ILT3 and CD3 binding agents and methods of use thereof

Abstract

The present disclosure relates to ILT3xCD3 binding agents, compositions comprising same, and methods of use thereof. The present disclosure also relates to polynucleotides and vectors encoding such ILT3xCD3 binding agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 325,101, filed March 29, 2022, and U.S. Provisional Application No. 63 / 386,634, filed December 8, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is titled "13370-172-228_SEQLISTING.xml", was created on March 24, 2023, and is 189,173 bytes in size.

[0003] 1.Technical Field The present disclosure relates to immunoglobulin-like transcript 3 (ILT3) and CD3 binding agents, compositions comprising same, and methods of use thereof. The present disclosure also relates to polynucleotides and vectors encoding such ILT3xCD3 binding agents. [Background technology]

[0004] 2. Background technology The basis of immunotherapy is the manipulation and / or regulation of the immune system, including both innate and adaptive immune responses. The general goal of immunotherapy is to treat disease by controlling the immune response to "foreign substances", such as pathogens or tumor cells. However, in some instances, immunotherapy is used to treat autoimmune diseases that may result from an abnormal immune response to proteins, molecules, and / or tissues that are normally present in the body. Immunotherapy may include methods for inducing or enhancing a specific immune response or for inhibiting or reducing a specific immune response.

[0005] The concept of cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, initiate an immune response, and suppress the development and / or growth of tumors. However, it is clear that many cancer cells have evolved mechanisms and / or hijacked normal inhibitory mechanisms to evade the immune system, which can allow tumors to grow uninhibited. Cancer / tumor immunotherapy (immuno-oncology) focuses on the development of new novel agents that can activate and / or enhance the immune system to achieve a more effective attack on cancer / tumor cells, resulting in increased killing of cancer / tumor cells and / or inhibition of cancer / tumor growth. There is still a need in the art for more effective molecules to treat various diseases or disorders. Summary of the Invention

[0006] 3. Overview of the Invention In one aspect, the disclosure provides a binding agent comprising a first binding region that binds to human ILT3 and a second binding region that binds to human CD3, wherein the CD3 binding region comprises an anti-CD3 scFv.

[0007] In certain embodiments, the first binding region comprises an anti-ILT3 Fab. In certain embodiments, the binding affinity of the first binding region to human ILT3 is higher than the binding affinity of the second binding region to human CD3. In certain embodiments, the binding affinity of the first binding region to human ILT3 is about 10-fold to about 100-fold higher than the binding affinity of the second binding region to human CD3. In certain embodiments, the binding agent further comprises an Fc region.

[0008] In certain embodiments, the binding agent comprises (i) a first polypeptide comprising an anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a VH domain, a CH1 domain, a second CH2 domain, and a second CH3 domain of the first binding region; and (iii) a third polypeptide comprising a VL domain and a CL domain of the first binding region, wherein the VH domain, the CH1 domain, the VL domain, and the CL domain of the first binding region form an anti-ILT3 Fab, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form an Fc region.

[0009] In certain embodiments, a first polypeptide comprises one or more amino acid mutations that form an engineered cavity, a second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via placement of the protuberance in the cavity.

[0010] In some embodiments, the second binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149; and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150.

[0011] In some embodiments, in the second binding region, the VH domain of the second binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 152, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 154; and the VL domain of the second binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 155, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 156, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 157.

[0012] In some embodiments, the first binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18.

[0013] In certain embodiments, in the first binding region, (a) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; (c) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 3. (d) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (e) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 13; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0014] In certain embodiments, (i) the first binding region comprises a VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; the second binding region comprises a VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and a VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150; or (ii) the first binding region comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; the second binding region comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 149, and a VL domain comprising the amino acid sequence of SEQ ID NO: 150.

[0015] In another aspect, the disclosure provides a binding agent comprising a first binding region that binds to human ILT3 and a second binding region that binds to human CD3, wherein the second binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150.

[0016] In some embodiments, the VH domain of the second binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 152, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 154; and the VL domain of the second binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 155; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 156; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 157.

[0017] In some embodiments, the first binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18.

[0018] In certain embodiments, in the first binding region, (a) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; (c) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 3. (d) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (e) the VH domain of the first binding region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 13; the VL domain of the first binding region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0019] In certain embodiments, (i) the first binding region comprises a VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; the second binding region comprises a VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and a VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150; or (ii) the first binding region comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; the second binding region comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 149, and a VL domain comprising the amino acid sequence of SEQ ID NO: 150.

[0020] In certain embodiments, the first binding region comprises an anti-ILT3 Fab. In certain embodiments, the second binding region comprises an anti-CD3 scFv. In certain embodiments, the binding agent further comprises an Fc region.

[0021] In certain embodiments, the binding agent comprises (i) a first polypeptide comprising an anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a VH domain, a CH1 domain, a second CH2 domain, and a second CH3 domain of the first binding region; and (iii) a third polypeptide comprising a VL domain and a CL domain of the first binding region, wherein the VH domain, the CH1 domain, the VL domain, and the CL domain of the first binding region form an anti-ILT3 Fab, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form an Fc region.

[0022] In certain embodiments, a first polypeptide comprises one or more amino acid mutations that form an engineered cavity, a second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via placement of the protuberance in the cavity.

[0023] In certain embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO:147, the second polypeptide comprises the amino acid sequence of SEQ ID NO:19, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:20, or (ii) the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:147, the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:19, and the third polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0024] In certain embodiments, the first binding region comprises two identical anti-ILT3 Fabs and the second binding region comprises an anti-CD3 scFv.

[0025] In certain embodiments, the binding agent comprises: (i) a first polypeptide comprising an anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a first VH domain, a second VH domain, a first CH1 domain, a second CH1 domain, a second CH2 domain, and a second CH3 domain, wherein each of the first and second VH domains comprises the VH domain of a first binding region; (iii) a third polypeptide comprising a first VL domain and a first CL domain, wherein the first VL domain comprises the VL domain of the first binding region; and (iv) a second VL domain and a second CL domain. a fourth polypeptide comprising a first binding domain and a second VL domain comprising a VL domain of a first binding domain, wherein the first VH domain and the first CH1 domain of the second polypeptide and the first VL domain and the first CL domain of the third polypeptide form a first Fab region, the second VH domain and the second CH1 domain of the second polypeptide and the second VL domain and the second CL domain of the fourth polypeptide form a second Fab region, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form an Fc region.

[0026] In certain embodiments, a first polypeptide comprises one or more amino acid mutations that form an engineered cavity, a second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via placement of the protuberance in the cavity.

[0027] In certain embodiments, (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO:147, the second polypeptide comprises the amino acid sequence of SEQ ID NO:169, the third polypeptide comprises the amino acid sequence of SEQ ID NO:20, and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:20; or (ii) the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:147, the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:169, the third polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, and the fourth polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0028] In certain embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 151. In certain embodiments, the binding agent is a humanized antibody.

[0029] In another aspect, the disclosure provides a binding agent comprising: (i) a first polypeptide comprising an scFv that binds human CD3, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a VH domain, a CH1 domain, a second CH2 domain, and a second CH3 domain that binds human ILT3; and (iii) a third polypeptide comprising a VL domain that binds human ILT3, and a CL domain, wherein the scFv that binds human CD3 comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150; the VH domain that binds human ILT3 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and the VL domain that binds human ILT3 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18.

[0030] In some embodiments, (a) HCDR1 of the scFv comprises the amino acid sequence of SEQ ID NO: 152, HCDR2 of the scFv comprises the amino acid sequence of SEQ ID NO: 153, HCDR3 of the scFv comprises the amino acid sequence of SEQ ID NO: 154, LCDR1 of the scFv comprises the amino acid sequence of SEQ ID NO: 155, LCDR2 of the scFv comprises the amino acid sequence of SEQ ID NO: 156, and LCDR3 of the scFv comprises the amino acid sequence of SEQ ID NO: 157; (b) in the VH domain that binds human ILT3 and the VL domain that binds human ILT3 (i ) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (ii) HCDR1 comprises the amino acid sequence of SEQ ID NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8; HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; LCDR (iii) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises the amino acid sequence of SEQ ID NO: 9; HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (iv) HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; HCD R3 comprises the amino acid sequence of SEQ ID NO:3; LCDR1 comprises the amino acid sequence of SEQ ID NO:4; LCDR2 comprises the amino acid sequence of SEQ ID NO:5; LCDR3 comprises the amino acid sequence of SEQ ID NO:6; or (v) HCDR1 comprises the amino acid sequence of SEQ ID NO:11; HCDR2 comprises the amino acid sequence of SEQ ID NO:12; HCDR3 comprises the amino acid sequence of SEQ ID NO:13; LCDR1 comprises the amino acid sequence of SEQ ID NO:14; LCDR2 comprises the amino acid sequence of SEQ ID NO:15; and LCDR3 comprises the amino acid sequence of SEQ ID NO:16.

[0031] In certain embodiments, the VH domain of the scFv that binds human CD3 comprises the amino acid sequence of SEQ ID NO: 149, the VL domain of the scFv that binds human CD3 comprises the amino acid sequence of SEQ ID NO: 150; the VH domain that binds human ILT3 comprises the amino acid sequence of SEQ ID NO: 17, and the VL domain that binds human ILT3 comprises the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 151.

[0032] In another aspect, the disclosure provides an isolated polynucleotide encoding a binding agent disclosed herein.

[0033] In another aspect, the disclosure provides a vector comprising a polynucleotide disclosed herein.

[0034] In another aspect, the disclosure provides an isolated cell comprising a polynucleotide or vector disclosed herein.

[0035] In another aspect, the present disclosure provides an isolated cell that produces the binding agents disclosed herein.

[0036] In another aspect, the present disclosure provides a pharmaceutical composition comprising a binding agent disclosed herein, an isolated polynucleotide disclosed herein, a vector disclosed herein, or an isolated cell disclosed herein, and a pharma- ceutically acceptable excipient.

[0037] In another aspect, the disclosure provides a method of directing T cells to cancer or tumor cells expressing ILT3, comprising contacting the T cells with an effective amount of a binding agent disclosed herein or a pharmaceutical composition disclosed herein.

[0038] In some embodiments, the T cells induce killing of cancer or tumor cells that express ILT3, hi some embodiments, the cancer or tumor cells are hematological cancer or tumor cells. In certain embodiments, the hematological cancer or tumor cell is selected from the group consisting of acute myeloid leukemia (AML) cells, M4 / M5 AML cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells. In certain embodiments, the T cells are unable to induce killing of normal hematopoietic stem cells (HSCs).

[0039] In another aspect, the disclosure provides a method of activating a T cell, the method comprising contacting the T cell with an effective amount of a binding agent disclosed herein or a pharmaceutical composition disclosed herein, wherein the second binding region binds the T cell.

[0040] In certain embodiments, the T cells are naive T cells. In certain embodiments, the T cells are polyclonally expanded from a population of PBMCs.

[0041] In another aspect, the disclosure provides a method of killing or inhibiting the growth of a cancer or tumor cell that expresses ILT3, comprising contacting the cancer or tumor cell with a binding agent disclosed herein or a pharmaceutical composition disclosed herein.

[0042] In certain embodiments, the binding agent activates the T cells, hi certain embodiments, the activated T cells induce the killing of cancer or tumor cells.

[0043] In certain embodiments, the cancer or tumor cell comprises a hematological cancer or tumor cell. In certain embodiments, the hematological cancer or tumor cell is selected from the group consisting of acute myeloid leukemia (AML) cells, M4 / M5 AML cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells.

[0044] In another aspect, the disclosure provides a method of treating an ILT3-expressing cancer or tumor in a subject, comprising administering to the subject an effective amount of a binding agent disclosed herein or a pharmaceutical composition disclosed herein.

[0045] In certain embodiments, the cancer or tumor comprises a hematological cancer or tumor. In certain embodiments, the hematological cancer or tumor is selected from the group consisting of acute myeloid leukemia (AML), M4 / M5 AML chronic myelomonocytic leukemia (CMML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), Hodgkin's lymphoma, lymphoplasmacytic lymphoma (LPL), follicular lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), marginal zone lymphoma, or mucosa-associated lymphoid tissue (MALT) lymphoma. [Brief description of the drawings]

[0046] 4. Brief description of the drawings [Figure 1]1 shows an exemplary ILT3×CD3 bispecific antibody (ABX1446) disclosed herein. The left arm (second binding region of the antibody) represents an anti-CD3 scFv provided herein, and the right arm (first binding region of the antibody) represents an anti-ILT3 Fab provided herein.

[0047] [Diagram 2] Figure 1 shows the results of a T-cell dependent cytotoxicity (TDCC) assay with different ILT3xCD3 bispecific antibodies (hz45G10-2B2, 16C5-2B2 and 12A12-2B2). Anti-KLH was used as a negative control.

[0048] [Diagram 3] Figure 1 shows the results of a T-cell dependent cytotoxicity (TDCC) assay with different ILT3xCD3 bispecific antibodies (hz45G10-1G4, 16C5-1G4 and 12A12-1G4). ​​Anti-KLH was used as a negative control.

[0049] [Figure 4] Figure 1 shows the results of a TNFα cytokine production assay with different ILT3xCD3 bispecific antibodies (hz45G10-2B2, 3A3-2B2 and 12A12-2B2). Anti-KLH was used as a negative control.

[0050] [Diagram 5] 1 shows the results of a T-cell dependent cytotoxicity (TDCC) assay in AML cells with ILT3xCD3 bispecific antibody. Anti-KLH was used as a negative control.

[0051] [Figure 6] 1 shows the results of a TNFα cytokine production assay with ILT3×CD3 bispecific antibody. Anti-KLH was used as a negative control.

[0052] [Figure 7] 1 shows various formats of exemplary ILT3xCD3 bispecific antibodies provided herein.

[0053] [Figure 8] Figure 7 shows the T cell dependent cytotoxicity (TDCC) activity of various formats of ILT3xCD3 bispecific antibodies on MOLM13 cells when expanded T cells were used as effectors. Anti-KLH represents a negative control and bibecotamab (CD123xCD3 bispecific) represents a positive control.

[0054] [Figure 9] FIG. 7 shows TNFα cytokine release when whole blood samples were added to plates pre-coated with ILT3×CD3 bispecific antibody in various formats (FIG. 7) at the indicated concentrations (for each treatment group, bars represent 50 μg / ml, 10 μg / ml, 1 μg / ml, and 0.1 μg / ml, respectively, from left to right).

[0055] [Figure 10] FIG. 7 shows TNFα cytokine release when whole blood samples were added to media containing soluble ILT3×CD3 bispecific antibody in various formats (FIG. 7) at the indicated concentrations (for each treatment group, bars represent, from left to right, 100 μg / ml, 10 μg / ml, 1 μg / ml, and 0.1 μg / ml, respectively).

[0056] [Figure 11] We show that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) induced potent apoptosis of ILT3-positive (ILT3+) AML cells (MOLM13) when expanded T cells were used as effectors. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0057] [Figure 12]We show that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) induced potent apoptosis of ILT3-positive (ILT3+) AML cells (MOLM13) when naive T cells were used as effectors. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0058] [Figure 13] Figure 1 shows that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) induced apoptosis in OCI-AML-2 cells with low ILT3 expression. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0059] [Figure 14] Figure 1 shows that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) induced apoptosis of NALM-1 cells with low ILT3 expression. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0060] [Figure 15] We show that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) induced apoptosis of OCI-AML-2 cells with low ILT3 expression when naive T cells were used as effectors. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0061] [Figure 16] We show that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) induced apoptosis of NALM-1 cells with low ILT3 expression when naive T cells were used as effectors. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0062] [Figure 17] This shows that ILT3xCD3 bispecific antibodies (ABX1446 and ABX1520) were unable to induce apoptosis in ILT3 knockout THP-1 cells. Bibecotamab (CD123xCD3 bispecific) was used as a positive control. Anti-KLH was used as a negative control.

[0063] [Figure 18] Shown are the results of TNFα cytokine production in PBMCs incubated with increasing concentrations (for each treatment group, bars represent, from left to right, 100 μg / ml, 10 μg / ml, 1 μg / ml, and 0.1 μg / ml, respectively) of plate-coated format anti-KLH (negative control), bibecotamab (CD123×CD3 bispecific), and ILT3×CD3 bispecific antibodies (ABX1446 and ABX1520). Soluble Staphylococcal Enterotoxin B (SEB) was included as a positive control.

[0064] [Figure 19] 1 shows the results of TNFα cytokine production in PBMCs incubated with increasing concentrations (for each treatment group, bars represent, from left to right, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, respectively) of soluble anti-KLH (negative control), SEB (Staphylococcal enterotoxin B; positive control), bibecotamab (CD123×CD3 bispecific), and ILT3×CD3 bispecific antibodies provided herein (ABX1446 and ABX1520).

[0065] [Figure 20]Figure 7 shows the results of a TNFα cytokine secretion assay in whole blood incubated with increasing concentrations (for each treatment group, bars represent, from left to right, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, respectively) of plate-coated anti-KLH (negative control), bibecotamab (CD123xCD3 bispecific), and two different formats of ILT3xCD3 bispecific antibodies including F0 (ABX1446) and F13 (ABX1520) (see Figure 7). Soluble Staphylococcal Enterotoxin B (SEB) was included as a positive control.

[0066] [Figure 21] 7 shows the results of a TNFα cytokine secretion assay in whole blood incubated with increasing concentrations (for each treatment group, bars represent, from left to right, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, respectively) of soluble anti-KLH (negative control), SEB (Staphylococcal enterotoxin B; positive control), bibecotamab (CD123×CD3 bispecific), and two different formats of ILT3×CD3 bispecific antibody, including F0 (ABX1446) and F14 (ABX1521) (see FIG. 7 ).

[0067] [Figure 22] Figure 1 shows that ILT3xCD3 bispecific antibody (ABX1446) induced less TNFα cytokine secretion in TDCC assays with ILT3 positive (ILT3+) AML cells (MOLM13) when naive T cells were used as effectors. Anti-KLH represents a negative control (Negative Control) and bibecotamab (CD123xCD3 bispecific) represents a positive control.

[0068] [Diagram 23]Figure 1 shows that ILT3xCD3 bispecific antibody (ABX1446) induced less IL6 cytokine secretion in TDCC assays with ILT3 positive (ILT3+) AML cells (MOLM13) when naive T cells were used as effectors. Anti-KLH represents the negative control and bibecotamab (CD123xCD3 bispecific) represents the positive control.

[0069] [Figure 24] 1 shows the results of a T-cell dependent cytotoxicity (TDCC) assay using anti-KLH (negative control), bibecotamab (CD123xCD3 bispecific), and ILT3xCD3 bispecific antibodies ABX1446 and flotetuzumab (CD123xCD3 DART).

[0070] [Diagram 25] Shown are the results of a TNFα cytokine secretion assay in whole blood at increasing concentrations (for each treatment group, bars represent, from left to right, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, respectively) of soluble anti-KLH (negative control), SEB (Staphylococcal enterotoxin B; positive control), bibecotamab (CD123×CD3 bispecific), ILT3×CD3 bispecific antibody ABX1446, and flotetuzumab (CD123×CD3 DART).

[0071] [Figure 26] Shown is the proliferation of CD3 positive (CD3+) T cells (as a percentage of total PBMCs) in PBMCs isolated from M5 AML patients with increasing concentrations (for each treatment group, bars represent, from left to right, 0.1 μg / ml, 1 μg / ml, and 10 μg / ml, respectively) of anti-KLH (negative control), bibecotamab (CD123×CD3 bispecific), and ILT3×CD3 bispecific antibody ABX1446.

[0072] [Figure 27]Shown is the proliferation of CD25 positive (CD25+) T cells (as a percentage of total T cells) in PBMCs isolated from M5 AML patients with increasing concentrations (for each treatment group, bars represent, from left to right, 0.1 μg / ml, 1 μg / ml, and 10 μg / ml, respectively) of anti-KLH (negative control), bibecotamab (CD123×CD3 bispecific), and ILT3×CD3 bispecific antibody ABX1446.

