or bind to the same epitope as any of the PD-L1 antibodies or antigen-binding fragments thereof described herein. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof competes for binding to PD-L1 with antibody 7H7 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7H7). In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof binds to the same epitope on PD-L1 as antibody 7H7 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7H7).

[0098] In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof competes with antibody 1B3 (or an antibody having heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 1B3) for binding to PD-L1. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof binds to the same epitope on PD-L1 as antibody 1B3 (or an antibody having heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 1B3).

[0099] In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof competes with antibody 3B6 (or an antibody having the heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 3B6) for binding to PD-L1. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof binds to the same epitope on PD-L1 as antibody 3B6 (or an antibody having the heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 3B6).

[0100] In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof competes with antibody 8B1 (or an antibody having heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 8B1) for binding to PD-L1. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof binds to the same epitope on PD-L1 as antibody 8B1 (or an antibody having heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 8B1).

[0101] In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof competes with antibody 4A3 (or an antibody having heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 4A3) for binding to PD-L1. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof binds to the same epitope on PD-L1 as antibody 4A3 (or an antibody having heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 4A3).

[0102] In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof competes with antibody 9H9 (or an antibody having the heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 9H9) for binding to PD-L1. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof binds to the same epitope on PD-L1 as antibody 9H9 (or an antibody having the heavy chain and light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 9H9).

[0103] In another embodiment, the PD-L1 antigen-binding fragment is a PD-L1 antibody or antigen-binding fragment thereof. In one embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively, and the CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:77 and a light chain variable region comprising SEQ ID NO:78, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences). In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively, and the CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:79 and a light chain variable region comprising SEQ ID NO:80, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences). In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 of a heavy chain variable region as shown in SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of a light chain variable region as shown in SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively.In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:81, and a light chain variable region comprising SEQ ID NO:82, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, respectively. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:83, and a light chain variable region comprising SEQ ID NO:84, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 of the heavy chain variable region depicted in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region depicted in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:85 and a light chain variable region comprising SEQ ID NO:86, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences).In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, and the CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, respectively. In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:87 and a light chain variable region comprising SEQ ID NO:88, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforesaid sequences).

[0104] In another embodiment, the PD-L1 antibody or antigen-binding fragment thereof has the following functional characteristics: (a) disrupts (e.g., partially or completely) the binding of PD1 to PD-L1; (b) induce NFAT pathway activation, and / or (c) induce a mixed lymphocyte reaction. The present invention has one or more of the following:

[0105] D binding substance Other binding agents (e.g., ligands, receptor / trap sequences, or antibodies and antigen-binding fragments thereof) for use with the ILT4 antibodies or antigen-binding fragments of the invention include, for example, binding agents that bind to immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, AXL, ILT2, or ILT3), immune co-stimulatory molecules (e.g., CD27, CD40, 4-1BB, OX40, or GITR), or tumor antigens (e.g., HER2, EGFR, ErB3, or CD24). Exemplary binding agents include antibodies or antigen-binding fragments thereof that bind to human PD-1, such as PD-1 antagonists. An exemplary PD-1 antibody is nivolumab (referred to as 5C4 in WO2006 / 121168; also known as BMS-936558, MDX-1106, or ONO-4538). Specific exemplary binding agents include PD-L1 antibodies and PD-1 antibodies (or antigen-binding fragments thereof), such as durvalumab, pembrolizumab (Keytruda®), cemiplimab (Libtaylo®), avelumab (Bavencio®), durvalumab (Imfinzi®), and atezolizumab (Tecentriq®).

[0106] E Bispecific and Multispecific Constructs Also provided herein is a bispecific construct comprising an ILT4 antibody or antigen-binding fragment thereof linked to a second binding agent, for example, a second binding agent that binds to an immune checkpoint molecule (e.g., PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, AXL, ILT2, or ILT3), an immune co-stimulatory molecule (e.g., CD27, CD40, 4-1BB, OX40, or GITR), or a tumor antigen (e.g., HER2, EGFR, ErB3, or CD24), such as a bispecific construct comprising an ILT4 antibody (or antigen-binding fragment thereof) linked to a PD-L1 antibody or a PD-1 antibody (or antigen-binding fragment thereof). Such bispecific constructs linked to one or more additional binding agents to form a multispecific construct are also described.

[0107] A "bispecific" or "bifunctional" construct is an artificial hybrid that has two different binding domain (e.g., heavy / light chain) pairs and two different binding sites. Bispecific constructs can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol.

[0108] As used herein, the term "linked" refers to the attachment of two or more molecules. Linkages can be covalent or non-covalent. Linkages can also be genetic (i.e., recombinantly fused). Such linkages can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0109] For chemical conjugation, suitable reagents and methods for coupling two or more moieties together, particularly two or more antibodies or fragments thereof, are known in the art. A variety of coupling or cross-linking agents are commercially available and can be used to conjugate an ILT4 antibody or its antigen-binding fragment with a PD-L1 antibody or PD-1 antibody or its antigen-binding fragment. Non-limiting examples include sulfo-SMCC, protein A, carboimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Sulfo-SMCC, SPDP, and DTNB are preferred agents, with sulfo-SMCC being particularly preferred. Other suitable procedures for cross-linking components (e.g., antibodies or antigen-binding fragments thereof) using cross-linking agents are known in the art. See, e.g., Karpovsky, B. et al., (1984) J. Exp. Med. 160:1686; Liu, MA et al., (1985) Proc. Natl. Acad. Sci USA 82:8648; Segal, DM and Perez, P., U.S. Patent No. 4,676,980; and Brennan, M. (1986) Biotechniques 4:42.

[0110] In the case of genetic engineering, a nucleic acid molecule encoding an ILT4 antibody or antigen-binding fragment thereof can be inserted into a suitable expression vector using standard recombinant DNA techniques. A nucleic acid molecule encoding a PD-L1 or PD-1 antibody or antigen-binding fragment thereof can also be inserted into the same expression vector so as to be operably linked (e.g., in-frame cloning) to an ILT4 antibody or antigen-binding fragment thereof, thereby generating an expression vector encoding a fusion protein that is a bispecific construct. Preferably, the PD-L1 or PD-1 antibody or antigen-binding fragment thereof is operably linked to the C-terminal region of the heavy chain of the ILT4 antibody or antigen-binding fragment thereof. Other suitable expression vectors and cloning strategies for preparing the bispecific constructs described herein are known in the art.

[0111] When expressing bispecific constructs in host cells, the coding region of antibody or its antigen-binding fragment is combined with cloned promoter sequence, leader sequence, translation initiation sequence, leader sequence, constant region sequence, 3' non-translation sequence, polyadenylation sequence, and transcription termination sequence to form an expression vector construct.These constructs can be used to express, for example, full-length human IgG1 κ antibody or full-length human IgG4 κ antibody.The fully human, humanized, and chimeric antibodies used in the bispecific constructs described herein also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies.Similar plasmids can be constructed to express other heavy chain isotypes or to express antibodies that contain λ light chains.

[0112] After preparing the expression vector encoding the bispecific construct, the bispecific construct can be recombinantly expressed in host cells using standard transfection methods.For example, in one embodiment, the nucleic acid encoding the bispecific construct can be ligated into an expression vector, such as a eukaryotic expression plasmid, for example, the one used by the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338 841, or other expression systems known in the art.The purified plasmid carrying the cloned bispecific construct gene can be introduced into eukaryotic host cells, such as CHO or NSO cells, or other eukaryotic cells, such as plant-derived cells, fungal cells, or yeast cells.The method used to introduce these genes can be the method described in the art, such as electroporation, lipofectin, or lipofectamine.After introducing the expression vector into the host cells, the cells expressing the bispecific construct can be identified and selected.

[0113] These cells represent transfectomas that can then be amplified and scaled up for expression levels to produce the bispecific constructs. Alternatively, these cloned bispecific constructs can be expressed in other expression systems, such as E. coli or whole organisms, or expressed synthetically. Recombinant bispecific constructs can be isolated and purified from these culture supernatants and / or cultured cells.

[0114] Regardless of whether the bispecific construct of the present invention is prepared by chemical conjugation or genetic engineering, it can be isolated and purified by one or more methods for protein purification that are well established in the art.Preferred methods for isolation and purification include, but are not limited to, gel filtration chromatography, affinity chromatography, and anion exchange chromatography.A particularly preferred method is gel filtration chromatography, for example, using a Superdex 200 column.The isolated and purified bispecific construct can be evaluated by standard methods, such as SDS-PAGE analysis.

[0115] Thus, in one embodiment, the ILT4 antibody or antigen-binding fragment thereof is genetically fused to the PD-L1 or PD-1 antibody or antigen-binding fragment thereof. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof and the PD-L1 antibody or antigen-binding fragment thereof are chemically conjugated. In one embodiment, the PD-L1 or PD-1 antibody or antigen-binding fragment thereof further comprises a human IgG1 constant domain. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof is linked to the C-terminus of the heavy chain of the PD-L1 or PD-1 antibody or antigen-binding fragment thereof. In another embodiment, the ILT4 antigen-binding fragment thereof is an scFv. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof further comprises a human IgG1 constant domain. In another embodiment, the PD-L1 or PD-1 antibody or antigen-binding fragment thereof is linked to the C-terminus of the heavy chain of the ILT4 antibody or antigen-binding fragment thereof. In another embodiment, the PD-L1 antigen-binding fragment thereof or PD-1 antigen-binding fragment thereof is an scFv.

