C19 C38 dual-specific antibody

Abstract

Targeting immunosuppressive B cell populations using bispecific or multivalent targeting molecules offers a potential route for therapeutic intervention that effectively modulates anti-tumor immune responses and improves treatment outcomes. Thus, there remains a need for engineered antibody-based therapeutic agents that can effectively and selectively target immunosuppressive B cell populations for the treatment of cancer. Provided are complex-binding molecules that target immunosuppressive B cells.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 981,990, filed February 26, 2020, U.S. Provisional Patent Application No. 62 / 990,330, filed March 16, 2020, and U.S. Provisional Patent Application No. 63 / 094,838, filed October 21, 2020, which applications are incorporated herein by reference. [Background technology]

[0002] While antibody therapeutics have been successfully used to treat a variety of diseases, their application can be limited in terms of clinical efficacy in complex diseases such as cancer. Engineering antibody-based therapeutics to alter target binding affinity and valency offers a potential pathway toward achieving increased efficacy and improving treatment outcomes. Bispecific or multivalent antibodies therefore offer a potential approach to solving challenges associated with the multifactorial nature of complex diseases. By binding to two different antigen molecules or different epitopes of the same antigen, bispecific antibodies offer greater functionality and diverse applications as targeting agents for the treatment of numerous diseases. Summary of the Invention

[0003] The dynamic relationship between cancer biology and the immune system is a factor associated with clinical outcomes. The immune response plays a significant role in regulating the tumor microenvironment during cancer development. Immune cells, such as T cells and B cells, therefore act as modulators and effectors of cancer progression or metastasis. Notably, immunosuppressive cells play a key role in antitumor immune responses, where immunosuppression is commonly associated with tumor growth and invasion and correlates with negative outcomes. While B cells are known to positively modulate immune responses, immunosuppressive B cell populations function to suppress antitumor immune responses and promote tumor growth.

[0004] Provided herein are certain binding molecules that target immunosuppressive B cell populations using bispecific or multivalent targeting molecules. Targeting immunosuppressive B cell populations offers a route for therapeutic intervention in cancer that effectively modulates antitumor immune responses and improves treatment outcomes (as opposed to, for example, selective depletion of epithelial cancer cell populations). The binding molecules provided herein can include bispecific antibodies that bind to B cell lineage surface markers (e.g., CD19, CD138, IgA, and / or CD20) and immunosuppressive B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, and / or latent TGF-beta (e.g., TGF-beta LAP)). In certain specific embodiments, the bispecific antibodies bind to CD19 and CD38, thereby providing selectivity for specific immunosuppressive B cell populations.

[0005] As provided and described herein, bispecific antibodies that bind to CD19 and CD38 offer advantages in selectively binding cells expressing CD19 and CD38 (e.g., immunosuppressive B cell populations). Furthermore, the bispecific antibodies disclosed herein that bind to CD19 and CD38 demonstrate advantages in that they do not promote hemolysis or erythrocyte aggregation, particularly when compared with monospecific CD19 or CD38 antibodies. Therefore, severe side effects, such as anemia, seen with monospecific CD19 or CD38 antibodies (e.g., SARCLISA® (isatuximab-irfc)) are overcome. Bispecific antibodies that bind to CD19 and CD38 also demonstrate advantages in that they effectively promote favorable target cell apoptosis of cells expressing CD19 and CD38, particularly when compared with monospecific controls. Moreover, bispecific antibodies that bind to CD19 and CD38 offer further advantages over the mere use of two separate monoclonal antibodies that independently target CD38 and CD19 in that they more effectively target specific immunosuppressive B cell populations, leading to greater efficacy and potentially fewer side effects, such as lymphopenia, seen with other B cell-targeting monoclonal antibodies (e.g., rituximab).

[0006] Described herein are complex-binding molecules comprising a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, wherein the first target comprises a B cell lineage surface marker and the second target comprises an immunosuppressive B cell surface marker, and the first and second targets are not identical. In some embodiments, the first or second binding moiety comprises a polypeptide. In some embodiments, the first or second binding moiety consists of a polypeptide. In some embodiments, the first and second binding moieties comprise a polypeptide. In some embodiments, the first and second binding moieties consist of a polypeptide. In some embodiments, the polypeptide of the first or second binding moiety comprises an amino acid sequence at least 100 amino acid residues in length. In some embodiments, the polypeptide of the first and second binding moieties comprises an amino acid sequence at least 100 amino acid residues in length.

[0007] In some embodiments, the B cell lineage surface marker comprises CD19, CD138, IgA, or CD45. In some embodiments, the B cell lineage surface marker comprises CD19. In some embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the immunoinhibitory B cell surface marker comprises IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP). In some embodiments, the immunoinhibitory B cell surface marker comprises CD38. In some embodiments, the immunoinhibitory B cell surface marker consists of CD38.

[0008] In some embodiments, the first or second binding moiety is an immunoglobulin heavy and light chain pair, an scFv, a F(ab), a F(ab')2, a single domain antibody, a variable region fragment (V) from an immunoglobulin neoantigen receptor, or a fragment thereof. NAR ), or a variable region (V) derived from a heavy chain antibody HIn some embodiments, the first and second binding components comprise an immunoglobulin heavy and light chain pair, an scFv, a F(ab), a F(ab')2, a single domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (V). NAR ), or a variable region (V) derived from a heavy chain antibody H H).

[0009] In some embodiments, the first or second binding component comprises an immunoglobulin heavy chain and light chain pair. In some embodiments, the first and second binding components comprise an immunoglobulin heavy chain and light chain pair. In some embodiments, the complex-binding molecule comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85, or 150 to 155, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41 to 45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51 to 55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61 to 65. In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 2. In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the complex-binding molecule is a common light chain bispecific IgG.

[0010] In some embodiments, the first or second binding component comprises an scFv. In some embodiments, the first and second binding components comprise scFv. In some embodiments, the complex binding molecule is a bispecific antibody or dual antigen-binding fragment thereof.

[0011] In some embodiments, the bispecific antibody is selected from one of the following formats: common light chain bispecific IgG, Fab-Fc:scFv-Fc bispecific IgG, Fab-Fc-Fab:Fc bispecific IgG, Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG, Fab-Fc-scFv:Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc bispecific IgG, scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG, Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG, scFv-Fab-Fc:Fc bispecific IgG, and Fab-Fc-scFv:Fab-Fc bispecific IgG. In some embodiments, the bispecific antibody is a Fab-Fc:scFv-Fc bispecific IgG. In some embodiments, the bispecific antibody is a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG. In some embodiments, the bispecific antibody is an scFv-Fab-Fc:Fc bispecific IgG. In some embodiments, the complex-binding molecule comprises an Fc region comprising an amino acid residue modified with a native or non-fucosylated carbohydrate. In some embodiments, the amino acid residue modified with a native or non-fucosylated carbohydrate corresponds to asparagine 297 according to EU numbering.

[0012] In some embodiments, the first binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35, an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41 to 45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51 to 55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61 to 65.

[0013] In some embodiments, the first binding component comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NO:1 and SEQ ID NO:2.

[0014] In some embodiments, the first binding component comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:1 and SEQ ID NO:2.

[0015] In some embodiments, the second binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85, or 150 to 155, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95, an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125.

[0016] In some embodiments, the second binding component comprises an amino acid sequence that comprises at least about 90%, 95%, 97%, 99% identity, or is 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the second binding component comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3 and SEQ ID NO: 4.

[0017] In some embodiments, the complex binding molecule binds to CD19+, CD38+ B cells.

[0018] Disclosed are cells comprising a nucleic acid encoding a complex-binding molecule. In some embodiments, the polynucleotide sequence encoding the complex-binding molecule is operably linked to a eukaryotic regulatory sequence. In some embodiments, the cell comprises a prokaryotic cell. In some embodiments, the prokaryotic cell is an Escherichia coli cell. In some embodiments, the cell comprises a eukaryotic cell. In some embodiments, the eukaryotic cell is a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a human PER.C6 cell.

[0019] Disclosed are compositions comprising a complex-binding molecule and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for subcutaneous administration.

[0020] Provided are complex-binding molecules for use in methods of treating tumors or cancer in individuals. In some embodiments, the cancer or tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In certain embodiments, the B-cell malignancy is B-cell acute lymphocytic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the preceding embodiments, the hematological cancer expresses CD19 and CD38 (e.g., cells of the cancer express CD19 and CD38).

[0021] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colorectal cancer, brain cancer, or head and neck cancer. In some embodiments, the breast cancer is triple-negative breast cancer, the lung cancer is non-small cell lung cancer, the head and neck cancer is head and neck squamous cell carcinoma, the kidney cancer is renal cell carcinoma, the brain cancer is glioblastoma multiforme, or the skin cancer is melanoma.

[0022] Provided are complex-binding molecules for use in methods for reducing immunoinhibitory B cells in, adjacent to, or surrounding an individual's tumor, or immunoinhibitory B cells that affect an individual's anti-tumor immune response distal to the tumor site. Provided are complex-binding molecules for use in methods for reducing immunoinhibitory B cells in, adjacent to, or surrounding an individual's tumor. In some embodiments, the tumor-infiltrating B cells or immunoinhibitory B cells comprise CD19+ CD38+ B cells. Also provided are complex-binding molecules for use in methods for reducing or inhibiting the function of immunoinhibitory B cells in, adjacent to, or surrounding an individual's tumor and / or immunoinhibitory B cells that affect an individual's anti-tumor immune response distal to the tumor site. In some embodiments, the function of immunoinhibitory B cells includes the release of anti-inflammatory or immunoinhibitory cytokines, such as IL-10, IL-35, TGF-beta, or a combination thereof.

[0023] Disclosed are methods of treating an individual suffering from cancer or a tumor, comprising administering a complex-binding molecule to the individual suffering from cancer or a tumor, thereby treating the cancer or tumor. In some embodiments, the cancer or tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In certain embodiments, the B-cell malignancy is B-cell acute lymphocytic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the preceding embodiments, the hematological cancer expresses CD19 and CD38 (e.g., cells of the cancer express CD19 and CD38).

[0024] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In some embodiments, the breast cancer is triple-negative breast cancer, the lung cancer is non-small cell lung cancer, the head and neck cancer is head and neck squamous cell carcinoma, the kidney cancer is renal cell carcinoma, the brain cancer is glioblastoma multiforme, or the skin cancer is melanoma.

[0025] Disclosed are methods for reducing immunoinhibitory B cells that affect the anti-tumor immune response to a tumor in an individual afflicted with a tumor or cancer, comprising administering a complex-binding molecule to the individual afflicted with a tumor or cancer, thereby reducing the immunoinhibitory B cells that affect the anti-tumor immune response. Also disclosed are methods for reducing immunoinhibitory B cells in, adjacent to, or surrounding the tumor in an individual afflicted with a tumor or cancer, comprising administering a complex-binding molecule to the individual afflicted with a tumor or cancer, thereby reducing the immunoinhibitory B cells in, adjacent to, or surrounding the tumor. In some embodiments, the tumor-infiltrating B cells or immunoinhibitory B cells comprise CD19+, CD38+ B cells.

[0026] Further disclosed is a method of preparing a cancer treatment for an individual, the method comprising mixing a complex binding molecule with a pharmaceutically acceptable diluent, carrier, or excipient.

[0027] Also disclosed are methods for producing a complex-binding molecule, comprising incubating cells containing an expression vector comprising a nucleic acid sequence encoding the complex-binding molecule in cell culture medium under conditions sufficient to allow expression, assembly, and secretion of the complex-binding molecule into the cell culture medium. In some embodiments, the method comprises isolating and purifying the molecule from the cell culture medium. Such isolation and purification can involve contacting the cell culture medium, or cell culture medium that has been subjected to one or more purification steps, with a resin or column containing Protein A, Protein G, Protein L, Protein A / G, or any combination thereof, and optionally washing the resin or column to remove one or more non-complex-binding molecules from the cell culture medium, or cell culture medium that has been subjected to one or more purification steps.

[0028] Provided herein is a complex-binding molecule comprising a CD19-binding component configured to bind to CD19 and a CD38-binding component configured to bind to CD38, wherein the CD19-binding component comprises an antibody or antigen-binding fragment thereof, and the CD38-binding component comprises an antibody or antigen-binding fragment thereof. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19- and / or CD38-binding component comprises an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab'), a single-domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19- or CD38-binding component comprises an immunoglobulin heavy and light chain pair. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19- and CD38-binding component comprises an immunoglobulin heavy and light chain pair.

[0029] In some embodiments, provided is a CD38-binding component comprising an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. and the CD19-binding component comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:3, and an immunoglobulin light chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:4, and / or the CD19-binding component comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:1, and an immunoglobulin light chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:4.In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3 or 5, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 4, and / or the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 1 or 6, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0030] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, which is a common light chain bispecific IgG. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an immunoglobulin heavy chain comprising an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. and wherein the CD19-binding component comprises an immunoglobulin heavy chain comprising an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, and wherein the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3 or 5, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4, and / or the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 7, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 2.

[0031] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:3 or 5, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:4, and the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:1 or 7, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component or the CD38-binding component comprises an scFv. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component comprises an scFv. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an scFv. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component or the CD38-binding component comprises an immunoglobulin heavy chain / light chain pair. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component comprises an immunoglobulin heavy / light chain pair. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an immunoglobulin heavy / light chain pair.

[0032] Further provided is a complex-binding molecule comprising a CD38 antigen-binding component that binds to CD38, the CD38 antigen-binding component comprising an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and a CD19 antigen-binding component that binds to CD19, the CD38 antigen-binding component comprising an anti-CD19 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, wherein the CD38 antigen-binding component comprises: a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-75; b) a heavy chain complementarity-determining region 2 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155. a) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; b) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; c) a heavy chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and / or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125.

[0033] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19 antigen-binding component comprises: g) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11-15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21-25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31-35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101-105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111-115; and / or l) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121-125.

[0034] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38 antigen-binding component comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3 or 5, and an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38 antigen-binding component comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 3 or 5, and the immunoglobulin light chain variable region comprises an amino acid sequence identical to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19 antigen-binding component comprises an anti-CD19 immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 6, and an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4.

[0035] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD19 antigen-binding component comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 1 or 6, and the immunoglobulin light chain variable region comprises an amino acid sequence identical to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises a first immunoglobulin heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD38 immunoglobulin light chain variable region further comprises an immunoglobulin light chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD19 immunoglobulin heavy chain variable region further comprises a second immunoglobulin heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the first immunoglobulin heavy chain constant region and / or the second immunoglobulin heavy chain constant region comprises one or more amino acid substitutions that disfavor homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote heterodimerization of the first heavy chain constant region and the second heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein one of the first or second immunoglobulin heavy chain constant regions comprises a T366W substitution (EU numbering) and the other of the first or second immunoglobulin heavy chain constant region comprises a T366S / L368A / Y407V substitution (EU numbering), such that heterodimerization of the first and second immunoglobulin heavy chain constant regions is promoted compared to homodimerization of the first or second immunoglobulin heavy chain constant region. In some embodiments, provided is the complex binding molecule of any of the preceding embodiments, wherein the single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component.