[0073] [Figure 28] The ILT3xCD3 bispecific antibody ABX1446 failed to induce T cell-dependent cytotoxicity (TDCC) against CD34 positive (CD34+) hematopoietic stem cells (HSCs), whereas bibecotamab (CD123xCD3 bispecific) induced TDCC against CD34+HSCs. For each treatment group, the bars represent 0.0006 μg / ml, 0.005 μg / ml, 0.04 μg / ml, 0.3 μg / ml, and 2.5 μg / ml, respectively, from left to right.

[0074] [Figure 29] The ILT3xCD3 bispecific antibody ABX1446 failed to induce apoptosis in CD34 positive (CD34+) hematopoietic stem cells (HSCs), whereas bibecotamab (CD123xCD3 bispecific) induced apoptosis in CD34+HSCs. For each treatment group, the bars represent 0.001 μg / ml, 0.01 μg / ml, 0.1 μg / ml, and 1.0 μg / ml, respectively, from left to right.

[0075] [Diagram 30] 13 shows that the ILT3×CD3 bispecific antibody ABX1446 was unable to induce apoptosis in non-monocytic KU812 basophils.

[0076] [Diagram 31] 13 shows that the ILT3×CD3 bispecific antibody ABX1446 was unable to induce apoptosis in non-monocytic LAMA84 basophils.

[0077] [Diagram 32] Figure 7 shows the T cell dependent cytotoxicity (TDCC) activity of various formats of ILT3xCD3 bispecific antibodies on MM1S cells when expanded T cells were used as effectors. Anti-KLH represents a negative control and BiLincyto (CD3xCD19 BiTe) represents a positive control.

[0078] [Diagram 33] Figure 7 shows the T cell dependent cytotoxicity (TDCC) activity of various formats of ILT3xCD3 bispecific antibodies on H929 cells when expanded T cells were used as effectors. Anti-KLH represents a negative control and BiLincyto (CD3xCD19 BiTe) represents a positive control.

[0079] [Diagram 34] Figure 7 shows the T-cell dependent cytotoxicity (TDCC) activity of various formats of ILT3xCD3 bispecific antibodies on U226B1 cells when expanded T cells were used as effectors. Anti-KLH represents a negative control and BiLincyto (CD3xCD19 BiTe) represents a positive control.

[0080] [Diagram 35] 1 shows the treatment scheme and tumor burden quantification in a mouse AML model. Mice were irradiated 48 hours prior to MOLM13 or MV4;11 cell injection. On day 2, expanded T cells were injected into mice receiving MOLM13 cells or whole PBMCs were injected into mice receiving MV4;11 cells. ABX1446 was then injected on days 7, 14, 21, and 28. Tumor burden in peripheral blood was then quantified using FACS and the equation # of cells retrieved / # of beads retrieved x 1000.

[0081] [Diagram 36]Figure 1 shows that ABX1446 and ABX1520 reduced circulating tumor burden in the MOLM13 murine AML model. NTB represents non-tumor-bearing mice, anti-KLH represents a negative control, hz45G10 represents an ILT3 antibody containing an N297G substitution in the Fc region, IO-202 represents a monoclonal antibody that antagonizes ILT3, ABX1559 corresponds to an antibody lacking the ILT3 Fab binding region and thus represents an anti-CD3 only control, and bibecotamab (CD123xCD3 bispecific) represents a positive control.

[0082] [Figure 37] Figure 1 shows that ABX1446 reduced circulating tumor burden in the MV4;11 murine AML model at week 2. NTB represents non-tumor bearing mice, anti-KLH represents the negative control, and bibecotamab (CD123xCD3 bispecific) represents the positive control (increasing concentrations of 0.01mpk, 0.1mpk and 1mpk from left to right).

[0083] [Figure 38] 1 shows that ABX1446 reduced circulating tumor burden in the MV4;11 murine AML model at week 3. NTB represents non-tumor bearing mice, anti-KLH represents the negative control, and bibecotamab (CD123xCD3 bispecific) represents the positive control (increasing concentrations of 0.01mpk, 0.1mpk and 1mpk from left to right).

[0084] [Figure 39] FIG. 1 shows the treatment scheme and tumor burden quantification in a mouse AML model. CD34+ hematopoietic stem cells (HSCs) were transplanted into mice 3 months prior to MV4;11 cell injection. ABX1446 was injected on days 7, 14, 21, and 28. Tumor burden was then quantified using FACS and the equation: # of cells retrieved / # of beads retrieved x 1000.

[0085] [Diagram 40]Figure 1 shows that ABX1446 reduced circulating tumor burden in CD34+ humanized mice. When anti-KLH (negative control), bibecotamab (CD123xCD3 bispecific; positive control) and ABX1446 were dosed at 1 mpk, the number of circulating MV4;11 cells per μL of blood was significantly reduced.

[0086] [Diagram 41] 1 shows that ABX1446 induced dose-dependent tumor cell depletion in primary M5 AML bone marrow cultures.

[0087] [Diagram 42] 1 shows that ABX1446 induced dose-dependent T cell activation in primary M5 AML bone marrow cultures.

[0088] [Diagram 43] We show that ABX1446 induced dose-dependent tumor cell depletion in primary MM bone marrow cultures.

[0089] [Diagram 44] 1 shows that ABX1446 induced dose-dependent T cell activation in primary MM bone marrow cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] 5. MODE FOR CARRYING OUT THEINVENTION The present disclosure is based, in part, on the novel binders and their surprising properties provided herein.

[0091] Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. Where appropriate, terms used in the singular also include the plural, and vice versa. In the event that any explanation of a term provided conflicts with any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0092] The term "binding agent," as used herein, refers to a molecule that binds a specific antigen or target (e.g., ILT3 and / or CD3). Binding agents may include proteins, peptides, nucleic acids, carbohydrates, lipids, or low molecular weight compounds. In some embodiments, binding agents include full-length antibodies. In some embodiments, binding agents are antigen-binding fragments of antibodies. In some embodiments, binding agents include alternative protein scaffolds or artificial scaffolds (e.g., non-immunoglobulin scaffolds). In some embodiments, binding agents are fusion proteins that include antigen-binding sites. In some embodiments, binding agents are bispecific molecules that include at least two antigen-binding sites.

[0093] The term "bind" or "binding" refers to interactions between molecules, including, for example, to form a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, e.g., an antigen, is the affinity of the antibody or functional fragment for that epitope. The binding rate (k) of a binding molecule (e.g., an antibody) to a monovalent antigen is determined by the binding rate (k) of the antibody or functional fragment. on ) to dissociation rate (k off ) ratio (k off / k on ) is the dissociation constant K D which correlates inversely with affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different complexes of antibody and antigen. on and k off The dissociation constant K for the antibodies provided herein depends on both Dcan be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody and the antigen at one site increases the possibility of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0094] With respect to the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen, e.g., a polypeptide. Binding molecules or antigen-binding domains that bind or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those skilled in the art. In some embodiments, a binding molecule or antigen-binding domain binds or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens, as determined using experimental techniques, e.g., enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise, and may exceed 10 times background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of a binding molecule or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its particular target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis. Binding molecules or antigen-binding domains that bind to an antigen include those that are capable of binding to an antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and / or diagnostic agent in targeting the antigen. In certain embodiments, a binding molecule or antigen-binding domain that binds to an antigen has a dissociation constant (K D In certain embodiments, the binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among antigens from different species.

[0095] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, immunoglobulin molecules that recognize and bind targets through at least one antigen-binding site, polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single-chain Fv (scFv) antibodies, and antibody fragments (so long as they exhibit the desired antigen-binding activity).

[0096] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For IgG, the four-chain unit is usually about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. The pairing of VH and VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5th ed. 1995). th Please refer to the following:

[0097] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. A conventional IgG usually contains two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0098] In some embodiments, the Fab is a single chain Fab (scFab), and the heavy and light chains of the Fab are connected by a polypeptide linker.

[0099] The term "variable region", "variable domain", "V region", or "V domain" refers to a portion of an antibody light or heavy chain, usually located at the amino terminus of the light or heavy chain, having a length of about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable region. Instead, V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of higher variability (e.g., hypervariability) called "hypervariable regions", each about 9-12 amino acids long. The variable regions of the heavy and light chains each contain four FRs that generally adopt a β-sheet configuration connected by three hypervariable regions that form loop connections, and in some cases, form part of a β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies, hi certain embodiments, the variable regions are human variable regions.

[0100] The term "variable region residue numbering according to Kabat" or "Kabat amino acid position numbering" and variants thereof refer to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence at the region of homology with the "standard" Kabat numbering sequence. The Kabat numbering system is typically used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is typically used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0101] The term "heavy chain", when used in reference to an antibody, refers to a polypeptide chain of about 50-70 kDa that includes a variable region of about 120-130 or more amino acids at the amino-terminal portion and a constant region at the carboxy-terminal portion. The constant region can be one of five different types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, whereas μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to the five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0102] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa that contains a variable region of about 100 to about 110 or more amino acids at the amino-terminal portion and a constant region at the carboxy-terminal portion. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0103] As used herein, the terms "hypervariable region", "HVR", "complementarity determining region" and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the β-sheet framework of an immunoglobulin (Ig or antibody) VH, or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the β-sheet framework of an antibody VL. CDR1, CDR2 and CDR3 in a VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively. CDR1, CDR2 and CDR3 in a VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0104] CDR regions are well known to those skilled in the art and are defined by well known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (see, for example, Kabat et al. (supra); Nick Deschacht et al., J Immunol 2010;184:5696-5704). Chothia, instead, refers to the position of structural loops (see, for example, Chothia and Lesk, J.Mol.Biol.196:901-17(1987)). The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2d ed. 2010)). The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system specific to human and other vertebrate immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complexes (MHC). As used herein, CDRs are referred to in terms of both amino acid sequence and location within a light or heavy chain.Since the "locations" of the CDRs within the structure of immunoglobulin variable domains are conserved between species and reside in structures called loops, the CDR and framework residues are easily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used in the transfer and replacement of CDR residues from one immunoglobulin into an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-70 (2001). Concordance between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are illustrated in the table below. [Table 1]

[0105] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof, e.g., a variable region, and the individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or region thereof should be understood to encompass the complementarity determining regions defined by any of the known schemes described herein above. In some instances, a scheme for the identification of a particular CDR(s) is specified, such as CDRs defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR is given. It should be noted that a CDR region may also be defined by a combination of various numbering systems, e.g., a combination of the Kabat and Chothia numbering systems, or a combination of the Kabat and IMGT numbering systems. Thus, a term such as "CDR1 shown in a particular VH" includes, but is not limited to, any CDR1 defined by the exemplary CDR numbering systems described above. Given a variable region (eg, VH or VL), one of skill in the art will understand that the CDRs within the region may be defined by different numbering systems, or a combination thereof.

[0106] The hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0107] The term "constant region" or "constant domain" refers to the carboxy-terminal portion of the light and heavy chains that is not directly involved in binding the antibody to the antigen, but exhibits various effector functions, such as interaction with the Fc receptor. The term refers to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence than the other part of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0108] The term "framework" or "FR" refers to those variable region residues which flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

[0109] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include an antibody population in which the entire K447 residue has been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require an Fc region in combination with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may retain at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0110] The term "antibody fragment" as used herein refers to a molecule other than an intact antibody that contains a portion of an antibody, typically an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain antibody molecules, scFv, sc(Fv)2, disulfide-linked scFv (dsscFv), nanobodies, diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antigen-binding antibody fragments.

[0111] The term "monoclonal antibody" as used herein refers to a substantially homogeneous antibody population involved in highly specific recognition and binding of a single antigenic determinant or epitope. The term "monoclonal antibody" encompasses intact and full-length antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, scFv, fusion proteins including antigen-binding antibody fragments, and any other modified immunoglobulin molecule that contains at least one antigen-binding site. Furthermore, "monoclonal antibody" refers to such antibodies produced by any number of techniques, including, but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0112] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen or target capable of being recognized and bound by a particular antibody. When the antigen or target is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids (also called linear epitopes) are typically retained upon protein denaturation, while epitopes formed by tertiary folding (also called conformational epitopes) are typically lost upon protein denaturation. An epitope typically includes at least 3, more usually at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a number of publicly available bioinformatics software tools. X-ray crystallography can be used to characterize epitopes on target proteins by analyzing amino acid residue interactions of antigen / antibody complexes.

[0113] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a first source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0114] The term "humanized antibody," as used herein, refers to an antibody comprising a human heavy chain variable region and a light chain variable region, in which native CDR amino acid residues have been replaced by residues from the corresponding CDRs from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), and the non-human antibody has a desired specificity, affinity, and / or activity. In some embodiments, one or more framework region amino acid residues of a human heavy or light chain variable region have been replaced by the corresponding residue from the non-human antibody. Furthermore, a humanized antibody may contain amino acid residues that are not found in a human antibody or a non-human antibody. In some embodiments, these modifications are made to further refine and / or optimize the characteristics of the antibody. In some embodiments, a humanized antibody comprises at least a portion of a human immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc, and / or hinge region).

[0115] The term "human antibody," as used herein, refers to an antibody that possesses an amino acid sequence that corresponds to an antibody produced by a human and / or an antibody produced using any of the techniques known to those of skill in the art for producing human antibodies, including, but not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma techniques.

[0116] The term "specifically binds" as used herein refers to an agent that interacts more frequently, more rapidly, for a longer duration, with higher affinity, or some combination of the above, with a particular antigen, epitope, protein, or target molecule than an alternative substance. The terms "specifically bind" and "bind" are used interchangeably in some embodiments. A binding agent that specifically binds an antigen can be identified, for example, by immunoassay, ELISA, surface plasmon resonance (SPR), or other techniques known to those skilled in the art. In some embodiments, an agent that specifically binds an antigen (e.g., human ILT3 or CD3) can bind a related antigen (e.g., cynoILT3 or CD3). Typically, a binding agent that specifically binds an antigen will bind the target antigen with a higher affinity than its affinity for a different antigen. The different antigen can be a related antigen. In some embodiments, a binding agent that specifically binds an antigen can bind a target antigen with an affinity that is at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, or at least 100 times higher than its affinity for a different antigen. In some embodiments, a binding agent that specifically binds a particular antigen binds a different antigen with such low affinity that the binding cannot be detected using the assays described herein or otherwise known in the art. In some embodiments, the affinity is measured using SPR technology in the Biacore system described herein or as known to those skilled in the art.

[0117] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, it may be interrupted by non-amino acids. These terms also include amino acid polymers modified naturally or through intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes polypeptides containing one or more analogs of an amino acid, including, but not limited to, for example, unnatural amino acids, and other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies, it is understood that the term "polypeptide" includes the polypeptide as a single chain and the polypeptide of two or more associated chains.

[0118] The terms "polynucleotide" and "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0119] The term "identical" or "percent identical" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and the like. In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid identity when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 20-40, at least about 40-60, at least about 60-80 nucleotides or amino acids in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 nucleotides or amino acids, e.g., at least about 80-100 nucleotides or amino acids, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., (i) the coding region of a nucleotide sequence or (ii) the entire length of an amino acid sequence.

[0120] The term "vector" as used herein means a construct capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0121] The term "isolated" as used herein refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An "isolated" antibody is substantially free of material from the cellular source from which it was derived. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition has been purified to the extent that it is no longer in the form in which it is found in nature. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure. A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition may be isolated from a natural source (e.g., tissue) or from a source, e.g., an engineered cell line.

[0122] The term "substantially pure," as used herein, refers to a material that is at least 50% pure (i.e., free from contaminating substances), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0123] The term "subject" refers to any animal (eg, a mammal), including, but not limited to, humans, non-human primates, dogs, cats, rabbits, rodents, and the like.

[0124] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other substance well known in the art for use in pharmaceutical formulations.It is understood that the characteristics of the carrier, excipient, or diluent depend on the route of administration for a particular application.The term "pharmaceutical acceptable" as used herein refers to a substance that is approved or approvable by a regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, including humans.

[0125] The term "pharmaceutically acceptable excipient, carrier, or adjuvant" or "acceptable pharmaceutical carrier" as used herein refers to an excipient, carrier, or adjuvant that may be administered to a subject together with at least one therapeutic agent and that is generally safe, non-toxic, and has no effect on the pharmacological activity of the therapeutic agent. Typically, those skilled in the art and governmental agencies consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation or any pharmaceutical composition.

[0126] The term "pharmaceutical composition" or "pharmaceutical formulation" as used herein refers to a preparation that is in a form such that the biological activity of the binding agent is effective. A pharmaceutical formulation or composition typically includes additional components, such as pharma- ceutical acceptable excipients, carriers, adjuvants, buffers, and the like.

[0127] The term "effective amount" or "therapeutically effective amount," as used herein, refers to an amount of an agent sufficient to (i) reduce and / or ameliorate a disease, disorder, or condition in a subject, and / or (ii) the severity and / or duration of symptoms in a subject. The term also encompasses the amount of an agent necessary to (i) reduce or ameliorate the development or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, progression, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).

[0128] The terms "treat" or "treatment" or "treating" or "to treat" or "alleviate" or "alleviating" or "alleviating" or "to alleviate," as used herein, refer to therapeutic measures aimed at curing, delaying, lessening the symptoms of, and / or halting the progression of a pathological condition or disorder. Thus, those in need of treatment include those already with the disorder.

[0129] The term "immune response" as used herein includes responses from both the innate and adaptive immune systems. It includes both cell-mediated and / or humoral immune responses. It includes both T cell and B cell responses, as well as responses from other cells of the immune system, such as natural killer (NK) cells, monocytes, macrophages, dendritic cells, etc.

[0130] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments related to that value or parameter. For example, reference to "about X" includes the description of "X."

[0131] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise.

[0132] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Further, whenever an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0133] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone), B (alone), and C (alone).

[0134] 5.1 ILT3 binding region The binding agents provided herein include a region that binds ILT3 (eg, human ILT3), and thus the binding agents include ILT3 binding agents.

[0135] The amino acid (aa) sequences of human ILT3 (UniProtKB number Q8NHJ6) and cynomolgus monkey ("cyno") ILT3 (NCBI reference number XP_015297198) are known. ILT3 is a single-pass type I transmembrane protein with a predicted molecular weight of approximately 47 kDa. ILT3 has been observed to be predominantly expressed in bone marrow antigen-presenting cells, such as normal monocytes, macrophages, and dendritic cells. ILT3 is characterized by an extracellular domain that includes two Ig-like C2-type domains, a transmembrane domain, and a long cytoplasmic domain that contains three ITIM domains (see, e.g., Cella et al., 1997, J. Exp. Med., 185:1743-1751). The two Ig-like C2-type domains may be referred to herein as domain 1 (D1) and domain 2 (D2). D1 is located in the N-terminal part of the protein and D2 is located closest to the transmembrane region. As characterized in UniProtKB, human ILT3 is a 448 amino acid (aa) protein (the signal sequence is aa 1-21, the extracellular domain is aa 22-259, the transmembrane region is aa 260-280, and the cytoplasmic domain is aa 281-448). Within the extracellular domain, D1 is aa 27-188, D2 is aa 124-218, and the "stem region" is aa 219-259. Within the cytoplasmic domain, the ITIMs are aa 358-363, 410-415, and 440-445.

[0136] The present disclosure provides agents that bind ILT3 (e.g., bispecific antibodies). In some embodiments, the ILT3-binding agents bind human ILT3 or a fragment thereof.