[0116] In another aspect, the bispecific and multispecific constructs of the invention comprise a modified human Fc domain (e.g. a modified IgG1 Fc domain), such as (a) a modified human IgG1 Fc domain comprising the non-natural amino acids 234A, 235Q, and 322Q, as numbered according to the EU index as set forth in Kabat; (b) a modified human IgG1 Fc domain comprising the non-natural amino acids 252Y, 254T, and 256E, as numbered according to the EU index as set forth in Kabat; and / or (c) a modified human IgG1 Fc domain comprising the non-natural amino acids 234A, 235Q, and 322Q, as numbered according to the EU index as set forth in Kabat.

[0117] Exemplary bispecific constructs are set out in Tables 2 and 3 below, in which antibodies or antigen-binding fragments thereof are defined by their CDR sequences (Table 2) or variable region sequences (Table 3).

[0118] Table 2: Exemplary bispecific constructs (CDRs) TIFF2024515560000003.tif238170

[0119] Table 3. Exemplary bispecific constructs (VR) TIFF2024515560000004.tif156170

[0120] Also provided herein are bispecific and multispecific constructs comprising sequences substantially identical to the aforementioned ILT4 and PD-L1 sequences (i.e., CDR and variable region sequences) (e.g., sequences with conservative sequence modifications and / or sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences).

[0121] In another embodiment, bispecific and multispecific constructs have the following properties: a. Interfering with ILT4 ligands (e.g., HLA-G ligands) binding to human ILT4; b enhancing or increasing the release of cytokines or chemokines by human macrophages; c enhancing the activating effects of LPS and IFNγ on macrophages; d promoting M1 macrophage polarization; e 10 -9 M or less, or alternatively, 10 +9 M -1 binds to human ILT4 with an equilibrium binding constant Ka of or greater; f lack of cross-reactivity with other ILT family members; g Cross-reactivity with cynomolgus ILT4; and / or h Inhibiting ILT4-expressing tumor cells indicates one or more of:

[0122] F composition Also provided herein are compositions, e.g., compositions comprising one or any combination of the antibodies or antigen-binding fragments thereof, bispecific constructs, or multispecific constructs described herein, formulated with a carrier (e.g., a pharma- ceutically acceptable carrier).

[0123] As used herein, the terms "carrier" and "pharmaceutical acceptable carrier" include any and all physiologically compatible solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, as well as isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous administration (e.g., by injection or infusion), intramuscular administration, subcutaneous administration, parenteral administration, spinal administration, or epidermal administration. Depending on the route of administration, the active compound (i.e., any of the antibodies or antigen-binding fragments thereof, bispecific constructs, or multispecific constructs described herein) may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0124] Examples of adjuvants that may be used with the antibodies or antigen-binding fragments thereof, bispecific constructs, or multispecific constructs described herein include, but are not limited to, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; cytokines such as GM-CSF, interleukin-2, interleukin-7, interleukin-12, and other similar factors; 3D-MPL; CpG oligonucleotides; and monophosphoryl lipid A, e.g., 3-de-O-acylated monophosphoryl lipid A.

[0125] MPL adjuvant is available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034, and 4,912,094).CpG-containing oligonucleotides (wherein CpG dinucleotides are unmethylated) are well known and are described, for example, in WO96 / 02555, WO99 / 33488, and U.S. Patent Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.

[0126] Other alternative adjuvants include, for example, saponins such as Quil A or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); escin; digitonin; or saponins from Gypsophila or Chenopodium quinoa; Montanide ISA 720 (Seppic, France); SAF (Chiron, California, United States); ISCOMS (CSL), MF-59 (Chiron); the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4 available from SmithKline Beecham, Rixensart, Belgium); Detox (Enhanzyn™) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs); WO polyoxyethylene ether adjuvants such as those described in J. Immunol. 99 / 52549A1; synthetic imidazoquinolines such as imiquimod [S-26308, R-837] (Harrison, et al., Vaccine 19: 1820-1826, 2001); and resiquimod [S-28463, R-848] (Vasilakos, et al., Cellular immunology 204: 64-74, 2000); Schiff bases of carbonyls and amines constitutively expressed on the surface of antigen-presenting cells and T cells, such as tucaresol (Rhodes, J. et al., Nature 377: 71-75, 1995); cytokines, chemokines, and costimulatory molecules, either as proteins or peptides (including, for example, proinflammatory cytokines, e.g., interferon, GM-CSF, IL-1α, IL-1β, TGF-α, and TGF-β, Th1 inducers (e.g., interferon γ, IL-2, IL-12, IL-15, IL-18, and IL-21), Th2 inducers (e.g., IL-4, IL-5, IL-6, IL-10, and IL-13), and other chemokines and costimulatory genes, e.g., MCP-1, MIP-1α, MIP-1β, RANTES, TCA-3, CD80, CD86, and CD40L); immune stimulators that target ligands (e.g., CTLA-4 and L-selectin), proteins and peptides that stimulate apoptosis (e.g., Fas); synthetic lipid-based adjuvants, e.g., vaxfectin (Reyes et al., Vaccine 19: 3778-3786, 2001), squalene, α-tocopherol, polysorbate 80, DOPC, and cholesterol; endotoxin, [LPS] (Beutler, B., Current Opinion in Microbiology 3: 23-30, 2000); ligands that trigger Toll receptors to produce Th1-inducing cytokines, such as synthetic mycobacterial lipoproteins, mycobacterial protein p19, peptidoglycan, teichoic acid, and lipid A; and CT (cholera toxin, subunits A and B) and LT (heat-labile enterotoxin from Escherichia coli, subunits A and B), the heat shock protein family (HSP), and LLO (listeriolysin O; WO 01 / 72329). These and various other Toll-like receptor (TLR) agonists are described, for example, in Kanzler et al, Nature Medicine, May 2007, Vol 13, No 5. .

[0127] "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, and those derived from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0128] The composition of the present invention can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0129] In order to administer the compound of the present invention by certain administration routes, it may be necessary to coat the compound with a material to prevent its inactivation, or to co-administer the compound with the material.For example, the compound may be mixed with suitable carrier, such as liposome, or diluent, and administered to a subject.Acceptable diluents include saline and aqueous buffer solutions.Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

[0130] Carrier includes sterile aqueous solution or dispersion, and sterile powder for preparing sterile injectable solution or dispersion immediately.The use of such media and agents for pharmaceutically active substances is known in the art.Except where any conventional media or agents are incompatible with active compounds, their use in the pharmaceutical compositions of the present invention is contemplated.Auxiliary active compounds can also be mixed in the composition.

[0131] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0132] Sterile injectable solution can be prepared by mixing the active compound in the required amount in suitable solvent with one or combination of the above-listed components as required, followed by sterilization filtration.Generally, dispersion is prepared by mixing active compound in sterile vehicle that contains basic dispersion medium and other components listed above.For the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which produces powder of active ingredient and any additional desired ingredients from its solution that has been previously sterilized and filtered.

[0133] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased according to the exigencies of the therapeutic situation. For example, the antibody of the present invention may be administered once or twice weekly by subcutaneous or intramuscular injection, or once or twice monthly by subcutaneous or intramuscular injection.

[0134] It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.Unit dosage form as used herein means a physically discrete unit suitable as a unitary dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of unit dosage form of the present invention is determined and directly depends on (a) the unique characteristics of active compound and the individual therapeutic effect to be achieved, and (b) the inherent constraints of the technology of compounding such active compound for treating individual susceptibility.

[0135] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0136] For therapeutic compositions, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be provided in unit dosage form and may be prepared by any method known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that provides a therapeutic effect. Generally, this amount will range from about 0.001 percent to about 90 percent of the active ingredient, preferably from about 0.005 percent to about 70 percent, and most preferably from about 0.01 percent to about 30 percent, out of one hundred percent.

[0137] The preparations of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or sprays, containing such carriers as are known in the art to be suitable.The dosage forms for topical or transdermal administration of the compositions of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0138] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0139] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, and polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0140] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, and dispersing agents. Prevention of the presence of microorganisms may be ensured by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0141] When the compounds of the present invention are administered to humans and animals as pharmaceuticals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, e.g., 0.01 to 30%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0142] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or pharmaceutical compositions of the present invention, are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0143] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for each patient, composition, and mode of administration without being toxic to the patient. The dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention or their esters, salts, or amides used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical art. A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start the dose of the compound of the present invention used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily amount of the composition of the present invention will be that amount of the compound that is the minimum dose effective to produce a therapeutic effect. Such an effective dose will generally vary depending on the factors mentioned above. Preferably, administration is intravenous, intramuscular, intraperitoneal or subcutaneous, and preferably administered proximal to target site.If desired, the effective daily amount of therapeutic composition can be administered as 2, 3, 4, 5, 6 or more divided doses, which are administered separately at appropriate intervals throughout the day, optionally in unit dosage form.Although it is possible to administer the compound of the present invention alone, it is preferred to administer the compound as a pharmaceutical preparation (composition).