[0036] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0037] The novel features of the invention are set forth with precision in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 illustrates the structure of a common light chain bispecific IgG. [Figure 2] FIG. 1 illustrates the structure of Fab-Fc:scFv-Fc bispecific IgG. [Figure 3] FIG. 1 illustrates the structure of Fab-Fc-Fab:Fc bispecific IgG. [Figure 4] FIG. 1 illustrates the structure of Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG. [Figure 5] FIG. 1 illustrates the structure of Fab-Fc-scFv:Fc bispecific IgG. [Figure 6] FIG. 1 illustrates the structure of Fab-Fc-Fab:Fab-Fc bispecific IgG. [Figure 7] FIG. 1 illustrates the structure of scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG. [Figure 8] FIG. 1 illustrates the structure of Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG. [Figure 9] FIG. 1 illustrates the structure of Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG. [Figure 10] FIG. 1 illustrates the structure of Fab-Fc-scFv:Fab-Fc bispecific IgG. [Figure 11]FIG. 1 illustrates the structure of scFv-Fab-Fc:Fc bispecific IgG. [Figure 12A] Figure 12 shows binding data for CD19 and CD38 antibodies. Figure 12A shows cell surface expression of CD19 and CD38. Figures 12B and 12C show binding profiles for CD19 and CD38 antibodies. Figures 12D and 12E show binding of CD19 and CD38 controls. Figure 12F shows the binding profile for cells that do not express CD19 or CD38. [Figure 12B] Figure 12 shows binding data for CD19 and CD38 antibodies. Figure 12A shows cell surface expression of CD19 and CD38. Figures 12B and 12C show binding profiles for CD19 and CD38 antibodies. Figures 12D and 12E show binding of CD19 and CD38 controls. Figure 12F shows the binding profile for cells that do not express CD19 or CD38. [Figure 12C] Figure 12 shows binding data for CD19 and CD38 antibodies. Figure 12A shows cell surface expression of CD19 and CD38. Figures 12B and 12C show binding profiles for CD19 and CD38 antibodies. Figures 12D and 12E show binding of CD19 and CD38 controls. Figure 12F shows the binding profile for cells that do not express CD19 or CD38. [Figure 12D] Figure 12 shows binding data for CD19 and CD38 antibodies. Figure 12A shows cell surface expression of CD19 and CD38. Figures 12B and 12C show binding profiles for CD19 and CD38 antibodies. Figures 12D and 12E show binding of CD19 and CD38 controls. Figure 12F shows the binding profile for cells that do not express CD19 or CD38. [Figure 12E] Figure 12 shows binding data for CD19 and CD38 antibodies. Figure 12A shows cell surface expression of CD19 and CD38. Figures 12B and 12C show binding profiles for CD19 and CD38 antibodies. Figures 12D and 12E show binding of CD19 and CD38 controls. Figure 12F shows the binding profile for cells that do not express CD19 or CD38. [Figure 12F]Figure 12 shows binding data for CD19 and CD38 antibodies. Figure 12A shows cell surface expression of CD19 and CD38. Figures 12B and 12C show binding profiles for CD19 and CD38 antibodies. Figures 12D and 12E show binding of CD19 and CD38 controls. Figure 12F shows the binding profile for cells that do not express CD19 or CD38. [Figure 13A] FIG. 1 shows antibody binding data to Daudi cells. [Figure 13B] FIG. 1 shows antibody binding data to Daudi cells. [Figure 14A] FIG. 1 shows binding data of antibodies to REH cells. [Figure 14B] FIG. 1 shows binding data of antibodies to REH cells. [Figure 15A] FIG. 1 shows antibody binding data to CD19-transfected HEK293 cells. [Figure 15B] FIG. 1 shows antibody binding data to CD19-transfected HEK293 cells. [Figure 16A] FIG. 1 shows antibody binding data to CD38-transfected HEK293 cells. [Figure 16B] FIG. 1 shows antibody binding data to CD38-transfected HEK293 cells. [Figure 17A] FIG. 1 shows antibody binding data to non-transfected CHO cells. [Figure 17B] FIG. 1 shows antibody binding data to non-transfected CHO cells. [Figure 18A] FIG. 1 shows data on direct apoptosis in Daudi cells for antibody test articles. [Figure 18B] FIG. 1 shows data on direct apoptosis in Daudi cells for antibody test articles. [Figure 19A] FIG. 1 shows data on crosslinking-induced apoptosis in Daudi cells for antibody test articles. [Figure 19B] FIG. 1 shows data on crosslinking-induced apoptosis in Daudi cells for antibody test articles. [Figure 20A] FIG. 1 shows ADCC data for three donors across antibody specimens. [Figure 20B] FIG. 1 shows ADCC data for three donors across antibody specimens. [Figure 20C] FIG. 1 shows ADCC data for three donors across antibody specimens. [Figure 21A] FIG. 1 shows ADCC data for three donors across antibody specimens. [Figure 21B] FIG. 1 shows ADCC data for three donors across antibody specimens. [Figure 21C] FIG. 1 shows ADCC data for three donors across antibody specimens. [Figure 22A] FIG. 1 shows CDC profiles across test articles. [Figure 22B] FIG. 1 shows CDC profiles across test articles. [Figure 23] FIG. 1 shows ADCP data across antibody specimens. [Figure 24] FIG. 1 shows RBC binding data across antibody specimens. [Figure 25A] FIG. 1 shows hemagglutination profiles for antibody specimens. [Figure 25B] FIG. 1 shows hemagglutination profiles for antibody specimens. [Figure 26] FIG. 1 shows hemolysis data across antibody specimens. DETAILED DESCRIPTION OF THE INVENTION

[0039] Immunosuppressive B cell populations that suppress anti-tumor immune responses can generally be defined by the presence of more than one cell surface biomarker. Therefore, therapeutic agents that effectively and specifically target immunosuppressive B cells can be used to prevent and / or remove immunosuppression in, adjacent to, or surrounding tumors, or within the tumor environment. Provided herein are complex-binding molecules that target immunosuppressive B cells. Also provided are complex-binding molecules comprising a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, wherein the first target comprises a B cell lineage surface marker and the second target comprises an inhibitory B cell surface marker. Disclosed herein are multivalent antibodies that specifically bind to B cell populations associated with negative modulation of anti-tumor responses or immunosuppression. Immunosuppressive B cells can comprise or be defined by the cell surface biomarkers CD19 and CD38. The bispecific antibodies provided herein can target both CD19 and CD38 to inhibit the function of immunosuppressive B cells. In certain cases, the function of immunosuppressive B cells includes the release of IL-10, IL-35, TGF-beta, or a combination thereof. Multivalent or bispecific antibodies targeting CD19 and CD38 can also be used to treat tumorigenic conditions and / or cancers associated with immunosuppressive B cells and / or immune dysfunction.

[0040] The terms "immunosuppression" or "immunodepression" or "negative immune modulation," as used herein, refer to a reduction or suppression of immune system function; i.e., immunosuppression generally refers to a state in which immune system function is reduced or absent. In certain cases, immunosuppression generally refers to a state in which immune system function is reduced or absent against a tumor or within, surrounding, or adjacent to the tumor microenvironment. The overall immune response may be suppressed, the immune response within a local or specific area may be reduced, or specific populations of immunologically active lymphocytes may be selectively affected. Antigen-specific immune suppression may be the result of the deletion or suppression of specific populations of antigen-specific cells, or the result of enhanced modulation of the immune response by antigen-specific suppressor cells. Reference to immunosuppressive B cells refers to B cells or B cell populations that exert negative modulation on the immune response and may be identified by specific surface markers associated with such populations, such as CD38. In certain instances, immunosuppression can be identified by the presence or release of IL-10, IL-35, TGF-beta, or a combination thereof. In certain instances, immunosuppression can be identified by the presence or release of IL-10, IL-35, TGF-beta, or a combination thereof by B cells.

[0041] As used herein, the term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell proliferation.Cancer can also include, but is not limited to, blood tumors and / or solid tumors.Cancer can refer to diseases of the blood, bones, organs, skin tissues and vascular system, including, but not limited to, bladder, blood, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, kidney, liver, lung, lymph nodes, mouth, cervix, ovary, pancreas, prostate, rectum, kidney, skin, stomach, testicles, pharynx and uterus. Specific cancers include leukemia (acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, mature B-cell tumors (small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's giant spheroid tumor)), and ball), proteinemia or indolent lymphoma), splenic marginal zone lymphoma, plasma cell myeloma, plasma cell leukemia, plasmacytoma, peri-implant immunoglobulin deposition, heavy chain disease, extranodal marginal zone B-cell lymphoma, MALT lymphoma), nodal marginal zone B-cell lymphoma (NMZL), gastrointestinal tumors (e.g., gastrointestinal stromal tumor (GIST)), follicular lymphoma, mantle cell lymphoma / leukemia, diffuse B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and Burkitt's lymphoma, mature T-cell and natural killer cell (NK) tumors (prolymphocytic leukemia, T-cell large lymphocytic leukemia, leukemia, invasive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathic T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides (Sézary syndrome), primary skin degenerative large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified, and degenerative large cell lymphomalymphoma), Hodgkin's lymphoma (nodular sclerosis, mixed cell type, lymphocyte-rich, lymphocyte-depleted or non-lymphocyte-reduced, nodular lymphocytic), myeloma (multiple myeloma, inert myeloma, smoldering myeloma), chronic myeloproliferative disorders, myelodysplastic syndromes / myeloproliferative disorders, myelodysplastic syndromes, lymphoproliferative disorders associated with immunodeficiency, histiocytic and dendritic cell neoplasms, leukocytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma-of-bone disease, osteosarcoma, breast cancer (hormone-dependent and non-hormone-dependent), gynecological cancers (pediatric cervix, endometrium, fallopian tube, gestational trophoblastic disease, ovary, peritoneum, uterus, vagina, and vulva), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, hairy cell astrocytoma, embryonic hair growth neuroepithelial neoplasia neoplasia, oligodendroglioma, ependymoma, glioblastoma multiforme, mixed glioma, oligodendroglial astrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, embryonal tissue tumor, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastric-entero-pancreatic or gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, colorectal cancer (knot rectal cancer), invasive neuroendocrine tumor, leiomyosarcoma, mucinous adenocarcinoma, signet ring cell adenocarcinoma, hepatocellular carcinoma, hepatobiliary liver cancer These include, but are not limited to, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung cancer (NSCLC) (squamous cell lung cancer, adenocarcinoma, large cell lung cancer), small cell lung cancer, thyroid cancer, prostate cancer (hormone refractory, non-androgen dependent sex, androgen dependent, hormone insensitive), renal cell carcinoma, and soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraskeletal osteosarcoma).

[0042] The term "CD19" or "cluster of differentiation 19" (also known as B4, T cell surface antigen Leu-12, and CVID3) refers to a B cell lineage surface biomarker or transmembrane protein that in humans is encoded by the gene CD19. CD19 can function as a coreceptor for the B cell antigen receptor complex (BCR) on B lymphocytes, lowering the threshold for activation of downstream signaling pathways and triggering a B cell response to antigen. Structurally, the CD19 amino acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over a sequence length of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids, or over the entire length of the polypeptide, to the amino acid sequence of, for example, GenBank Accession No. NM_001178098.2→NP_001171569.1 or NM_001770.6→NP_001761.3. Structurally, the CD19 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 300, 500, 750, 1000, 1250, 1500 nucleic acids, or over the entire length of the polynucleotide, to the nucleic acid sequence of, for example, GenBank Accession No. NG_007275.1 or NCBI Gene ID 930. Sequence alignment can be performed using any alignment algorithm known in the art, for example, BLAST, ALIGN set to default settings.

[0043] The term "CD38" or "cluster of differentiation 38" (also known as ADPRC1) refers to a B cell surface biomarker or transmembrane protein encoded by the gene CD38 in humans. CD38 can function in B cell signaling leading to cell activation and proliferation. Structurally, the CD38 amino acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 50, 100, 150, 200, 250 amino acids, or over the entire length of the polypeptide, with, for example, the amino acid sequence of GenBank Accession No. NM_001775.4→NP_001766.2. Structurally, the CD38 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 300, 500, 750 nucleic acids, or over the entire length of the polynucleotide, to the nucleic acid sequence of, for example, GenBank Accession No. NC_000004.12 or NCBI Gene ID 952. Sequence alignment can be performed using any alignment algorithm known in the art, for example, BLAST, ALIGN set to default settings.

[0044] The term "antibody" is used herein in the broadest sense and includes multivalent or bispecific antibodies and monoclonal antibodies, such as intact antibodies and functional (antigen-binding) antibody fragments thereof, such as fragments, antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, such as single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, e.g., antibodies of any class or subclass, e.g., IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgG1 constant region. The antibody can comprise a human IgG4 constant region.

[0045] Among the antibodies provided are multispecific or multivalent antibodies (e.g., bispecific and polyreactive antibodies) and antibody fragments thereof. Antibodies include antibody conjugates and antibody-containing molecules, such as chimeric molecules. Thus, antibodies include full-length and native antibodies, as well as fragments and portions thereof that retain their binding specificity, including any specific-binding portion thereof, including those of any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), and biologically relevant (antigen-binding) fragments or specific-binding portions thereof, such as, but not limited to, Fab, F(ab'), Fv, and scFv (single-chain or related entities). Monoclonal antibodies generally reside in a substantially homogeneous antibody composition, such that any individual antibody contained within a monoclonal antibody composition is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies can include a human IgG1 constant region or a human IgG4 constant region.

[0046] The terms "complementarity-determining region," synonymous with "hypervariable region" or "HVR," and "CDR" are known in the art and refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known in the art and refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70 (the "Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modeling antibodies on the WEB", Protein Eng. 2000 Dec;13(12):819-24 (the "AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0047] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat scheme and the Chothia scheme numbering are based on the most common antibody region sequence length, and insertions are applied by inserting letters, such as "30a", and deletions appear in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0048] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). H or V L The V domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen may have a V domain from the antibody that binds to the antigen. H or V L Each complementary V domain is used L or V HThey may also be isolated by screening libraries of domains (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0049] Among the antibodies provided are antibody fragments. "Antibody fragment" can refer to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv or sFv), and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, containing a variable heavy chain region and / or a variable light chain region. Antibody fragments can be produced by various techniques, including, but not limited to, proteolytic digestion of an intact antibody and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment containing a non-naturally occurring construct, such as two or more antibody regions or chains joined by a synthetic linker, e.g., a polypeptide linker, and / or not produced by enzymatic digestion of a naturally occurring intact antibody.

[0050] As used herein, molecules, peptides, polypeptides, antibodies, or antibody fragments may be referred to as "bispecific" or "bispecific," including grammatical equivalents. Bispecific molecules have the ability to specifically bind to at least two structurally distinct targets. Specific binding can occur through two distinct binding moieties that are structurally distinct at the molecular level, including, but not limited to, distinct, non-identical amino acid sequences, or through high affinity (e.g., about 1x10 -6This may be the result of a single binding moiety capable of specifically binding to two structurally distinct targets with a KD of less than 0.01. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as "multispecific" refers to a molecule having the ability to specifically bind to at least three structurally distinct targets. A "bispecific antibody," including grammatical equivalents, refers to a bispecific molecule that preserves at least one fragment of an antibody, e.g., a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule, that is capable of specifically binding to a target. A "multispecific antibody," including grammatical equivalents, refers to a multispecific molecule that preserves at least one fragment of an antibody, e.g., a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule, that is capable of specifically binding to a target.

[0051] A "linker" as referred to herein is also referred to as a "linker sequence," "spacer," "tethering sequence," or grammatical equivalents thereof. A "linker," as referred to herein, connects two distinct molecules, e.g., two distinct binding moieties or heavy / light chain pairs, that themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides. Numerous strategies can be used to covalently link molecules. These include, but are not limited to, a polypeptide linkage between the N- and C-termini of a protein or protein domain, linkage via a disulfide bond, and linkage via a chemical cross-linking reagent. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant technology or peptide synthesis. The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of sufficient length to link the two molecules so that they adopt the correct conformation relative to each other and thereby retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length or about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used. Useful linkers include glycine-serine polymers, such as (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Exemplary linkers for linking antibody fragments or single-chain variable fragments can include AAEPKSS, AAEPKSSDKTHTCPPCP, GGGG, or GGGGDKTHTCPPCP. Alternatively, various nonproteinaceous polymers, such as, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may have utility as linkers.

[0052] A "fragment-based" bispecific antibody or a bispecific antibody comprising a "single-chain variable fragment" or "scFv" of the present disclosure can refer to a single-chain antibody, or a fragment thereof, comprising two binding moieties and a linker connecting the two binding moieties. The linker may be a polypeptide linker or other linker of suitable flexibility that does not interfere with the binding of either targeting moiety. Fragment-based bispecific antibody formats include tandem V HH These fragment-based antibodies include antibodies, tandem scFvs, scFv-Fabs, F(ab)2s, dual affinity retargeting antibodies (DARTs), etc. Such fragment-based antibodies can be further manipulated to include additional binding moieties with specificity for a given target, such as A2:B1, A1:B2, or A2:B2, or with fragments of the Fc region to improve pharmacokinetics or promote ADCC, ADCP, or CDC.

[0053] "Binding portion" refers to the portion of a molecule, peptide, polypeptide, antibody, or antibody fragment that mediates specific binding to a specified target or antigen or epitope. By way of example, the binding portion of an antibody may comprise a heavy / light chain variable region pair or one or more complementarity determining regions (CDRs).

[0054] As referred to herein, a "target" refers to the portion of a molecule that associates with the binding portion of a molecule, peptide, polypeptide, antibody, or antibody fragment. A target can include an amino acid sequence and / or a carbohydrate, lipid, or other chemical entity. An "antigen" is a target that includes a portion that can be bound by an adaptive immune molecule, such as an antibody or antibody fragment, a B-cell receptor, or a T-cell receptor.