[0137] In some embodiments, the ILT3 binding region in the binding agent is an antibody or a binding domain derived from an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody comprises an IgG heavy chain. In some embodiments, the antibody comprises an IgG1 heavy chain. In some embodiments, the antibody comprises an IgG2 heavy chain. In some embodiments, the antibody comprises an IgG4 heavy chain. In some embodiments, the antibody comprises a kappa light chain. In some embodiments, the antibody comprises a kappa light chain constant region. In some embodiments, the antibody comprises a lambda light chain. In some embodiments, the antibody comprises a lambda light chain constant region. In some embodiments, the antibody is an antibody fragment comprising an antigen binding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide linked scFv. In some embodiments, the antibody is a disulfide linked sc(Fv)2. In some embodiments, the antibody is a Fab, Fab', or F(ab)2 antibody. In some embodiments, the antibody is a single chain Fab (scFab). In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a nanobody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a multivalent antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is a tetravalent antibody.

[0138] In some embodiments, the ILT3 binding region is derived from a monoclonal antibody. The monoclonal antibody may be prepared by any method known to those skilled in the art. In some embodiments, the monoclonal antibody is prepared using hybridoma methods known to those skilled in the art. For example, using hybridoma methods, a mouse, rat, rabbit, hamster, or other suitable host animal is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a mouse protein or a fragment thereof. In some embodiments, the immunizing antigen is a cyno protein or a fragment thereof.

[0139] After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol. Hybridoma cells are selected using special media as known in the art, and unfused lymphocytes and myeloma cells are not retained in the selection process. Hybridomas producing monoclonal antibodies specifically directed against the selected antigen can be identified by a variety of methods, including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution techniques. In some embodiments, high-throughput methods are used to distribute single cell hybridoma cells to plates. Hybridomas can be grown either in vitro culture using standard methods or in vivo as ascites tumors in animals. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art, including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

[0140] In some embodiments, monoclonal antibodies are produced using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding the antibodies are isolated from mature B cells or hybridoma cells, e.g., by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody, and their sequences are determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector that produces the monoclonal antibody when transfected into a host cell, e.g., E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

[0141] In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of the desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

[0142] In some embodiments, monoclonal antibodies are modified by using recombinant DNA techniques to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of the monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region(s) is used to optimize the specificity and affinity of the monoclonal antibody.

[0143] In some embodiments, the ILT3 binding region is derived from a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, a humanized antibody contains one or more amino acid residues introduced into it from a source that is non-human. In some embodiments, humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody. In some embodiments, a humanized antibody is constructed by substituting all six CDRs of a non-human antibody (e.g., a mouse antibody) for the corresponding CDRs of a human antibody.

[0144] The selection of which human heavy and / or light chain variable regions to use to generate a humanized antibody can be made based on a variety of factors and by a variety of methods known in the art. In some embodiments, a "best-fit" method is used in which the sequence of a variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to that of the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody. In some embodiments, a particular variable region framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from the consensus sequence of the most abundant human subclass. In some embodiments, human germline genes are used as the source of variable region framework sequences.

[0145] Other methods for humanization include, but are not limited to, the method termed "superhumanization", described as the direct transfer of CDRs into human germline frameworks, the method termed human string content (HSC), which is based on a measure of "antibody humanness", methods based on the generation of large libraries of humanized variants (including phage, ribosomal, and yeast display libraries), and methods based on framework region shuffling.

[0146] In some embodiments, the ILT3 binding region is derived from a human antibody. Human antibodies can be prepared using a variety of techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In either case, cells producing antibodies directed against a target antigen can be generated and isolated. In some embodiments, human antibodies are selected from phage libraries, which express human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires from unimmunized human donors. Techniques for the generation and use of antibody phage libraries are well known in the art. Once an antibody is identified, affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis, can be used to generate higher affinity human antibodies. In some embodiments, human antibodies are generated in transgenic mice containing human immunoglobulin loci. Upon immunization, these mice are capable of generating a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[0147] In some embodiments, the ILT3 binding region is an antibody fragment. As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an antibody, typically an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), nanobodies, diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antibody fragments.

[0148] In some specific embodiments, the ILT3 binding region comprises an scFv that binds ILT3. In some specific embodiments, the ILT3 binding region comprises one or more Fabs that bind ILT3. In some specific embodiments, the ILT3 binding region comprises a Fab. In other specific embodiments, the ILT3 binding region comprises two Fabs. In other specific embodiments, the ILT3 binding region comprises two Fabs in tandem.

[0149] Antibody fragments can be generated by a variety of techniques, including, but not limited to, proteolytic digestion of an intact antibody. The antibody fragments described herein can be produced using recombinant techniques known in the art (e.g., E. coli or phage expression).

[0150] In some embodiments, the ILT3 binding regions provided herein have a binding activity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D) to ILT3 (e.g., human ILT3). In some embodiments, the ILT3 binding regions provided herein bind to ILT3 (e.g., human ILT3) with a dissociation constant of ≦0.1 nM. In some embodiments, the ILT3 binding regions provided herein bind to ILT3 (e.g., human ILT3) with a dissociation constant of ≦0.2 nM. In some embodiments, the ILT3 binding regions provided herein bind to ILT3 (e.g., human ILT3) with a dissociation constant of ≦0.3 nM. In some embodiments, the ILT3 binding regions provided herein bind to ILT3 (e.g., human ILT3) with a dissociation constant of ≦0.8 nM. In some embodiments, the ILT3 binding regions provided herein bind to ILT3 (e.g., human ILT3) with a dissociation constant of ≦3 nM. In some embodiments, the ILT3 binding regions provided herein bind to ILT3 (e.g., human ILT3) with a dissociation constant of ≦9 nM. A variety of methods are known in the art for measuring binding affinity, including, for example, by RIA performed with a Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by Octet®, for example, using the Octet® Red96 system, or by Biacore®, for example, using the Biacore® TM-2000 or Biacore® TM-3000, any of which may be used for the purposes of this disclosure. "On-rate" or "rate of binding" or "on-rate" or "kon" may also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, for example, using the Octet® Red96, Biacore® TM-3000, or Biacore® TM-8000 systems.

[0151] Any ILT3 binding agent (e.g., anti-ILT3 antibody) known in the art can be used to induce the ILT3 binding region disclosed herein. In certain embodiments, the ILT3 binding region disclosed herein is derived from any of the ILT3 antibodies disclosed in International Publication No. WO2021 / 183839, the contents of which are incorporated herein by reference. For example, the ILT3 binding region disclosed herein is derived from H7K3 or a variant thereof disclosed in WO2021 / 183839. In certain embodiments, the H7K3 variant comprises a VH variant selected from the group consisting of H7m1, H7m2, H7m3, and H7m4, and / or a VL variant selected from the group consisting of K3m1, K3m2, K3m3, K3m4, K3m5, K3m6, K3m7, and K3m8, as disclosed in WO2021 / 183839. The amino acid sequences of the CDRs, VL and VH of H7K3 and variants thereof are disclosed, for example, in Table 1 and paragraph

[0155] of WO2021 / 183839. In certain embodiments, the ILT3 binding region disclosed herein is derived from any anti-ILT3 antibody described in any of the following patent publications: US20190153093, WO2020056077, WO2021183839, US20200031926, US20210221887, US20150110714, US20200031926, US20190241655, WO2020180789, and WO2020056077, the contents of each of which are incorporated herein by reference.

[0152] In some embodiments, the ILT3 binding region provided herein is derived from an antibody in International Publication No. WO2021 / 127200, the contents of which are incorporated herein by reference. In some embodiments, the ILT3 binding region is any one of those in Tables 1-8.

[0153] In some embodiments, the ILT3 binding region provided herein comprises one or more CDR sequences of the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:145, and SEQ ID NO:146. The CDR sequences may be determined and defined according to any well-known numbering system. In some embodiments, the CDRs are determined and defined according to the IMGT numbering. In some embodiments, the CDRs are determined and defined according to the Kabat numbering. In some embodiments, the CDRs are determined and defined according to the AbM numbering. In other embodiments, the CDRs are determined and defined according to the Chothia numbering. In other embodiments, the CDRs are determined and defined according to the Contact numbering. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0154] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18.

[0155] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 37, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 38.

[0156] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 55, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 56.

[0157] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 73, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 74.

[0158] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 91, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 92.

[0159] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 110.

[0160] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 127. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 128. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 127, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 128.

[0161] In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, an ILT3 binding region provided herein comprises an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 146. In some embodiments, an ILT3 binding region provided herein comprises an HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 145, and an LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 146.

[0162] CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, CDRs are defined according to IMGT numbering. In some embodiments, CDRs are defined according to Kabat numbering. In some embodiments, CDRs are defined according to AbM numbering. In other embodiments, CDRs are defined according to Chothia numbering. In other embodiments, CDRs are defined according to Contact numbering.

[0163] In other embodiments, the ILT3 binding region comprises (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 7, 10, and 11; HCDR2 comprising an amino acid sequence having 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 or 13; (iii) an HCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 or 13. (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4 or 14; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% sequence identity to SEQ ID NO:5 or 15. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 or 16. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0164] In some specific embodiments, the ILT3 binding regions provided herein comprise one or more CDRs in Table 1.

[0165] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 7, 10, and 11; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 8, 9, and 12; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 13; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 14; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or 15; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 16.

[0166] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0167] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0168] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0169] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0170] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of SEQ ID NO: 12, HCDR3 comprises the amino acid sequence of SEQ ID NO: 13, LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0171] In other embodiments, the ILT3 binding region comprises (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 27, 30, and 31; , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23 or 33; (iii) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23 or 33. (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24 or 34; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 or 35; %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26 or 36. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0172] In some specific embodiments, the ILT3 binding regions provided herein comprise one or more CDRs in Table 2.

[0173] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 21, 27, 30, and 31; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 22, 28, 29, and 32; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23 or 33; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 34; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 25 or 35; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 26 or 36.

[0174] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 21, HCDR2 comprises the amino acid sequence of SEQ ID NO: 22, HCDR3 comprises the amino acid sequence of SEQ ID NO: 23, LCDR1 comprises the amino acid sequence of SEQ ID NO: 24, LCDR2 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0175] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 27, HCDR2 comprises the amino acid sequence of SEQ ID NO: 28, HCDR3 comprises the amino acid sequence of SEQ ID NO: 23, LCDR1 comprises the amino acid sequence of SEQ ID NO: 24, LCDR2 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0176] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 21, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, HCDR3 comprises the amino acid sequence of SEQ ID NO: 23, LCDR1 comprises the amino acid sequence of SEQ ID NO: 24, LCDR2 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0177] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 30, HCDR2 comprises the amino acid sequence of SEQ ID NO: 22, HCDR3 comprises the amino acid sequence of SEQ ID NO: 23, LCDR1 comprises the amino acid sequence of SEQ ID NO: 24, LCDR2 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0178] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises the amino acid sequence of SEQ ID NO: 32, HCDR3 comprises the amino acid sequence of SEQ ID NO: 33, LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 36.

[0179] In other embodiments, the ILT3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 39, 45, 48, and 49; , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41 or 51; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42 or 52; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43 or 53. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 44 or 54. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0180] In some particular embodiments, the ILT3 binding regions provided herein comprise one or more CDRs in Table 3.

[0181] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39, 45, 48 and 49; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 40, 46, 47 and 50; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 41 or 51; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 42 or 52; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 43 or 53; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 44 or 54.

[0182] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 39, HCDR2 comprises the amino acid sequence of SEQ ID NO: 40, HCDR3 comprises the amino acid sequence of SEQ ID NO: 41, LCDR1 comprises the amino acid sequence of SEQ ID NO: 42, LCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 44.

[0183] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, HCDR3 comprises the amino acid sequence of SEQ ID NO: 41, LCDR1 comprises the amino acid sequence of SEQ ID NO: 42, LCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 44.

[0184] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 39, HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, HCDR3 comprises the amino acid sequence of SEQ ID NO: 41, LCDR1 comprises the amino acid sequence of SEQ ID NO: 42, LCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 44.

[0185] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 48, HCDR2 comprises the amino acid sequence of SEQ ID NO: 40, HCDR3 comprises the amino acid sequence of SEQ ID NO: 41, LCDR1 comprises the amino acid sequence of SEQ ID NO: 42, LCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 44.

[0186] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 49, HCDR2 comprises the amino acid sequence of SEQ ID NO: 50, HCDR3 comprises the amino acid sequence of SEQ ID NO: 51, LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, LCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 54.

[0187] In other embodiments, the ILT3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 57, 63, 66, and 67; , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 59 or 69; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60 or 70; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 61 or 71; %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 62 or 72. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0188] In some particular embodiments, the ILT3 binding regions provided herein comprise one or more CDRs in Table 4.

[0189] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 57, 63, 66 and 67; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 58, 64, 65 and 68; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 59 or 69; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 60 or 70; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 61 or 71; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 62 or 72.

[0190] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 57, HCDR2 comprises the amino acid sequence of SEQ ID NO: 58, HCDR3 comprises the amino acid sequence of SEQ ID NO: 59, LCDR1 comprises the amino acid sequence of SEQ ID NO: 60, LCDR2 comprises the amino acid sequence of SEQ ID NO: 61, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 62.

[0191] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 63, HCDR2 comprises the amino acid sequence of SEQ ID NO: 64, HCDR3 comprises the amino acid sequence of SEQ ID NO: 59, LCDR1 comprises the amino acid sequence of SEQ ID NO: 60, LCDR2 comprises the amino acid sequence of SEQ ID NO: 61, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 62.

[0192] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 57, HCDR2 comprises the amino acid sequence of SEQ ID NO: 65, HCDR3 comprises the amino acid sequence of SEQ ID NO: 59, LCDR1 comprises the amino acid sequence of SEQ ID NO: 60, LCDR2 comprises the amino acid sequence of SEQ ID NO: 61, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 62.

[0193] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 66, HCDR2 comprises the amino acid sequence of SEQ ID NO: 58, HCDR3 comprises the amino acid sequence of SEQ ID NO: 59, LCDR1 comprises the amino acid sequence of SEQ ID NO: 60, LCDR2 comprises the amino acid sequence of SEQ ID NO: 61, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 62.

[0194] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 67, HCDR2 comprises the amino acid sequence of SEQ ID NO: 68, HCDR3 comprises the amino acid sequence of SEQ ID NO: 69, LCDR1 comprises the amino acid sequence of SEQ ID NO: 70, LCDR2 comprises the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 72.

[0195] In other embodiments, the ILT3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 75, 81, 84, and 85; , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 77 or 87; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 78 or 88; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 79 or 89. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 80 or 90. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0196] In some particular embodiments, the ILT3 binding regions provided herein comprise one or more CDRs in Table 5.

[0197] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 81, 84 and 85; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 76, 82, 83 and 86; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 77 or 87; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 78 or 88; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 79 or 89; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 80 or 90.

[0198] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 75, HCDR2 comprises the amino acid sequence of SEQ ID NO: 76, HCDR3 comprises the amino acid sequence of SEQ ID NO: 77, LCDR1 comprises the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 80.

[0199] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 81, HCDR2 comprises the amino acid sequence of SEQ ID NO: 82, HCDR3 comprises the amino acid sequence of SEQ ID NO: 77, LCDR1 comprises the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 80.

[0200] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 75, HCDR2 comprises the amino acid sequence of SEQ ID NO: 83, HCDR3 comprises the amino acid sequence of SEQ ID NO: 77, LCDR1 comprises the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 80.

[0201] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 84, HCDR2 comprises the amino acid sequence of SEQ ID NO: 76, HCDR3 comprises the amino acid sequence of SEQ ID NO: 77, LCDR1 comprises the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 80.

[0202] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 85, HCDR2 comprises the amino acid sequence of SEQ ID NO: 86, HCDR3 comprises the amino acid sequence of SEQ ID NO: 87, LCDR1 comprises the amino acid sequence of SEQ ID NO: 88, LCDR2 comprises the amino acid sequence of SEQ ID NO: 89, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 90.

[0203] In other embodiments, the ILT3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 93, 99, 102, and 103; (iii) an HCDR2 comprising an amino acid sequence having 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 95 or 105; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 96 or 106; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 97 or 107; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 98 or 108. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0204] In some particular embodiments, the ILT3 binding region provided herein comprises one or more CDRs in Table 6.

[0205] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 93, 99, 102 and 103; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 94, 100, 101 and 104; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 95 or 105; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 96 or 106; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 97 or 107; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 98 or 108.

[0206] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 93, HCDR2 comprises the amino acid sequence of SEQ ID NO: 94, HCDR3 comprises the amino acid sequence of SEQ ID NO: 95, LCDR1 comprises the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 98.

[0207] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 99, HCDR2 comprises the amino acid sequence of SEQ ID NO: 100, HCDR3 comprises the amino acid sequence of SEQ ID NO: 95, LCDR1 comprises the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 98.

[0208] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 93, HCDR2 comprises the amino acid sequence of SEQ ID NO: 101, HCDR3 comprises the amino acid sequence of SEQ ID NO: 95, LCDR1 comprises the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 98.

[0209] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 102, HCDR2 comprises the amino acid sequence of SEQ ID NO: 94, HCDR3 comprises the amino acid sequence of SEQ ID NO: 95, LCDR1 comprises the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 98.

[0210] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 103, HCDR2 comprises the amino acid sequence of SEQ ID NO: 104, HCDR3 comprises the amino acid sequence of SEQ ID NO: 105, LCDR1 comprises the amino acid sequence of SEQ ID NO: 106, LCDR2 comprises the amino acid sequence of SEQ ID NO: 107, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 108.

[0211] In other embodiments, the ILT3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 111, 117, 120, and 121; , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 113 or 123; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 114 or 124; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 116 or 126. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0212] In some particular embodiments, the ILT3 binding regions provided herein comprise one or more CDRs in Table 7.

[0213] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 111, 117, 120, and 121; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 112, 118, 119, and 122; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 113 or 123; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 114 or 124; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 115 or 125; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 116 or 126.

[0214] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 111, HCDR2 comprises the amino acid sequence of SEQ ID NO: 112, HCDR3 comprises the amino acid sequence of SEQ ID NO: 113, LCDR1 comprises the amino acid sequence of SEQ ID NO: 114, LCDR2 comprises the amino acid sequence of SEQ ID NO: 115, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 116.

[0215] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 117, HCDR2 comprises the amino acid sequence of SEQ ID NO: 118, HCDR3 comprises the amino acid sequence of SEQ ID NO: 113, LCDR1 comprises the amino acid sequence of SEQ ID NO: 114, LCDR2 comprises the amino acid sequence of SEQ ID NO: 115, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 116.

[0216] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 111, HCDR2 comprises the amino acid sequence of SEQ ID NO: 119, HCDR3 comprises the amino acid sequence of SEQ ID NO: 113, LCDR1 comprises the amino acid sequence of SEQ ID NO: 114, LCDR2 comprises the amino acid sequence of SEQ ID NO: 115, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 116.

[0217] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 120, HCDR2 comprises the amino acid sequence of SEQ ID NO: 112, HCDR3 comprises the amino acid sequence of SEQ ID NO: 113, LCDR1 comprises the amino acid sequence of SEQ ID NO: 114, LCDR2 comprises the amino acid sequence of SEQ ID NO: 115, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 116.

[0218] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 121, HCDR2 comprises the amino acid sequence of SEQ ID NO: 122, HCDR3 comprises the amino acid sequence of SEQ ID NO: 123, LCDR1 comprises the amino acid sequence of SEQ ID NO: 124, LCDR2 comprises the amino acid sequence of SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 126.

[0219] In other embodiments, the ILT3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 129, 135, 138, and 139; , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 131 or 141; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 132 or 142; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 134 or 144. In some embodiments, the ILT3 binding region is humanized. In some embodiments, the ILT3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0220] In some particular embodiments, the ILT3 binding region provided herein comprises one or more CDRs in Table 8.

[0221] In some embodiments, the ILT3 binding region provided herein comprises an HCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 129, 135, 138, and 139; (ii) an HCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 130, 136, 137, and 140; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 131 or 141; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 132 or 142; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 133 or 143; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 134 or 144.