[0144] The therapeutic composition can be administered using medical devices known in the art.For example, in preferred embodiments, the therapeutic composition of the present invention can be administered using needleless hypodermic injection devices, such as the devices disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Examples of well-known implants and modules useful in the present invention include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for administering medical agents at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering pharmaceutical agents through the skin; U.S. Patent No. 4,447,233, which discloses a medical agent infusion pump for delivering medical agents at precise infusion rates; U.S. Patent No. 4,447,224, which discloses an implantable variable flow rate infusion device for sustained drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0145] In certain embodiments, the antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if desired), they can be formulated, for example, in the form of liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thereby facilitating targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species of which may constitute components of the formulations and molecules of the invention; p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in the form of liposomes; in a more preferred embodiment, the liposomes contain a targeting moiety.In the most preferred embodiment, the therapeutic compound in liposome is delivered by bolus injection to the site proximal to tumor or infection.The composition must be fluid to the extent that it can be easily injected.It must be stable under the conditions of manufacture and storage, and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0146] The ability of a compound to inhibit cancer can be evaluated in an animal model system that is useful for predicting efficacy in human tumors. Alternatively, this property of a composition can be evaluated by investigating the inhibitory ability of the compound, such inhibition in vitro, by assay methods known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise improve symptoms in a subject. Those skilled in the art will be able to determine such amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the individual composition or route of administration selected.

[0147] These compositions must be sterile and fluid enough to allow the composition to be delivered by syringe.Besides water, the carrier can be isotonic buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, for example, aluminum monostearate or gelatin in the composition.

[0148] When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an edible digestible carrier.

[0149] G nucleic acid Also provided herein are isolated nucleic acid molecules that code antibody or its antigen-binding fragment, bispecific construct, and multispecific construct, as well as expression vectors that contain such nucleic acid and host cells that contain such expression vectors.In one embodiment, a nucleic acid molecule is provided that codes any of the antibody or its antigen-binding fragment, bispecific construct, or multispecific construct described herein.In another embodiment, the nucleic acid molecule is in the form of an expression vector.In another embodiment, the nucleic acid molecule is in the form of an expression vector that expresses antibody or its antigen-binding fragment, bispecific construct, or multispecific construct when administered to a subject in vivo.

[0150] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO: 108, or an amino acid sequence that is at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the foregoing sequences).

[0151] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody chain, wherein the chain comprises an amino acid sequence set forth in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the aforesaid sequences).

[0152] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and light chain variable regions of an antibody, wherein the heavy and light chain variable regions comprise the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:19 and SEQ ID NO:20, respectively, or amino acid sequences that are at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the foregoing sequences.

[0153] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and light chains of the antibody, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, respectively, or amino acid sequences that are at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the foregoing sequences.

[0154] The term "vector", as used herein, is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which means a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors have the ability to replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. In addition, certain vectors have the ability to direct the expression of a gene to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are often in the form of a plasmid. Since the plasmid is the most commonly used form of vector, herein, "plasmid" and "vector" may be used interchangeably. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0155] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to mean a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to mean not only the particular subject cell, but also the progeny of such a cell. Because some modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0156] H Combination therapy Any of the antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein can be administered in combination with another therapeutic agent, i.e., in combination with other agents. As used herein, the term "co-administered" includes any or all of the simultaneous, separate, or sequential administration of the antibodies, antigen-binding fragments thereof, bispecific constructs, or multispecific constructs described herein with adjuvants and other agents, including administration as part of a dosing regimen. For example, combination therapy can include administering any of the antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein with at least one or more additional therapeutic agents, such as anti-inflammatory agents, DMARDs (disease-modifying antirheumatic drugs), immunosuppressants, chemotherapeutic agents, radiation therapy, other antibodies, cytotoxins, and / or drugs, as well as adjuvants, immunostimulants, and / or immunosuppressants.

[0157] Chemotherapeutic agents suitable for administration in combination with the antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein in the treatment of tumors include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Other agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclosporine, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine and vinblastine), and temozolomide.

[0158] For example, agents that eliminate or inhibit immunosuppressive activity by immune cells (e.g., regulatory T cells, NKT cells, macrophages, myeloid-derived suppressor cells, immature dendritic cells or suppressive dendritic cells) or suppressive factors (e.g., TGFβ, indoleamine 2,3 dioxygenase (IDO)) produced by tumor cells or host cells in the local tumor microenvironment may also be administered with the binding domains, antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein. Such agents include antibodies and small molecule drugs, e.g., IDO inhibitors such as 1-methyltryptophan or derivatives.

[0159] Suitable agents for administration in combination with the antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein for the treatment of such immune disorders include, for example, immunosuppressants such as rapamycin, cyclosporine, and FK506; anti-TNF agents such as etanercept, adalimumab, and infliximab; and steroids. Examples of specific natural and synthetic steroids include, for example, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoxymethasone, dexamethasone, difluorocortolone, fluclorone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol, and triamcinolone.

[0160] Agents suitable for co-administration with the antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein for the purpose of inducing or enhancing an immune response include, for example, adjuvants and / or immunostimulants, non-limiting examples of which are disclosed above. In one embodiment, the immunostimulant is a TLR3 agonist, such as PolyIC.

[0161] As used herein, the term "immunostimulant" includes, but is not limited to, compounds that can stimulate antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages.For example, immune stimulants suitable for use in the present invention can stimulate APCs so that the maturation process of APCs is accelerated, the proliferation of APCs is increased, and / or the recruitment or release of costimulatory molecules (e.g., CD80, CD86, ICAM-1, MHC molecules, and CCR7) and proinflammatory cytokines (e.g., IL-1β, IL-6, IL-12, IL-15, and IFN-γ) is upregulated.Suitable immune stimulants can also increase T cell proliferation. Such immune stimulants include, but are not limited to, CD40 ligand; FLT3 ligand; cytokines such as IFN-α, IFN-β, IFN-γ, and IL-2; colony stimulating factors such as G-CSF (granulocyte colony stimulating factor) and GM-CSF (granulocyte macrophage colony stimulating factor); CTLA-4 antibody, PD-1 antibody, 41BB antibody, or OX-40 antibody; LPS (endotoxin); ssRNA; dsRNA; Bacille Calmette-Guerin (BCG); levamisole hydrochloride; and intravenous immunoglobulin. In one embodiment, the immune stimulant may be a Toll-like receptor (TLR) agonist.For example, the immune stimulant may be a TLR3 agonist, such as a double stranded inosine:cytosine polynucleotide (poly I:C, available, for example, from Hemispherx Bipharma (PA, US) as Ampligen™ or from Oncovir as poly IC:LC) or poly A:U; a TLR4 agonist, such as monophosphoryl lipid A (MPL) or RC-529 (available, for example, from GSK (UK)); a TLR5 agonist, such as flagellin; a TLR7 or TLR8 agonist, such as an imidazoquinoline-based TLR7 or TLR8 agonist, such as imiquimod (e.g. Aldara™) or resiquimod and related imidazoquinoline-based substances (available, for example, from 3M Corporation); or a TLR9 agonist, such as deoxynucleotides containing unmethylated CpG motifs (so-called "CpGs," e.g., Coley Such immune stimulants may be administered simultaneously, separately or sequentially with the antibodies, antigen-binding fragments thereof, bispecific constructs, and / or multispecific constructs described herein.

[0162] I Uses and Methods of the Invention Also provided herein are methods of inducing or enhancing an immune response and methods of treating cancer by administering to a patient in need thereof a bispecific construct, a multispecific construct, an antibody or antigen-binding fragment thereof, or a composition described herein.

[0163] The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response (ie, either passive or adaptive) to a particular antigen.

[0164] The terms "treat", "treating" and "treatment" as used herein refer to therapeutic or prophylactic measures as described herein. The method of "treatment" employs administration of a bispecific construct, a multispecific construct, an antibody, an antigen-binding fragment thereof, or a composition as described herein to a subject in need of such treatment, e.g., a subject in need of an enhanced immune response to a particular antigen or a subject who may ultimately develop such a disorder, to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurrent disorder, or to extend the survival of the subject longer than would be expected in the absence of such treatment.

[0165] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially halt the progression of the disease and its complications in a patient already suffering from the disease. The amount effective for this use will vary depending on the severity of the disorder being treated and the general condition of the patient's own immune system.

[0166] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0167] As used herein, the term "inhibiting proliferation" (e.g., in relation to cells) is intended to include any measurable decrease in cell proliferation, e.g., inhibition of cell proliferation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0168] In another aspect, a method for inducing or enhancing an immune response (e.g., against an antigen) in a subject comprises administering to the subject any one of the antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein in an amount effective to induce or enhance an immune response (e.g., against an antigen) in the subject.

[0169] In another aspect, a method for treating cancer in a subject is provided, the method comprising administering to the subject any one of the antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein in an amount effective to treat the condition or disease.

[0170] In another aspect, a method for treating cancer in a subject is provided, the method comprising administering to the subject any one of the ILT4 antibodies or antigen-binding fragments thereof described herein in combination with another antibody or antigen-binding fragment thereof, such as any one of the PD-L1 or PD-1 antibodies or antigen-binding fragments thereof described herein.