[0055] The "valency" of a bispecific or multispecific molecule refers to the number of targets that the described molecule, peptide, polypeptide, antibody, or antibody fragment can bind to. For example, a monovalent molecule can bind to one molecule of a specific target, a bivalent molecule can bind to two molecules, and a tetravalent molecule can bind to four targets. For example, a bispecific bivalent molecule is a molecule that can bind to two targets and two structurally different targets. For example, when a bispecific bivalent molecule is contacted with a solution containing target A and target B, it can bind to A2, B2, or A:B.

[0056] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody can optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized" non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0057] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a non-human source using a human antibody repertoire, including a human antibody library, or other human antibody coding sequence. The term excludes humanized versions of non-human antibodies containing non-human antigen-binding regions, e.g., those in which all or substantially all CDRs are non-human. Human antibodies may be prepared by administering an immunogen to transgenic animals engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Human antibodies may also be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody coding sequences from the human repertoire.

[0058] "ADCC" or "antibody-dependent cell-mediated cytotoxicity," as used herein, refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, subsequently causing lysis of the target cells. ADCC can be correlated with binding to FcγRIIIa, and increased binding to FcγRIIIa leads to an increase in ADCC activity. "ADCP" or antibody-dependent cell-mediated phagocytosis, as used herein, can refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, subsequently causing phagocytosis of the target cells.

[0059] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, such as linkers and binding peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, a polypeptide can contain modifications to the native or native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts producing the protein or errors due to PCR amplification.

[0060] The percent sequence identity (%) to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared, can be determined. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, together with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: multiply the ratio X / Y by 100 (where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B). It is recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0061] Amino acid sequence variants of the antibodies provided herein can be envisioned and envisioned. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above-described polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of numerous known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody; amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics, e.g., antigen binding. Antibody variants with one or more amino acid substitutions can be provided. Sites of interest for substitutional mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into an antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0062] The present disclosure also provides "immunoconjugates" or "antibody conjugates" or "antibody-drug conjugates," which refer to antibodies conjugated to one or more heterologous molecules. For example, an immunoconjugate can comprise an antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, protein domains, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. In some embodiments, an immunoconjugate can comprise a complex-binding molecule disclosed herein, or a fragment thereof (e.g., an scFv).

[0063] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, nucleic acids are components of vectors that can be used to transfer a polynucleotide encoding a polypeptide into cells. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a genome-integrating vector, or "integrating vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. Vectors capable of directing the expression of operably linked genes are referred to herein as "expression vectors." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and viral vectors. In expression vectors, regulatory elements such as promoters, enhancers, and polyadenylation signals for use in controlling transcription can be derived from mammalian, microorganism, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene for facilitating recognition of transformants may also be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses, may also be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors can contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or a limited set of sites in the genome. Additionally, vectors can contain sequences derived from transposable elements.

[0064] As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence compared to a reference sequence, may be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; modified in Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Such formula has been incorporated into the basic local alignment search tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent sequence homology may be determined using the most recent version of BLAST available as of the filing date of this application.

[0065] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise transfect suitable cells transgenic for the nucleic acid to enable production of the antibody for commercial or therapeutic use. Standard cell lines and methods for large-scale antibody production from cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing antibodies, such as Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In certain embodiments, the antibody-encoding nucleic acid is integrated into a genomic locus of a cell useful for producing the antibody. In certain embodiments, described herein is a method of producing an antibody, comprising culturing cells containing an antibody-encoding nucleic acid under in vitro conditions sufficient to allow for the production and secretion of the antibody.

[0066] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease for which the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0067] As used herein, the term "about" when used to modify a specific number refers to that number plus or minus 10% of that number. The term "about" when used to modify a range refers to a range extending from minus 10% of the minimum value to plus 10% of the maximum value.

[0068] As used herein, the term "treatment" or "treating" refers to a pharmaceutical or other intervention regimen used to obtain beneficial or desired results in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or preventative benefit. Therapeutic benefit can refer to the eradication or amelioration of symptoms or the underlying disorder being treated. Therapeutic benefit can also be achieved with the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in a subject, even though the subject may still be suffering from the underlying disorder. A preventative effect includes delaying, preventing, or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping, or reversing the progression of a disease or condition, or any combination thereof. For preventative benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease can receive treatment even if the disease has not been diagnosed. Those skilled in the art will recognize that not all of a given population of individuals potential for treatment will respond, or will respond equally, to the treatment; such individuals will be considered treated.

[0069] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0070] bispecific molecule Provided herein are bispecific, multivalent, or complex-binding molecules comprising a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, wherein the first target comprises a B cell lineage surface marker and the second target comprises an inhibitory B cell surface marker. The immunosuppressive B cells or B cell population can comprise a B cell lineage surface biomarker and an inhibitory B cell surface biomarker. The B cell lineage surface marker can comprise CD19, CD138, IgA, or CD45. The immunosuppressive B cell surface marker can comprise IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38. In certain embodiments, the complex binding molecule binds to CD38 and CD19.

[0071] Multivalent, bispecific, or complex-binding molecules have the ability to specifically bind to at least two structurally distinct targets. Specific binding can be the result of two distinct binding moieties that are structurally distinct at the molecular level, including, but not limited to, distinct, non-identical amino acid sequences, or a single binding moiety that can specifically bind to two structurally distinct targets. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as "multispecific," "multivalent," or "bispecific" can refer to a molecule capable of specifically binding to at least two structurally distinct targets. In some embodiments, the first or second binding component of a complex-binding molecule comprises a polypeptide. In certain embodiments, the first or second binding component consists of a polypeptide. In some embodiments, the first and second binding components of a complex-binding molecule comprise a polypeptide. In certain embodiments, the first and second binding components consist of a polypeptide. In certain embodiments, the polypeptide of the first or second binding component comprises an amino acid sequence at least 100 amino acid residues in length. In certain embodiments, the polypeptides of the first and second binding moieties comprise amino acid sequences at least 100 amino acid residues in length.

[0072] A bispecific molecule can be a bispecific antibody that preserves at least one fragment of an antibody capable of specifically binding to a target, such as a variable region, a heavy or light chain, or one or more complementarity-determining regions from an antibody molecule. In some embodiments, the complex-binding molecules described herein are bispecific antibodies and / or dual antigen-binding fragments thereof. Bispecific antibodies have the ability to bind to two structurally distinct targets or antigens. In some embodiments, a bispecific antibody comprises a first binding component configured to bind to a first target and a second binding component configured to bind to a second target, wherein the first target comprises a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45), and the second target comprises an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0073] The immunosuppressive B cells or immunosuppressive B cell population can comprise the cell surface biomarkers CD19 and CD38. Further disclosed herein are bispecific antibodies that target CD19 and CD38. In some embodiments, the CD19-binding component comprises a variable heavy chain (VH) comprising SEQ ID NO: 1. In certain embodiments, the CD19-binding component comprises a VH CDR1 region comprising any one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In certain embodiments, the CD19-binding component comprises a VH CDR2 region comprising any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. In certain embodiments, the CD19-binding component comprises a VH CDR3 region comprising any one of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.

[0074] In some embodiments, the CD19 binding component comprises a variable light chain (VL) comprising SEQ ID NO: 2. In certain embodiments, the CD19 binding component comprises a VL CDR1 region comprising any one of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 45. In certain embodiments, the CD19 binding component comprises a VL CDR2 region comprising any one of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. In certain embodiments, the CD19 binding component comprises a VL CDR3 region comprising any one of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65.

[0075] In some embodiments, the bispecific antibody comprises a first binding component, and the first binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35, an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41 to 45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51 to 55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61 to 65.

[0076] In some embodiments, the bispecific antibody comprises a CD19 binding component, wherein the CD19 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 amino acid sequence set forth in SEQ ID NO: 21, the HCDR3 amino acid sequence set forth in SEQ ID NO: 31, the LCDR1 amino acid sequence set forth in SEQ ID NO: 41, the LCDR2 amino acid sequence set forth in SEQ ID NO: 51, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO: 61.

[0077] In some embodiments, the bispecific antibody comprises a CD19 binding component, and the first binding component for CD19 comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 amino acid sequence set forth in SEQ ID NO: 22, the HCDR3 amino acid sequence set forth in SEQ ID NO: 32, the LCDR1 amino acid sequence set forth in SEQ ID NO: 42, the LCDR2 amino acid sequence set forth in SEQ ID NO: 52, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO: 62.

[0078] In some embodiments, the bispecific antibody comprises a CD19 binding component, wherein the CD19 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 15, the HCDR2 amino acid sequence set forth in SEQ ID NO: 25, the HCDR3 amino acid sequence set forth in SEQ ID NO: 35, the LCDR1 amino acid sequence set forth in SEQ ID NO: 45, the LCDR2 amino acid sequence set forth in SEQ ID NO: 55, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO: 65.

[0079] In some embodiments, the CD19 binding component comprises variable heavy and light chains or CDRs corresponding to or derived from inebilizumab, tafasitamab, taplitumomab, obexelimab, blinatumomab, coltuximab, denintuzumab, or loncastuximab, MOR208, MEDI-551, XmAb 5871, MDX-1342, or AFM11.

[0080] In some embodiments, the CD38 binding component comprises a variable heavy chain (VH) comprising SEQ ID NO: 3. In certain embodiments, the CD19 binding component comprises a VH CDR1 region comprising any one of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 75, or SEQ ID NO: 75. In certain embodiments, the CD19 binding component comprises a VH CDR2 region comprising any one of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 85. In certain embodiments, the CD19 binding component comprises a VH CDR3 region comprising any one of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95.

[0081] In some embodiments, the CD38 binding component comprises a variable light chain (VL) comprising SEQ ID NO: 4. In certain embodiments, the CD19 binding component comprises a VL CDR1 region comprising any one of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 105. In certain embodiments, the CD19 binding component comprises a VL CDR2 region comprising any one of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In certain embodiments, the CD19 binding component comprises a VL CDR3 region comprising any one of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 125.

[0082] In some embodiments, the bispecific antibody comprises a CD38 binding component, wherein the CD38 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:71, the HCDR2 amino acid sequence set forth in SEQ ID NO:81, the HCDR3 amino acid sequence set forth in SEQ ID NO:91, the LCDR1 amino acid sequence set forth in SEQ ID NO:101, the LCDR2 amino acid sequence set forth in SEQ ID NO:111, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:121.

[0083] In some embodiments, the bispecific antibody comprises a CD38 binding component, wherein the CD38 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:72, the HCDR2 amino acid sequence set forth in SEQ ID NO:82, the HCDR3 amino acid sequence set forth in SEQ ID NO:92, the LCDR1 amino acid sequence set forth in SEQ ID NO:102, the LCDR2 amino acid sequence set forth in SEQ ID NO:112, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:122.

[0084] In some embodiments, the bispecific antibody comprises a CD38 binding component, wherein the CD38 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:75, the HCDR2 amino acid sequence set forth in SEQ ID NO:85, the HCDR3 amino acid sequence set forth in SEQ ID NO:95, the LCDR1 amino acid sequence set forth in SEQ ID NO:105, the LCDR2 amino acid sequence set forth in SEQ ID NO:115, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:125.

[0085] In some embodiments (e.g., any of the preceding embodiments), the CDR-H2 of the CD38 binding component comprises amino acid residues P(X1)LG(X2)A, where X1 and X2 tolerate amino acid substitutions while maintaining binding to CD38. In certain embodiments, X1 and X2 are selected from amino acids that reduce the hydrophobicity of the CDRH2 amino acid sequence. In certain embodiments, amino acids that reduce hydrophobicity include H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T.

[0086] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:3, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:4, and the CD19-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:2.

[0087] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO:3, and the VL comprises an amino acid sequence identical to SEQ ID NO:4, and the CD19-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO:1, and the VL comprises an amino acid sequence identical to SEQ ID NO:2.

[0088] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 215, or 218-223, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 4 or 223, and the CD19-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 201, or 216-217, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the CD19 binding component comprises a VH amino acid sequence comprising substitutions at A84 and A108, hi some embodiments, the substitutions comprise A84S and A108L.

[0089] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO: 3, 215, or 218-223, and the VL comprises an amino acid sequence identical to SEQ ID NO: 4 or 223, and the CD19-binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO: 1, 201, 216-217, and the VL comprises an amino acid sequence identical to SEQ ID NO: 2. In some embodiments, the CD19-binding component comprises a VH amino acid sequence comprising substitutions at A84 and A108. In some embodiments, the substitutions comprise A84S and A108L.

[0090] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:71, the HCDR2 amino acid sequence set forth in SEQ ID NO:81, the HCDR3 amino acid sequence set forth in SEQ ID NO:91, the LCDR1 amino acid sequence set forth in SEQ ID NO:101, the LCDR2 amino acid sequence set forth in SEQ ID NO:111, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:121, and the CD19-binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:11, the HCDR2 amino acid sequence set forth in SEQ ID NO:21, the HCDR3 amino acid sequence set forth in SEQ ID NO:31, the LCDR1 amino acid sequence set forth in SEQ ID NO:41, the LCDR2 amino acid sequence set forth in SEQ ID NO:51, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:61.

[0091] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:72, the HCDR2 amino acid sequence set forth in SEQ ID NO:82, the HCDR3 amino acid sequence set forth in SEQ ID NO:92, the LCDR1 amino acid sequence set forth in SEQ ID NO:102, the LCDR2 amino acid sequence set forth in SEQ ID NO:112, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:122, and the CD19-binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:12, the HCDR2 amino acid sequence set forth in SEQ ID NO:22, the HCDR3 amino acid sequence set forth in SEQ ID NO:32, the LCDR1 amino acid sequence set forth in SEQ ID NO:42, the LCDR2 amino acid sequence set forth in SEQ ID NO:52, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:62.

[0092] In some embodiments, when the bispecific comprises a Fab or other structure requiring a light chain constant region for a bispecific format, the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% or identical to any one of SEQ ID NOs: 210 and / or 211. In some embodiments, when the bispecific comprises a Fab or other structure requiring a light chain constant region for a bispecific format, the VL comprises an amino acid sequence identical to any one of SEQ ID NOs: 210 and / or 211.

[0093] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, wherein the CD38-binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:75, the HCDR2 amino acid sequence set forth in SEQ ID NO:85, the HCDR3 amino acid sequence set forth in SEQ ID NO:95, the LCDR1 amino acid sequence set forth in SEQ ID NO:105, the LCDR2 amino acid sequence set forth in SEQ ID NO:115, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:125, and the CD19-binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:15, the HCDR2 amino acid sequence set forth in SEQ ID NO:25, the HCDR3 amino acid sequence set forth in SEQ ID NO:35, the LCDR1 amino acid sequence set forth in SEQ ID NO:45, the LCDR2 amino acid sequence set forth in SEQ ID NO:55, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:65.

[0094] In some embodiments, the CD38 binding component comprises variable heavy and light chains or CDRs corresponding to or derived from daratumumab or isatuximab.

[0095] Substitutions, insertions, or deletions may occur within one or more CDRs, and the substitutions, insertions, or deletions do not substantially reduce the binding of the antibody to the antigen. For example, conservative substitutions that do not substantially reduce binding affinity may be made in the CDRs. Such changes may be outside the CDR "hot spots." Variant V H and V LIn some embodiments of the sequence, each CDR is unaltered. Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion of an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody. An example of an intrasequence insertional variant of an antibody molecule is an insertion of three amino acids in the light chain. An example of a terminal deletion is an antibody with a deletion of seven or fewer amino acids at the end of the light chain.

[0096] Alterations (e.g., substitutions) may be made in CDRs, for example, to improve antibody affinity. Such alterations may be made in CDRs encoding codons with high mutation rates during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting variants may be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) may be used to improve antibody affinity (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178:1-37 (2001)). CDR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, e.g., Cunningham and Wells Science 244:1081-1085 (1989)). CDR-H3 and CDR-L3 are particularly often targeted.Alternatively, or in addition, there is a crystal structure of the antigen-antibody complex to identify the contact points between antibody and antigen.Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution.Variants can be screened to determine whether they contain desired properties.