[0222] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 129, HCDR2 comprises the amino acid sequence of SEQ ID NO: 130, HCDR3 comprises the amino acid sequence of SEQ ID NO: 131, LCDR1 comprises the amino acid sequence of SEQ ID NO: 132, LCDR2 comprises the amino acid sequence of SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0223] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 135, HCDR2 comprises the amino acid sequence of SEQ ID NO: 136, HCDR3 comprises the amino acid sequence of SEQ ID NO: 131, LCDR1 comprises the amino acid sequence of SEQ ID NO: 132, LCDR2 comprises the amino acid sequence of SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0224] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 129, HCDR2 comprises the amino acid sequence of SEQ ID NO: 137, HCDR3 comprises the amino acid sequence of SEQ ID NO: 131, LCDR1 comprises the amino acid sequence of SEQ ID NO: 132, LCDR2 comprises the amino acid sequence of SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0225] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 138, HCDR2 comprises the amino acid sequence of SEQ ID NO: 130, HCDR3 comprises the amino acid sequence of SEQ ID NO: 131, LCDR1 comprises the amino acid sequence of SEQ ID NO: 132, LCDR2 comprises the amino acid sequence of SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0226] In some specific embodiments, in the ILT3 binding region provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 139, HCDR2 comprises the amino acid sequence of SEQ ID NO: 140, HCDR3 comprises the amino acid sequence of SEQ ID NO: 141, LCDR1 comprises the amino acid sequence of SEQ ID NO: 142, LCDR2 comprises the amino acid sequence of SEQ ID NO: 143, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 144.

[0227] In some embodiments, the ILT3 binding region further comprises one or more framework regions of the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 145 and SEQ ID NO: 146. The framework regions described herein are determined based on the boundaries of the CDR numbering system, i.e., when the CDRs are determined by, for example, Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the amino acid residues of CDR1, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the amino acid residues C-terminal to the CDR3, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0228] In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37, and a VL domain comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 55, and a VL domain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 73, and a VL domain comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 91, and a VL domain comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 109, and a VL domain comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 127, and a VL domain comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, an ILT3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 145, and a VL domain comprising the amino acid sequence of SEQ ID NO: 146.

[0229] In certain embodiments, the ILT3 binding regions provided herein comprise an amino acid sequence having a predetermined percent identity compared to any of the ILT3 binding regions provided herein (e.g., those in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8).

[0230] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215: 403 (1990). BLAST nucleotide searches can be performed, for example, using the NBLAST nucleotide program parameters set for score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using XBLAST program parameters set, for example, to score=50 and word length=3 to obtain amino acid sequences homologous to the molecular proteins described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (supra). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., those of XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, typically only exact matches are counted.

[0231] In some embodiments, the ILT3 binding regions provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an ILT3 binding region comprising the sequence retains the ability to bind to ILT3. In some embodiments, a total of 1-10 amino acids may be substituted, inserted, and / or deleted in the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the ILT3 binding regions provided herein include post-translational modifications of the reference sequence.

[0232] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0233] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0234] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:55, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:56.

[0235] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74.

[0236] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:91, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:92.

[0237] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 109, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 110.

[0238] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 127, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 128.

[0239] In some embodiments, an ILT3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 145, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 146.

[0240] In some embodiments, the ILT3 binding regions provided herein bind to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the ILT3 binding regions provided herein bind to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the ILT3 binding regions provided herein bind to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 55 and a VL domain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the ILT3 binding regions provided herein bind to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 73 and a VL domain comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the ILT3 binding regions provided herein bind to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 91 and a VL domain comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, an ILT3 binding region provided herein binds to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 109, and a VL domain comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, an ILT3 binding region provided herein binds to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 127, and a VL domain comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, an ILT3 binding region provided herein binds to the same epitope as an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 145, and a VL domain comprising the amino acid sequence of SEQ ID NO: 146.

[0241] In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 55 and a VL domain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 73 and a VL domain comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 91 and a VL domain comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 109 and a VL domain comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, the ILT3 binding regions provided herein specifically bind to ILT3 (e.g., human ILT3) competitively with an ILT3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 145 and a VL domain comprising the amino acid sequence of SEQ ID NO: 146. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

[0242] 5.2 CD3 binding region The binding agents provided herein include a region that binds CD3 (eg, human CD3), and thus the binding agents include CD3 binding agents.

[0243] The amino acid (aa) sequences of human CD3 (UniProtKB number P07766) and cynomolgus monkey ("cyno") CD3 (e.g., isoforms X1 (NCBI reference number XP_015290838.2) and X2 (NCBI reference number XP_015290839.2) are known). CD3 is a single-pass type I transmembrane protein with a predicted molecular weight of approximately 23 kDa. CD3 has been observed to be expressed on T cells, among other tissues. CD3 is characterized by an extracellular domain containing paired Ig-fold domains, a transmembrane domain, and a long cytoplasmic domain containing one ITAM domain (see, e.g., Kuhns et al. (see, e.g., et al., 2006, Immunity, 24.2:133-139). As characterized in UniProtKB, human CD3 is a 207 amino acid (aa) protein (the signal sequence is aa 1-22, the extracellular domain is aa 23-126, the transmembrane region is aa 127-152, and the cytoplasmic domain is aa 153-207). Within the extracellular domain, the Ig-like domain is aa 32-112. Within the cytoplasmic domain, the ITAM is aa 178-205.

[0244] The present disclosure provides agents that bind CD3 (e.g., bispecific antibodies). In some embodiments, the CD3 binding agents bind human CD3 or a fragment thereof.

[0245] In some embodiments, the CD3 binding region in the binding agent is an antibody or a binding domain derived from an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody comprises an IgG heavy chain. In some embodiments, the antibody comprises an IgG1 heavy chain. In some embodiments, the antibody comprises an IgG2 heavy chain. In some embodiments, the antibody comprises an IgG4 heavy chain. In some embodiments, the antibody comprises a kappa light chain. In some embodiments, the antibody comprises a kappa light chain constant region. In some embodiments, the antibody comprises a lambda light chain. In some embodiments, the antibody comprises a lambda light chain constant region. In some embodiments, the antibody is an antibody fragment comprising an antigen binding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide linked scFv. In some embodiments, the antibody is a disulfide linked sc(Fv)2. In some embodiments, the antibody is a Fab, Fab', or F(ab)2 antibody. In some embodiments, the antibody is a single chain Fab (scFab). In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a nanobody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a multivalent antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is a tetravalent antibody.

[0246] In some embodiments, the CD3 binding region is derived from a monoclonal antibody. The monoclonal antibody may be prepared by any method known to those skilled in the art. In some embodiments, the monoclonal antibody is prepared using hybridoma techniques as described in the section above. In some embodiments, the monoclonal antibody is prepared using recombinant DNA techniques as described in the section above.

[0247] In some embodiments, the CD3 binding region is derived from a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, a humanized antibody contains one or more amino acid residues introduced into it from a source that is non-human. In some embodiments, humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody. In some embodiments, a humanized antibody is constructed by substituting all six CDRs of a non-human antibody (e.g., a mouse antibody) for the corresponding CDRs of a human antibody. Other methods for humanization include those described in the above section.

[0248] In some embodiments, the CD3 binding region is derived from a human antibody. Human antibodies can be prepared using a variety of techniques known in the art, including those described in the sections above.

[0249] In some embodiments, the CD3 binding region is an antibody fragment. For example, antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), nanobodies, diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antibody fragments. Antibody fragments can be produced by a variety of techniques, including but not limited to those described in the sections above.

[0250] In certain embodiments, the CD3 binding region comprises an anti-CD3 scFv. In certain embodiments, the CD3 binding region comprises one or more Fabs. In certain embodiments, the CD3 binding region comprises a Fab. In other certain embodiments, the CD3 binding region comprises two Fabs.

[0251] In some embodiments, the CD3 binding regions provided herein have an affinity for ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D ) to CD3 (e.g., human CD3). In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a dissociation constant of ≦0.1 nM. In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a dissociation constant of ≦0.01 nM. In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a dissociation constant of ≦2 nM. In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a dissociation constant of ≦3 nM. In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a dissociation constant of ≦4 nM. In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a dissociation constant of ≦300 nM.

[0252] In some embodiments, the CD3 binding regions provided herein have a K DIn some embodiments, the CD3 binding region provided herein binds to CD3 (e.g., human CD3) at about 1 nM to about 500 nM, about 1 nM to about 250 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 5 nM to about 1 μM, about 5 nM to about 500 nM, about 5 nM to about 25 ... Approximately 100nM, approximately 5nM to approximately 50nM, approximately 5nM to approximately 25nM, approximately 5nM to approximately 20nM, approximately 5nM to approximately 15nM, approximately 5nM to approximately 10nM, approximately 10nM to approximately 1μM, approximately 10nM to approximately 500nM , about 10nM to about 250nM, about 10nM to about 100nM, about 10nM to about 50nM, about 10nM to about 25nM, about 10nM to about 20nM, about 10nM to about 15nM, about 15nM to about 1μM, Approximately 15nM to approximately 500nM, approximately 15nM to approximately 250nM, approximately 15nM to approximately 100nM, approximately 15nM to approximately 50nM, approximately 15nM to approximately 25nM, approximately 15nM to approximately 20nM, approximately 20nM to approximately 1μM , about 20nM to about 500nM, about 20nM to about 250nM, about 20nM to about 100nM, about 20nM to about 50nM, about 20nM to about 25nM, about 25nM to about 1μM, about 25nM to about 500n M, about 25 nM to about 250 nM, about 25 nM to about 100 nM, about 25 nM to about 50 nM, about 50 nM to about 1 μM, about 50 nM to about 500 nM, about 50 nM to about 250 nM, about 50 nM to about 100 nM, about 100 nM to about 1 μM, about 100 nM to about 500 nM, about 100 nM to about 250 nM, about 250 nM to about 1 μM, about 250 nM to about 500 nM, or about 500 nM to about 1 μM D and binds to CD3 (e.g., human CD3).

[0253] In some embodiments, the CD3 binding regions provided herein have a K D In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a K D In some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a K of 6 nM as measured by SRP. DIn some embodiments, the CD3 binding regions provided herein bind to CD3 (e.g., human CD3) with a K of 6 nM to 13 nM, as measured by different methods of measuring binding affinity. D and binds to CD3 (e.g., human CD3).

[0254] A variety of methods are known in the art for measuring binding affinity, including, for example, by RIA performed with a Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by Octet®, for example, using the Octet® Red96 system, or by Biacore®, for example, using the Biacore® TM-2000 or Biacore® TM-3000, any one of which may be used for the purposes of this disclosure. "On-rate" or "rate of binding" or "on-rate" or "kon" may also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, for example, using the Octet® Red96, Biacore® TM-3000, or Biacore® TM-8000 systems.

[0255] Any anti-CD3 antibody known in the art, for example, the anti-CD3 antibodies disclosed in Kuhn & Weiner, Immunotheray, 2016 Jul;8(8):889-906, International Patent Publication Nos. WO2016204966, WO2017053856, WO2015095392, WO2016116626, WO2018114748, WO2005118635, and WO2014047231 (the contents of each of which are incorporated herein by reference), may be used to induce the CD3 binding regions disclosed herein.

[0256] In some embodiments, the CD3 binding region is derived from an antibody described in International Patent Publication No. WO2008 / 119567 and U.S. Patent No. 10,066,016, the contents of each of which are incorporated herein by reference. In some embodiments, the CD3 binding region is as described in the Examples section below (see Section 7). In some embodiments, the CD3 binding region is 2B2 or a derivative thereof. In some embodiments, the CD3 binding region is 1G4 or a derivative thereof.

[0257] In some embodiments, the CD3 binding region provided herein comprises one or more CDR sequences of the amino acid sequences set forth in SEQ ID NOs: 149, 150, 158 and 159. The CDR sequences may be determined according to well-known numbering systems. In some embodiments, the CDRs follow the IMGT numbering. In some embodiments, the CDRs follow the Kabat numbering. In some embodiments, the CDRs follow the AbM numbering. In other embodiments, the CDRs follow the Chothia numbering. In other embodiments, the CDRs follow the Contact numbering. In some embodiments, the CD3 binding region is humanized. In some embodiments, the CD3 binding region comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0258] In some embodiments, the CD3 binding regions provided herein comprise HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149. In some embodiments, the CD3 binding regions provided herein comprise LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150. In some embodiments, the CD3 binding regions provided herein comprise HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150. The CDR sequences may be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs follow the IMGT numbering. In some embodiments, the CDRs follow the Kabat numbering. In some embodiments, the CDRs follow the AbM numbering. In other embodiments, the CDRs follow the Chothia numbering. In other embodiments, the CDRs follow the Contact numbering.

[0259] In other embodiments, the CD3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 152; (ii) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 153; (iii) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 154; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:155; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:156; %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 157; and / or (vi) an LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 157. In some embodiments, the CD3 binding region is humanized. In some embodiments, the CD3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0260] In some specific embodiments, in the CD3 binding regions provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 152, HCDR2 comprises the amino acid sequence of SEQ ID NO: 153, HCDR3 comprises the amino acid sequence of SEQ ID NO: 154, LCDR1 comprises the amino acid sequence of SEQ ID NO: 155, LCDR2 comprises the amino acid sequence of SEQ ID NO: 156, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 157.

[0261] In some embodiments, the CD3 binding region further comprises one or more framework regions of the amino acid sequences of SEQ ID NOs: 149 and 150. The framework regions described herein are determined based on the boundaries of the CDR numbering system, i.e., when the CDRs are determined by, for example, Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the amino acid residues of CDR1, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the amino acid residues C-terminal to the CDR3, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0262] In some embodiments, the CD3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 149 and a VL domain comprising the amino acid sequence of SEQ ID NO: 150.

[0263] In certain embodiments, the CD3 binding regions provided herein comprise an amino acid sequence having a predetermined percent identity relative to any of the CD3 binding regions provided herein. Determining the percent identity may be accomplished using the mathematical algorithm described in the section above.

[0264] In some embodiments, the CD3 binding regions provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but the CD3 binding regions comprising the sequence retain the ability to bind to CD3. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). Optionally, the CD3 binding regions provided herein include post-translational modifications of the reference sequence.

[0265] In some embodiments, the CD3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 149, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 150. In some embodiments, the CD3 binding regions provided herein comprise a VH domain having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 149, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 150. In some embodiments, the CD3 binding regions provided herein comprise a VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and a VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150. In some embodiments, the CD3 binding regions provided herein comprise a VH domain having 95%-96% sequence identity to the amino acid sequence of SEQ ID NO: 149, and a VL domain having 90%-91% sequence identity to the amino acid sequence of SEQ ID NO: 150.

[0266] In some embodiments, the CD3 binding regions provided herein bind to the same epitope as a CD3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 149 and a VL domain comprising the amino acid sequence of SEQ ID NO: 150.

[0267] In some embodiments, the CD3 binding regions provided herein specifically bind to CD3 (e.g., human CD3) in a competitive manner with a CD3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 149 and a VL domain comprising the amino acid sequence of SEQ ID NO: 150.

[0268] In some particular embodiments, the CD3 binding region is an scFv. In some embodiments, the scFv comprises one or more amino acid substitutions, e.g., those that stabilize the scFv. In a particular embodiment, the scFv stabilizing mutation is a G44C mutation. In another particular embodiment, the scFv stabilizing mutation is a G100C mutation. In yet another embodiment, the scFv comprises both a G44C and a G100C mutation. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 151.

[0269] In some embodiments, the anti-CD3 scFv comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence having 92%-93% sequence identity to the amino acid sequence of SEQ ID NO: 151.

[0270] In some embodiments, the CD3 binding regions provided herein comprise HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 158. In some embodiments, the CD3 binding regions provided herein comprise LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 159. In some embodiments, the CD3 binding regions provided herein comprise HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 158, and LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 159. The CDR sequences may be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs follow the IMGT numbering. In some embodiments, the CDRs follow the Kabat numbering. In some embodiments, the CDRs follow the AbM numbering. In other embodiments, the CDRs follow the Chothia numbering. In other embodiments, the CDRs follow the Contact numbering.

[0271] In other embodiments, the CD3 binding region comprises: (i) an HCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 161; (ii) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 162; (iii) an HCDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 163; (iv) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 164; (v) an LCDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 165; %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and / or (vi) LCDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 166. In some embodiments, the CD3 binding region is humanized. In some embodiments, the CD3 binding region comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0272] In some specific embodiments, in the CD3 binding regions provided herein, HCDR1 comprises the amino acid sequence of SEQ ID NO: 161, HCDR2 comprises the amino acid sequence of SEQ ID NO: 162, HCDR3 comprises the amino acid sequence of SEQ ID NO: 163, LCDR1 comprises the amino acid sequence of SEQ ID NO: 164, LCDR2 comprises the amino acid sequence of SEQ ID NO: 165, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 166.

[0273] In some embodiments, the CD3 binding region further comprises one or more framework regions of the amino acid sequences of SEQ ID NOs: 158 and 159. The framework regions described herein are determined based on the boundaries of the CDR numbering system, i.e., when the CDRs are determined by, for example, Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the amino acid residues of CDR1, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the amino acid residues C-terminal to the CDR3, e.g., as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0274] In some embodiments, the CD3 binding region provided herein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 158 and a VL domain comprising the amino acid sequence of SEQ ID NO: 159.

[0275] In some embodiments, the CD3 binding region provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 158, and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the CD3 binding regions provided herein comprise a VH domain having at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 158, and a VL domain having at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the CD3 binding regions provided herein comprise a VH domain having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 158, and a VL domain having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the CD3 binding regions provided herein comprise a VH domain having 97%-98% sequence identity to the amino acid sequence of SEQ ID NO: 158, and a VL domain having 98%-99% sequence identity to the amino acid sequence of SEQ ID NO: 159.

[0276] In some embodiments, the CD3 binding regions provided herein bind to the same epitope as a CD3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 158 and a VL domain comprising the amino acid sequence of SEQ ID NO: 159.

[0277] In some embodiments, the CD3 binding regions provided herein specifically bind to CD3 (e.g., human CD3) in a competitive manner with a CD3 binding region comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 158 and a VL domain comprising the amino acid sequence of SEQ ID NO: 159.

[0278] In some specific embodiments, the CD3 binding region is an scFv. In some embodiments, the scFv comprises one or more amino acid substitutions, for example, those that stabilize the scFv. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 160.

[0279] 5.3 Polyspecific Binding Agents to ILT3 and CD3 The ILT3xCD3 binding agents provided herein comprise an ILT3 binding domain and a CD3 binding domain, each of which are described in more detail in Sections 5.1 and 5.2 above.

[0280] In certain embodiments, the ILT3 binding region has a binding affinity for human ILT3 that is greater than the binding affinity of the CD3 binding region for human CD3. In certain embodiments, the binding affinity of the ILT3 binding region for human ILT3 is about 10-fold to about 100-fold greater than the binding affinity of the CD3 binding region for human CD3. In certain embodiments, the binding affinity of the ILT3 binding region for human ILT3 is about 10-fold to about 100-fold, about 10-fold to about 90-fold, about 10-fold to about 80-fold, about 10-fold to about 70-fold, about 10-fold to about 60-fold, about 10-fold to about 50-fold, about 10-fold to about 40-fold, about 10-fold to about 30-fold, about 10-fold to about 20-fold, about 20-fold to about 100-fold, about 20-fold to about 90-fold, about 20-fold to about 80-fold, about 20-fold to about 70-fold, about 20-fold to about 60-fold, about 20-fold to about 50-fold, about 20-fold to about 40-fold, about 20-fold to about 30-fold, about 30-fold to about 100-fold, about 30-fold to about 90-fold, about 30-fold to about 80-fold, times, approximately 30 times to approximately 70 times, approximately 30 times to approximately 60 times, approximately 30 times to approximately 50 times, approximately 30 times to approximately 40 times, approximately 40 times to approximately 100 times, approximately 40 times to approximately 90 times, approximately 4 0x to 80x, 40x to 70x, 40x to 60x, 40x to 50x, 50x to 100x, 50x to 90x, 50x to 8 0 fold, about 50-fold to about 70-fold, about 50-fold to about 60-fold, about 60-fold to about 100-fold, about 60-fold to about 90-fold, about 60-fold to about 80-fold, about 60-fold to about 70-fold, about 70-fold to about 100-fold, about 70-fold to about 90-fold, about 70-fold to about 80-fold, about 80-fold to about 100-fold, about 80-fold to about 90-fold, or about 90-fold to about 100-fold higher. Such a difference in binding affinity between the two binding regions allows the ILT3×CD3 binding agent to bind ILT3-expressing target cells before engaging any T cells, thus reducing off-target effects and increasing the safety profile of the ILT3×CD3 binding agents disclosed herein.