[0171] In one embodiment, the ILT4 antibody or antigen-binding fragment thereof and the PD-L1 or PD-1 antibody or antigen-binding fragment thereof are administered separately. In one embodiment, the ILT4 antibody or antigen-binding fragment thereof and the PD-L1 or PD-1 antibody or antigen-binding fragment thereof are administered sequentially. For example, the ILT4 antibody or antigen-binding fragment thereof can be administered first, followed by (e.g., immediately after) the PD-L1 or PD-1 antibody or antigen-binding fragment thereof, or vice versa. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof and the PD-L1 or PD-1 antibody or antigen-binding fragment thereof are administered together. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof and the PD-L1 or PD-1 antibody or antigen-binding fragment thereof are administered simultaneously. In another embodiment, the ILT4 antibody or antigen-binding fragment thereof and the PD-L1 or PD-1 antibody or antigen-binding fragment thereof are administered simultaneously in a single formulation. Alternatively, the ILT4 antibody or antigen-binding fragment thereof, and the PD-L1 or PD-1 antibody or antigen-binding fragment thereof, are formulated for separate administration and administered either concurrently or sequentially, such concurrent or sequential administration preferably resulting in the simultaneous presence of both antibodies in the treated patient.

[0172] In certain embodiments, administration of any of the ILT4 antibodies, or antigen-binding fragments thereof, described herein in combination with any of the PD-L1 or PD-1 antibodies, or antigen-binding fragments thereof, described herein results in a synergistic effect (e.g., in enhancing immune responses in vivo) compared to the use of either antibody alone.

[0173] The subject may be, for example, a subject suffering from a condition or disease in which stimulating an immune response is desirable. In one embodiment, the condition or disease is cancer. Types of cancer include, but are not limited to, leukemia, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia), chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's disease, and leukemia. Rehm's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma Cancer, Renal Cell Carcinoma, Hepatoma, Cholangiocarcinoma, Choriocarcinoma, Seminoma, Embryonal Carcinoma, Embryonal Carcinoma, Wilms' Tumor, Cervical Cancer, Uterine Cancer, Testicular Tumor, Lung Cancer, Small Cell Lung Cancer, Non-Small Cell Lung Cancer, Bladder Cancer, Epithelial Carcinoma, Glioma, Astrocytoma, Medulloblastoma, Craniopharyngioma, Ependymoma, Pinealoma, Hemangioblastoma, Acoustic Neuroma, Oligodendroglioma, Meningioma, Melanoma, Neuroblastoma, Retinoblastoma, Nasopharyngeal Carcinoma, Esophageal Cancer, Basal Cell Carcinoma, Biliary Tract Cancer, Bladder Cancer, Bone Cancer, Cancer of the Brain and Central Nervous System (CNS) , cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer. Particular cancers include tumors that express ILT4 selected from the group consisting of chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, and marginal zone B-cell lymphoma.Other indications include bacterial, fungal, viral, and parasitic infections.

[0174] The method of inducing or enhancing an immune response (e.g., against an antigen) in a subject described herein can further include administering the antigen to the subject.As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, hapten, polysaccharide, and / or lipid.The bispecific construct, multispecific construct, antibody, antigen-binding fragment thereof, or composition described herein and the antigen can be administered simultaneously, or the bispecific construct, multispecific construct, antibody, antigen-binding fragment thereof, or composition can be administered before or after the antigen is administered.

[0175] In one embodiment, the bispecific construct, multispecific construct, antibody, antigen-binding fragment thereof, or composition described herein is administered in combination with a vaccine to enhance the immune response to vaccine antigens, such as tumor antigens (to enhance the immune response to tumors) or antigens derived from infectious disease pathogens (to enhance the immune response to infectious disease pathogens).Thus, in one embodiment, the vaccine antigen can include, for example, an antigen or antigenic composition capable of eliciting an immune response to tumors or infectious disease pathogens, such as viruses, bacteria, parasites, or fungi.The one or more antigens are derived from tumors, such as various tumor antigens disclosed herein above.Alternatively, the one or more antigens can be derived from pathogens, such as viruses, bacteria, parasites, and / or fungi.

[0176] Preferred antigens to be administered in combination with the antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein include tumor antigens and vaccine antigens (e.g., antigens of bacteria, viruses, or other pathogens against which it is desirable to generate protective immunity in a subject for vaccination purposes). Other examples of suitable pathogen antigens include tumor-associated antigens (TAA), including, but not limited to, EGFR, EGFRvIII, gp100 or Pmel17, HER2 / neu, mesothelin, CEA, MART1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MUC-1, GPNMB, HMW-MAA, TIM1, ROR1, CD19, and sequences that include all or part of the sequences of germline-derived tumor antigens.

[0177] Other suitable antigens include viral antigens for preventing or treating viral diseases. Examples of viral antigens include, but are not limited to, HIV-1 env antigen, HBsAg antigen, HPV antigen, FAS antigen, HSV-1 antigen, HSV-2 antigen, p17 antigen, ORF2 antigen, and ORF3 antigen.Furthermore, viral antigens or viral antigenic determinants can be derived, for example, from: cytomegalovirus (particularly human, e.g. gB or derivatives thereof); Epstein-Barr virus (e.g. gp350); Flavivirus (e.g. yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus); Hepatitis viruses, e.g. hepatitis B virus (e.g. PreSl antigen, PreS2 antigen, and S antigen as described in EP-A-414374; EP-A-0304578, and EP-A-198474, etc. hepatitis B surface antigen from Gluck et al., Hepatitis A virus, Hepatitis C virus, and Hepatitis E virus; HIV-1 (e.g., tat, nef, gpl20, or gpl60); human herpes viruses, such as gD or derivatives thereof, or immediate early proteins (e.g., ICP27 from HSV1 or HSV2); human papilloma viruses (e.g., HPV6, HPV11, HPV16, HPV18); influenza viruses (eggs or MDCK cells, or Vero cells or whole influenza virosomes (Gluck, Vaccine, 1992, 10, 915-920), whole live or inactivated viruses, split influenza viruses, or purified or recombinant proteins thereof, such as the NP, NA, HA, or M proteins; measles viruses; mumps viruses; parainfluenza viruses; rabies viruses; respiratory syncytial viruses (e.g., F and G proteins); rotaviruses (including live attenuated viruses); smallpox viruses; varicella-zoster viruses (e.g., gpI, gpII, and IE63); and HPV viruses involved in cervical cancer (e.g., early proteins E6 or E7 fused to a protein D carrier to form a protein D-E6 fusion or a protein D-E7 fusion or a combination thereof from HPV16; or a combination of E6 or E7 with L2 (see, e.g., WO96 / 26277).

[0178] Examples of bacterial antigens include, but are not limited to, Toxoplasma gondii or Treponema pallidum. Bacterial antigens can be present in the treatment or prevention of various bacterial diseases, such as anthrax, botulism, tetanus, chlamydia infection, cholera, diphtheria, Lyme disease, syphilis, and tuberculosis. Bacterial antigens or antigenic determinants can be derived, for example, from Bacillus species, including B. anthracis (e.g., botulinum toxin); Bordetella species, including B. pertussis (e.g., pertactin, pertussis toxin, filamentous hemagglutinin, adenylate cyclase, fimbriae); Borrelia species, including B. burgdorferi (e.g., OspA, OspC, DbpA, DbpB), B. garinii (e.g., OspA, OspC, DbpA, DbpB), B. afzelii (e.g., OspA, OspC, DbpA, DbpB), B. andersonii (e.g., OspA, OspC, DbpA, DbpB), B. hermsii; Campylobacter species, including C. jejuni (e.g., toxins, adhesins, and invasins) and C. coli; Chlamydia species, including C. trachomatis (e.g., MOMP, heparin binding protein), C. pneumoniae (e.g., MOMP, heparin binding protein), C. psittaci (e.g., psittaci ... the Chlamydia species, including C. psittaci; the Clostridium species, including C. tetani (e.g., tetanus toxin), C. botulinum (e.g., botulinum toxin), C. difficile (C.difficile (e.g., Clostridium toxins A or B); Corynebacterium species, including C. diphtheriae (e.g., diphtheria toxin); Ehrlichia species, including E. equi and the etiological agent of human granulocytic ehrlichiosis; Rickettsia species, including R. rickettsii; Enterococcus species, including E. faecalis, E. faecium, E. faecium; Escherichia species, including enterotoxigenic E. coli (e.g., colonization factors, heat labile toxins or derivatives thereof, or heat stable toxins), enterohemorrhagic E. coli, enteropathogenic E. coli (e.g., Shiga-like toxins); Haemophilus species, including H. influenzae type B (e.g., PRP), non-capsulated H. influenzae, e.g., OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin-derived peptides (see, e.g., U.S. Pat. No. 5,843,464); Helicobacter species, including H. pylori (H. pylori (e.g., urease, catalase, vacuolating toxin); Pseudomonas species, including P. aeruginosa; Legionella species, including L. pneumophila; Leptospira species, including L. interrogans; Listeria species, including L. monocytogenes (e.g., L.the Listeria species, including M. monocytogenes; the Moraxella species, including M. catarrhalis, also known as Branhamella catarrhalis (e.g., high and low molecular weight adhesins and invasins); Morexella catarrhalis, including its outer membrane vesicles and OMP106 (see, e.g., WO 97 / 41731); the Mycobacterium species, including M. tuberculosis (e.g., ESAT6, antigen 85A, antigen 85B, or antigen 85C), M. bovis, M. leprae, M. avium, M. paratuberculosis (M. Mycobacterium species, including M. paratuberculosis, M. smegmatis; Neisseria species, including N. gonorrhea and N. meningitidis (e.g., capsular polysaccharides and conjugates thereof, transferrin binding proteins, lactoferrin binding proteins, PilC, adhesins); Neisseria mengitidis B, including its outer membrane vesicles and NspA (see, e.g., WO 96 / 29412); Salmonella species, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis (S. the Salmonella spp., including S. enteritidis; the Shigella spp., including S. sonnei, S. dysenteriae, S. flexnerii; the Staphylococcus spp., including S. aureus, S. epidermidis, S.epidermidis; Streptococcus species, including S. pneumoniae (e.g., capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins), and the protein antigen pneumolysin (Biochem Biophys Acta, 1989,67,1007; Rubins et al., Microbial Pathogenesis, 25,337-342) and detoxified mutant derivatives thereof (see, e.g., WO 90 / 06951; WO 99 / 03884); Treponema species, including T. pallidum (e.g., outer membrane proteins), T. denticola, T. hyodysenteriae (T. hyodysenteriae; Vibrio species, including V. cholera (e.g., cholera toxin); and Yersinia species, including Y. enterocolitica (e.g., Yop protein), Y. pestis, Y. pseudotuberculosis.