[0097] Antibodies can be altered to increase or decrease their glycosylation (e.g., by altering the amino acid sequence to create or remove one or more glycosylation sites). The carbohydrate attached to the Fc region of the antibody may also be altered. Native antibodies from mammalian cells typically have a nucleotide sequence at the Asn of the CH2 domain of the Fc region. 297(See, e.g., Wright et al., TIBTECH 15:26-32 (1997)). Oligosaccharides can be various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, or fucose attached to a GlcNAc in the base of the biantennary oligosaccharide structure. Modification of the oligosaccharides in antibodies can be performed, for example, to generate antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by the ratio of the amount of fucose attached to Asn297 relative to the sum of all glycan structures attached to Asn297. 297 The average amount of fucose in the glycan is determined by calculating the average amount of fucose in the glycan (see, for example, WO 08 / 077546). 297 refers to the asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region (see, e.g., Edelman et al., Proc Natl Acad Sci U S A. 1969 May; 63(1):78-85). However, Asn 297may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function (see, e.g., Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004)). Cell lines, e.g., knockout cell lines, and methods for their use can be used to produce defucosylated antibodies, e.g., Lec13 CHO cells deficient in protein fucosylation and alpha-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)). Other antibody glycosylation variants are also included (see, e.g., U.S. Patent No. 6,602,684).

[0098] In some embodiments, the complex binding molecules provided herein have an affinity for the antibody target of about 10 μM, 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D The target of the antibody can be a CD19 target, a CD38 target, or a target that includes both CD19 and CD38. Dcan be measured by any suitable assay. In certain embodiments, KD can be measured using a surface plasmon resonance assay (e.g., using a BIACORE®-2000 or BIACORE®-3000 or Octet).

[0099] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. As used herein, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0100] In some cases, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), which result in target cell killing, albeit through different mechanisms. Thus, in some embodiments, the antibodies described herein comprise a variable domain of the invention combined with a constant domain comprising a different Fc region selected based on the biological activity of the antibody for its intended use. In certain cases, human IgG can be classified into, for example, four subclasses, IgG1, IgG2, IgG3, and IgG4, each of which comprises an Fc region with a unique profile for binding to one or more of the Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32), FcγRIIIA and FcγRIIIB (CD16), and the inhibitory receptor FcγRIIB) and the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors, whereas IgG2 binds to FcγRIIA H131, and at lower affinity FcγRIIA R131 , FcγRIIIA V158 IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 The inhibitory receptor FcγRIIB binds to IgG1, IgG2, and IgG3, whereas the inhibitory receptor FcγRIIB has lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to either IgG2 or IgG4. Ibid. Generally, with regard to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2.

[0101] In certain embodiments, the anti-CD19 or anti-CD38 variable regions described herein are linked to an Fc that binds to one or more activating Fc receptors (FcγRI / CD64, FcγRIIa / CD32, or FcγRIIIa / CD16), thereby stimulating ADCC and, in some cases, causing target depletion. In certain embodiments, the anti-CD19 or anti-CD38 variable regions described herein are linked to a human IgG1 or IgG3 Fc, i.e., the antibody is an IgG1 or IgG3 isotype. In some cases, modifications in the Fc region generate Fc variants with (a) increased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased complement-mediated cytotoxicity (CDC), (c) increased affinity for C1q, and / or (d) increased affinity for Fc receptors, compared to the parent Fc. In some embodiments, the Fc region variants comprise at least one amino acid modification in the Fc region. Combinations of amino acid modifications are also useful. For example, the variant Fc region may comprise two, three, four, five, etc. substitutions, eg, at the unique Fc region positions identified herein.

[0102] In some embodiments, ADCC activity can be increased by modifying the Fc region. Regarding ADCC activity, in some cases, human IgG1 and IgG3 show increased ADCC activation compared to IgG4 and IgG2, so IgG1 or IgG3 constant domains are selected for use in antibodies where ADCC is desired, rather than IgG2 or IgG4. In some embodiments, IgG3 is selected for activating FcγRIIIA-expressing NK cells, monocytes, and macrophages. In certain cases, different IgG isotypes also exhibit differential CDC activity, with IgG3 and IgG1 showing greater CDC activation compared to IgG2 or IgG4. Alternatively, in some embodiments, the Fc region may comprise any of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 34 70, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, One or more amino acids at 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 (Kabat numbering) are modified to increase antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-mediated cytotoxicity (CDC), affinity for C1q, and / or affinity for Fcγ receptors. Non-limiting examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods may be used (eg, ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and natural killer (NK) cells.

[0103] The antibody can have an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., U.S. Patent Application Publication No. 2005 / 0014934). Such an antibody can include an Fc region with one or more substitutions that improve binding of the Fc region to FcRn, and can include an Fc region with substitutions at one or more of the following Fc region residues according to the EU numbering system: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988), U.S. Pat. Nos. 5,648,260 and 5,624,821, and WO 94 / 29351).

[0104] In some embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residues are present at accessible sites of the antibody. The reactive thiol group may be positioned at a site for conjugation to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region.

[0105] In some embodiments, the antibodies provided herein may be further modified to contain additional known and available non-proteinaceous moieties. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules.

[0106] Complex-binding molecules or bispecific antibodies can vary based on the binding moieties associated with these molecules, and several different formats are applicable and contemplated herein. Complex-binding molecules or bispecific antibodies can comprise antibody fragments, substantially intact antibodies, or combinations thereof. In some embodiments, the first or second binding component comprises an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab'), a single domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In certain embodiments, the first and second binding components comprise an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab'), a single domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In some embodiments, the first or second binding component comprises an immunoglobulin heavy and light chain pair. In certain embodiments, the first and second binding components comprise an immunoglobulin heavy and light chain pair. In some embodiments, the first or second binding component comprises an scFv. In certain embodiments, the first and second binding components comprise an scFv.

[0107] Bispecific antibodies according to the present disclosure comprise intact or substantially fully intact antibody molecules, and may be asymmetric or symmetric.

[0108] Asymmetric bispecific antibodies generally contain a heavy chain / light chain (HC / LC) pair from an antibody specific for target A and a HC / LC pair from an antibody specific for target B, creating a heterobifunctional antibody. Heterobifunctional antibodies such as these face the problem of non-productive formation of molecules during production. HC / LC-A:HC / LC-B is desirable, but is usually thermodynamically or statistically unfavorable from all possible combinations. Several schemes have been introduced to circumvent this problem. In some cases, the HC / LC pair from an antibody with specificity for A and the HC / LC pair from an antibody with specificity for B further contain mutations to the Fc region to increase the probability of forming an antibody with HC / LC-A:HC / LC-B. This can be achieved by engineering structural features that promote heterodimer formation between HC-A and HC-B, such as a "knob" in the Fc region of HC-A and a "hole" in the HC-B, or vice versa. Another scheme for promoting HC-A:HC-B heterodimers is to engineer amino acid residues in the FC portions of the HC-A and HC-B to contain charge pairs that promote electrostatic interactions between the HC-B and HC-A. Another scheme for addressing the chain association problem is to combine the variable region of one of the HC / LC pairs with a single-chain binding molecule (e.g., V HHThe goal is to replace one half of the molecule with a classical HC / LC pair and the other half contains the HC constant region fused or otherwise connected to a single-chain binding molecule. Further modifications are possible to facilitate proper HC / LC pairing, including engineering mutations into the HC and LC of either A or B to favor proper HC / LC pairing, and CrossMab technology, which utilizes the exchange of the corresponding constant regions of the HC / LC pair. Symmetric bispecific antibodies avoid the problem of chain association by not relying on the formation of heterobifunctional molecules. Examples include dual variable domain molecules containing stacked variable regions of different specificities, IgG-scFv molecules containing scFvs of different specificities fused to the c-terminus of the heavy chain of a classical antibody molecule, (scFV)4-FC (the Fc dimerizes to create a bispecific tetravalent molecule), DART-Fc, and two-in-one, among others, which contain two scFvs connected by the Fc region of an Ig.

[0109] The structure of the complex-binding molecule or bispecific antibody can be envisioned and designed to alter the functionality or binding properties of the complex-binding molecule or bispecific antibody (see, e.g., "Bispecific antibodies: a mechanistic review of the pipeline." Nat Rev Drug Discovery. 2019 Aug;18(8):585-608) (see, e.g., "The making of bispecific antibodies," MAbs. 2017 Feb-Mar;9(2):182-212). For example, the bispecific antibody may be selected from one of the following formats: common light chain bispecific IgG, Fab-Fc:scFv-Fc bispecific IgG, Fab-Fc-Fab:Fc bispecific IgG, Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG, Fab-Fc-scFv:Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc bispecific IgG, scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG, Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and Fab-Fc-scFv:Fab-Fc bispecific IgG.

[0110] Common light chain bispecific IgG Bispecific antibodies with a common light chain bispecific IgG structure can be used in the present invention. Figure 1 illustrates a bispecific antibody with a common light chain bispecific IgG structure. The structure includes a first and a second IgG heavy chain. Each heavy chain includes a VH, CH1, CH2, and CH3 domain. The first heavy chain includes a VH 102, a CH1 104, a CH2 106, and a CH3 108. The second heavy chain includes a VH 112, a CH1 114, a CH2 116, and a CH3 118. The common light chain bispecific IgG structure also includes a light chain including a VL domain 120 and a CL domain 122. Generally, the first heavy chain includes a sequence derived from the heavy chain of an antibody with a first specificity, and the second heavy chain includes a heavy chain from an antibody with a second specificity. The light chains paired with the first and second heavy chains can be identical and derived from the light chains of antibodies with either specificity or distinct specificities. A heavy chain can be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 130). A heavy chain can be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 134 and / or 136). A common light chain bispecific IgG structure can include first and second heavy chain molecules that further contain mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can prevent linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance, "knob" into "hole") or biochemically (e.g., electrostatic interactions). Exemplary knob-into-hole mutations can include T366W (EU numbering) in one heavy chain and T366S / L368A / Y407V (EU numbering) in the second heavy chain. Exemplary mutations that facilitate linkage of first and second heavy chain molecules are disclosed, for example, in WO 2009089004, U.S. Pat. No. 8,642,745, U.S. Pat. App. Pub. No. 20140322756, and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar;9(2):182-212.The common light chain bispecific IgG structure may also include a carbohydrate molecule 140 linked thereto or additional modifications thereof.

[0111] Bispecific antibodies having a common light chain bispecific IgG structure can target a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45) and an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the first heavy chain is configured to bind to a B cell lineage surface marker, and the second heavy chain is configured to bind to an inhibitory B cell surface marker. In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0112] In some embodiments, the first heavy chain comprises a VH sequence comprising a CD19-binding component, and the second heavy chain comprises a VH sequence comprising a CD38-binding component. In certain embodiments, the heavy chain CD19-binding component comprises SEQ ID NO: 201, 1, or a variant comprising mutations at one or both of A84 and A108 of SEQ ID NO: 201, and the heavy chain CD38-binding component comprises SEQ ID NO: 202, 215, 218-221. In certain embodiments, the variant comprises mutations A84S and A108L. In some embodiments, the bispecific antibody comprises a common light chain. In certain embodiments, the common light chain sequence comprises a CD19-binding component (e.g., SEQ ID NO: 2). In certain embodiments, the common light chain sequence comprises a CD38-binding component (e.g., SEQ ID NO: 4 or SEQ ID NO: 222).

[0113] BS1 as described herein comprises a common light chain format having a CD19 binding component configured to bind to CD19 and a CD38 binding component configured to bind to CD38, wherein the CD19 binding component comprises an antibody or antigen-binding fragment thereof, and the CD38 binding component comprises an antibody or antigen-binding fragment thereof, wherein the CD38 antibody or antigen-binding fragment comprises an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and wherein the CD19 antibody or antigen-binding fragment comprises an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region. a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91-95; d) a heavy chain complementarity determining region 4 (HCDR4) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101-105; a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and / or c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125, and the CD19 antigen-binding component comprises: g) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25. i) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and / or l) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125.In some embodiments, the CD38 antigen-binding component comprises an HCDR2 amino acid sequence comprising the sequence P-X1-LG-X2-A, where X1 and X2 are each selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T. In some embodiments, the CD19 heavy chain sequence comprises an A84S and / or A108L substitution. In some embodiments, the CD38 light chain comprises a W32H substitution.

[0114] Fab-Fc: scFv-Fc bispecific IgG Bispecific antibodies having a Fab-Fc:scFv-Fc bispecific IgG structure can be used in the present invention. Figure 2 illustrates a bispecific antibody having a Fab-Fc:scFv-Fc bispecific IgG structure. The structure comprises a first heavy chain molecule and a modified second IgG heavy chain molecule comprising a single-chain variable fragment. The first heavy chain comprises, from N- to C-terminus, VH 202, CH1 204, CH2 206, and CH3 208, respectively. The modified second heavy chain comprises, from N- to C-terminus, a single-chain variable fragment (scFv) 210, CH2 216, and CH3 218, respectively. The single-chain variable fragment (scFv) can comprise a first domain 212 corresponding to the variable light chain domain, or a fragment thereof, a second domain 214 corresponding to the variable heavy chain, or a fragment thereof, and a linker polypeptide 215. The Fab-Fc:scFv-Fc bispecific IgG structure also includes a light chain comprising a VL domain 220 and a CL domain 222. The first heavy chain can be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 230). The first heavy chain can be linked to the modified second heavy chain via one or more covalent bonds (e.g., disulfide bonds 234 and / or 236). The Fab-Fc:scFv-Fc bispecific IgG structure can include first and modified second heavy chain molecules that further include mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can prevent linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions). Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in U.S. Patent Application Publication No. 20140322756 and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2): 182-212. The Fab-Fc:scFv-Fc bispecific IgG structure can also include a carbohydrate molecule 240 linked thereto or additional modifications thereof.

[0115] Bispecific antibodies having a Fab-Fc:scFv-Fc bispecific IgG structure can target a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD138, IgA, or CD45) and an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0116] A Fab-Fc:scFv-Fc bispecific IgG structure can be engineered such that the first antigen-binding site targets CD19 and the second antigen-binding site targets CD38. In some embodiments, the first heavy chain comprises a VH sequence comprising a CD19-binding component, and the second heavy chain comprises a single-chain variable fragment (scFv) sequence comprising a CD38-binding component. In certain embodiments, the heavy chain comprising a CD38 single-chain variable fragment comprises SEQ ID NO: 205 or SEQ ID NO: 206. In certain embodiments, the VL sequence comprises a CD19-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD38-binding component comprises a CD38-binding component corresponding to antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof. In some embodiments, the first heavy chain comprises a VH sequence comprising a CD38-binding component, and the second heavy chain comprises a single-chain variable fragment (scFv) sequence comprising a CD19-binding component. In certain embodiments, the heavy chain comprising the CD19 single chain variable fragment comprises SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 217. In certain embodiments, the single chain variable fragment (scFv) sequence comprising a CD19 binding component comprises a CD19 binding component corresponding to an antibody heavy and light chain variable sequence, or a CD19-binding fragment thereof.

[0117] A Fab-Fc:scFv-Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the first heavy chain comprises a VH sequence comprising a CD38-binding component, and the second heavy chain comprises a single-chain variable fragment (scFv) sequence comprising a CD19-binding component. In certain embodiments, the VL sequence comprises a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof.

[0118] The BS2 described herein comprises a CD19-binding component configured to bind to CD19 and a CD38-binding component configured to bind to CD38, wherein the CD19-binding component comprises an antibody or antigen-binding fragment thereof, and the CD38-binding component comprises an antibody or antigen-binding fragment thereof, wherein the CD38-antigen-binding component comprises a Fab that binds to CD38 comprising an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and the CD19-antigen-binding component comprises an scFv that binds to CD19 comprising an anti-CD19 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and wherein the CD38-binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOS: 71 to 75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOS: 81 to 85, or 150 to 155, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOS: 91 to 95, the immunoglobulin light chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; the CD19-binding component comprises an immunoglobulin heavy chain comprising an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41 to 45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51 to 55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61 to 65. In some embodiments, the CD38 antigen-binding component comprises an HCDR2 amino acid sequence comprising the sequence P-X1-LG-X2-A, where X1 and X2 are selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T. In some embodiments, the CD19 heavy chain sequence comprises an A84S and / or A108L substitution.In some embodiments, the CD38 light chain comprises a W32H substitution.