[0281] In some embodiments, the ILT3xCD3 binding agent is a multispecific antibody, e.g., a bispecific antibody. Any technique known in the art for constructing a multispecific antibody or any multispecific format known in the art can be used in constructing the present multispecific antibodies provided herein. Non-limiting exemplary techniques and formats are described below.

[0282] The binding agents provided herein may include antibodies having a full-length antibody structure. A "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) consists of the well-known heavy chain variable and constant domains VH, CH1, hinge, CH2, and CH3. A full-length antibody light chain (LC) consists of the well-known light chain variable and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine (K) in either one or both heavy chains. A "Fab arm" or "half molecule" refers to one heavy-light chain pair that specifically binds an antigen.

[0283] Full-length bispecific antibodies can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example, by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimerization of two antibody half molecules with different specificities in a cell-free environment in vitro or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-binding of the CH3 domain. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an internal heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released by dissociation-binding and reforms. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules that bind different epitopes, ie, an epitope on ILT3 and an epitope on CD3, respectively.

[0284] "Homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. "Homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences. "Heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. "Heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0285] In some embodiments, binding agents provided herein include designs such as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-in-Hole (Genentech), CrossMAb (Roche) and Electrostatic Match (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (SEEDbody) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A / S).

[0286] In some embodiments, the ILT3xCD3 binding agents provided herein are in a knob-in-hole format: in some embodiments, the CD3 binding region (e.g., anti-CD3 scFv) side of the Fc region carries the hole, and the ILT3 binding region (e.g., anti-ILT3 Fab) side of the Fc region carries the knob.

[0287] In some embodiments, the ILT3xCD3 binding agents provided herein are in a DuoBody format.

[0288] Triomab quadroma technology can be used to generate full-length bispecific antibodies provided herein. Triomab technology promotes Fab arm exchange between two parent chimeric antibodies, one parent mAb with IgG2a and a second parent mAb with rat IgG2b constant regions, to generate chimeric bispecific antibodies.

[0289] The "knob-in-hole" strategy (see, e.g., International Publication No. WO2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of the antibody that specifically binds the first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of the antibody that specifically binds the second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form knobs and holes (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0290] CrossMAb technology can be used to generate full-length bispecific antibodies provided herein that, in addition to utilizing a "knobs-in-holes" strategy to facilitate Fab arm exchange, have the CH1 and CL domains in one of the half-arms swapped to ensure correct light chain pairing in the resulting bispecific antibody (see, e.g., U.S. Patent No. 8,242,247).

[0291] Other crossover strategies can be used to generate full-length bispecific antibodies provided herein by exchanging variable or constant domains, or both, between the heavy and light chains or within the heavy chains in the bispecific antibody, either in one or both arms. These exchanges include, for example, VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1, as described in International Publication Nos. WO2009 / 080254, WO2009 / 080251, WO2009 / 018386, and WO2009 / 080252.

[0292] Other strategies, such as promoting heavy chain heterodimerization using electrostatic interactions by replacing positively charged residues on one CH3 surface with negatively charged residues on the second CH3 surface, can be used as described in U.S. Patent Publication No. US2010 / 0015133; U.S. Patent Publication No. US2009 / 0182127; U.S. Patent Publication No. US2010 / 028637; or U.S. Patent Publication No. US2011 / 0123532, the contents of each of which are incorporated herein by reference. In another strategy, heterodimerization can be promoted by the following substitutions (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W (as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849, the contents of each of which are incorporated herein by reference).

[0293] LUZ-Y technology can be utilized to generate the bispecific antibodies provided herein, in which a leucine zipper is added to the C-terminus of the CH3 domain to induce heterodimer assembly from the parent mAb, which is removed after purification as described in Wranik et al., (2012) J Biol Chem 287(52):42221-9.

[0294] SEEDbody technology can be utilized to generate the bispecific antibodies provided herein. SEEDbodies have select IgG residues in their constant domains substituted with IgA residues to promote heterodimerization as described in U.S. Patent No. US20070287170, the contents of which are incorporated herein by reference.

[0295] In addition to the methods described above, the binding agents provided herein can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization according to the methods described in PCT Patent Publication No. WO2011 / 131746.

[0296] In some embodiments described herein, the ILT3xCD3 bispecific antibody comprises a first binding region that binds ILT3 and a second binding region that binds CD3, and further comprises at least one substitution in the antibody CH3 constant domain, which is typically made at the DNA level for molecules such as the constant domain of an antibody using standard methods.

[0297] The antibodies provided herein can be engineered into a variety of well-known antibody formats.

[0298] In some embodiments, the bispecific antibody is a diabody or a crossbody.

[0299] In some embodiments, bispecific antibodies include IgG-like molecules with complementary CH3 domains that promote heterodimerization; recombinant IgG-like dual targeting molecules, in which each of the two sides of the molecule contains at least two different antibody Fab fragments or portions of Fab fragments; IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; Fc fusion molecules, in which a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc region or portion thereof; Fab fusion molecules, in which different Fab fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies), in which different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0300] In some embodiments, recombinant IgG-like dual targeting molecules include dual targeting (DT)-Ig (GSK / Domantis), Two-in-One Antibody (Genentech), Cross-Linking Mab (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer).

[0301] In some embodiments, IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), IgG-like bispecific (ImClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).

[0302] In some embodiments, Fc fusion molecules can include ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART) (MacroGenics), and dual (ScFv)2-Fab (National Research Center for Antibody Medicine--China).

[0303] In some embodiments, Fab-fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecifics (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-based, diabody-based and domain antibodies include, but are not limited to, bispecific T-cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (Affimed), dual affinity retargeting technology (DART) (MacroGenics), single chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain single domain antibodies. Various formats of bispecific antibodies are described, for example, in Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276 and Nunez-Prado et al., (2015) Drug Discovery Today 20(5):588-594.

[0304] Any of the VH and VL domains specified herein (e.g., those that bind CD3) can be engineered into an scFv format. In some embodiments, the scFv format is in a VH-linker-VL orientation. In other embodiments, the scFv format is in a VL-linker-VH orientation. Any of the VH and VL domains specified herein can also be used to generate an sc(Fv)2 structure. In some embodiments, the sc(Fv)2 structure is VH-linker-VL-linker-VL-linker-VH. In some embodiments, the sc(Fv)2 structure is VH-linker-VL-linker-VH-linker-VL. In some embodiments, the sc(Fv)2 structure is VH-linker-VH-linker-VL-linker-VL. In some embodiments, the sc(Fv)2 structure is VL-linker-VH-linker-VH-linker-VL. In some embodiments, the sc(Fv)2 structure is VL-linker-VH-linker-VL-linker-VH. In some embodiments, the sc(Fv)2 structure is VL-linker-VL-linker-VH-linker-VH.

[0305] In certain embodiments, the linker is a peptide linker. In some embodiments, the linker comprises a naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. In some embodiments, the linker has a length appropriate to link the VH and VL or heavy and light chains of the Fab in a manner that allows them to form the correct conformation relative to each other so as to retain the desired activity, e.g., binding to a target (e.g., ILT3 or CD3).

[0306] In some embodiments, the linker is about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long.In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that may be used are Gly-rich linkers, Gly and Se-containing linkers, Gly and Ala-containing linkers, Ala and Ser-containing linkers, and other flexible linkers. Exemplary linkers include the sequence (G4S). n (wherein n=1 to 10, for example, 1 to 5 or 2 to 5, for example, 2, 3, 4, or 5).

[0307] Exemplary linkers that may be used may include, for example, any one of the linkers described in International Patent Application No. WO2019 / 060695, the contents of which are incorporated herein by reference. In certain embodiments, antibodies, including those provided herein, include two linkers. In other embodiments, antibodies provided herein include three linkers. In still other embodiments, antibodies provided herein include four or more linkers. In certain embodiments, the antibody is an antigen-binding fragment thereof.

[0308] In some specific embodiments, the ILT3×CD3 binding agents provided herein are configured in any one of the formats disclosed in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F0 as shown in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F1 as shown in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F5 as shown in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F13 as shown in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F7 as shown in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F2 as shown in FIG. 7. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F6 as shown in FIG. In some specific embodiments, the ILT3×CD3 binding agents provided herein have a format of F3 as shown in FIG. 7. In some specific embodiments, an ILT3xCD3 binding agent provided herein has a format of F14 as shown in Figure 7. In some specific embodiments, an ILT3xCD3 binding agent provided herein has a format of F4 as shown in Figure 7.

[0309] In some embodiments, an ILT3xCD3 binding agent provided herein comprises an scFv that binds CD3 and a Fab that binds ILT3, and the binding agent further comprises an Fc region. In some embodiments, having the CD3 binding region in an scFv format improves cytotoxicity and reduces cytokine release of the ILT3xCD3 binding agents provided herein.

[0310] In some embodiments, the Fc regions disclosed herein are modified to have reduced Fc-mediated effector function, e.g., via reduced Fc receptor binding. In some embodiments, the Fc region is modified at one or more of the following amino acid positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), and Ala327 (A327). In certain embodiments, the Fc region comprises one or more of the following amino acid substitutions: Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), and Ala327Ser (A327S). In some embodiments, the Fc region is altered at both amino acids 234 and 235. For example, Leu234Ala and Leu235Ala (L234A / L235A). References to amino acid substitutions in the Fc region are according to the EU numbering according to Kabat. The EU numbering follows the known and most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information System®) and the EU index reported in Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0311] In some embodiments, an ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising a scFv that binds CD3 linked to one arm of an Fc region, (ii) a second polypeptide comprising a VH domain that binds ILT3 linked to the other arm of the Fc region, and (iii) a third polypeptide comprising a VL domain that binds ILT3, wherein the VH domain and the VL domain form a Fab that binds ILT3, and the first and second polypeptides form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In some embodiments, the first polypeptide comprises one or more of the T366S, L368A and Y407V mutations (e.g., all of the T366S, L368A and Y407V mutations) in a domain forming the Fc region (e.g., the CH3 domain), and the second polypeptide comprises a T366W mutation in a domain forming the Fc region (e.g., the CH3 domain).

[0312] In some embodiments, the ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising a scFv that binds CD3 linked to one arm of an Fc region, (ii) a second polypeptide comprising a VH domain that binds ILT3 and a CH1 domain linked to the other arm of the Fc region, and (iii) a third polypeptide comprising a VL domain and a CL domain that bind ILT3, wherein the VH domain, the CH1 domain, the CL domain, and the VL domain form a Fab that binds ILT3, and the first and second polypeptides form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In some embodiments, the first polypeptide comprises one or more of the T366S, L368A and Y407V mutations (e.g., all of the T366S, L368A and Y407V mutations) in a domain forming the Fc region (e.g., the CH3 domain), and the second polypeptide comprises a T366W mutation in a domain forming the Fc region (e.g., the CH3 domain).

[0313] In some embodiments, the ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising an scFv, a CH2 domain, and a CH3 domain that binds CD3, (ii) a second polypeptide comprising a VH domain, a CH2 domain, and a CH3 domain that binds ILT3, and (iii) a third polypeptide comprising a VL domain that binds ILT3, wherein the VH domain and the VL domain form a Fab that binds ILT3, and the first and second polypeptides form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In some embodiments, the CH3 domain of the first polypeptide comprises one or more of a T366S, L368A and Y407V mutation (e.g., all of a T366S, L368A and Y407V mutation), and the CH3 domain of the second polypeptide comprises a T366W mutation.

[0314] In some embodiments, the ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising an scFv, a CH2 domain, and a CH3 domain that binds CD3, (ii) a second polypeptide comprising a VH domain, a CH1 domain, a CH2 domain, and a CH3 domain that binds ILT3, and (iii) a third polypeptide comprising a VL domain and a CL domain that binds ILT3, wherein the VH domain, the CH1 domain, the CL domain, and the VL domain form a Fab that binds ILT3, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In certain embodiments, the CH3 domain of the first polypeptide comprises one or more of the T366S, L368A and Y407V mutations (e.g., all of the T366S, L368A and Y407V mutations) and the CH3 domain of the second polypeptide comprises the T366W mutation. In certain embodiments, the ILT3xCD3 binder has the structure shown in F0 of Figure 7.

[0315] In some specific embodiments, an ILT3xCD3 binding agent provided herein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 20.

[0316] In other embodiments, an ILT3xCD3 binding agent provided herein comprises an scFv that binds CD3 and two Fabs that each bind ILT3, and the binding agent further comprises an Fc region. In some embodiments, the two Fabs are identical and linked to each other.

[0317] In some embodiments, an ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising an scFv that binds CD3 linked to one arm of an Fc region, (ii) a second polypeptide comprising two identical VH domains in tandem, each binding ILT3 linked to the other arm of the Fc region, (iii) a third polypeptide comprising a VL domain that binds ILT3, and (iv) a fourth polypeptide comprising a VL domain that binds ILT3, wherein the two VH domains and the two VL domains form two Fabs that bind ILT3, and the first and second polypeptides form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In some embodiments, the first polypeptide comprises one or more of the T366S, L368A and Y407V mutations (e.g., all of the T366S, L368A and Y407V mutations) in a domain that forms the Fc region (e.g., the CH3 domain), and the second polypeptide comprises a T366W mutation in a domain that forms the Fc region (e.g., the CH3 domain).

[0318] In some embodiments, the ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising an scFv that binds CD3 linked to one arm of an Fc region, (ii) a second polypeptide comprising two identical VH domains and two identical CH1 domains, each of which binds ILT3, one of the CH1 domains being linked to the other arm of the Fc region, (iii) a third polypeptide comprising a VL domain and a CL domain that binds ILT3, and (iv) a fourth polypeptide comprising a VL domain and a CL domain that binds ILT3, wherein the two VH domains, the two VL domains, the two CH1 domains, and the two CL domains form a Fab that binds ILT3, and the first and second polypeptides form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises a L234A / L235A mutation. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In certain embodiments, the first polypeptide comprises one or more of the T366S, L368A and Y407V mutations (e.g., all of the T366S, L368A and Y407V mutations) in a domain that forms the Fc region (e.g., the CH3 domain), and the second polypeptide comprises a T366W mutation in a domain that forms the Fc region (e.g., the CH3 domain).

[0319] In some embodiments, the ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising an scFv that binds CD3, a CH2 domain, and a CH3 domain, (ii) a second polypeptide comprising a first VH domain that binds ILT3, a second VH domain that binds ILT3, a CH2 domain, and a CH3 domain, (iii) a third polypeptide comprising a first VL domain that binds ILT3, and (iv) a fourth polypeptide comprising a second VH domain that binds ILT3, wherein the first VH domain and the first VL domain form a first Fab that binds ILT3, and the second VH domain and the second VL domain form a second Fab that binds ILT3, and the first polypeptide and the second polypeptide form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In some embodiments, the first polypeptide comprises one or more of T366S, L368A and Y407V mutations (e.g., all of T366S, L368A and Y407V mutations) in a domain that forms the Fc region (e.g., the CH3 domain), and the second polypeptide comprises a T366W mutation in a domain that forms the Fc region (e.g., the CH3 domain).

[0320] In some embodiments, an ILT3xCD3 binding agent provided herein comprises (i) a first polypeptide comprising an scFv that binds CD3, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a first VH domain that binds ILT3, a first CH1 domain, a second VH domain that binds ILT3, a second CH1 domain, a second CH2 domain, and a second CH3 domain; (iii) a third polypeptide comprising a first VL domain and a first CL domain that binds ILT3; and (iv) a fourth polypeptide comprising a second VL domain and a second CL domain that binds ILT3, wherein the first VH domain, the first CH1 domain, the first VL domain, and the first CL domain form a first Fab that binds ILT3, the second VH domain, the second CH1 domain, the second VL domain, and the second CL domain form a second Fab that binds ILT3, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form an Fc region. In some embodiments, the first polypeptide comprises a full or partial hinge domain. In some embodiments, the second polypeptide comprises a full or partial hinge domain. In some embodiments, the Fc region comprises one or more amino acid mutations that reduce or eliminate Fc effector function. In some embodiments, the Fc region comprises L234A / L235A mutations. In some embodiments, the Fc region comprises one or more amino acid mutations that facilitate dimerization of the two arms of the Fc region. In certain embodiments, the first polypeptide comprises one or more of the T366S, L368A and Y407V mutations (e.g., all of the T366S, L368A and Y407V mutations) in a domain that forms the Fc region (e.g., the CH3 domain), and the second polypeptide comprises a T366W mutation in a domain that forms the Fc region (e.g., the CH3 domain). In certain embodiments, the ILT3xCD3 binder has the structure shown in F13 of Figure 7.In some specific embodiments, an ILT3xCD3 binding agent provided herein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 169, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 20, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 20.

[0321] 5.4 Pharmaceutical Compositions In another general aspect, there is provided a pharmaceutical composition comprising an ILT3xCD3 binding agent provided herein and a pharma- ceutically acceptable excipient. In another general aspect, there is provided a pharmaceutical composition comprising a nucleic acid encoding an ILT3xCD3 binding agent provided herein or a fragment or portion thereof and a pharma- ceutically acceptable excipient. In another general aspect, there is provided a pharmaceutical composition comprising an engineered cell expressing an ILT3xCD3 binding agent provided herein and a pharma- ceutically acceptable excipient.

[0322] In some embodiments, the pharmaceutical compositions provided herein are prepared for storage by mixing a binder having the desired purity with any physiologically acceptable excipient (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)) in the form of an aqueous solution or lyophilized or other dried form.

[0323] In another general aspect, provided herein is a method of producing a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein, the method comprising combining the antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable carrier to obtain the pharmaceutical composition.

[0324] 5.5 How to use The functional activity of the binding agents provided herein can be characterized by methods known in the art and as described herein. Methods for characterizing binding agents include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and OctetRed analysis; binding assays for detecting binding of antibodies to target cells by FACS; binding assays for detecting binding of antibodies to target antigens on cells. According to certain embodiments, methods for characterizing binding agents include those described below.

[0325] The ILT3xCD3 binding agents of the present disclosure are useful in a variety of applications, including, but not limited to, therapeutic treatment methods, such as the treatment of cancers expressing human ILT3. In some embodiments, the therapeutic treatment methods include immunotherapy for cancers expressing ILT3 (e.g., human ILT3). In some embodiments, the ILT3xCD3 binding agents are useful for activating, promoting, increasing, and / or improving an immune response against a cancer or cancer cells expressing ILT3 (e.g., human ILT3). In some embodiments, the ILT3xCD3 binding agents are useful for activating, promoting, increasing, and / or improving an immune response against a tumor or tumor cells expressing ILT3 (e.g., human ILT3). In some embodiments, the ILT3xCD3 binding agents are useful for activating, promoting, increasing, and / or improving a T cell response against a cancer or cancer cells expressing ILT3 (e.g., human ILT3). In some embodiments, the ILT3xCD3 binding agents are useful for activating, promoting, increasing and / or enhancing T cell responses against tumors or tumor cells expressing ILT3 (e.g., human ILT3). Methods of use may be in vitro, ex vivo, or in vivo methods.