[0179] Parasitic / fungal antigens or antigenic determinants can be derived, for example, from: Babesia species, including B. microti; Candida species, including C. albicans; Cryptococcus species, including C. neoformans; Entamoeba species, including E. histolytica; Giardia species, including G. lamblia; Leshmania species, including L. major; Plasmodium falciparum. faciparum) (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27 / 25, Pfs16, Pfs48 / 45, Pfs230, and their analogs in Plasmodium species); Pneumocystis species, including P. carinii; Schisostoma species, including S. mansoni; Trichomonas species, including T. vaginalis; T. gondii gondii (e.g., SAG2, SAG3, Tg34); Trypanosoma species, including T. cruzi.

[0180] It will be appreciated that according to this aspect of the present invention, antigens and antigenic determinants can be used in many different forms.For example, antigens or antigenic determinants can be present as isolated proteins or peptides (e.g., in so-called "subunit vaccines"), or as cell-associated or virus-associated antigens or antigenic determinants (e.g., in either live or killed pathogen strains).Live pathogens are preferably attenuated in known manners.Alternatively, antigens or antigenic determinants can be generated in situ in subjects by using polynucleotides that code for antigens or antigenic determinants (as in so-called "DNA vaccination"), but it will be appreciated that the polynucleotides that can be used with this approach are not limited to DNA, but can also include RNA and the modified polynucleotides described above.

[0181] In one embodiment, vaccine antigens can also be targeted, for example, to specific cell types or specific tissues.For example, vaccine antigens can be targeted to antigen-presenting cells (APCs), for example, by using agents such as antibodies that target APC surface receptors, for example, DEC-205, as discussed in WO 2009 / 061996 (Celldex Therapeutics, Inc.), or mannose receptors (CD206), as discussed in WO 03040169 (Medarex, Inc.).

[0182] J kit Also provided are kits (e.g., diagnostic kits) that include one or more ILT4 antibodies or antigen-binding fragments thereof, bispecific constructs, multispecific constructs, or compositions described herein, optionally with instructions for use. The kits may also include informational brochures, such as brochures that inform how to use the reagents to practice the methods disclosed herein. The term "brochure" includes any written, marketing, or recorded material provided on or with the kit, or that otherwise accompanies the kit.

[0183] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0184] V. Working Example Example 1: Generation of mouse antibodies Mouse ILT4 monoclonal antibodies were generated by immunizing BALB / c mice with soluble human ILT4 antigen. The antigen used was a soluble fusion protein containing the ILT4 extracellular domain with a HIS tag (R&D Systems® or AcroBiosystems®). The antigen (i.e., 5-20 micrograms of soluble recombinant ILT4 antigen in PBS) was mixed with MPL+TDM adjuvant system (Sigma®) at a 1:1 ratio. Mice were injected into the peritoneal cavity with 200 microliters of prepared antigen approximately every 14 days. Animals that showed anti-ILT4 titers were given an intravenous injection of 1-10 micrograms of soluble recombinant ILT4 antigen 3-4 days prior to fusion. Mouse spleens were harvested and isolated splenocytes were used for hybridoma preparation.

[0185] P3x63Ag8.653 mouse myeloma cell line (ATCC CRL 1580) cultured in RPMI 1640 (Invitrogen®) containing 10% FBS was used for fusion. Additional media supplements were added to the hybridoma growth medium, including: HAT (Sigma; 1.0×10 4 M Hypoxanthine, 4.0 x 10 -7 M aminopterin, 1.6 × 10 -5 M-thymidine medium) with up to 10% Hybridoma Cloning Supplement (Sigma), 10% FBS (Sigma), L-glutamine (Gibco®), 0.1% gentamicin (Gibco), 2-mercaptoethanol (Gibco).

[0186] Spleen cells were mixed with P3x63Ag8.653 myeloma cells at a 6:1 ratio and pelleted by centrifugation. Polyethylene glycol was added dropwise with careful mixing to promote fusion. Hybridomas were cultured for 2-3 weeks until visible colonies were established. Supernatants were collected and used for initial screening of mouse IgG by ELISA using human soluble ILT4 fusion protein and mouse Fc-specific detector. IgG-positive supernatants were then assessed for ILT4 specificity by flow cytometry. Hybridomas were also screened for cross-reactivity with cynomolgus ILT4 and all were positive for binding.

[0187] Hybridoma cells were grown and cell pellets were frozen for RNA isolation and sequencing. H and V LThe coding regions of were identified using RNA from the corresponding hybridomas. The RNA was reverse transcribed into cDNA, the V coding regions were amplified by PCR, the PCR products were sequenced, inserted into a human IgG4 vector, transiently expressed as IgG4 chimeric antibodies, and purified by Protein A column chromatography to isolate two antibodies of particular interest. These variable region sequences were designated 7A3 (SEQ ID NO: 9 and SEQ ID NO: 10) and 7B1 (SEQ ID NO: 19 and SEQ ID NO: 20).

[0188] Example 2: Generation of humanized antibodies A computer model of the parent heavy and light chain variable domains (i.e., VH and VL domains) of antibodies 7A3 and 7B1 from Example 1 was created and used to guide the humanization process. The original mouse variable sequences of antibodies 7A3 and 7B1 were aligned against all human germline sequences. The original mouse sequences and the best matching germline sequences were analyzed for sequence liability, and the optimal germline framework was selected. The complementarity determining regions (CDRs) from the parent antibodies were grafted onto the appropriate number of human frameworks, and back mutations were introduced as necessary.

[0189] For antibody 7A3, four heavy chain humanized variants and four light chain humanized variants were designed. The 7A3 heavy chain variants were designated 7A3-H1 (SEQ ID NO: 97), 7A3-H2 (SEQ ID NO: 9), 7A3-H3 (SEQ ID NO: 98), and 7A3-H4 (SEQ ID NO: 99). The 7A3 light chain variants were designated 7A3-L1 (SEQ ID NO: 10), 7A3-L2 (SEQ ID NO: 100), 7A3-L3 (SEQ ID NO: 101), and 7A3-L4 (SEQ ID NO: 102).

[0190] Four heavy chain humanized variants and four light chain humanized variants were designed for antibody 7B1. The 7B1 heavy chain variants were designated 7B1-H1 (SEQ ID NO: 103), 7B1-H2 (SEQ ID NO: 19), 7B1-H3 (SEQ ID NO: 104), and 7B1-H4 (SEQ ID NO: 105). The 7B1 light chain variants were designated 7B1-L1 (SEQ ID NO: 20), 7B1-L2 (SEQ ID NO: 106), 7B1-L3 (SEQ ID NO: 107), and 7B1-L4 (SEQ ID NO: 108). The pairing of these variable domain sequences is shown in Table 4. Antibodies 7A3 VH6-L17 and 7B1 VH10-L21 were Protein A purified, their Fc domains mutated (AQQ), and selected for further investigation as described below.

[0191] Table 4. Heavy and light chain pairing TIFF2024515560000005.tif50150

[0192] Example 3: Measurement of affinity and kinetic constants of a humanized monoclonal antibody by Biolayer Interferometry (BLI) The binding affinity and binding kinetics of various humanized ILT4 antibodies were analyzed using Octet™ QK e The samples were examined by Biolayer Interferometry (BLI) using an instrument (ForteBio Sartorius®, Fremont, Calif.) following the manufacturer's guidelines.

[0193] Purified chimeric (7A3-huG4 and 7B1-huG4) and humanized antibodies (7A3 VH6-L17, 7A3 VH6-L18, 7A3 VH6-L20, 7B1 VH10-L21, 7B1 VH10-L22, and 7B1 VH10-L24) were captured on an anti-human Fc capture (AHC) biosensor (Fortebio product number 18-5060). Each antibody was prepared at 0.5 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and added to a freshly hydrated and preconditioned AHC biosensor at 30° C. and a plate shaking speed of 1000 rpm for 300 seconds. The same antibody was added to eight biosensors in one assay.