[0119] Fab-Fc-Fab:Fc bispecific IgG Engineered bispecific antibodies having a Fab-Fc-Fab:Fc bispecific IgG structure can be used in the present invention. Figure 3 illustrates a bispecific antibody having a Fab-Fc-Fab:Fc bispecific IgG structure. The structure comprises a first heavy chain molecule and a modified IgG heavy chain molecule. The first heavy chain comprises, from N- to C-terminus, a VH domain 302, a CH1 domain 304, a CH2 domain 306, a CH3 domain 308, a linker 310, a second VH domain 312, and a second CH1 domain 314. The modified heavy chain comprises, from N- to C-terminus, a CH2 domain 316 and a CH3 domain 318. The Fab-Fc-Fab:Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 320 and a CL domain 322. The Fab-Fc-Fab:Fc bispecific IgG structure also comprises a second light chain comprising a VL domain 324 and a CL domain 326. The heavy chain can be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 330). The first heavy chain can also be covalently linked to the first and second chain molecules via a covalent bond (e.g., disulfide bond 332). The heavy and light chains can be linked in such a manner that the VH and CH1 domains of the first heavy chain pair with the VL and CL domains of the first light chain. The first heavy and second light chains can be linked in such a manner that the second VH and second CH1 domains of the first heavy chain pair with the VL and CL domains of the second light chain. The first heavy chain can be linked to the modified second heavy chain via one or more covalent bonds (e.g., disulfide bonds 334 and / or 336). The Fab-Fc-Fab:Fc bispecific IgG structure can comprise first and modified second heavy chain molecules that further comprise mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of a first heavy chain to another first heavy chain or a second heavy chain to another second heavy chain. The mutations can prevent linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions).Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in U.S. Patent Application Publication No. 20140322756 and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2): 182-212. The Fab-Fc-Fab:Fc bispecific IgG structure can also include a carbohydrate molecule 340 or additional modifications thereof linked thereto.

[0120] Bispecific antibodies having a Fab-Fc-Fab:Fc bispecific IgG structure can target a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD138, IgA, or CD45) and an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0121] A Fab-Fc-Fab:Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 302) and VL domain (e.g., 320) comprise CD19-binding components, and the second VH domain (e.g., 312) and VL domain (e.g., 324) comprise CD38-binding components. In some embodiments, the Fab-Fc-Fab heavy chain comprises SEQ ID NO: 207, and the Fc heavy chain comprises SEQ ID NO: 208.

[0122] Fab-Fc-Fab:Fc bispecific IgG structures can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD 19. In some embodiments, the first heavy chain VH domain (e.g., 302) and VL domain (e.g., 320) comprise a CD38-binding component, and the second VH domain (e.g., 312) and VL domain (e.g., 324) comprise a CD19-binding component.

[0123] Fab-Fc-scFv: Fab-Fc-scFv bispecific IgG Engineered bispecific antibodies having a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure can be used in the present invention. Figure 4 illustrates a bispecific antibody having a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure. The structure comprises two first heavy chain molecules. The first heavy chains each comprise, from N- to C-terminus, a VH domain 402, a CH1 domain 404, a CH2 domain 406, a CH3 domain 408, a linker 410, and a single-chain variable fragment (scFv) 412. The single-chain variable fragment (scFv) can comprise a first domain 414, or a fragment thereof, corresponding to the variable light chain domain, a second domain 416, or a fragment thereof, corresponding to the variable heavy chain, and a second linker polypeptide 415. The Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure also comprises a first light chain comprising a VL domain 420 and a CL domain 422. A heavy chain can be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 430). A heavy chain can be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 434 and / or 436). The Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure can also include a carbohydrate molecule 440 or additional modifications thereof linked thereto.

[0124] Bispecific antibodies having a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure can target a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD138, IgA, or CD45) and an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0125] Fab-Fc-scFv: A Fab-Fc-scFv bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 402) and VL domain (e.g., 420) comprise a CD19-binding component, and the single-chain variable fragment (scFv) (e.g., 412) sequence comprises a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD38-binding component comprises a CD38-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof.

[0126] Fab-Fc-scFv: Fab-Fc-scFv bispecific IgG structures can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 402) and VL domain (e.g., 420) comprise a CD38-binding component, and the single-chain variable fragment (scFv) (e.g., 412) sequence comprises a CD19-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof. In some embodiments, the Fab-Fc-scFv heavy chain comprises SEQ ID NO: 209.

[0127] Fab-Fc-scFv:Fc bispecific IgG Engineered bispecific antibodies having a Fab-Fc-scFv:Fc bispecific IgG structure can be used in the present invention. Figure 5 illustrates a bispecific antibody having a Fab-Fc-scFv:Fc bispecific IgG structure. The structure comprises a first heavy chain molecule and a second IgG heavy chain molecule. The first heavy chain comprises, from N- to C-terminus, a VH domain 502, a CH1 domain 504, a CH2 domain 506, a CH3 domain 508, a linker 510, and a single-chain variable fragment (scFv) 512. The single-chain variable fragment (scFv) can comprise a first domain 514, or a fragment thereof, corresponding to the variable light chain domain, a second domain 516, or a fragment thereof, corresponding to the variable heavy chain, and a second linker polypeptide 515. The Fab-Fc-scFv:Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 520 and a CL domain 522. The Fab-Fc-scFv:Fc bispecific IgG structure also includes a second light chain comprising a VL domain 524 and a CL domain 526. The heavy chain can be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 530). The heavy chain can be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 534 and / or 536). The Fab-Fc-scFv:Fc bispecific IgG structure can include first and modified second heavy chain molecules that further include mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can prevent linkage of the two heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions). Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in U.S. Patent Application Publication No. 20140322756 and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2): 182-212. The Fab-Fc-scFv:Fc bispecific IgG structure can also include a carbohydrate molecule 540 or additional modifications thereof linked thereto.

[0128] Bispecific antibodies having a Fab-Fc-scFv:Fc bispecific IgG structure can target a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD138, IgA, or CD45) and an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0129] A Fab-Fc-scFv:Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 502) and VL domain (e.g., 520) comprise a CD19-binding component, and the single-chain variable fragment (scFv) (e.g., 512) sequence comprises a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD38-binding component comprises a CD38-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof.

[0130] Fab-Fc-scFv:Fc bispecific IgG structures can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 502) and VL domain (e.g., 520) comprise a CD38-binding component, and the single-chain variable fragment (scFv) (e.g., 512) sequence comprises a CD19-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof.

[0131] Fab-Fc-Fab:Fab-Fc bispecific IgG Engineered bispecific antibodies having a Fab-Fc-Fab:Fab-Fc bispecific IgG structure can be used in the present invention. Figure 6 illustrates a bispecific antibody having a Fab-Fc-Fab:Fab-Fc bispecific IgG structure. The structure comprises a first heavy chain molecule and a second IgG heavy chain molecule. The first heavy chain comprises, from N- to C-terminus, a VH domain 602, a CH1 domain 604, a CH2 domain 606, a CH3 domain 608, a linker 610, a second VH domain 612, and a second CH1 domain 614. The second heavy chain comprises, from N- to C-terminus, a VH domain 652, a CH1 domain 654, a CH2 domain 656, and a CH3 domain 658, as in the first heavy chain. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 620 and a CL domain 622. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure also includes a second light chain comprising a VL domain 624 and a CL domain 626. The heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 630). The first heavy chain and first light chain may be linked in such a manner that the VH domain and CH1 domain of the first heavy chain pair with the VL domain and CL domain of the first light chain. The first heavy chain and second light chain may be linked in such a manner that the second VH domain and second CH1 domain of the first heavy chain pair with the VL domain and CL domain of the second light chain. The heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 634 and / or 636). The Fab-Fc-Fab:Fab-Fc bispecific IgG structure can comprise first and second heavy chain molecules that further comprise mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can prevent linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions).Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in U.S. Patent Application Publication No. 20140322756 and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2): 182-212. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure can also include a carbohydrate molecule or additional modifications thereof linked thereto.

[0132] Bispecific antibodies having a Fab-Fc-Fab:Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0133] A Fab-Fc-Fab:Fab-Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD 19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 602) and VL domain (e.g., 620) comprise a CD19-binding component, and the second VH domain (e.g., 612) and VL domain (e.g., 624) comprise a CD38-binding component.

[0134] Fab-Fc-Fab:Fab-Fc bispecific IgG structures can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD 19. In some embodiments, the first heavy chain VH domain (e.g., 602) and VL domain (e.g., 620) comprise a CD38-binding component, and the second VH domain (e.g., 612) and VL domain (e.g., 624) comprise a CD19-binding component.

[0135] scFv-Fab-Fc: scFv-Fab-Fc bispecific IgG Engineered bispecific antibodies having an scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can be used in the present invention. Figure 7 illustrates a bispecific antibody having an scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure. The structure comprises two first heavy chain molecules. The first heavy chains each comprise, from N- to C-terminus, a single-chain variable fragment (scFv) 712, a linker 710, a VH domain 702, a CH1 domain 704, a CH2 domain 706, and a CH3 domain 708. The single-chain variable fragment (scFv) can comprise a first domain 714, or a fragment thereof, corresponding to the variable light chain domain, a second domain 716, or a fragment thereof, corresponding to the variable heavy chain, and a second linker polypeptide 715. The ScFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 720 and a CL domain 722. A heavy chain can be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 730). A heavy chain can be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 734 and / or 736). The ScFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can also include a carbohydrate molecule 740 or additional modifications thereof linked thereto.

[0136] Bispecific antibodies having an scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0137] The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 702) and VL domain (e.g., 720) comprise a CD19-binding component, and the single-chain variable fragment (scFv) (e.g., 712) sequence comprises a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD38-binding component comprises a CD38-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof.

[0138] The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 702) and VL domain (e.g., 720) comprise a CD38-binding component, and the single-chain variable fragment (scFv) (e.g., 712) sequence comprises a CD19-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof.

[0139] Fab-Fab-Fc: Fab-Fab-Fc bispecific IgG Engineered bispecific antibodies having a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can be used in the present invention. Figure 8 illustrates a bispecific antibody having a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure. The structure comprises two heavy chain molecules. The heavy chains each comprise, from N- to C-terminus, an additional VH domain 812, an additional CH1 domain 814, a linker 810, a VH domain 802, a CH1 domain 804, a CH2 domain 806, and a CH3 domain 808. The Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 820 and a CL domain 822. The Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure also comprises a second light chain comprising a VL domain 824 and a CL domain 826. The heavy chain molecules can be covalently linked to the light chain molecules via a covalent bond (e.g., disulfide bond 830). The heavy chain and a first light chain can be linked in such a manner that the VH and CH1 domains of the heavy chain pair with the VL and CL domains of the first light chain. The heavy chain and a second light chain can be linked in such a manner that an additional VH and an additional CH1 domain of the heavy chain pair with the VL and CL domains of the second light chain. The heavy chain can be linked to a modified second heavy chain via one or more covalent bonds (e.g., disulfide bonds 834 and / or 836). The Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can also include a carbohydrate molecule 840 linked thereto or additional modifications thereof.

[0140] Bispecific antibodies having a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0141] A Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD 19 and a second antigen-binding site targets CD38. In some embodiments, the first VH domain (e.g., 802) and VL domain (e.g., 820) comprise a CD19-binding component, and the second VH domain (e.g., 812) and VL domain (e.g., 824) comprise a CD38-binding component.

[0142] Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structures can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD 19. In some embodiments, the VH domain (e.g., 802) and VL domain (e.g., 820) comprise CD38-binding components, and the second VH domain (e.g., 812) and VL domain (e.g., 824) comprise CD19-binding components.

[0143] Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG Engineered bispecific antibodies having a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can be used in the present invention. Figure 9 illustrates a bispecific antibody having a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure. The structure comprises two heavy chain molecules and two light chain molecules. The heavy chains each comprise, from N- to C-terminus, a VH domain 902, a CH1 domain 904, a CH2 domain 906, a CH3 domain 908, a linker 910, a second VH domain 912, and a second CH1 domain 914. The Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure also comprises a first light chain comprising a VL domain 920 and a CL domain 922. The Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure also comprises a second light chain comprising a VL domain 924 and a CL domain 926. The heavy chain can be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 930). The heavy chain and a first light chain can be linked in such a manner that the VH and CH1 domains of the heavy chain pair with the VL and CL domains of the first light chain. The heavy chain and a second light chain can be linked in such a manner that the second VH and second CH1 domains of the heavy chain pair with the VL and CL domains of the second light chain. The heavy chain can also be covalently linked to another heavy chain molecule via a covalent bond (e.g., disulfide bonds 934 and 936). The Fab-Fc-Fab bispecific IgG structure can also include a carbohydrate molecule 940 linked thereto or additional modifications thereof.

[0144] Bispecific antibodies having a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0145] A Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can be engineered such that a first antigen-binding site targets CD 19 and a second antigen-binding site targets CD38. In some embodiments, the first VH domain (e.g., 902) and VL domain (e.g., 920) comprise a CD19-binding component, and the second VH domain (e.g., 912) and VL domain (e.g., 924) comprise a CD38-binding component.

[0146] A Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD 19. In some embodiments, the VH domain (e.g., 902) and the VL domain (e.g., 920) comprise a CD38-binding component, and the second VH domain (e.g., 912) and the VL domain (e.g., 924) comprise a CD19-binding component.

[0147] Fab-Fc-scFv: Fab-Fc bispecific IgG Engineered bispecific antibodies having a Fab-Fc-scFv:Fab-Fc bispecific IgG structure can be used in the present invention. Figure 10 demonstrates a bispecific antibody having a Fab-Fc-scFv:Fab-Fc bispecific IgG structure. The structure comprises a first heavy chain molecule and a second IgG heavy chain molecule. The first heavy chain comprises, from N- to C-terminus, a VH domain 1002, a CH1 domain 1004, a CH2 domain 1006, a CH3 domain 1008, a linker 1010, and a single-chain variable fragment (scFv) 1012. The single-chain variable fragment (scFv) can comprise a first domain 1014, or a fragment thereof, corresponding to the variable light chain domain, a second domain 1016, or a fragment thereof, corresponding to the variable heavy chain, and a second linker polypeptide 1015. The second heavy chain comprises, from N-terminus to C-terminus, a VH domain 1002, a CH1 domain 1004, a CH2 domain 1004, and a CH3 domain 1008, respectively, as in the first heavy chain. The Fab-Fc-scFv:Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 1020 and a CL domain 1022. The heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 1030). The heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 1034 and / or 1036). The Fab-Fc-scFv:Fab-Fc bispecific IgG structure can include first and second heavy chain molecules that further comprise mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can prevent linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions). Exemplary mutations that facilitate linkage of first and second heavy chain molecules are disclosed, for example, in U.S. Patent Application Publication No. 20140322756 and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2): 182-212.The Fab-Fc-scFv:Fab-Fc bispecific IgG structure may also include a carbohydrate molecule 1040 linked thereto or additional modifications thereof.

[0148] Bispecific antibodies having a Fab-Fc-scFv:Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0149] Fab-Fc-scFv: A Fab-Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 1002) and VL domain (e.g., 1020) comprise a CD19-binding component, and the single-chain variable fragment (scFv) (e.g., 1012) sequence comprises a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD38-binding component comprises a CD38-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof.

[0150] Fab-Fc-scFv:Fab-Fc bispecific IgG structures can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 1002) and VL domain (e.g., 1020) comprise a CD38-binding component, and the single-chain variable fragment (scFv) (e.g., 1012) sequence comprises a CD19-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof.

[0151] scFv-Fab-Fc:Fc bispecific IgG Engineered bispecific antibodies having an scFv-Fab-Fc:Fc bispecific IgG structure can be used in the present invention. Figure 11 demonstrates a bispecific antibody having an scFv-Fab-Fc:Fc bispecific IgG structure. The structure includes a first heavy chain molecule comprising an scFv, VH, and Fc region and a second heavy chain molecule comprising an Fc region. The scFv-Fab-Fc:Fc bispecific IgG structure can include first and second heavy chain molecules that further include mutations in the CH3 domain that facilitate linkage of the first and second heavy chains and / or prevent linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can facilitate physical (e.g., knob-in-hole structure) or biochemical (e.g., electrostatic interaction) association of the first heavy chain molecule with the second heavy chain molecule. The scFv-Fab-Fc:Fc bispecific IgG structure comprises a light chain molecule associated with a first heavy chain molecule, creating a first antigen-binding site. The second antigen-binding site is provided by an scFv fragment linked to the N-terminus of the first heavy chain. Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in U.S. Patent Application Publication No. 20140322756 and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2): 182-212. The scFv-Fab-Fc:Fc bispecific IgG structure can also include a carbohydrate molecule 1140 linked thereto or additional modifications thereof.