[0326] In one aspect, provided herein is a method of targeting T cells to cancer or tumor cells expressing ILT3 (e.g., human ILT3), comprising contacting the T cells with an effective amount of an ILT3xCD3 binding agent provided herein, wherein the CD3 binding region binds the T cells. In another aspect, provided herein is a method of targeting T cells to cancer or tumor cells expressing ILT3 (e.g., human ILT3), comprising contacting the T cells with a pharmaceutical composition comprising an effective amount of an ILT3xCD3 binding agent provided herein, wherein the CD3 binding region binds the T cells. In some embodiments, the targeted T cells induce apoptosis in the cancer or tumor cells. In some embodiments, when the T cells are targeted to cancer or tumor cells expressing ILT3 (e.g., human ILT3), the T cells induce distinct cytotoxicity and cytokine release. That is, the method of directing T cells to cancer or tumor cells expressing ILT3 (e.g., human ILT3) results in T cell dependent cytotoxicity (TDCC) that is inversely correlated with T cell cytokine release. For example, in some embodiments, TDCC is increased compared to a reference and cytokine release is decreased compared to a reference. In some embodiments, the TDCC reference is (a) TDCC measured in a corresponding normal cell or tissue; (b) TDCC measured in an adjacent non-cancer cell or tissue in the same subject; or (c) TDCC measured in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, the TDCC is determined by measuring apoptosis. In some embodiments, caspase-mediated apoptosis is increased. In some embodiments, the cytokine reference is (a) cytokines measured in a corresponding normal cell or tissue; (b) cytokines measured in an adjacent non-cancer cell or tissue in the same subject; or (c) cytokines measured in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, the cytokine release is determined by measuring TNFα. In some embodiments, TNFα cytokine release is decreased.

[0327] In some embodiments, the cancer or tumor cell comprises a hematological cancer or tumor cell. In some embodiments, the hematological cancer or tumor cell is an acute myeloid leukemia (AML) cell. In some embodiments, the AML is M4 / M5 AML. In some embodiments, the cancer or tumor cells are acute myeloid leukemia (AML) cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, or marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells. In some embodiments, the cancer or tumor cells include solid tumor cells.

[0328] In some embodiments, the cancer cells express a high level of ILT3 compared to a reference expression level. In some embodiments, the cancer cells express a low level of ILT3 compared to a reference expression level. In some embodiments, the reference expression level of ILT3 is (a) a predefined expression level of ILT3; (b) an ILT3 expression level in a corresponding normal cell or tissue; (c) an ILT3 expression level measured in adjacent non-cancerous cells or tissues in the same subject; or (d) an ILT3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, the expression level of ILT3 is determined by measuring the protein expression level of ILT3. In some embodiments, the cancer cells express a low level of ILT3 compared to a reference expression level, and the reference expression level is a known ILT3 expression level. 高ILT3 expression levels in cancer cells or tissues. Cancer cells expressing low levels of ILT3 include OCI-AML-2 or NALM-1 cells. Cancer cells expressing high levels of ILT3 include M4 and M5 AML, MM, and B-ALL, THP-1, and MOLM13 cells.

[0329] In some embodiments, the binding agents provided herein do not induce T cell-mediated killing of normal bone marrow hematopoietic stem cells (HSCs).

[0330] In one aspect, provided herein is a method of activating T cells, comprising contacting T cells with an effective amount of an ILT3xCD3 binding agent provided herein, wherein the CD3 binding domain binds the T cells. In another aspect, provided herein is a method of activating T cells, comprising contacting T cells with a pharmaceutical composition comprising an ILT3xCD3 binding agent provided herein. In some embodiments, the T cells are naive T cells. In some embodiments, the T cells are polyclonally expanded from a population of PBMCs.

[0331] In one aspect, provided herein is a method of targeting an antigen on the surface of a target cell expressing ILT3 (e.g., human ILT-3), the method comprising contacting the target cell with an effective amount of an ILT3×CD3 binding agent provided herein, wherein the ILT3 binding region binds to the target cell. In another aspect, provided herein is a method of targeting an antigen on the surface of a target cell, the method comprising contacting the target cell with a pharmaceutical composition comprising an effective amount of an ILT3×CD3 binding agent provided herein, wherein the ILT3 binding region binds to the target cell. In some embodiments, provided herein is a method of targeting an antigen on the surface of a target cell, the method comprising contacting the target cell with a pharmaceutical composition comprising an effective amount of an ILT3×CD3 binding agent provided herein. In some embodiments, the target cell expresses a high level of ILT3 compared to a reference expression level. In some embodiments, the target cell expresses a low level of ILT3 compared to a reference expression level. In some embodiments, said reference expression level of ILT3 is (a) a predefined expression level of ILT3; (b) an ILT3 expression level in a corresponding normal cell or tissue; (c) an ILT3 expression level measured in adjacent non-cancerous cells or tissues in the same subject; or (d) an ILT3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said expression level of ILT3 is determined by measuring the protein expression level of ILT3. In some embodiments, the target cells express a low level of ILT3 compared to the reference expression level, and the reference expression level is a known ILT3 expression level. 高ILT3 expression levels in cancer cells or tissues. Cancer cells expressing low levels of ILT3 include OCI-AML-2 or NALM-1 cells. Cancer cells expressing high levels of ILT3 include M4 and M5 AML, MM, and B-ALL, THP-1, and MOLM13 cells. In some embodiments, the target cells are from a cancer (e.g., a hematological cancer). In some embodiments, the target cells include cells from a B-cell malignancy or leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML), including M4 / M5 AML, chronic myelomonocytic leukemia (CMML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), Hodgkin's lymphoma, lymphoplasmacytic lymphoma (LPL), follicular lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or marginal zone lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma). In some embodiments, the cancer is a solid tumor.

[0332] In one aspect, provided herein is a method of killing or inhibiting the growth of a cancer or tumor cell expressing ILT3 (e.g., human ILT3), comprising contacting the cancer or tumor cell with an ILT3xCD3 binding agent provided herein. In another aspect, provided herein is a method of killing or inhibiting the growth of a cancer or tumor cell expressing ILT3 (e.g., human ILT3), comprising contacting the cancer or tumor cell with a pharmaceutical composition comprising an ILT3xCD3 binding agent provided herein. In some embodiments, the ILT3xCD3 binding agent activates T cells. In some embodiments, the CD3 binding region activates T cells. In some embodiments, the activated T cells induce apoptosis in the cancer or tumor cells. In some embodiments, the cancer or tumor cells comprise a hematological cancer or tumor cell. In some embodiments, the hematological cancer or tumor cell is an acute myeloid leukemia (AML) cell. In some embodiments, the AML is M4 / M5 AML. In some embodiments, the cancer or tumor cells are acute myeloid leukemia (AML) cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, or marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells. In some embodiments, the cancer or tumor cells include solid tumor cells. In some embodiments, the cancer or tumor cells express a high level of ILT3 compared to a reference expression level. In some embodiments, the cancer or tumor cells express a low level of ILT3 compared to a reference expression level.In some embodiments, the reference expression level of ILT3 is (a) a predefined expression level of ILT3; (b) an ILT3 expression level in a corresponding normal cell or tissue; (c) an ILT3 expression level measured in adjacent non-cancer cells or tissues in the same subject; or (d) an ILT3 expression level measured in a corresponding cell or tissue in a cohort of healthy subjects. In some embodiments, the expression level of ILT3 is determined by measuring the protein expression level of ILT3. In some embodiments, cancer cells express a low level of ILT3 compared to the reference expression level, and the reference expression level is a known ILT3. 高 ILT3 expression levels in cancer cells or tissues. Cancer cells expressing low levels of ILT3 include OCI-AML-2 or NALM-1 cells. Cancer cells expressing high levels of ILT3 include M4 and M5 AML, MM, and B-ALL, THP-1, and MOLM13 cells.

[0333] In one aspect, provided herein is a method of treating a cancer or tumor expressing ILT3 (e.g., human ILT3) in a subject, comprising administering an effective amount of an ILT3xCD3 binding agent provided herein. In another aspect, provided herein is a method of treating a cancer or tumor expressing ILT3 (e.g., human ILT3) in a subject, comprising administering an effective amount of a pharmaceutical composition comprising an ILT3xCD3 binding agent provided herein or a pharmaceutical composition provided herein. In some embodiments, the cancer or tumor is a blood cancer or tumor. In some embodiments, the cancer or tumor is a leukemia. In some embodiments, the blood cancer or tumor is acute myeloid leukemia (AML). In some embodiments, the cancer or tumor is a myelodysplastic syndrome. Myelodysplastic syndrome (MDS) is a group of cancers in which immature blood cells in the bone marrow do not mature and therefore do not become healthy blood cells. In some embodiments, the myelodysplastic syndrome progresses to AML. In some embodiments, the cancer or tumor is acute myeloid leukemia (AML), including M4 / M5 AML, chronic myelomonocytic leukemia (CMML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), Hodgkin's lymphoma, lymphoplasmacytic lymphoma (LPL), follicular lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or marginal zone lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma). In some embodiments, the cancer or tumor comprises a solid tumor. In some embodiments, the cancer or tumor cells express a high level of ILT3 compared to a reference expression level. In some embodiments, the cancer or tumor cells express a low level of ILT3 compared to a reference expression level.In some embodiments, the reference expression level of ILT3 is (a) a predefined expression level of ILT3; (b) an ILT3 expression level in a corresponding normal cell or tissue; (c) an ILT3 expression level measured in adjacent non-cancer cells or tissues in the same subject; or (d) an ILT3 expression level measured in a corresponding cell or tissue in a cohort of healthy subjects. In some embodiments, the expression level of ILT3 is determined by measuring the protein expression level of ILT3. In some embodiments, cancer cells express a low level of ILT3 compared to the reference expression level, and the reference expression level is a known ILT3. 高 ILT3 expression levels in cancer cells or tissues. Cancer cells expressing low levels of ILT3 include OCI-AML-2 or NALM-1 cells. Cancer cells expressing high levels of ILT3 include M4 and M5 AML, MM, and B-ALL, THP-1, and MOLM13 cells.

[0334] In another aspect, provided herein is the use of an ILT3xCD3 binding agent provided herein in the manufacture of a medicament for the treatment of a cancer or tumor expressing ILT3 (e.g., human ILT3) in a subject. In yet a further aspect, provided herein is a binding agent for use in the treatment of a cancer or tumor expressing ILT3 (e.g., human ILT3). In some embodiments, the cancer or tumor is a hematological cancer or tumor. In some embodiments, the cancer or tumor is acute myeloid leukemia (AML), including M4 / M5 AML, chronic myelomonocytic leukemia (CMML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), Hodgkin's lymphoma, lymphoplasmacytic lymphoma (LPL), follicular lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or marginal zone lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma). In some embodiments, the cancer or tumor is myelodysplastic syndrome. Myelodysplastic syndrome (MDS) is a group of cancers in which immature blood cells in the bone marrow do not mature and therefore do not become healthy blood cells. In some embodiments, the myelodysplastic syndrome progresses to AML. In certain embodiments, the cancer or tumor comprises a hematological cancer. In some embodiments, the hematological cancer or tumor is acute myeloid leukemia (AML). In some embodiments, the AML is M4 / M5 AML. In some embodiments, the cancer or tumor comprises a solid tumor.

[0335] In some embodiments, the cancer or tumor cells express a high level of ILT3 compared to a reference expression level. In some embodiments, the cancer or tumor cells express a low level of ILT3 compared to a reference expression level. In some embodiments, the reference expression level of ILT3 is (a) a predefined expression level of ILT3; (b) an ILT3 expression level in a corresponding normal cell or tissue; (c) an ILT3 expression level measured in adjacent non-cancer cells or tissues in the same subject; or (d) an ILT3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, the expression level of ILT3 is determined by measuring the protein expression level of ILT3. In some embodiments, the cancer cells express a low level of ILT3 compared to a reference expression level, and the reference expression level is a known ILT3 expression level. 高 ILT3 expression levels in cancer cells or tissues. Cancer cells expressing low levels of ILT3 include OCI-AML-2 or NALM-1 cells. Cancer cells expressing high levels of ILT3 include M4 and M5 AML, MM, and B-ALL, THP-1, and MOLM13 cells.

[0336] In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject is a human. In certain embodiments, the subject is administered an effective amount of a binding agent or pharmaceutical composition disclosed herein.

[0337] According to certain embodiments, the pharmaceutical compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the pharmaceutical compositions described herein may be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0338] In some embodiments, an ILT3xCD3 binding agent provided herein is used in combination with replacement therapy.

[0339] As used herein, the term "in combination" in the context of administering two or more therapies to a subject refers to the use of multiple therapies.The use of the term "in combination" does not limit the order in which the therapies are administered to a subject.For example, a first therapy (e.g., a composition described herein) can be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.

[0340] 5.6 Kits In another general aspect, the disclosure relates to a kit comprising an isolated bispecific antibody or antigen-binding fragment thereof provided herein and instructions for use.

[0341] In one embodiment, a kit is provided that includes the ILT3xCD3 binding agent provided herein. The described kits can be used to carry out the methods of using the ILT3xCD3 binding fragments provided herein or other methods known to those skilled in the art. In some embodiments, the described kits can include an antibody or antigen-binding fragment described herein and a reagent for use in detecting the presence of an ILT3xCD3 binding agent in a biological sample. Thus, the described kits can include an antibody, or antigen-binding fragment(s) thereof, as described herein, and one or more of a container for containing the antibody or fragment when not in use, instructions for using the antibody or fragment, an antibody or fragment immobilized on a solid support, and / or a detectably labeled form of the antibody or fragment, as described herein.

[0342] In another embodiment, there is provided a kit comprising an ILT3xCD3 binding agent comprising a first binding domain that specifically binds ILT3 as provided herein and a second binding domain that specifically binds CD3.

[0343] In some embodiments, the kit comprises an antibody as described herein and a reagent for detecting the antibody. The kit may further comprise one or more other components, including instructions for use; other reagents, such as a label, a therapeutic agent, or an agent useful for chelating or otherwise linking the antibody to a label or therapeutic agent, or a radioprotective composition; a device or other material for preparing the antibody for administration; a pharma- ceutically acceptable carrier; and a device or other material for administration to a subject.

[0344] In some embodiments, the kit comprises an ILT3xCD3 binding agent provided herein in a container and instructions for use of the kit.

[0345] In some embodiments, the ILT3xCD3 binding agent in the kit is labeled.

[0346] In case of conflict, the specification, including definitions, will control. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a "peptide sequence" or a "treatment" includes a plurality of such sequences, treatments, etc. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a predicate basis for the use of such exclusive terms, such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0347] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0348] As used herein, numerical values ​​are often presented in a range format throughout this document. The use of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Thus, the use of a range explicitly includes all possible subranges, all individual numerical values ​​within that range, as well as integers within such ranges and fractions of values ​​or integers within the range, unless the context clearly indicates otherwise. This configuration applies in all contexts throughout this patent document, regardless of the breadth of the range. Thus, for example, a reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, etc. References to the range of 90-100% also include 91%, 92%, 93%, 94%, 95%, 96%, 97% etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5% etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5% etc. Additionally, references to the ranges 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, and 225-250 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In further examples, references to the ranges 25-250, 250-500, 500-1000, 1000-2500, 2500-5000, 5000-25,000, or 5000-50,000 include any numbers or ranges within or including such values, for example, 25, 26, 27, 28, 29...250, 251, 252, 253, 254...500, 501, 502, 503, 504... etc. Use of consecutive ranges includes combinations of higher and lower ranges to provide additional ranges.This configuration applies in all contexts throughout this patent document, regardless of the breadth of the ranges. Thus, for example, references to a series of ranges, e.g., 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, include ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, etc.

[0349] For purposes of brevity, certain abbreviations are used herein. One example is the one-letter abbreviations to represent amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: [Table 10]

[0350] The present invention is generally disclosed herein using positive language to describe a number of embodiments. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, etc., is entirely or partially excluded. Thus, even if the present invention is generally not expressly stated herein with respect to what the present invention does not include, aspects not expressly included in the present invention are nevertheless disclosed herein.

[0351] Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. It is anticipated that upon reading the foregoing description, variations of the disclosed embodiments may become apparent to individuals working in the art, and that those skilled in the art may employ such variations where appropriate. It is therefore intended that the invention be practiced otherwise than as specifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0352] All publications, patent applications, accession numbers, and other references cited herein are incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0353] Numerous embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the description in the experimental section is intended to illustrate, but not limit, the scope of the invention as set forth in the claims.