[0194] Binding was measured by exposing seven of the antibody-loaded biosensors to the analyte, soluble human ILT4-HIS (His-tagged ILT4 extracellular domain). Affinity measurements were taken using two-fold serial dilutions of analyte in dilution buffer ranging from 25 to 0.4 nM at 30°C and a plate shaking speed of 1000 rpm. Binding of the antibody-loaded biosensors in the analyte wells was carried out for 300 seconds, and then the biosensors were transferred to the dilution buffer wells for 1500 seconds for dissociation measurements.

[0195] A corresponding control was performed in each case by keeping the remaining biosensor with captured antibody in the dilution buffer well for the binding and dissociation steps. The data from the control biosensor were used to subtract the background and to account for biosensor drift and antibody dissociation from the biosensor.

[0196] Kinetic parameters were derived from the concentration series of analyte in dilution buffer that bound to the captured antibody using Fortebio's data analysis software version 10.0.3.1 (ForteBio Sartorius, Fremont, CA) in each case. Binding and dissociation curves were fitted to a 1:1 binding model using the data analysis software according to the manufacturer's guidelines.

[0197] The measured affinity and kinetic parameters (subtracted background) are shown in Figure 1A and Figure 1B, where k on = binding rate, k dis = dissociation rate, and K D = affinity constant (ratio k dis / k on A representative trace is shown in FIG. 2.

[0198] Example 4: Binding of chimeric and humanized monoclonal antibodies to human ILT4 using ELISA Microtiter plates were coated with recombinant human ILT4-κ in PBS and then blocked with 5% bovine serum albumin in PBS. Protein A purified chimeric monoclonal antibodies (7A3-huG4, 7B1-huG4), some of their humanized versions (7A3 VH6-L17, 7A3 VH6-L18, 7A3 VH6-L20, 7B1 VH10-L21, 7B1 VH10-L22, and 7B1 VH10-L24), and isotype controls were added at various concentrations and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG Fc specific polyclonal reagent conjugated with horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD450 using a microtiter plate reader. Representative binding curves are shown in Figures 3A and 3B.

[0199] Example 5: Binding of chimeric and humanized monoclonal antibodies to cells expressing human ILT4 Antibodies were tested for binding to human HEK293 cell line expressing human ILT4 on the cell surface. Protein A purified chimeric monoclonal antibodies (7A3-huG4, 7B1-huG4), various humanized versions (7A3 VH6-L17, 7A3 VH6-L18, 7A3 VH6-L20, 7B1 VH10-L21, 7B1 VH10-L22, and 7B1 VH10-L24), and isotype controls were incubated with HEK293 cells expressing human ILT4 at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and bound antibodies were detected by incubating the cells with a PE-labeled goat anti-human IgG Fc specific probe. Excess probe was washed from the cells with PBA, and cell-bound fluorescence was measured by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's instructions. Representative binding curves are shown in Figures 4A and 4B.

[0200] Example 6: Induction of TNF-α production in macrophages by chimeric and humanized monoclonal antibodies Macrophages were derived from human monocytes as follows: PBMCs were cultured at T175cm 2 The monocytes were added to the flask and allowed to adhere for approximately 2 hours at 37° C. and 6% CO. Non-adherent cells were removed and the monocytes were cultured for 7 days in RPMI containing 10% FBS and 50 ng / mL M-CSF (R&D Systems®).

[0201] These cells were then incubated at 37° C., 6% CO2 in the presence of protein A purified chimeric monoclonal antibodies (7A3-huG4, 7B1-huG4), their humanized versions (7A3 VH6-L17, 7A3 VH6-L18, 7A3 VH6-L20, 7B1 VH10-L21, 7B1 VH10-L22, and 7B1 VH10-L24) and isotype controls, as well as 50 ng / mL LPS (Invivogen). After 24 hours, the cells were harvested and the supernatants were collected and saved for cytokine analysis. Induction of TNF-α in the harvested supernatants was assessed by ELISA (R&D Systems). Figures 5A and 5B show the increase in TNF-α production by various ILT4 antibodies.

[0202] Example 7: Induction of TNF-α and MIP1-γ production in macrophages by humanized monoclonal antibodies Human PBMCs were differentiated with MCSF (100 ng / mL) for 7 days. After differentiation, human macrophages were cultured at 1.5 × 10 6 Cells were seeded at a concentration of 1000 x 1000 cells / well and allowed to adhere overnight. The next day, the medium was removed and the cells were treated with monoclonal antibodies (100 nM) with or without LPS (10 ng / mL) or IFNγ (10 ng / mL) for 24 h. After treatment, conditioned supernatants were removed and stored at -80°C until ready to perform ELISAs for human TNF-α and MIP1-α (R&D Systems) according to the manufacturer's protocol. Experiments were performed in triplicate. Results are shown in Figures 6A-6F.

[0203] Example 8: Gene expression analysis of humanized monoclonal antibodies in macrophages Human PBMCs were differentiated with MCSF (100 ng / mL) for 7 days. After differentiation, human macrophages were cultured at 1.5 × 10 6Cells were seeded at a concentration of 10000 cells / well and allowed to adhere overnight. The next day, the medium was removed and cells were treated with monoclonal antibodies (100 nM) in the presence of LPS (10 ng / mL) for 24 h. After treatment, cells were lysed with RLT buffer and RNA was extracted using RNEasy MiniKit Plus (Qiagen®) according to the manufacturer's protocol. cDNA synthesis was performed using Superscript IV VILO Mastermix according to the manufacturer's protocol with 1 μg of input total RNA. Gene expression was measured (HPRT, CD86, iNOS, CD54) by quantitative real-time PCR using SYBR Green Mastermix (Applied Biosystems®) and plates were run on a 7900 HT Fast Real-Time PCR system (ThermoFisher®). Relative gene expression was measured using the 2-ΔΔCt method and HPRT as housekeeping gene. Experiments were performed in duplicate. The results are shown in Figures 7A, 7B, and 7C.

[0204] Example 9: Cross-reactivity of humanized monoclonal antibodies: Binding to cells expressing ILT family members Protein A purified humanized monoclonal antibodies 7A3 VH6-L17 and 7B1 VH10-L21 and isotype controls were incubated with CHO cells expressing the human ILT family members with the highest homology to ILT4, such as LILRA1, LILRA2 (ILT1), LILRA4 (ILT7), LILRA5 (ILT11), LILRB1 (ILT2), and LILRB2, at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and bound antibodies were detected by incubating the cells with a PE-labeled goat anti-human IgG Fc-specific probe. Excess probe was washed off the cells with PBA and cell-bound fluorescence was measured by analysis using a FACSCanto II™ instrument (BD Biosciences®, NJ, USA) according to the manufacturer's instructions. Representative binding is shown in Figures 8A and 8B.

[0205] Example 10: Binding of humanized monoclonal antibodies to myeloid cells Macrophages were prepared as previously described in Example 6. Dendritic cells were prepared as follows: PBMCs were cultured at T175cm 2 The cells were added to the flask and allowed to adhere for approximately 2 hours at 37° C. and 6% CO. Nonadherent cells were removed and the monocytes were cultured for 7 days in RPMI containing 10% FBS, 100 ng / mL GM-CSF (R&D Systems), and 10 ng / mL IL-4 (R&D Systems).

[0206] Protein A purified monoclonal antibodies 7A3 VH6-L17 and 7B1 VH10-L21 and isotype controls were incubated with human monocytes, macrophages, and dendritic cells at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and bound antibodies were detected by incubating the cells with a PE-labeled goat anti-human IgG Fc-specific probe. Excess probe was washed off the cells with PBA and cell-bound fluorescence was measured by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's instructions. Representative binding is shown in Figure 9A, Figure 9B, and Figure 9C.

[0207] Example 11: Construction and production of bispecific antibodies A tetravalent bispecific antibody construct was developed using a mutated fully human IgG1 scaffold for the PD-L1 monoclonal antibody sequence (9H9; see SEQ ID NO: 87 and SEQ ID NO: 88 of WO2019204462 for heavy and light chain sequences) and an scFv of the ILT4 monoclonal antibody genetically linked to the C-terminus of the 9H9 heavy chain via a linker. Such a bispecific antibody was made using the scFv versions of both the 7A3 and 7B1 antibodies. The humanized antibody scFv sequences used in these bispecifics were obtained from 7A3 VH6-L17 and 7B1 VH10-L21, respectively. The Fc domain was mutated (AQQ) and several other amino acid residues were also modified. These constructs are designated 9H9-7A3 HL and 9H9-7B1 HL, respectively, in which the scFv is in a VH-VL orientation.

[0208] Alternative bispecific antibody constructs were generated in which the heavy and light chains of the anti-ILT4 scFv are in the opposite orientation. In this construct, the scFv is in the VL-VH orientation. These constructs are designated 9H9-7A3 LH and 9H9-7B1 LH, respectively.