[0152] Bispecific antibodies having an scFv-Fab-Fc:Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0153] The scFv-Fab-Fc:Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH and VL domains comprise a CD19-binding component, and the single-chain variable fragment (scFv) sequence comprises a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprises a CD38-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof.

[0154] The scFv-Fab-Fc:Fc bispecific IgG structure can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the heavy chain VH and VL domains comprise a CD38-binding component, and the single-chain variable fragment (scFv) sequence comprises a CD19-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence comprising a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof.

[0155] In certain embodiments, the first heavy chain molecule comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 212. In certain embodiments, the first heavy chain molecule comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 212.

[0156] In certain embodiments, the light chain molecule comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 213. In certain embodiments, the light chain molecule comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 213.

[0157] In certain embodiments, the second heavy chain molecule comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 214. In certain embodiments, the first heavy chain molecule comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 214.

[0158] Framework Area Mutations or backmutations to germline sequences made within the framework regions of the heavy and light chains can be advantageous for improving the pharmacokinetic and pharmacodynamic properties of the CD19 and CD38 binding molecules described herein. In certain cases, mutations or backmutations to germline sequences made within the heavy and / or light chains improve the stability of the CD19 and CD38 binding molecules (e.g., the bispecific antibodies described herein). In certain cases, mutations or backmutations to germline sequences made within the heavy and / or light chains reduce the immunogenicity of the CD19 and CD38 binding molecules (e.g., the bispecific antibodies described herein). Thus, in some embodiments, the framework regions of the heavy and / or light chains contain 1, 2, 3, 4, 5, 8, or 10 mutations or backmutations back to germline sequences. In some embodiments, the framework regions of the heavy and / or light chains contain from 1 mutation or backmutation back to germline sequences to 10 mutations or backmutations back to germline sequences. In some embodiments, the framework regions of the heavy and / or light chains comprise at least one mutation or backmutation back to the germline sequence. In some embodiments, the framework regions of the heavy and / or light chains comprise up to 10 mutations or backmutations back to the germline sequence. In some embodiments, the framework regions of the heavy and / or light chains comprise from 1 mutation or backmutation back to the germline sequence to 2 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 3 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 4 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 5 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 8 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 10 mutations or backmutations back to the germline sequence.Two mutations or reversions back to a germline sequence up to three mutations or reversions back to a germline sequence, two mutations or reversions back to a germline sequence up to four mutations or reversions back to a germline sequence, two mutations or reversions back to a germline sequence up to five mutations or reversions back to a germline sequence, two mutations or reversions back to a germline sequence up to eight mutations or reversions back to a germline sequence, two mutations or reversions back to a germline sequence up to ten mutations or reversions back to a germline sequence, three mutations or reversions back to a germline sequence up to four mutations or reversions back to a germline sequence, three mutations or reversions back to a germline sequence up to five mutations or reversions back to a germline sequence, three mutations or reversions back to a germline sequence up to five mutations or reversions back to a germline sequence In some embodiments, the framework regions of the heavy and / or light chains contain up to 8 mutations or backmutations back to the germline sequence, 3 mutations or backmutations back to the germline sequence up to 10 mutations or backmutations back to the germline sequence, 4 mutations or backmutations back to the germline sequence up to 5 mutations or backmutations back to the germline sequence, 4 mutations or backmutations back to the germline sequence up to 8 mutations or backmutations back to the germline sequence, 4 mutations or backmutations back to the germline sequence up to 10 mutations or backmutations back to the germline sequence, 5 mutations or backmutations back to the germline sequence up to 8 mutations or backmutations back to the germline sequence, 5 mutations or backmutations back to the germline sequence up to 10 mutations or backmutations back to the germline sequence, or 8 mutations or backmutations back to the germline sequence up to 10 mutations or backmutations back to the germline sequence. In some embodiments, the framework regions of the heavy and / or light chains contain up to 1 mutation or backmutation back to the germline sequence, 2 mutations or backmutations back to the germline sequence, 3 mutations or backmutations back to the germline sequence,The CD38-binding portion comprises a heavy chain framework region set forth in SEQ ID NO: 5. In some embodiments, the CD19-binding portion comprises a heavy chain framework region set forth in SEQ ID NO: 6 or 7.

[0159] Pharmaceutically Acceptable Excipients, Carriers, and Diluents The composition comprising the complex-binding molecule of the present disclosure is contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibody of the present disclosure is administered suspended in a sterile and / or isotonic solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of a buffering agent, such as acetic acid (salt), citric acid (salt), histidine, succinic acid (salt), phosphoric acid (salt), bicarbonate (salt), and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine; or a chelating agent, such as EDTA or EGTA.

[0160] Subcutaneous formulations for administration of antibodies can include one or more of the following: a buffering agent, such as acetic acid (salt), citric acid (salt), histidine, succinic acid (salt), phosphoric acid (salt), bicarbonate (salt), and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine, or a chelating agent, such as EDTA or EGTA. Additionally, a compound or molecule that relieves pain at the injection site, such as hyaluronidase at a concentration of about 2,000 U / ml to about 12,000 U / ml, can be included.

[0161] In certain embodiments, the complex-binding molecules of the present disclosure are lyophilized for transport / storage and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation includes a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation may be contained in a vial made of glass or other suitable non-reactive material. The antibody, when formulated, whether reconstituted or not, may be buffered to a specific pH, generally a pH below 7.0. In certain embodiments, the pH may be 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0162] Also described herein are kits comprising one or more of the complex binding molecules described herein in a suitable container and one or more additional components selected from instructions for use, diluents, excipients, carriers, and devices for administration.

[0163] In certain embodiments, described herein are methods of preparing a cancer treatment, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents and a complex-binding molecule of the present disclosure. In certain embodiments, described herein are methods of preparing a cancer treatment for storage or transport, comprising lyophilizing one or more antibodies of the present disclosure.

[0164] Manufacturing and Production Nucleic acids encoding the complex-binding molecules (e.g., bispecific antibodies) described herein can be used to infect, transfect, transform, or otherwise transgenic suitable cells for the nucleic acid to enable production of the complex-binding molecules for commercial or therapeutic use. Standard cell lines and methods for the production of antibodies from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct; 2(5): 466-477.

[0165] In certain embodiments, the nucleic acid sequence encodes a complex-binding molecule or a bispecific antibody disclosed herein. In certain embodiments, the polynucleotide sequence encoding the complex-binding molecule is operably linked to a eukaryotic regulatory sequence. In some embodiments, a cell comprises the nucleic acid sequence.

[0166] In some embodiments, the cell comprises a nucleic acid encoding a complex-binding molecule disclosed herein. In certain embodiments, the cell comprises a prokaryotic cell. In certain embodiments, the prokaryotic cell is an E. coli cell. In certain embodiments, the cell comprises a eukaryotic cell. In certain embodiments, the eukaryotic cell is a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a human PER.C6 cell.

[0167] In certain embodiments, described herein are methods of producing a complex-binding molecule, the methods comprising culturing a cell comprising a nucleic acid encoding the complex-binding molecule under in vitro conditions sufficient to allow for the production and secretion of the complex-binding molecule.

[0168] In certain embodiments, described herein is a master cell bank comprising (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank comprises (a) a CHO cell line comprising a nucleic acid encoding a complex-binding molecule integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing with liquid nitrogen.

[0169] Also described herein are methods for producing the complex-binding molecules described herein. Such methods include incubating cells or cell lines containing nucleic acids encoding the complex-binding molecules under conditions sufficient to allow expression and secretion of the complex-binding molecules in cell culture medium, and further harvesting the complex-binding molecules from the cell culture medium. Harvesting can further include one or more purification steps to remove viable cells, cell debris, uncomplex-binding molecule proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, Protein A, Protein G, Protein A / G, or Protein L purification, and / or ion exchange chromatography.

[0170] How to use Suppression of the immune response by immunoregulatory cells can promote tumor growth, migration, and metastasis. Immunosuppression or negative immune modulation can include processes or pathways that result in a total or partial reduction of the immune response. Immunosuppression can be systemic or localized to a specific site (e.g., tumor microenvironment), tissue, or region of a subject's or patient's body. While B cells are primarily known as positive immune modulators through the production of antibodies that promote pathogen neutralization, certain populations of B cells can function to suppress or negatively regulate immune responses. Such populations of B cells can be defined by the expression of more than one cell surface biomarker. Immunosuppressive B cells or B cell populations can include B cell lineage surface biomarkers and inhibitory B cell surface biomarkers. B cell lineage surface markers can include CD19, CD138, IgA, or CD45. B cell surface markers can include IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP). Immunosuppressive B cells or immunosuppressive B cell populations can function to suppress immune responses by suppressing a diverse set of cell subtypes, including T cells, through the secretion of anti-inflammatory mediators, such as cytokines. Immunosuppressive B cells can also function in attenuating immune responses by negatively modulating lymphoid structures and / or promoting the conversion of T cells into regulatory T cells. Thus, disclosed herein are methods for targeting immunosuppressive B cell populations to effectively modulate responses.

[0171] Targeting immunosuppressive B cells or B cell populations can result in immune activation or positive modulation of immune responses against tumors or tumorigenic cells. Provided herein is a method of treating an individual suffering from cancer or a tumor, comprising administering a complex-binding molecule disclosed herein to the individual suffering from cancer or a tumor. Also provided herein is a method of reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor or tumor in an individual suffering from cancer, comprising administering a complex-binding molecule disclosed herein to the tumor or individual suffering from cancer, thereby reducing immunosuppressive B cells in, adjacent to, or surrounding the tumor. Also disclosed is a method of contacting immunosuppressive B cells in a subject with a complex-binding molecule, comprising administering the complex-binding molecule to the subject. In certain embodiments, the subject has a tumor or cancer.

[0172] The type, subtype, or form of tumor or cancer may be an important factor in treatment strategies and methods. In some embodiments, the cancer or tumor is a blood cancer. In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer.

[0173] Immunosuppressive B cells can suppress anti-tumor immune responses. In some embodiments, the tumor or cancer comprises B cells comprising a B cell lineage surface biomarker and an inhibitory B cell surface biomarker. The B cell lineage surface marker can include CD19, CD138, IgA, or CD45. The B cell surface marker can include IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or TGFB. In some embodiments, the B cell surface marker includes CD19 (e.g., CD19+) and CD38 (e.g., CD38+). In some embodiments, the tumor-infiltrating B cells or immunosuppressive B cells comprise CD19+, CD38+ B cells.

[0174] In certain embodiments, disclosed herein are bispecific antibodies useful for the treatment of cancer or tumors. Treatment refers to a method that seeks to improve or ameliorate the condition being treated. With respect to cancer, treatment includes, but is not limited to, a reduction in tumor volume, a reduction in tumor volume growth, progression-free survival, or an increase in overall life expectancy. In certain embodiments, treatment affects the remission of the cancer being treated. In certain embodiments, treatment encompasses use as a prophylactic or maintenance dose intended to prevent the recurrence or progression of a previously treated cancer or tumor. It will be understood by those skilled in the art that not all individuals will respond equally or reliably to an administered treatment, but that these individuals will nonetheless be considered to be treated.

[0175] In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer or tumor. In certain embodiments, the cancer or tumor includes breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and liver tumors. In certain embodiments, tumors that can be treated with the antibodies of the present invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, sarcoma, focal nodular hyperplasia, gastrinoma, germ line tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, liver adenoma, liver adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, nerve sheath, medulloblastoma, medulloepithelioma , mesothelioma, mucosal epithelial carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian carcinoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate carcinoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar carcinoma, VIPpoma, and Wilms' tumor.In certain embodiments, tumors / cancers treated with one or more antibodies of the present disclosure include brain cancer, head and neck cancer, colorectal cancer, acute myeloid leukemia, pre-B-cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate cancer, and breast cancer, preferably breast ductal carcinoma, and / or breast cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include glioblastoma. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include pancreatic cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include ovarian cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include lung cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include prostate cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present disclosure comprises colon cancer. In certain embodiments, the cancer to be treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In certain embodiments, the cancer to be treated is recurrent. In certain embodiments, the cancer is recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

[0176] In certain embodiments, the cancer and or tumor treated with the complex-binding molecules herein is a mature B-cell neoplasm: chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma), mucosa-associated lymphoid tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma and Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, or primary central nervous system lymphoma.

[0177] In certain embodiments, the cancer and / or tumor treated with the complex-binding molecules herein is a T-cell neoplasm, such as T-cell non-Hodgkin's lymphoma, T-cell ALL, mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, T-lymphocytic leukemia (T-ALL), acute myeloblastic leukemia, acute monocytic leukemia, and others.

[0178] In certain embodiments, the antibody may be administered to a subject in need thereof by any route suitable for administering an antibody-containing pharmaceutical composition, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral administration. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered according to a suitable dosing schedule, such as weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In certain embodiments, the antibody is administered once every three weeks. The antibody may be administered in any therapeutically effective amount. In certain embodiments, a therapeutically acceptable amount is about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, a therapeutically acceptable amount is about 1 mg / kg to about 40 mg / kg. In certain embodiments, a therapeutically acceptable amount is about 5 mg / kg to about 30 mg / kg. A therapeutically effective amount includes an amount sufficient to ameliorate one or more symptoms associated with the disease or condition being treated.

[0179] Illustrative Embodiments Provided herein is a complex-binding molecule comprising a CD19-binding component configured to bind to CD19 and a CD38-binding component configured to bind to CD38, wherein the CD19-binding component comprises an antibody or antigen-binding fragment thereof, and the CD38-binding component comprises an antibody or antigen-binding fragment thereof. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19- and / or CD38-binding component comprises an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab'), a single-domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19- or CD38-binding component comprises an immunoglobulin heavy and light chain pair. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19- and CD38-binding component comprises an immunoglobulin heavy and light chain pair.

[0180] In some embodiments, provided is a CD38-binding component comprising an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. and the CD19-binding component comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:3, and an immunoglobulin light chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:4, and / or the CD19-binding component comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:1, and an immunoglobulin light chain comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:4.In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3 or 5, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 4, and / or the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 1 or 6, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0181] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the complex-binding molecule is a common light chain bispecific IgG. In some embodiments, provided is the CD38-binding component comprises an immunoglobulin heavy chain comprising an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. and wherein the CD19-binding component comprises an immunoglobulin heavy chain comprising an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, and wherein the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3 or 5, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4, and / or the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 7, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 2.

[0182] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:3 or 5, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:4, and the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:1 or 7, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component or the CD38-binding component comprises an scFv. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component comprises an scFv. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an scFv. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component or the CD38-binding component comprises an immunoglobulin heavy chain / light chain pair. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component comprises an immunoglobulin heavy / light chain pair. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an immunoglobulin heavy / light chain pair.

[0183] Further provided is a complex-binding molecule comprising a CD38 antigen-binding component that binds to CD38, the CD38 antigen-binding component comprising an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and a CD19 antigen-binding component that binds to CD19, the CD38 antigen-binding component comprising an anti-CD19 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, wherein the CD38 antigen-binding component comprises: a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-75; b) a heavy chain complementarity-determining region 2 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155. a) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; b) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; c) a heavy chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and / or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125.

[0184] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19 antigen-binding component comprises: g) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11-15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21-25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31-35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101-105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111-115; and / or l) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121-125.

[0185] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38 antigen-binding component comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3 or 5, and an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38 antigen-binding component comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 3 or 5, and the immunoglobulin light chain variable region comprises an amino acid sequence identical to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19 antigen-binding component comprises an anti-CD19 immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 6, and an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4.

[0186] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD19 antigen-binding component comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 1 or 6, and the immunoglobulin light chain variable region comprises an amino acid sequence identical to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises a first immunoglobulin heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD38 immunoglobulin light chain variable region further comprises an immunoglobulin light chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the anti-CD19 immunoglobulin heavy chain variable region further comprises a second immunoglobulin heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the first immunoglobulin heavy chain constant region and / or the second immunoglobulin heavy chain constant region comprises one or more amino acid substitutions that disfavor homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote heterodimerization of the first heavy chain constant region and the second heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein one of the first or second immunoglobulin heavy chain constant regions comprises a T366W substitution (EU numbering) and the other of the first or second immunoglobulin heavy chain constant region comprises a T366S / L368A / Y407V substitution (EU numbering), such that heterodimerization of the first and second immunoglobulin heavy chain constant regions is promoted compared to homodimerization of the first or second immunoglobulin heavy chain constant region. In some embodiments, provided is the complex binding molecule of any of the preceding embodiments, wherein the single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component.