[0354] 6. Embodiment The present invention provides the following non-limiting embodiments: 1. A binding agent comprising a first binding region that binds to human ILT3 and a second binding region that binds to human CD3, wherein said CD3 binding region comprises an anti-CD3 scFv. 2. The binding agent of embodiment 1, wherein the first binding region comprises an anti-ILT3 Fab. 3. The binding agent of embodiment 1 or 2, wherein the binding affinity of said first binding region to human ILT3 is higher than the binding affinity of said second binding region to human CD3. 4. The binding agent of embodiment 3, wherein the binding affinity of the first binding region for human ILT3 is about 10- to about 100-fold higher than the binding affinity of the second binding region for human CD3. 5. The binding agent according to any one of embodiments 1 to 4, further comprising an Fc region. 6. (i) a first polypeptide comprising the anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising the VH domain, the CH1 domain, a second CH2 domain, and a second CH3 domain of the first binding region; and (iii) a third polypeptide comprising the VL domain and the CL domain of the first binding region; wherein the VH domain, the CH1 domain, the VL domain of the first binding region, and the CL domain form the anti-ILT3 Fab, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form the Fc region. 7. The binding agent of embodiment 6, wherein the first polypeptide comprises one or more amino acid mutations that form an engineered cavity and the second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via positioning of the protuberance into the cavity. 8. The binding agent of any one of embodiments 1 to 7, wherein the second binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149; and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150. 9. In the second binding region, The binding agent of embodiment 8, wherein the VH domain of the second binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 152, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 154; and the VL domain of the second binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 155, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 156, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 157. 10. A binding agent described in any of embodiments 1 to 9, wherein the first binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 17, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 18. 11. In the first binding region, (a) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (b) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:7, the HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (c) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:9, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (d) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (e) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 13; and the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; 11. The binding agent according to embodiment 10. 12. (i) the first binding region comprises the VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; the second binding region comprises the VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150; or (ii) the first binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 17, and the VL domain comprising the amino acid sequence of SEQ ID NO: 18; and the second binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 149, and the VL domain comprising the amino acid sequence of SEQ ID NO: 150; 12. The binding agent according to embodiment 10 or 11. 13. A binding agent comprising a first binding region that binds to human ILT3 and a second binding region that binds to human CD3, wherein the second binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150. 14. The VH domain of the second binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 152, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 154; the VL domain of the second binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 155; the LCDR2 comprising the amino acid sequence of SEQ ID NO: 156; and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 157. 14. A binding agent according to embodiment 13. 15. The binding agent of embodiment 13 or 14, wherein the first binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18. 16. In the first binding region, (a) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (b) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:7, the HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (c) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:9, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (d) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (e) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 13; and the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; 16. The binder according to any one of embodiments 13 to 15. 17. (i) the first binding region comprises the VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; the second binding region comprises the VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150; or (ii) the first binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 17, and the VL domain comprising the amino acid sequence of SEQ ID NO: 18; and the second binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 149, and the VL domain comprising the amino acid sequence of SEQ ID NO: 150; 17. The binding agent according to embodiment 15 or 16. 18. The binding agent of any one of embodiments 13-17, wherein the first binding region comprises an anti-ILT3 Fab. 19. The binding agent of any one of embodiments 13 to 18, wherein the second binding region comprises an anti-CD3 scFv. 20. The binding agent according to any one of embodiments 13 to 19, wherein the binding agent further comprises an Fc region. 21. The binder is (i) a first polypeptide comprising the anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising the VH domain, a CH1 domain, a second CH2 domain and a second CH3 domain of the first binding region; and (iii) a third polypeptide comprising the VL domain and the CL domain of the first binding region. wherein the VH domain of the first binding region, the CH1 domain, the VL domain of the first binding region, and the CL domain form the anti-ILT3 Fab, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form the Fc region. 21. The binding agent according to embodiment 20. 22. The binding agent of embodiment 21, wherein the first polypeptide comprises one or more amino acid mutations that form an engineered cavity, and the second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via positioning of the protuberance into the cavity. twenty three. (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the third polypeptide comprises the amino acid sequence of SEQ ID NO: 20, or (ii) the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, and the third polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; 23. The binding agent according to embodiment 21 or 22. 24. The binding agent according to embodiments 13-17, wherein the first binding region comprises two identical anti-ILT3 Fabs and the second binding region comprises an anti-CD3 scFv. 25. The binder is (i) a first polypeptide comprising the anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a first VH domain, a second VH domain, a first CH1 domain, a second CH1 domain, a second CH2 domain, and a second CH3 domain, wherein each of the first and second VH domains comprises the VH domain of the first binding region; (iii) a third polypeptide comprising a first VL domain and a first CL domain, wherein the first VL domain comprises the VL domain of the first binding region; and (iv) a fourth polypeptide comprising a second VL domain and a second CL domain, wherein the second VL domain comprises the VL domain of the first binding region. wherein the first VH domain and the first CH1 domain of the second polypeptide and the first VL domain and the first CL domain of the third polypeptide form a first Fab region, the second VH domain and the second CH1 domain of the second polypeptide and the second VL domain and the second CL domain of the fourth polypeptide form a second Fab region, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form the Fc region. 26. The binding agent of embodiment 25, wherein the first polypeptide comprises one or more amino acid mutations that form an engineered cavity, and the second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via positioning of the protuberance into the cavity. 27. (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 169, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 20, and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 20; or (ii) the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 169, the third polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20, and the fourth polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; 27. The binding agent according to embodiment 25 or 26. 28. The binding agent according to any one of embodiments 1 to 12 and 19 to 27, wherein the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 151. 29. The binding agent according to any one of embodiments 1 to 28, wherein the binding agent is a humanized antibody. 30. A binder comprising: (i) a first polypeptide comprising an scFv that binds human CD3, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a VH domain, a CH1 domain, a second CH2 domain, and a second CH3 domain that binds human ILT3; and (iii) a third polypeptide comprising a VL domain that binds to human ILT3 and a CL domain. wherein the scFv that binds to human CD3 comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150; The VH domain that binds to human ILT3 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 17, and the VL domain that binds to human ILT3 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 18. The binder. 31. (a) the HCDR1 of the scFv comprises the amino acid sequence of SEQ ID NO: 152, the HCDR2 of the scFv comprises the amino acid sequence of SEQ ID NO: 153, the HCDR3 of the scFv comprises the amino acid sequence of SEQ ID NO: 154, the LCDR1 of the scFv comprises the amino acid sequence of SEQ ID NO: 155, the LCDR2 of the scFv comprises the amino acid sequence of SEQ ID NO: 156, and the LCDR3 of the scFv comprises the amino acid sequence of SEQ ID NO: 157; (b) a VH domain that binds to human ILT3 and a VL domain that binds to human ILT3 (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (ii) the HCDR1 comprises the amino acid sequence of SEQ ID NO:7; the HCDR2 comprises the amino acid sequence of SEQ ID NO:8; the HCDR3 comprises the amino acid sequence of SEQ ID NO:3; the LCDR1 comprises the amino acid sequence of SEQ ID NO:4; the LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6; (iii) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (iv) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or (v) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 12; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 13; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16. 31. The binding agent according to embodiment 30. 32. The binding agent of embodiment 30 or embodiment 31, wherein the VH domain of the scFv that binds to human CD3 comprises the amino acid sequence of SEQ ID NO: 149, and the VL domain of the scFv that binds to human CD3 comprises the amino acid sequence of SEQ ID NO: 150; the VH domain that binds to human ILT3 comprises the amino acid sequence of SEQ ID NO: 17, and the VL domain that binds to human ILT3 comprises the amino acid sequence of SEQ ID NO: 18. 33. The binding agent according to any one of embodiments 30 to 32, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 151. 34. An isolated polynucleotide encoding a binding agent according to any one of embodiments to 33. 35. A vector comprising the polynucleotide of embodiment 34. 36. An isolated cell comprising a polynucleotide according to embodiment 34 or a vector according to embodiment 35. 37. An isolated cell that produces a binding agent according to any one of embodiments 1 to 33. 38. A pharmaceutical composition comprising a binding agent according to any one of embodiments 1 to 33, an isolated polynucleotide according to embodiment 34, a vector according to embodiment 35, or an isolated cell according to embodiment 36 or embodiment 37, and a pharma- ceutical acceptable excipient. 39. A method for directing T cells to cancer or tumor cells expressing ILT3, comprising contacting said T cells with an effective amount of a binding agent according to any one of embodiments 1 to 33, or a pharmaceutical composition according to embodiment 38. 40. The method of embodiment 39, wherein the T cells induce killing of cancer or tumor cells that express the ILT3. 41. The method of embodiment 40, wherein the cancer or tumor cells are blood cancer or tumor cells. 42. The method of embodiment 41, wherein the blood cancer or tumor cell is selected from the group consisting of acute myeloid leukemia (AML) cells, M4 / M5 AML cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells. 43. The method of any one of embodiments 39-42, wherein the T cells are unable to induce killing of normal hematopoietic stem cells (HSCs). 44. A method for activating T cells, comprising contacting the T cells with an effective amount of a binding agent according to any one of embodiments 1 to 33 or a pharmaceutical composition according to embodiment 38, wherein the second binding region binds the T cells. 45. The method of embodiment 44, wherein the T cells are naive T cells. 46. ​​The method of embodiment 44 or embodiment 45, wherein the T cells are polyclonally expanded from a population of PBMCs. 47. A method for killing or inhibiting the growth of a cancer or tumor cell expressing ILT3, comprising contacting said cancer or tumor cell with a binding agent according to any one of embodiments 1 to 33 or a pharmaceutical composition according to embodiment 38. 48. The method of embodiment 47, wherein the binding agent activates T cells. 49. The method of embodiment 48, wherein the activated T cells induce killing of cancer or tumor cells. 50. The method of any one of embodiments 47 to 49, wherein the cancer or tumor cells comprise hematological cancer or tumor cells. 51. The method of embodiment 50, wherein the blood cancer or tumor cell is selected from the group consisting of acute myeloid leukemia (AML) cells, M4 / M5 AML cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells. 52. A method for treating a cancer or tumor expressing ILT3 in a subject, comprising administering to the subject an effective amount of a binding agent according to any one of embodiments 1 to 33 or a pharmaceutical composition according to embodiment 38. 53. The method of embodiment 52, wherein the cancer or tumor comprises a blood cancer or tumor. 54. The method of embodiment 53, wherein the hematological cancer or tumor is selected from the group consisting of acute myeloid leukemia (AML), M4 / M5 AML chronic myelomonocytic leukemia (CMML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), Hodgkin's lymphoma, lymphoplasmacytic lymphoma (LPL), follicular lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), marginal zone lymphoma, or mucosa-associated lymphoid tissue (MALT) lymphoma. EXAMPLES

[0355] 7. Working Example The following is a description of various methods and materials used in the workup, presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and is not intended to limit the scope of what the inventors regard as their invention, nor is it intended to represent that the following experiments are all that may be performed. It is to be understood that the exemplary descriptions written in the present tense have not necessarily been performed, but rather the descriptions may be performed to generate data and the like relevant to the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0356] Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric. Standard abbreviations are used, including: bp = base pair(s); kb = kilobase(s); s or sec = second(s); min = minute(s); h or hr = hour(s); aa = amino acid(s); kb = kilobase(s); nt = nucleotide(s); pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; pl or pL = picoliter(s); dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromole. Concentration; mM = molar; M = molar; kDa = kilodaltons; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = daily; BID = twice a day; QW = weekly; TIW = three times a week; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = units; ns = not statistically significant; PBS = phosphate buffered saline; PCR = polymerase chain reaction; NHS = N-hydroxysuccinimide; HSA = human serum albumin; BSA = bovine serum albumin; DMEM = Dulbecco's modified Eagle's medium; GC = genome copies; EDTA = ethylenediaminetetraacetic acid.

[0357] The following experimental methods were used throughout the examples described herein.

[0358] Protocol for generation of expanded T cells. T cells were purified from human PBMCs by negative selection using a pan T cell isolation kit (Miltenyi) and activated with human anti-CD3 / CD28 antibody (Gibco / ThermoFisher Scientific) coated Dynabeads at a 1:1 cell:bead ratio for 2 days in X-Vivo15 medium (Lonza) containing 5% normal human serum (MilliporeSigma), 2 mM GlutaGro (Corning), 10 mM HEPES (Corning), and 5 ng / mL IL-17, 5 ng / mL IL-15, and 25 ng / mL IL-2 (all from Peprotech). After removal of the Dynabeads by passage through a magnetic column, activated T cells were cultured in the same medium for an additional 8 days and viably frozen in CryoStor CS10 cell storage medium (StemCell Technologies) for future use in T cell-dependent cytotoxicity (TDCC) assays.

[0359] Protocol for the generation of red fluorescent cell lines for use in T cell-dependent cytotoxicity (TDCC) assays. Target cell lines were stably transfected with a red nuclear dye using Nuclight Red lentiviral reagent (Sartorius) at a multiplicity of infection (MOI) of 1-3. Red fluorescent cells were sorted and expanded on an Aria II flow cytometer.

[0360] Protocol for assessing T cell-dependent cytotoxicity (TDCC) in expanded T cells. One day prior to the T cell toxicity assay, cryopreserved expanded T cells (generated as described above) were thawed and cultured overnight in X-Vivo15 medium (Lonza). The following day, wells of a 96-well flat-bottom plate were pre-filled with 100 μL / well of serial dilutions of test antibodies diluted in X-Vivo15 to 4× the final concentration. The highest final concentration of each test article at 1 μg / mL was used. Cryopreserved expanded T cells were thawed viably and cultured at 1×10 in X-Vivo15 medium. 6 The fluorescently labeled cell lines were counted and diluted to a density of 2 × 10 cells / mL and added to the plate at 50 μL / well. 5 Cells were resuspended to a density of 1000 cells / mL and added to the plate at 50 μL / well. The final effector:target cell ratio was 5:1. Caspase 3 / 7 Green Reagent (Sartorius), a caspase cleavage domain (DEVD) connected to a green DNA-binding fluorescent label that is released upon DEVD cleavage by activated caspase 3 / 7, was added to the wells at a final concentration of 1:1000. Cultures were then imaged over a 24-h period using an Incucyte ZOOM live cell imager (Sartorius). The percentage of apoptotic target cells was determined by the overlap of red and green (caspase 3 / 7+) signals. Data were analyzed using Incucyte ZOOM software, version 2018A (Sartorius). In some experiments, cell-free culture supernatants were harvested after 24 h and analyzed for cytokine levels by Luminex (ProcartaPlex system; ThermoFisher Scientific).

[0361] Protocol for assessing PBMC cytotoxicity. Wells of a 96-well flat-bottom plate were pre-filled with 100 μL / well of serial dilutions of test antibodies diluted in X-Vivo15 medium (Lonza) to 4× the final concentration. The highest final concentration of each test article of 10 μg / mL was used. Cryopreserved human PBMCs were viably thawed and diluted at 1×10 in X-Vivo15 medium. 6The fluorescently labeled cell lines were counted and diluted to a density of 2 × 10 cells / mL and added to the plate at 50 μL / well. 5 Cells were resuspended to a density of 1000 cells / mL and added to the plate at 50 μL / well. The final effector:target cell ratio was 5:1. Caspase 3 / 7 Green Reagent (Sartorius), a caspase cleavage domain (DEVD) connected to a green DNA-binding fluorescent label that is released upon DEVD cleavage by activated caspase 3 / 7, was added to the wells at a final concentration of 1:1000. Cultures were then imaged over a 24-h period using an Incucyte ZOOM live cell imager (Sartorius) and supernatants were harvested for cytokine measurements after 24 h by Luminex (ProcartaPlex system; ThermoFisher Scientific). The percentage of apoptotic target cells was determined by overlap of red and green (caspase 3 / 7+) signals. Data were analyzed using Incucyte ZOOM software, version 2018A (Sartorius).

[0362] Whole blood cytokine release assay. Immune activation by antibodies and reference molecules was assessed by cytokine release assay. Antibodies were tested in both plate-coated and soluble formats. For plate-coated formats, antibody dilutions were prepared in PBS at the indicated concentrations and added at 50 μL / well to 96-well flat-bottom tissue culture plates. Plates were incubated overnight at 4°C on a shaker at 300 rpm, followed by two washes with PBS. For soluble antibody formats, antibody dilutions were prepared at 20× the indicated final concentrations and 7.5 μL / well were added to the bottom of 96-well U-bottom tissue culture plates. Freshly collected Na 2+Heparinized whole blood was added to the plate at 150 μl / well, and samples were incubated overnight at 37°C. After centrifugation at 1800 × g for 5 min at room temperature, plasma was collected from each well for cytokine analysis by Luminex assay (ProcartaPlex system; ThermoFisher Scientific). Reference molecules used included Staphylococcal enterotoxin B (1 μg / ml, soluble only), anti-CD3 (Biolegend), and anti-CD28 superagonist antibody (clone ANC28.1, AnCell).

[0363] PBMC cytokine release assay. Cryopreserved PBMCs were thawed viably and plated at 2 × 10 in X-Vivo15 medium (Lonza) in a final volume of 150 μL / well. 5 Cytokine release assays using PBMCs were performed similarly to the whole blood cytokine release assays, except that 10 cells / well were seeded.

[0364] T cell activation assay with primary AML samples. Cryopreserved bone marrow or PBMCs (Reprocell) from M5 AML patients were thawed in HBSS at room temperature and resuspended in RPMI1640 (Corning) containing 10% heat-inactivated FBS, 1% GlutaGro (Corning), 50 mM β-mercaptoethanol (Gibco / ThermoFisher Scientific) and 1% penicillin-streptomycin (Corning). Primary AML cells (2 × 10 in 100 μL of medium) were cultured at 4°C for 1 h. 5 Cells (1000 cells / well) were added to 100 μL of test antibody prepared at 2× concentration in X-Vivo15 medium. Cells were incubated with test antibody for 5 days at 37° C. On day 5, cells were harvested and stained with fluorochrome-conjugated antibodies listed below for 30 minutes at 4° C. Data was collected on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software, v.10. [Table 11]

[0365] First generation CD34 + TDCC assay using HSCs. One day prior to the T cell cytotoxicity assay, cryopreserved expanded T cells (generated as described above) were thawed and cultured overnight in X-Vivo15 medium (Lonza). On the day of the assay, cryopreserved CD34 + Bone marrow cells (StemCell Technologies) were thawed in HBSS (Corning) at room temperature and resuspended in X-Vivo15 medium. Wells of a 96-well plate were preloaded with 100 μL of test antibody at 2× concentration. CD34 + Hematopoietic stem cells (1 x 10 in a volume of 50 µL) 4 cells / well) and expanded T cells (5 × 10 in a volume of 50 μL 4 1000 cells / well) were added to each well containing the test antibody and caspase 3 / 7 green reagent (Sartorius) was added at a final concentration of 1:1000. Cells were cultured overnight at 37°C. After centrifugation, supernatants were collected for cytokine secretion analysis and cells were stained with antibodies against CD45, CD25, CD34, ILT3, and CD123 for 30 min at 4°C using the reagents listed above. Data were collected on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software, v.10.

[0366] 7.1 Example 1: Generation of ILT3xCD3 bispecific molecules The aim of this study was to design cytotoxic T cell engagers with enhanced selectivity for tumor cells and improved therapeutic index (i.e., high affinity for binding to ILT3 expressing cells and good safety profile combined with efficient tumor cell killing). The safety of the T cell engager depends on minimizing cytokine release and maximizing tumor cell killing. The therapeutic index is measured using the ratio between tumor cell killing and cytokine release induced by the T cell engager. The higher the ratio, the better the therapeutic index for the T cell engager.

[0367] A panel of anti-ILT3 antibodies, and anti-CD3 antibodies with different binding affinities (high and low) were tested. The main criterion for the selection of the ILT3 targeting arm of the bispecific was high affinity. The criterion for selecting the CD3 targeting arm was a good therapeutic index. Furthermore, the rationale for the design was to have an ILT3 targeting arm that binds ILT3 with 10-100 times higher affinity than the CD3 targeting arm that binds CD3, so that the T cell engager binds ILT3-expressing cancer cells before engaging any T cells. As a result, this design reduces off-target effects and increases safety profile. Tumor cell cytotoxicity and cytokine production were evaluated with various antibody combinations and formats.

[0368] Selection of ILT3 targeting arms. Various ILT3 antibody clones (see Tables 1-8) were coupled to either high affinity CD3 scFv (2B2) or low affinity CD3 scFv (1G4). ​​The binding affinity of various ILT3 antibodies to ILT3 is shown in Table 9. The T-cell dependent cytotoxicity (TDCC) of each anti-ILT3 Fab when coupled to either CD3 scFv 2B2 or CD3 scFv 1G4 is shown in Table 9. [Table 12]

[0369] All ILT3xCD3 bispecific molecules tested induced TDCC and cytokine release. Among all anti-ILT3 clones tested, Hz45G10 had the highest affinity for ILT3. Hz45G10 potently induced apoptosis compared to other ILT3 antibody clones (16C5 or 12A12) when coupled with CD3 scFv 2B2 (Figure 2) or CD3 scFv 1G4 (Figure 3). Furthermore, Hz45G10 Fab induced less TNFα release when coupled with CD3 scFv 2B2 (Figure 4, Table 10). Modifying the ILT3 antibody had no effect on the ratio between cytotoxicity and cytokine production (see Table 10). [Table 13]

[0370] Selection of CD3 targeting arm. The affinity of the CD3 binding arm of the T cell engager can vary depending on the tumor antigen binding arm. For the ILT3-CD3 T cell engager, an optimal CD3 scFv was generated based on the selected Hz45G10 clone. First, two CD3 scFv clones with different CD3 binding affinities were tested when linked with Hz45G10 (CD3 scFv 2B2 with high affinity to CD3, and r CD3 scFv 1G4 with low affinity to CD3 (see International Publication No. WO2008 / 119567 and US Patent No. 10,066,016)). CD3 scFv 2B2 showed 35-fold higher affinity to CD3 than scFv 1G4. 2B2 scFv showed 160-fold increase in T cell dependent cytotoxicity (TDCC) compared to scFv IG4, but only increased cytokine production by 20-fold. See Figure 5, Figure 6 and Table 11. [Table 14]

[0371] In view of the above results, an anti-CD3 scFv clone with high binding affinity to CD3 was developed (VH as SEQ ID NO: 149, VL as SEQ ID NO: 150, K determined by SPR). D = 6 nM). The binding affinity of Hz45G10 to human ILT3 was 38-fold higher than that of CD3 scFv.

[0372] Bispecific antibodies incorporating Hz45G10 and CD3 scFv were then generated and evaluated in multiple formats as shown in Figure 7. All bispecific formats were screened in cytotoxicity and cytokine release assays.