[0209] The same 9H9 light chain was used in all four cases. The complete sequence of the tetravalent bispecific antibody includes:

[0210] Symbol Explanation : Bold double underline: 9H9 variable region Bold single underline: Constant domain (including embedded dotted underline) Italics: anti-ILT4 scFv Underlined italics: linker Dotted underline: modified amino acid residue TIFF2024515560000006.tif92150TIFF2024515560000007.tif229150

[0211] The bispecific constructs were expressed in a CHO cell line. Figure 10A shows a schematic of the bispecific constructs. Figure 10B shows an alternative bispecific construct using an anti-PD-1 antibody instead of an anti-PD-L1 antibody.

[0212] Example 12: Measurement of affinity and kinetic constants of humanized bispecific antibodies by Biolayer Interferometry (BLI) The binding affinity and binding kinetics of various human ILT4 bispecific antibodies were analyzed using Octet™ QK e The samples were examined by Biolayer Interferometry (BLI) using an instrument (ForteBio Sartorius, Fremont, CA) according to the manufacturer's guidelines.

[0213] Purified bispecific antibodies (9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH as described in Example 11) were captured on an anti-human Fc capture (AHC) biosensor (Fortebio product number 18-5060). Each antibody was prepared at 0.5 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and added to a freshly hydrated and preconditioned AHC biosensor at 30° C. and a plate shaking speed of 1000 rpm for 300 seconds. In one assay, the same antibody was added to eight biosensors.

[0214] Binding was measured by exposing six of the antibody-loaded biosensors to the analyte, soluble human ILT4-HIS (Celldex in-house reagent). Affinity measurements were taken using two-fold serial dilutions of analyte in dilution buffer ranging from 25 to 0.4 nM at 30° C. and a plate shaking speed of 1000 rpm. Binding of the antibody-loaded biosensors in the analyte wells was carried out for 300 seconds, and then the biosensors were transferred to the dilution buffer wells for 1500 seconds for dissociation measurements.

[0215] A corresponding control was performed in each case by keeping the remaining biosensor with captured antibody in the dilution buffer well for the binding and dissociation steps. The data from the control biosensor were used to subtract the background and to account for biosensor drift and antibody dissociation from the biosensor.

[0216] Kinetic parameters were derived from the concentration series of analyte in dilution buffer that bound to the captured antibody using Fortebio's data analysis software version 8.2.0.7 (ForteBio Sartorius, Fremont, CA) in each case. Association and dissociation curves were fitted to a 1:1 binding model using the data analysis software according to the manufacturer's guidelines.

[0217] The measured affinity and kinetic parameters (subtracted background) are shown in FIG. 11, where k on = binding rate, k dis = dissociation rate, and K D = affinity constant (ratio k dis / k on (defined by:

[0218] Example 13: Binding of humanized bispecific antibodies to human PD-L1 using ELISA Microtiter plates were coated with recombinant human PD-L1-msFc in PBS and then blocked with 5% bovine serum albumin in PBS. Protein A purified anti-PD-L1 monoclonal antibody 9H9, bispecific antibodies 9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH (described in Example 11), and isotype control were added at various concentrations and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG Fc specific polyclonal reagent conjugated with horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD450 using a microtiter plate reader. Representative binding curves are shown in Figure 12A and Figure 12B.

[0219] Example 14: Binding of humanized bispecific antibodies to cells expressing human PD-L1 Protein A purified monoclonal antibodies, bispecific antibodies (9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH as described in Example 11), and isotype controls were incubated with HEK293 cells expressing human PD-L1 at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and bound antibodies were detected by incubating the cells with a PE-labeled goat anti-human IgG Fc-specific probe. Excess probe was washed off the cells with PBA and cell-bound fluorescence was measured by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's instructions. Representative binding curves are shown in Figure 13A and Figure 13B.

[0220] Example 15: Binding of humanized bispecific antibodies to cells expressing human ILT4 Protein A purified monoclonal antibodies, bispecific monoclonal antibodies (9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH as described in Example 11), and isotype controls were incubated with HEK293 cells expressing human ILT4 at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and bound antibodies were detected by incubating the cells with a PE-labeled goat anti-human IgG Fc-specific probe. Excess probe was washed off the cells with PBA and cell-bound fluorescence was measured by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's instructions. Representative binding curves are shown in Figures 14A and 14B.

[0221] Example 16: Bifunctional binding of humanized bispecific antibodies to human ILT4 and human PD-L1 expressed in cells The binding of the bispecific constructs to ILT4 and PD-L1 was evaluated using HEK293 cells expressing human ILT4. Briefly, dilutions of the bispecific constructs were bound to ILT4-expressing cells, followed by the addition of human PD-L1-msFc, which was detected using a PE-labeled goat anti-mouse IgG Fc-specific probe. Representative binding curves of the four bispecific constructs (9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, and 9H9-7B1 LH, as described in Example 11) are shown in Figure 15A and Figure 15B. All four bispecific antibodies showed significant binding to both ILT4 and PD-L1.

[0222] Example 17: Humanized bispecific antibody T cell PD1 / PD-L1 blockade The effect of bispecific antibodies (9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH described in Example 11) on blocking PD1 / PD-L1 interaction was measured using a commercially available PD-1 / PD-L1 blocking assay from Promega®. Two engineered cell lines, namely PD1 effector cells and PD-L1 aAPC / CHO-K1 cells, were co-cultured in the presence of these antibodies for 6 hours. Disruption of PD1 / PD-L1 interaction leads to TCR activation and induces luminescence via the NFAT pathway. Luminescence was detected by addition of Bio-Glo reagent and quantified using a Perkin Elmer® Victor X4 luminometer. As shown in Figure 16A and Figure 16B, anti-PD-L1 antibodies and bispecific antibodies effectively blocked PD1 / PD-L1 interaction between cells, resulting in activation of the NFAT pathway.

[0223] Example 18: Induction of TNF-α production in macrophages by humanized bispecific antibodies Macrophages were derived from human monocytes as follows: PBMCs were cultured at T175cm 2The cells were added to the flask and allowed to adhere for approximately 2 hours at 37° C. and 6% CO. Non-adherent cells were removed and the monocytes were cultured for 7 days in RPMI containing 10% FBS and 50 ng / mL M-CSF (R&D Systems).

[0224] The cells were then incubated with 50 ng / mL LPS (Invivogen) at 37° C., 6% CO2 in the presence of bispecific antibodies (9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH as described in Example 11) and appropriate antibody controls. After 24 hours, the cells were harvested and the supernatants were collected and saved for cytokine analysis. Induction of TNF-α in the harvested supernatants was assessed by ELISA (R&D Systems). Figures 17A and 17B show the increase in TNF-α production by the bispecific antibodies.

[0225] Example 19: Inhibition of HLA-G binding to ILT4 by humanized bispecific antibodies Dilutions of bispecific antibodies (bispecific antibodies 9H9-7A3 HL, 9H9-7A3 LH, 9H9-7B1 HL, 9H9-7B1 LH described in Example 11) and antibody controls were incubated on HEK293 cells expressing human ILT4 at room temperature on a plate shaker. After 30 minutes, cells were washed and PE-labeled HLA-G tetramers (FredHutch) were added. After another 30 minutes, cells were washed with PBA and cell-associated fluorescence was measured by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's instructions. Representative interference curves are shown in Figures 18A and 18B.

[0226] (Table 5) Sequence Listing Summary Humanized sequences of 7A3 and 7B1 TIFF2024515560000008.tif94163TIFF2024515560000009.tif223163TIFF2024515560000010.tif141163 PD-L1 antibody sequence TIFF2024515560000011.tif75163TIFF2024515560000012.tif234163TIFF20245155600 00013.tif29163TIFF2024515560000014.tif185163TIFF2024515560000015.tif111163 ILT4 nucleotide sequence Explanation of symbols for SEQ ID NO: 90~93 Bold: variable regions Italics: constant domains Dotted underline: modified amino acid / base TIFF2024515560000016.tif88163TIFF2024515560000017.tif231163TIFF2024515560000018.tif66163 Other ILT4 humanized sequences TIFF2024515560000019.tif168163 Mouse Layout TIFF2024515560000020.tif236163 PD-L1 NA sequence TIFF2024515560000021.tif166163TIFF2024515560000022.tif226163TIFF20245155600 00023.tif212163TIFF2024515560000024.tif208163TIFF2024515560000025.tif102163 Bispecific antibody sequences Explanation of symbols for SEQ ID NO: 141~145 Bold double underline: 9H9 variable region Bold single underline: constant domain (including embedded dotted underline) Italics: anti-ILT4 scFv Underlined italics: linker Dotted underline: modified amino acid residue TIFF2024515560000026.tif115163TIFF2024515560000027.tif210163 Explanation of symbols for SEQ ID NO: 146~ Double underline: 9H9 variable region Single underline: constant domain (including embedded dotted underline) Italics: scFv Underlined italics: linker Dotted underline: modified base TIFF2024515560000028.tif210163TIFF2024515560000029.tif229163TIFF2024515560000030.tif226163TIFF2024515560000031.tif170163

[0227] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the scope of the claims appended hereto.