[0187] Also provided is a complex-binding molecule comprising a CD19-binding component that binds to CD19 and a CD38-binding component that binds to CD38, wherein the CD19-binding component comprises an scFv that binds to CD19, and the CD38-binding component comprises an immunoglobulin variable region comprising a light chain variable region and a heavy chain variable region that binds to CD38. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the scFv that binds to CD19 is linked to a first immunoglobulin heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the heavy chain variable region of the CD38-binding component further comprises a second immunoglobulin heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the light chain variable region of the CD38-binding component further comprises an immunoglobulin light chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, or an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65.

[0188] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, or an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19-binding component comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 7, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 2. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38 binding component comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3 or 5, and an amino acid sequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 4. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD19 binding component comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 1 or 7, and an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38 binding component comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3 or 5, and an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0189] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the first immunoglobulin heavy chain constant region and / or the second immunoglobulin heavy chain constant region comprises one or more amino acid substitutions that disfavor homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote heterodimerization of the first heavy chain constant region and the second heavy chain constant region. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein one of the first or second immunoglobulin heavy chain constant regions comprises a T366W substitution (EU numbering) and the other of the first or second immunoglobulin heavy chain constant region comprises a T366S / L368A / Y407V substitution (EU numbering), such that heterodimerization of the first and second immunoglobulin heavy chain constant regions is favored over homodimerization of the first or second immunoglobulin heavy chain constant region. In some embodiments, provided is the complex binding molecule of any of the preceding embodiments, wherein the single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component.

[0190] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, which is a bispecific antibody or dual antigen-binding fragment thereof. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, which comprises an Fc region comprising an amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate corresponds to asparagine 297 according to EU numbering.

[0191] In some embodiments, provided is a complex-binding molecule of any of the preceding embodiments that binds to CD19+, CD38+ B cells. In some embodiments, provided is a complex-binding molecule of any of the preceding embodiments that exhibits reduced hemagglutination compared to a CD19 or CD38 monospecific antibody comprising an Fc region.

[0192] Provided is a nucleic acid comprising a polynucleotide sequence encoding any one of the complex-binding molecules of the preceding embodiments. Embodiment 49: The nucleic acid of embodiment 47, wherein the polynucleotide sequence encoding the complex-binding molecule is operably linked to a eukaryotic regulatory sequence. In some embodiments, provided is a cell comprising the nucleic acid of any one of the preceding embodiments. In some embodiments, provided is a cell of any of the preceding embodiments, comprising a prokaryotic cell. In some embodiments, provided is a cell of any of the preceding embodiments, wherein the prokaryotic cell is an E. coli cell. In some embodiments, provided is a cell of any of the preceding embodiments, comprising a eukaryotic cell. In some embodiments, provided is a cell of any of the preceding embodiments, wherein the eukaryotic cell is a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a human PER.C6 cell.

[0193] Also provided is a pharmaceutical composition, for example, a composition comprising the complex-binding molecule of any one of the preceding embodiments and a pharmaceutically acceptable diluent, carrier, or excipient.In some embodiments, the composition is formulated for intravenous administration.In some embodiments, the composition is formulated for subcutaneous administration.

[0194] Provided is a complex-binding molecule of any one of the preceding embodiments or a pharmaceutical composition of any one of the preceding embodiments for use in a method of treating a tumor or cancer in an individual. In some embodiments, the tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In certain embodiments, the B-cell malignancy is B-cell acute lymphocytic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the preceding embodiments, the hematological cancer expresses CD19 and CD38 (e.g., cells of the cancer express CD19 and CD38).

[0195] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the solid tissue cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colorectal cancer, brain cancer, or head and neck cancer. In some embodiments, the breast cancer is triple-negative breast cancer, the lung cancer is non-small cell lung cancer, the head and neck cancer is head and neck squamous cell carcinoma, the kidney cancer is renal cell carcinoma, the brain cancer is glioblastoma multiforme, or the skin cancer is melanoma.

[0196] Also provided is a complex-binding molecule of any one of the preceding embodiments or a pharmaceutical composition of any one of the preceding embodiments for use in a method for reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual. In some embodiments, further provided is a complex-binding molecule of any one of the preceding embodiments or a pharmaceutical composition of any one of the preceding embodiments for use in a method for reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual. In some embodiments, the tumor-infiltrating B cells or immunosuppressive B cells comprise CD19+, CD38+ B cells.

[0197] In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises an HCDR2 amino acid sequence comprising the sequence P-X1-LG-X2-A, where X1 and X2 are each selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein X1 is H and X2 is T. In some embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the heavy chain constant region of the CD19-binding component comprises an A84S and / or A108L modification. In certain embodiments, provided is the complex-binding molecule of any of the preceding embodiments, wherein the CD38-binding component comprises a light chain sequence comprising a W32H substitution.

[0198] Also provided is a method of treating an individual suffering from cancer or a tumor, comprising administering to the individual suffering from cancer or a tumor a complex-binding molecule of any one of the preceding embodiments or a pharmaceutical composition of any one of the preceding embodiments, thereby treating the cancer or tumor. In some embodiments, the cancer or tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In certain embodiments, the B-cell malignancy is B-cell acute lymphocytic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the preceding embodiments, the hematological cancer expresses CD19 and CD38 (e.g., cells of the cancer express CD19 and CD38).

[0199] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the solid tissue cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In some embodiments, the breast cancer is triple-negative breast cancer, the lung cancer is non-small cell lung cancer, the head and neck cancer is head and neck squamous cell carcinoma, the kidney cancer is renal cell carcinoma, the brain cancer is glioblastoma multiforme, or the skin cancer is melanoma.

[0200] Provided is a method of reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual afflicted with a tumor or cancer, comprising administering to the tumor or individual afflicted with cancer a complex-binding molecule of any one of the preceding embodiments or a pharmaceutical composition of any one of the preceding embodiments, thereby reducing immunosuppressive B cells in the tumor.

[0201] Also provided is a method of reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual afflicted with a tumor or cancer, comprising administering to the individual afflicted with the tumor or cancer a complex-binding molecule of any one of the preceding embodiments or a pharmaceutical composition of any one of the preceding embodiments, thereby reducing immunosuppressive B cells in the tumor. In some embodiments, the tumor-infiltrating B cells or immunosuppressive B cells comprise CD19+, CD38+ B cells.

[0202] Also provided herein is a method for producing a complex-binding molecule of any one of the preceding embodiments, comprising incubating the cells of the preceding embodiments in a cell culture medium under conditions sufficient to allow expression, assembly, and secretion of the complex-binding molecule into the cell culture medium. In some embodiments, the method comprises isolating and purifying the molecule from the cell culture medium. Also provided is a method for preparing a cancer treatment for an individual, comprising mixing a complex-binding molecule of any one of the preceding embodiments with a pharmaceutically acceptable diluent, carrier, or excipient.

[0203] Thus, provided herein is a complex-binding molecule comprising a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, wherein the first target comprises a B cell lineage surface marker and the second target comprises an inhibitory B cell surface marker, and the first target and the second target are not identical. In some embodiments, the first or second binding moiety comprises a polypeptide. In certain embodiments, the first or second binding moiety consists of a polypeptide. In some embodiments, the first and second binding moieties comprise a polypeptide. In certain embodiments, the first and second binding moieties consist of a polypeptide. In some embodiments, the polypeptide of the first or second binding moiety comprises an amino acid sequence at least 100 amino acid residues in length. In some embodiments, the polypeptide of the first and second binding moieties comprises an amino acid sequence at least 100 amino acid residues in length.

[0204] B cell lineage surface markers can include CD19, CD138, IgA, or CD45. In some embodiments, the B cell lineage surface marker includes CD19. In certain embodiments, the B cell lineage surface marker is CD19. In some embodiments, the B cell lineage surface marker is IgA. In certain embodiments, the B cell lineage surface marker is IgA. In some embodiments, the B cell lineage surface marker is CD138. In certain embodiments, the B cell lineage surface marker is CD138. In some embodiments, the B cell lineage surface marker is CD45. In certain embodiments, the B cell lineage surface marker is CD45. In some embodiments, the B cell lineage surface marker is selected from the group consisting of IgA, CD19, CD138, CD45, and any combination thereof.

[0205] Inhibitory B cell surface markers can include IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP). In some embodiments, the inhibitory B cell surface marker includes IgD. In certain embodiments, the inhibitory B cell surface marker is IgD. In some embodiments, the inhibitory B cell surface marker includes CD1. In certain embodiments, the inhibitory B cell surface marker is CD1. In some embodiments, the inhibitory B cell surface marker includes CD5. In certain embodiments, the inhibitory B cell surface marker is CD5. In certain embodiments, the inhibitory B cell surface marker includes CD21. In certain embodiments, the inhibitory B cell surface marker is CD21. In some embodiments, the inhibitory B cell surface marker includes CD24. In certain embodiments, the inhibitory B cell surface marker is CD24. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker is CD38. In some embodiments, the B cell surface marker is selected from the group consisting of IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, latent TGF-beta (e.g., TGF-beta LAP), and any combination thereof.

[0206] The complex-binding molecule can comprise an antibody or a target-binding fragment thereof. In some embodiments, the first or second binding component comprises an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab'), a single domain antibody, a variable region fragment from an immunoglobulin neo-antigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In some embodiments, the first and second binding components comprise an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab'), a single domain antibody, a variable region fragment from an immunoglobulin neo-antigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In certain embodiments, the first or second binding component comprises an immunoglobulin heavy and light chain pair. In certain embodiments, the first and second binding components comprise an immunoglobulin heavy and light chain pair. In certain embodiments, the first or second binding component comprises an immunoglobulin heavy and light chain pair. In certain embodiments, the first or second binding component comprises an scFv. In certain embodiments, the first and second binding components comprise scFvs.

[0207] A complex-binding molecule as described herein that is a bispecific antibody or dual antigen-binding fragment thereof.

[0208] In some embodiments, the complex-binding molecule comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOS: 71 to 75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOS: 81 to 85, or 150 to 155, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOS: 91 to 95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOS: 41 to 45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOS: 51 to 55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOS: 61 to 65. In certain embodiments, the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, or 99% identity to SEQ ID NO: 3, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, or 99% identity to SEQ ID NO: 2. In certain embodiments, the immunoglobulin heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3, and the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the complex binding molecule is a common light chain bispecific IgG.

[0209] The complex-binding molecule can be a bispecific antibody. In some embodiments, the bispecific antibody is selected from one of the following formats: common light chain bispecific IgG, Fab-Fc:scFv-Fc bispecific IgG, Fab-Fc-Fab:Fc bispecific IgG, Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG, Fab-Fc-scFv:Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc bispecific IgG, scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG, Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and Fab-Fc-scFv:Fab-Fc bispecific IgG. In certain embodiments, the bispecific antibody is a common light chain bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fc:scFv-Fc bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fc-Fab:Fc bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fc-Fab:Fab-Fc bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fc-Fab:Fab-Fc bispecific IgG. In certain embodiments, the bispecific antibody is a scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG. In certain embodiments, the bispecific antibody is a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG. In certain embodiments, the bispecific antibody is an IgG-scFv.

[0210] The complex-binding molecule can include post-translational modifications. In some embodiments, the complex-binding molecule includes an Fc region comprising an amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate. In certain embodiments, the amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate corresponds to asparagine 297 according to EU numbering.

[0211] In some embodiments, the first binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65. In certain embodiments, the first binding component comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1 and 2. In certain embodiments, the first binding component comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 1 and 2.

[0212] In some embodiments, the second binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85, or 150 to 155, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95, an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125. In certain embodiments, the second binding component comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3 and 4. In certain embodiments, the second binding component comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3 and SEQ ID NO:4.

[0213] The complex-binding molecule is capable of binding to a first target and a second target, wherein the first target comprises a B-cell lineage surface marker and the second target comprises an inhibitory B-cell surface marker. In some embodiments, the complex-binding molecule is CD19-positive (CD19+ or CD19 high ) and CD38 positive (CD38+ or CD19 high ) binds to B cells.

[0214] The complex-binding molecule can be encoded by a nucleic acid molecule. Disclosed herein is a nucleic acid comprising a polynucleotide sequence encoding the complex-binding molecule disclosed herein. In some embodiments, the polynucleotide sequence encoding the complex-binding molecule is operably linked to a eukaryotic regulatory sequence.

[0215] The cell can comprise a nucleic acid encoding the complex-binding molecule. In some embodiments, the cell comprises a prokaryotic cell. In certain embodiments, the prokaryotic cell is an E. coli cell. In some embodiments, the cell comprises a eukaryotic cell. In certain embodiments, the eukaryotic cell is a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a human PER.C6 cell.

[0216] Also disclosed herein is a composition comprising a complex-binding molecule and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for subcutaneous administration.

[0217] The complex-binding molecules disclosed herein can inhibit and / or reduce the number of immunosuppressive B cells that suppress anti-tumor immune responses. As such, the complex-binding molecules herein can be used in methods for treating tumors or cancer in individuals. In some embodiments, the cancer or tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In certain embodiments, the B-cell malignancy is a B-cell acute lymphocytic leukemia. In certain embodiments, the B-cell malignancy is a chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the preceding embodiments, the hematological cancer expresses CD19 and CD38 (e.g., cells of the cancer express CD19 and CD38).

[0218] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the cancer comprises breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, gastric cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In some embodiments, the cancer is breast cancer. In certain embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the cancer is lung cancer. In certain embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cancer is head and neck cancer. In certain embodiments, the head and neck cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is kidney cancer. In certain embodiments, the kidney cancer is renal cell carcinoma. In some embodiments, the cancer is brain cancer. In some embodiments, the brain cancer is glioblastoma multiforme. In some embodiments, the cancer is skin cancer. In certain embodiments, the skin cancer is melanoma.

[0219] The complex-binding molecules herein can be used in methods for reducing tumor-infiltrating B cells and / or immunosuppressive B cells that suppress an individual's anti-tumor immune response to a tumor. The complex-binding molecules herein can be used in methods for inhibiting the function of tumor-infiltrating B cells and / or immunosuppressive B cells that suppress an individual's anti-tumor immune response to a tumor. The complex-binding molecules can be used in methods for reducing suppressive B cells in, adjacent to, or surrounding an individual's tumor. In some embodiments, the tumor-infiltrating B cells or immunosuppressive B cells comprise CD19+, CD38+ B cells.

[0220] Further disclosed herein are methods of treating an individual suffering from cancer or a tumor, comprising administering a complex-binding molecule disclosed herein to the individual suffering from cancer or a tumor, thereby treating the cancer or tumor. In some embodiments, the cancer or tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In certain embodiments, the B-cell malignancy is B-cell acute lymphocytic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the preceding embodiments, the hematological cancer expresses CD19 and CD38 (e.g., cells of the cancer express CD19 and CD38).

[0221] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the cancer comprises breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, gastric cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In some embodiments, the cancer is breast cancer. In certain embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the cancer is lung cancer. In certain embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cancer is head and neck cancer. In certain embodiments, the head and neck cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is kidney cancer. In certain embodiments, the kidney cancer is renal cell carcinoma. In some embodiments, the cancer is brain cancer. In some embodiments, the brain cancer is glioblastoma multiforme. In some embodiments, the cancer is skin cancer. In certain embodiments, the skin cancer is melanoma.

[0222] Also disclosed are methods for reducing tumor-infiltrating B cells in, adjacent to, or surrounding a tumor in an individual afflicted with a tumor or cancer, comprising administering a complex-binding molecule disclosed herein to the individual afflicted with the tumor or cancer, thereby reducing tumor-infiltrating B cells in the tumor. Also disclosed are methods for reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual afflicted with a tumor or cancer, comprising administering a complex-binding molecule disclosed herein to the individual afflicted with the tumor or cancer, thereby reducing immunosuppressive B cells in the tumor. In some embodiments, the tumor-infiltrating B cells or immunosuppressive B cells comprise CD19+, CD38+ B cells. In some embodiments, reducing tumor-infiltrating B cells comprises reducing and / or blocking and / or preventing and / or inhibiting the recruitment of immunosuppressive B cells into the tumor environment or microenvironment. In some embodiments, reducing tumor-infiltrating B cells comprises reducing and / or blocking and / or preventing and / or inhibiting cell-to-cell contact-induced immunosuppression mediated by immunosuppressive B cells, hi some embodiments, reducing tumor-infiltrating B cells comprises reducing and / or blocking and / or preventing and / or inhibiting differentiation of immunosuppressive B cells.