[0373] The F0 (i.e., ABX1446) ILT3xCD3 bispecific antibody format demonstrated strong cytolytic activity in the AML cell line, MOLM13 (see Table 12 and Figure 8). The F0 ILT3xCD3 bispecific antibody format also induced low cytokine secretion in whole blood cytokine release assays when whole blood was added to F0 pre-coated plates (Figure 9) or to media with soluble F0 (Figure 10). See Table 13. Similarly, among the formats tested, F13 (i.e., ABX1520) demonstrated strong cytolytic activity but low cytokine secretion. [Table 15] [Table 16]

[0374] 7.2 Example 2: In vitro characterization of ILT3xCD3 bispecific molecules The aim of this study was to evaluate the in vitro activity of F0 (i.e., ABX1446) and F13 (i.e., ABX1520) in cell lines expressing ILT3. Cytotoxicity assays and cytokine secretion were performed as outlined in Example 1.

[0375] ABX1446 and ABX1520 inhibited ILT3-positive (ILT3 + ) potently induced apoptosis in AML cells (MOLM13). See Table 14. [Table 17]

[0376] ABX1446 and ABX1520 potently induced apoptosis in AML cells with low expression of ILT3 (OCI-AML-2 and NALM-1 cells) when expanded T cells (Figure 13, Figure 14 and Table 15) or naive T cells (from PBMC; Figure 15, Figure 16 and Table 16) were used as effectors. [Table 18] [Table 19]

[0377] ABX1446 and ABX1520 were unable to induce TDCC in ILT3 knockout THP-1 cells. See Figure 17 and Table 17. [Table 20]

[0378] ABX1446 and ABX1520 induced less cytokine release in a PBMC cytokine secretion assay when PBMCs were incubated with ABX1446 or ABX1520 in either a plate coated format (FIG. 18) or a soluble format (FIG. 19). See Table 18. [Table 21]

[0379] ABX1446 and ABX1520 induced less cytokine release in a whole blood cytokine secretion assay when whole blood was incubated with ABX1446 in plate coated (FIG. 20) or soluble (FIG. 21) format. See Table 19. ABX1446 and bibecotamab also showed similar potency in the PBMC cytotoxicity assay, but ABX1446 induced less cytokine secretion in the PBMC cytotoxicity assay. See FIG. 22, FIG. 23 and Table 20. [Table 22] [Table 23]

[0380] When compared to CD123xCD3 DART (Flotetuzumab), ABX1446 induced less cytokine secretion (Figure 25), but induced potent apoptosis in MOLM13 cells (Figure 24). ABX1446 induced less cytokine secretion compared to Flotetuzumab. See Table 21. [Table 24]

[0381] To study T cell proliferation and activation in PBMCs isolated from M5 AML patients, PBMCs were incubated with ABX1446 and proliferation and activation were measured by flow cytometry. ABX1446 induced T cell proliferation (Figure 26) and activation (Figure 27) in M5 PBMCs.

[0382] ABX1446 was unable to induce depletion (FIG. 28) and apoptosis (FIG. 29) in primary HSCs.

[0383] Furthermore, ABX1446 was unable to eliminate non-monocytic immune cells. CD123 is expressed on many immune cell types, whereas ILT3 is expressed on only a subset. The use of bispecific antibody bibecotamab may represent a safety risk in a clinical setting. Therefore, cytotoxicity was measured in KU812 basophils and LAMA84 basophils. Both cell types are CD123 positive and ILT3 negative. Thus, ABX1446 had no effect on these basophils. See Figures 30 and 31.

[0384] Multiple myeloma cells also express ILT3. The F0 ILT3xCD3 bispecific antibody format (ABX1446) showed strong cytolytic activity against MM1S (Figure 32), H929 (Figure 33) and U226B1 (Figure 34) multiple myeloma cell lines. See Table 22. [Table 25]

[0385] The above studies demonstrated that ABX1446 exhibited potent cytotoxicity with lower levels of cytokine release compared to the T cell engager bispecific bibecotamab. Also, ABX1446 did not eliminate HSCs or mature immune cells. Thus, ABX1446 had a safety profile that distinguished it from current T cell engaging bispecific antibodies on the market.

[0386] 7.3 Example 3: In vivo characterization of ILT3xCD3 bispecific molecules The objective of this study was to evaluate the in vivo activity of F0 (i.e., ABX1446) and F13 (i.e., ABX1520) in three human AML mouse models, including the MOLM13 AML model (Figure 35), the MV4;11 AML model (Figure 35), and a model using CD34+ humanized mice engrafted with MV4;11 AML cells (Figure 39).

[0387] In the MOLM13 mouse model, ABX1446 and ABX1520 reduced the number of circulating tumor cells similarly to bibecotamab. See Figure 36 and Table 23. [Table 26]

[0388] In the MV4;11 mouse model, mice receiving increasing concentrations of ABX1446 (0.01 mpk, 0.1 mpk and 1 mpk) had reduced numbers of circulating tumor cells at weeks 2 (Figure 37) and 3 (Figure 38), similar to bibecotamab. See Table 24, which shows the number of MV4;11 cells per μL of blood. [Table 27]

[0389] MV4;11 AML cells were transplanted with CD34 + In a model using humanized mice, ABX1446 reduced the number of circulating MV4;11 cells per μL of blood. See Figure 40 and Table 25, which show the number of MV4;11 cells per μL of blood. Mice received ABX1446, anti-KLH and bibecotamab at 1 mpk. [Table 28]

[0390] The studies described above demonstrated that ABX1446 inhibited tumor growth in three different mouse models of human AML.

[0391] 7.4 Example 4: Characterization of ILT3xCD3 bispecific molecules in primary tumor cell cultures The activity of the ILT3xCD3 bispecific molecule was evaluated in a native tumor microenvironment platform (Vivia Biotech). In this platform, whole bone marrow samples from human patients diagnosed with M5 AML were cultured with dose titrations of ABX1446. T cell activation and tumor cell depletion were evaluated. Briefly, whole bone marrow from three patients with M5 AML was evaluated in the Vivia Biotech Native Tumor Microenvironment platform. The demographics of the bone marrow donors were as follows: [Table 29]

[0392] For each sample, cryopreserved whole bone marrow was thawed viably and baseline ILT3 receptor density was quantified by flow cytometry using Quantibrite beads (BD Biosciences, 340495) and PE-conjugated ILT3 antibody (clone ZM4.1, BD Biosciences, 333007). Flow cytometry was also used to count baseline tumor and T cell numbers. Baseline effector:target (E:T) ratios were calculated for each sample. [Table 30]

[0393] To evaluate the activity of the ILT3xCD3 bispecific molecule, bone marrow samples were plated in serum-free medium supplemented with fetal bovine serum (FBS) and a proprietary cocktail of growth factors. ABX1446 was administered at 8-point dose titration (final concentrations, 0.3–3 × 10 -6ABX1446 was added to the cultures at 0.3 mg / mL. Control conditions included bone marrow treated with PBS and bone marrow treated with isotype control antibody at 0.3 mg / mL. Cells were harvested at 72 and 120 hours for evaluation of tumor cell depletion and T cell activation by flow cytometry. Results were normalized to baseline values ​​for each donor. ABX1446 induced dose-dependent tumor cell depletion and T cell activation in primary M5 AML bone marrow samples as shown in Figure 41 and Figure 42 in one representative donor (out of three donors evaluated).

[0394] ILT3 + Similar studies were initiated to evaluate the activity of the ILT3xCD3 bispecific molecule in primary cultures of multiple myeloma samples. Fresh multiple myeloma bone marrow samples were first analyzed for ILT3 expression and identified ILT3 cells with sufficient cell numbers and viability. + Sample (CD138 + ≥60% of myeloma blasts are LT3 + ABX1446 was evaluated in a native tumor microenvironment platform. + It induced a dose-dependent depletion of multiple myeloma cells and concomitant T cell activation. Results from a representative donor are shown in Figures 43 and 44. [Table 31] TIFF2025510232000033.tif198165TIFF2025510232000034.tif150165TIFF2025510232000035.tif197165TIFF2025510232000036.tif233165TIFF2025510232000037.tif233165TIFF2025510232000038.tif233165TIFF2025510232000039.tif233165TIFF2025510232000040.tif213165TIFF2025510232000041.tif219165TIFF2025510232000042.tif195165TIFF2025510232000043.tif217165TIFF2025510232000044.tif110165

Claims

1. 1. A binding agent comprising a first binding region that binds to human ILT3 and a second binding region that binds to human CD3, wherein the CD3 binding region comprises an anti-CD3 scFv.

2. The binding agent of claim 1 , wherein the first binding region comprises an anti-ILT3 Fab.

3. The binding agent of claim 1 or 2, wherein the binding affinity of the first binding region to human ILT3 is higher than the binding affinity of the second binding region to human CD3.

4. 4. The binding agent of claim 3, wherein the binding affinity of the first binding region to human ILT3 is about 10-fold to about 100-fold higher than the binding affinity of the second binding region to human CD3.

5. The binding agent of any one of claims 1 to 4, further comprising an Fc region.

6. (i) a first polypeptide comprising the anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising the VH domain, the CH1 domain, a second CH2 domain, and a second CH3 domain of the first binding region; and (iii) a third polypeptide comprising the VL domain and the CL domain of the first binding region.

6. The binding agent of claim 5, wherein the VH domain, the CH1 domain, the VL domain, and the CL domain of the first binding region form the anti-ILT3 Fab, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form the Fc region.

7. 7. The binding agent of claim 6, wherein the first polypeptide comprises one or more amino acid mutations that form an engineered cavity, the second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via positioning of the protuberance into the cavity.

8. The binding agent of any one of claims 1 to 7, wherein the second binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149; and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO:

150.

9. In the second binding region, The binding agent of claim 8, wherein the VH domain of the second binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 152, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 154; and the VL domain of the second binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 155, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 156, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:

157.

10. The binding agent of any one of claims 1 to 9, wherein the first binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 17, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

18.

11. In the first bonding region, (a) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (b) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:7, the HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (c) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:9, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (d) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (e) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 13; and the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:

16. The binder of claim 10.

12. (i) the first binding region comprises the VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; the second binding region comprises the VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150; or (ii) the first binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 17, and the VL domain comprising the amino acid sequence of SEQ ID NO: 18; the second binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 149, and the VL domain comprising the amino acid sequence of SEQ ID NO: 150; A binder according to claim 10 or 11.

13. A binding agent comprising a first binding region that binds to human ILT3 and a second binding region that binds to human CD3, wherein the second binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO:

150.

14. The VH domain of the second binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 152, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 154; the VL domain of the second binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 155; the LCDR2 comprising the amino acid sequence of SEQ ID NO: 156; and the LCDR3 comprising the amino acid sequence of SEQ ID NO:

157. The binder of claim 13.

15. The binding agent of claim 13 or 14, wherein the first binding region comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO:

18.

16. In the first bonding region, (a) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (b) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:7, the HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (c) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO:1, the HCDR2 comprising the amino acid sequence of SEQ ID NO:9, and the HCDR3 comprising the amino acid sequence of SEQ ID NO:3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO:4, the LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:6; (d) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (e) the VH domain of the first binding region comprises the HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 13; and the VL domain of the first binding region comprises the LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprising the amino acid sequence of SEQ ID NO:

16. The binder according to any one of claims 13 to 15.

17. (i) the first binding region comprises the VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; the second binding region comprises the VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 149, and the VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 150; or (ii) the first binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 17, and the VL domain comprising the amino acid sequence of SEQ ID NO: 18; the second binding region comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 149, and the VL domain comprising the amino acid sequence of SEQ ID NO: 150; 17. The binder according to claim 15 or 16.

18. The binding agent of any one of claims 13 to 17, wherein the first binding region comprises an anti-ILT3 Fab.

19. The binding agent of any one of claims 13 to 18, wherein the second binding region comprises an anti-CD3 scFv.

20. The binding agent of any one of claims 13 to 19, further comprising an Fc region.

21. The binder is (i) a first polypeptide comprising the anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising the VH domain, the CH1 domain, a second CH2 domain and a second CH3 domain of the first binding region; and (iii) a third polypeptide comprising the VL domain and the CL domain of the first binding region. wherein the VH domain of the first binding region, the CH1 domain, the VL domain of the first binding region, and the CL domain form the anti-ILT3 Fab, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form the Fc region. The binder of claim 20.

22. 22. The binding agent of claim 21 , wherein the first polypeptide comprises one or more amino acid mutations that form an engineered cavity, and the second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via positioning of the protuberance into the cavity.

23. (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the third polypeptide comprises the amino acid sequence of SEQ ID NO: 20; or (ii) the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, and the third polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; 23. The binder according to claim 21 or 22.

24. The binding agent of claims 13 to 17, wherein the first binding region comprises two identical anti-ILT3 Fabs and the second binding region comprises an anti-CD3 scFv.

25. The binder is (i) a first polypeptide comprising the anti-CD3 scFv, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a first VH domain, a second VH domain, a first CH1 domain, a second CH1 domain, a second CH2 domain, and a second CH3 domain, wherein each of the first and second VH domains comprises the VH domain of the first binding region; (iii) a third polypeptide comprising a first VL domain and a first CL domain, wherein the first VL domain comprises the VL domain of the first binding region; and (iv) a fourth polypeptide comprising a second VL domain and a second CL domain, wherein the second VL domain comprises the VL domain of the first binding region. wherein the first VH domain and the first CH1 domain of the second polypeptide and the first VL domain and the first CL domain of the third polypeptide form a first Fab region, the second VH domain and the second CH1 domain of the second polypeptide and the second VL domain and the second CL domain of the fourth polypeptide form a second Fab region, and the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain form the Fc region.

26. 26. The binding agent of claim 25, wherein the first polypeptide comprises one or more amino acid mutations that form an engineered cavity and the second polypeptide comprises one or more amino acid mutations that form an engineered protuberance, and the first polypeptide dimerizes with the second polypeptide via placement of the protuberance in the cavity.

27. (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 169, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 20, and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 20; or (ii) the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 147, the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 169, the third polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20, and the fourth polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; 27. The binder according to claim 25 or 26.

28. The binding agent of any one of claims 1 to 12 and 19 to 27, wherein the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO:

151.

29. The binding agent of any one of claims 1 to 28, wherein the binding agent is a humanized antibody.

30. A binder comprising: (i) a first polypeptide comprising an scFv that binds human CD3, a first CH2 domain, and a first CH3 domain; (ii) a second polypeptide comprising a VH domain, a CH1 domain, a second CH2 domain, and a second CH3 domain that binds human ILT3; and (iii) a third polypeptide comprising a VL domain that binds to human ILT3 and a CL domain. and the scFv that binds to human CD3 comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 149, and a VL domain comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 150; The VH domain that binds to human ILT3 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 17, and the VL domain that binds to human ILT3 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

18. The binder.

31. (a) the HCDR1 of the scFv comprises the amino acid sequence of SEQ ID NO: 152, the HCDR2 of the scFv comprises the amino acid sequence of SEQ ID NO: 153, the HCDR3 of the scFv comprises the amino acid sequence of SEQ ID NO: 154, the LCDR1 of the scFv comprises the amino acid sequence of SEQ ID NO: 155, the LCDR2 of the scFv comprises the amino acid sequence of SEQ ID NO: 156, and the LCDR3 of the scFv comprises the amino acid sequence of SEQ ID NO: 157; (b) the VH domain and VL domain that bind to human ILT3 (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (ii) the HCDR1 comprises the amino acid sequence of SEQ ID NO:7; the HCDR2 comprises the amino acid sequence of SEQ ID NO:8; the HCDR3 comprises the amino acid sequence of SEQ ID NO:3; the LCDR1 comprises the amino acid sequence of SEQ ID NO:4; the LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6; (iii) the HCDR1 comprises the amino acid sequence of SEQ ID NO:1; the HCDR2 comprises the amino acid sequence of SEQ ID NO:9; the HCDR3 comprises the amino acid sequence of SEQ ID NO:3; the LCDR1 comprises the amino acid sequence of SEQ ID NO:4; the LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6; (iv) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or (v) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 12; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 13; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15; and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

16. The binder of claim 30.

32. The binding agent of claim 30 or claim 31, wherein the VH domain of the scFv that binds to human CD3 comprises the amino acid sequence of SEQ ID NO: 149, and the VL domain of the scFv that binds to human CD3 comprises the amino acid sequence of SEQ ID NO: 150; the VH domain that binds to human ILT3 comprises the amino acid sequence of SEQ ID NO: 17, and the VL domain that binds to human ILT3 comprises the amino acid sequence of SEQ ID NO:

18.

33. The binding agent of any one of claims 30 to 32, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

151.

34. An isolated polynucleotide encoding the binding agent of any one of claims 1 to 33.

35. A vector comprising the polynucleotide of claim 34.

36. 36. An isolated cell comprising the polynucleotide of claim 34 or the vector of claim 35.

37. An isolated cell producing the binding agent of any one of claims 1 to 33.

38. A pharmaceutical composition comprising a binding agent according to any one of claims 1 to 33, an isolated polynucleotide according to claim 34, a vector according to claim 35, or an isolated cell according to claim 36 or claim 37, and a pharma- ceutically acceptable excipient.

39. 39. A method of directing T cells to cancer or tumor cells that express ILT3, comprising contacting the T cells with an effective amount of the binding agent of any one of claims 1 to 33 or the pharmaceutical composition of claim 38.

40. 40. The method of claim 39, wherein the T cells induce killing of cancer or tumor cells that express the ILT3.

41. 41. The method of claim 40, wherein the cancer or tumor cell is a hematological cancer or tumor cell.

42. 42. The method of claim 41, wherein the hematological cancer or tumor cell is selected from the group consisting of acute myeloid leukemia (AML) cells, M4 / M5 AML cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells.

43. The method of any one of claims 39 to 42, wherein the T cells are unable to induce killing of normal hematopoietic stem cells (HSCs).

44. 39. A method of activating a T cell, comprising contacting the T cell with an effective amount of the binding agent of any one of claims 1 to 33 or the pharmaceutical composition of claim 38, wherein the second binding region binds to the T cell.

45. 45. The method of claim 44, wherein the T cells are naive T cells.

46. 46. ​​The method of claim 44 or claim 45, wherein the T cells are polyclonally expanded from a population of PBMCs.

47. 39. A method of killing or inhibiting the growth of a cancer or tumor cell that expresses ILT3, comprising contacting the cancer or tumor cell with a binding agent of any one of claims 1 to 33 or a pharmaceutical composition of claim 38.

48. 48. The method of claim 47, wherein the binding agent activates T cells.

49. 49. The method of claim 48, wherein the activated T cells induce killing of cancer or tumor cells.

50. 50. The method of any one of claims 47 to 49, wherein the cancer or tumor cells comprise hematological cancer or tumor cells.

51. 51. The method of claim 50, wherein the hematological cancer or tumor cell is selected from the group consisting of acute myeloid leukemia (AML) cells, M4 / M5 AML cells, chronic myelomonocytic leukemia (CMML) cells, B-cell acute lymphoblastic leukemia (B-ALL) cells, chronic lymphocytic leukemia (CLL) cells, diffuse large B-cell lymphoma (DLBCL) cells, mantle cell lymphoma (MCL) cells, multiple myeloma (MM) cells, myelodysplastic syndrome (MDS) cells, Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma (LPL) cells, follicular lymphoma cells, Burkitt's lymphoma cells, blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, marginal zone lymphoma cells, or mucosa-associated lymphoid tissue (MALT) lymphoma cells.

52. A method of treating an ILT3-expressing cancer or tumor in a subject, comprising administering to the subject an effective amount of a binding agent of any one of claims 1 to 33 or a pharmaceutical composition of claim 38.

53. 53. The method of claim 52, wherein the cancer or tumor comprises a hematological cancer or tumor.

54. 54. The method of claim 53, wherein the hematological cancer or tumor is selected from the group consisting of acute myeloid leukemia (AML), M4 / M5 AML chronic myelomonocytic leukemia (CMML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndromes (MDS), Hodgkin's lymphoma, lymphoplasmacytic lymphoma (LPL), follicular lymphoma, Burkitt's lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), marginal zone lymphoma, or mucosa-associated lymphoid tissue (MALT) lymphoma.

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