Claims

1. below: (i) a heavy chain variable region CDR1 amino acid sequence selected from the consensus sequence GYT(I,M)H (SEQ ID NO: 21) or a conservative sequence variant thereof; (ii) the heavy chain variable region CDR2 amino acid sequence shown in SEQ ID NO: 3, or a conservative sequence variant thereof; (iii) a heavy chain variable region CDR3 amino acid sequence selected from the consensus sequence ERPGGSQFIYYY(P,A)(M,L)DY (SEQ ID NO:22) or a conservative sequence variant thereof; (iv) a light chain variable region CDR1 amino acid sequence selected from the consensus sequence RAS(A,E)NIYSYLA (SEQ ID NO: 23) or a conservative sequence variant thereof; (v) a light chain variable region CDR2 amino acid sequence selected from the consensus sequence NA(I,D)TLAE (SEQ ID NO: 24) or a conservative sequence variant thereof; and (vi) the light chain variable region CDR3 amino acid sequence shown in SEQ ID NO: 8 or a conservative sequence variant thereof An isolated monoclonal antibody that binds to human ILT4, or an antigen-binding fragment thereof, comprising the amino acid sequences of heavy and light chain variable regions CDR1, CDR2, and CDR3 selected from the group consisting of:

2. below: (a) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, or conservative sequence variants thereof, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, or conservative sequence variants thereof; (b) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, or conservative sequence variants thereof, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or conservative sequence variants thereof; (c) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 9, or a sequence that is at least 80% identical thereto, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 10, or a sequence that is at least 80% identical thereto; (d) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 19, or a sequence that is at least 80% identical thereto, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 20, or a sequence that is at least 80% identical thereto; (e) a heavy chain amino acid sequence set forth in SEQ ID NO: 25, or a sequence at least 80% identical thereto, and a light chain amino acid sequence set forth in SEQ ID NO: 26, or a sequence at least 80% identical thereto; (f) a heavy chain amino acid sequence set forth in SEQ ID NO: 27, or a sequence at least 80% identical thereto, and a light chain amino acid sequence set forth in SEQ ID NO: 28, or a sequence at least 80% identical thereto; (g) a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or a sequence at least 80% identical thereto; or (h) a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, or a sequence at least 80% identical thereto. An isolated monoclonal antibody that binds to human ILT4, or an antigen-binding fragment thereof, comprising:

3. The following characteristics: a. Interfering with ILT4 ligands (e.g., HLA-G ligands) binding to human ILT4; b enhancing or increasing the release of cytokines or chemokines by human macrophages; c enhancing the activating effects of LPS and IFNγ on macrophages; d promoting M1 macrophage polarization; e 10 -9 M or less, or alternatively, 10 +9 M -1 binds to human ILT4 with an equilibrium binding constant Ka of or greater; f lack of cross-reactivity with other ILT family members; g Cross-reactivity with cynomolgus ILT4; and / or h Inhibiting ILT4-expressing tumor cells 2. The antibody or antigen-binding fragment thereof of claim 1, which exhibits one or more of the following:

4. The antigen-binding fragment is Fab, Fab', F(ab') 2 2. The antibody or antigen-binding fragment thereof of claim 1, which is an Fv, Fv, or single-chain Fv.

5. 13. A bispecific construct comprising the ILT4 antibody or antigen-binding fragment thereof of claim 1 linked to a second binding agent.

6. 6. The bispecific construct of claim 5, wherein the second binding agent binds to an immune checkpoint molecule, an immune co-stimulatory molecule, or a tumor antigen.

7. (a) the immune checkpoint molecule is PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, AXL, ILT2, or ILT3; (b) the immune costimulatory molecule is CD27, CD40, 4-1BB, OX40, or GITR; or (c) the tumor antigen is HER2, EGFR, ErB3, or CD24; 7. The bispecific construct of claim 6.

8. the binding agent binds to PD-L1 and (a) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64, respectively; (b) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; (c) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; (d) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; (e) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; and (f) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; 7. The bispecific construct of claim 6, comprising the amino acid sequences of heavy and light chain CDR1, CDR2, and CDR3 selected from the group consisting of:

9. The PD-L1 binding substance is as follows: (a) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 88; (b) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 79 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 80; (c) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 81 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 82; (d) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 83 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 84; (e) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 85 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 86; and (f) the heavy chain variable region amino acid sequence shown in SEQ ID NO: 77 and the light chain variable region amino acid sequence shown in SEQ ID NO: 78 9. The bispecific construct of claim 8, comprising heavy and light chain variable region sequences selected from the group consisting of: (a) the ILT4 antibody or antigen-binding fragment thereof is an scFv; or (b) the second antibody or antigen-binding fragment thereof is an scFv; 6. The bispecific construct of claim 5.

11. The bispecific construct of claim 5, wherein the ILT4 antibody or antigen-binding fragment thereof and the second binding agent comprise a human IgG1 constant domain.

12. (a) the ILT4 antibody or antigen-binding fragment thereof is linked to the C-terminus of the heavy chain of the second binding agent; or (b) the second binding agent is linked to the C-terminus of the heavy chain of the ILT4 antibody or antigen-binding fragment thereof; 6. The bispecific construct of claim 5.

13. The ILT4 antibody or antigen-binding fragment thereof and the second binding agent: (a) genetically fused; or (b) chemically conjugated; 6. The bispecific construct of claim 5.

14. 1. A bispecific construct comprising an antibody or antigen-binding fragment thereof that binds to human PD-L1 linked to an ILT4 scFv, comprising: (a) the PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequences of CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, respectively; and (b) the ILT4 scFv (i) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: NO:1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; or (ii) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; Including, The bispecific construct.

15. (a) the PD-L1 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence set forth in SEQ ID NO:87 and a light chain variable region amino acid sequence set forth in SEQ ID NO:88; and (b) ILT4 scFv, (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 10; or (ii) a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 19 and a light chain variable region amino acid sequence as set forth in SEQ ID NO: 20 Including, 15. The bispecific construct of claim 14.

16. The bispecific construct of claim 14, wherein the ILT4 scFv further comprises a disulfide stabilizing modification having Cys substitutions in VH44 and VL100.

17. 15. The bispecific construct of claim 14, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises a human IgG1 constant domain.

18. 1. A bispecific construct comprising an antibody or antigen-binding fragment thereof that binds human ILT4 linked to PD-L1 scFv, (a) the ILT4 antibody or antigen-binding fragment thereof, (i) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region as set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; or (ii) the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; Including, (b) the PD-L1 scFv comprises the amino acid sequences of CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, respectively; The bispecific construct.

19. (a) an ILT4 antibody or antigen-binding fragment thereof, (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 10; or (ii) a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 19 and a light chain variable region amino acid sequence as set forth in SEQ ID NO: 20 and (b) the PD-L1 scFv comprises a heavy chain variable region amino acid sequence set forth in SEQ ID NO:87 and a light chain variable region amino acid sequence set forth in SEQ ID NO:88; 19. The bispecific construct of claim 18.

20. 19. The bispecific construct of claim 18, wherein the ILT4 antibody or antigen-binding fragment thereof comprises a human IgG1 constant domain.

21. A multispecific construct comprising the bispecific construct of claim 5 and a third binding substance.

22. 6. The bispecific construct of claim 5, comprising: (i) a modified IgG1 domain having a mutated human IgG1 Fc domain comprising the unnatural amino acids 234A, 235Q, and 322Q, as numbered according to the EU index as set forth in Kabat.

23. 23. The bispecific construct of claim 22, wherein the modified human IgG1 Fc domain further comprises the unnatural amino acids 252Y, 254T, and 256E, as numbered according to the EU index as set forth in Kabat.

24. (a) the unnatural amino acids 234A, 235Q, and 322Q, as numbered according to the EU index as set forth in Kabat; and / or (b) the unnatural amino acids 252Y, 254T, and 256E, as numbered according to the EU index as set forth in Kabat A bispecific or multispecific construct comprising a modified human IgG1 Fc domain comprising:

25. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a bispecific or multispecific construct according to any one of claims 5 to 24, and a pharma- ceutically acceptable carrier.

26. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a bispecific or multispecific construct according to any one of claims 5 to 24, and instructions for use.

27. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region and / or the light chain variable region of the antibody or antigen-binding fragment thereof described in claim 1.

28. 25. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific or multispecific construct of claim 24.

29. 28. A vector comprising at least one nucleic acid molecule of claim 27.

30. 30. A host cell comprising the vector of claim 29.

31. 25. An in vitro method of activating macrophages comprising contacting said macrophages with the antibody or antigen-binding fragment thereof of any one of claims 1 to 4 or the bispecific or multispecific construct of any one of claims 5 to 24.

32. 25. A pharmaceutical composition for inducing or enhancing an immune response in a subject, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or a bispecific or multispecific construct according to any one of claims 5 to 24.

33. 25. A pharmaceutical composition for treating cancer in a subject, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a bispecific or multispecific construct according to any one of claims 5 to 24.

34. 34. The pharmaceutical composition of claim 33, wherein the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, renal cell carcinoma, head and neck squamous cell carcinoma, breast cancer, lung cancer, bladder cancer, prostate cancer, melanoma, gynecological cancer, sarcoma, lymphoma, and glioblastoma.

35. 25. A pharmaceutical composition for treating a tumor in a subject, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or a bispecific or multispecific construct according to any one of claims 5 to 24.

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