[0223] Disclosed herein are methods of producing a complex-binding molecule disclosed herein, comprising incubating a cell disclosed herein in a cell culture medium under conditions sufficient to allow expression, assembly, and secretion of the complex-binding molecule into the cell culture medium. In some embodiments, the method included isolating and purifying the molecule from the cell culture medium.

[0224] The complex-binding molecules disclosed herein can be used in the treatment of cancer or tumors. Thus, disclosed is a method of preparing a cancer treatment for an individual, comprising mixing a complex-binding molecule of the present disclosure with a pharmaceutically acceptable diluent, carrier, or excipient. [Example]

[0225] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0226] Example 1: Cell-binding properties of CD19 and CD38 antibodies To illustrate the disclosure herein, a complex-binding molecule comprising a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, where the first target comprises a B cell lineage surface marker and the second target comprises an inhibitory B cell surface marker, was tested for binding to cells expressing CD19 and CD38. The binding characteristics of an antibody comprising CD19 and CD38 light and heavy chains to Raji cells expressing CD19 and CD38 are shown in Figure 12A. Raji cells expressing CD19 and CD38 were incubated with an antibody comprising CD19 and CD38 light and heavy chains. Cells were incubated with 30 μg / mL of antibody across 11 different concentrations to generate a binding profile for each sample. CD19 and CD38 expression was verified using commercially available antibodies. The samples tested were: (A) matched CD19 heavy and light chains, where the CD19 heavy chain comprises SEQ ID NO: 1 and the CD19 light chain comprises SEQ ID NO: 2; (B) swapped CD19 heavy and CD38 light chains, where the CD19 heavy chain comprises SEQ ID NO: 1 and the CD38 light chain comprises SEQ ID NO: 4; (C) swapped CD38 heavy and CD19 light chains, where the CD38 heavy chain comprises SEQ ID NO: 3 and the CD19 light chain comprises SEQ ID NO: 2; (D) matched CD38 heavy and light chains, where the CD38 heavy chain comprises SEQ ID NO: 3 and the CD38 light chain comprises SEQ ID NO: 4; (E) CD19 single chain variable fragments (scFv) comprising SEQ ID NOs: 1-2; (F) CD38 single chain variable fragments (scFv) comprising SEQ ID NOs: 3-4; Darzalex (CD38 control); anti-CD19 PE (CD19 control); anti-CD38 PE (CD38 control) as well as an IgG1 isotype control are included.

[0227] Figures 12B and 12C show the binding profiles of Samples A-F, Darzalex, and the IgG1 isotype control. Tables 1 and 2 show the EC20 binding profiles of Samples A-F, Darzalex, and the IgG1 isotype control. 50The values ​​and maximum mean fluorescence intensity (MFI) are shown. Each of samples A-F demonstrated binding to Raji cells expressing CD19 and CD38, and the binding profiles of samples A-F varied between samples. Figures 12D and 12E show binding of control anti-CD19 (Figure 11D) and anti-CD38 antibodies (Figure 11E). Figure 12F shows that the tested antibodies did not bind to CHO cells, which do not express CD19 and CD38.

[0228] [Table 1]

[0229] [Table 2]

[0230] Example 2: Octet binding data Biolayer interferometry was used to determine the binding affinities of the parent and bispecific antibodies. Binding experiments were performed on an Octet Red 96 at 25°C using an assay buffer consisting of 0.1% BSA, 1X PBS, 0.02% Tween-20, and 0.05% NaN3. Antibodies were loaded onto an anti-hIgG Fc Capture biosensor for 300 seconds. The ligand-loaded sensor was immersed in serial dilutions of antigen (starting at 300 nM: 2-fold serial dilutions for CD19 and 3-fold serial dilutions for CD38) for association (200 seconds for CD19, 150 seconds for CD38) followed by dissociation (600 seconds for CD19, 400 seconds for CD38). Rate constants were calculated using a monovalent (1:1) binding model.

[0231] The parent test article included:

[0232] 851A=Anti-CD19 3C10

[0233] 851B = Anti-CD19 3C10 heavy chain & anti-CD38 003 light chain

[0234] 851C = anti-CD38 003 heavy chain & anti-CD19 3C10 light chain

[0235] 851D=Anti-CD38 003

[0236] 851E=anti-CD19 3C10(scFv-Fc)2

[0237] 851F=Anti-CD38 003(scFv-Fc)2

[0238] Two parental antibodies (851A / 851E) with anti-CD19 3C10 VH and VL bound to CD19 with similar KD. Substitution of anti-CD19 3C10 VL with anti-CD38 VL (851B) resulted in an approximately five-fold reduction in binding to CD19. As expected, parental antibodies (851D / 851F) with anti-CD38 003 VH and VL did not bind to CD19.

[0239] Table 3 shows the binding data. Two parent antibodies (851D / 851F) with anti-CD38 003 VH and VL bound to CD38 with similar KDs. Substitution of anti-CD38 003 VL with anti-CD19 VL (851C) resulted in a significant reduction in binding to CD38. As expected, parent antibodies (851A / 851E) with anti-CD19 VH and VL did not bind to CD38, and 851B also did not bind to CD38. This data indicates that only anti-CD38 003 VL can function as a common light chain for anti-CD19 3C10 VH.

[0240] [Table 3]

[0241] Bispecific antibody (format) test articles included:

[0242] BS1 = 003HC:3C10HC:003LC (common light chain) in a ratio of 1:1:2

[0243] BS1b = 003HC:3C10HC:003LC (common light chain) in a ratio of 2:1:2

[0244] BS2 = 1:1:1 ratio of 003Knob:3C10scFvHole:003LC (Fab-Fc:scFv-Fc bispecific IgG1)

[0245] BS2b = 4:1:4 ratio of 003Knob:3C10scFvHole:003LC (Fab-Fc:scFv-Fc bispecific IgG1)

[0246] BS3 = 3C10 scFv-003Fab-FcKnob:FcHole:003LC) at a ratio of 1:1:1 (scFv-Fab-Fc:Fc bispecific IgG1)

[0247] BS4 = 003Fab-FcKnob-3C10scFv:FcHole (Fab-Fc-scFv:Fc bispecific IgG1) at a ratio of 1:1:1

[0248] BS4b = 003Fab-FcKnob-3C10scFv:FcHole (Fab-Fc-scFv:Fc bispecific IgG1) at a ratio of 4:1:4

[0249] CM1 = 1:1:2 ratio of 3C10Hole:VZVKnob:003LC anti-CD19 control antibody

[0250] CM1b=1:3:3 ratio 3C10Hole:VZVKnob:003LC

[0251] CM2 = 1:1:2 ratio of 003Knob:VZVHole:003LC anti-CD38 control antibody

[0252] CM2b=3:1:3 ratio 003Knob:VZVHole:003LC

[0253] Table 4 shows binding data for bispecific test articles in a single antigen format. Bispecific antibody BS1 / BS2 / BS4 bound both target antigens with KDs within 4-fold of the parent antibodies (shown in gray shading). BS3 bound only to CD19 and not to CD38, suggesting that either the anti-CD38 Fab binding site was blocked by the anti-CD19 scFv N-terminal fusion or that anti-CD38 requires a free VH N-terminus for binding. One-arm control antibodies (CM1, CM2) bound only to their intended target antigens.

[0254] [Table 4]

[0255] For the two-antigen format, antibodies were loaded onto an anti-hIgG Fc Capture biosensor for 300 seconds. The ligand-loaded sensor was saturated with 500 nM of the first antigen for 500 seconds, followed by 300 nM of the second antigen for 240 seconds. Rate constants were calculated using a monovalent (1:1) binding model. Table 5 shows that the bispecific antibody BS1 / BS2 / BS4 was able to simultaneously bind both target antigens with kA (1 / Ms) within twofold of the parent antibodies (851B, 851D, and 851E). As with the single-antigen format, BS3 bound only to CD19 and not to CD38.

[0256] [Table 5]

[0257] The variants were further tested for their ability to bind to CD19 and / or CD38. Binding experiments were performed on Octet Red at 25°C. Antibodies were loaded onto anti-hIgG Fc Capture (AHC) biosensors for 300 seconds. The ligand-loaded sensors were immersed in two-fold serial dilutions of antigen (CD19 and CD38) (starting at 300 nM) for 240 seconds for CD19 and 150 seconds for CD38 for association, followed by 600 seconds for CD19 and 130 seconds for CD38 for dissociation. Rate constants were calculated using a monovalent (1:1) binding model. Table 6 shows the binding of anti-CD38 CDRH2 variants. Table 7 shows the binding of the CD38 light chain W32H variant. Table 8 shows the binding of the CD19 heavy chain framework mutant A84S A108L.

[0258] [Table 6]

[0259] [Table 7]

[0260] [Table 8]

[0261] Example 3: Cell binding studies Cell Binding Study Protocol: Five cell lines (HEK293-CD19, HEK293-CD38, HEK293-CD19 / CD38, Daudi, and REH) were transiently transfected into HEK293 cell lines and incubated in triplicate with test articles at 133 nM followed by a 3-fold dilution series (7 dilutions total), in addition to an untreated control.

[0262] A study was conducted to evaluate cell surface expression of CD19 and CD38 on Daudi, Raji, and REH cell lines. Cells were stained in triplicate with commercially available PE-conjugated antibodies, washed, and acquired via flow cytometry. To quantify molecular expression on the surface of cells, a standard curve was generated to interpolate MFI to molecule / cell values ​​using the Quantum Simply Cellular anti-mouse IgG kit (Cat. #815-A) from Bangs Laboratories (Table 9).

[0263] [Table 9]

[0264] Figure 13A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (each with one arm directed against CD19 or CD38 and the other arm directed against varicella-zoster virus) to Daudi cells. Considering that Daudi cells have approximately 1 million copies of CD38 but only approximately 200,000 copies of CD19 on their surface, Figure 13A shows that anti-CD38 851D and 38K-VZVH bind efficiently, while anti-CD19 851A, 851B, and 19H-VZVK bind only moderately. Note that 851D, which has two CD38-binding Fabs, binds approximately five times better than 38K-VZVH, which has only one binding Fab for CD38.

[0265] Figure 13B shows the binding of bispecific antibodies BS1, BS2, and BS4 to Daudi cells. The avidity of the bispecific antibodies, which bind to both CD38 and CD19, is revealed by comparing their binding to 38K-VZVH, which binds only to CD38.

[0266] Figure 14A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (each with one arm directed against CD19 or CD38 and the other arm directed against varicella-zoster virus) to REH cells. Considering that REH cells have approximately 300,000 copies of CD38 but only approximately 50,000 copies of CD19 on their surface, Figure 14A shows that anti-CD38 851D and 38K-VZVH bind efficiently, whereas anti-CD19 851A, 851B, and 19H-VZVK bind only moderately. The magnitude of MFI is significantly lower compared to Daudi cells (Figures 2A and 2B), due to the lower expression levels of both CD38 and CD19 on REH cells. Furthermore, 851D, which has two CD38-binding Fabs, binds approximately 5 times better than 38K-VZVH, which has only one binding Fab for CD38.

[0267] Figure 14B shows the binding of bispecific antibodies BS1, BS2, and BS4 to REH cells. The avidity of the bispecific antibodies that bind to both CD38 and CD19 is evident by comparing their binding to 38K-VZVH, which binds only to CD38.

[0268] Figure 15A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (38K-VZVH, 19H-VZVK) to CD19-transfected HEK293 cells. As expected, the two anti-CD38 antibodies do not bind to these cells. Note that 851A and 851B, each containing two CD19-binding Fabs, bind significantly better than 19H-VZVK, which contains only one binding Fab for CD19.

[0269] Figure 15B shows the binding of bispecific antibodies BS1, BS2, and BS4 to CD19-transfected HEK293 cells. BS2 and BS4 bind slightly better than BS1, and because BS1 has an anti-CD38 light chain, BS2 and BS4 bind CD19 approximately 10-fold better than BS1 (see Table Octet data).

[0270] Figure 16A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (38K-VZVH, 19H-VZVK) to CD38-transfected HEK293 cells. As expected, the three anti-CD19 antibodies do not bind to these cells. Note that 851D, which has two CD38-binding Fabs, binds better than 38K-VZVH, which has only one binding Fab for CD38.

[0271] FIG. 16B shows the binding of bispecific antibodies BS1, BS2 and BS4 to CD38-transfected HEK293 cells.

[0272] Cell Binding Study Protocol - Nonspecific Background Binding: A study was conducted to evaluate the binding of three parental monoclonal antibodies (anti-CD19 clones 851A and 851B and anti-CD38 clone 851D), a human IgG1 isotype control, and daratumumab to CHO-S and Expi293T cell lines. The two cell lines were stained with a viability dye and then incubated in triplicate with the test article at a top concentration of 1,250 nM followed by a 5-fold dilution series (four total points), in addition to an untreated control, no treatment, and no secondary control.

[0273] Figure 17A shows the binding of parental antibodies (851A, 851B, 851D) to non-transfected CHO-S cells. Non-specific binding was seen for all three parental antibodies starting at 250 nM and was more pronounced with anti-CD38 851D.

[0274] Figure 17B shows the binding of parental antibodies (851A, 851B, 851D) to non-transfected Expi293T cells. Non-specific binding was seen for all three parental antibodies starting at 250 nM and was more pronounced with anti-CD38 851D.

[0275] Example 4: Direct and cross-linking apoptosis For assessment of direct apoptosis, cells were treated with test article and incubated for 48 hours at 37°C / 5% CO2. For assessment of crosslinking-induced apoptosis, cells were incubated with test article for 30 minutes on ice, followed by the addition of 5 μg / mL of rabbit anti-human Fc gamma-specific F(ab')2. Cells were then incubated for 48 hours at 37°C / 5% CO2. o Cells were incubated at 37°C / 5% CO2. After incubation, cells were washed, stained with Annexin V, and then resuspended in Annexin V buffer containing a viability dye (propidium iodide; PI) before flow cytometry acquisition. Early apoptotic cells were defined as Annexin V+ / PI- single cells, and late apoptotic / necrotic cells were defined as Annexin V+ / PI+ single cells. The sum of Annexin V+ / PI- and Annexin V+ / PI- was defined as total apoptotic / necrotic cells. The percentage of Annexin V+ / PI- cells or Annexin V+ / PI+ cells was plotted to compare various apoptotic conditions.

[0276] For direct apoptosis assessment, test articles were tested in triplicate at a final top concentration of 33 nM, followed by seven 5-fold dilutions, in addition to an untreated control. For crosslinking-induced apoptosis, individual test articles (BS1, BS2, BS4, 851A, 851B, and 851D) and test article combinations (851A and 851D, 851B and 851D, and 38K-VZVH and 19H-VZVK), in addition to daratumumab and an IgG1 isotype control, were tested in triplicate at a final top concentration of 33 nM, followed by seven 5-fold dilutions, in addition to an untreated control. As a positive control for Annexin V staining, cells were treated with 5 mM staurosporine.

[0277] Figure 18A shows direct apoptosis in Daudi cells for parental antibodies (851A, 851B, 851D), two control bispecific antibodies (38K-VZVH, 19H-VZVK), daratumumab, and an IgG1 isotype control. Daratumumab exhibited the highest level of apoptosis. Both anti-CD19 parental antibodies (851A, 851B) exhibited lower levels of apoptosis compared to daratumumab. The two bispecific controls and the anti-CD38 parental antibody 851D did not exhibit appreciable direct apoptosis.

[0278] Figure 18B shows direct apoptosis in Daudi cells for bispecific antibodies BS1, BS2, BS4, daratumumab, and the IgG1 isotype control. The BS1 and BS2 formats showed significantly higher levels of direct apoptosis compared to daratumumab. The bispecific format BS4 showed a level of direct apoptosis comparable to the parent anti-CD19 851A / 851B antibody (compare Figure 12A), which may be due to the BS4 format's inability to bring CD19 and CD38 into close proximity to initiate apoptosis.

[0279] Figure 19A shows cross-linking-induced apoptosis in Daudi cells for parental antibodies (851A, 851B, 851D), two combinations of parental antibodies (851A + 851D, 851B + 851D), daratumumab, and an IgG1 isotype control. Cross-linking increased the level of daratumumab-driven apoptosis (compare Figure 12A and Figure 7A). Cross-linking significantly increased the level of apoptosis for anti-CD38 851D, which did not show direct apoptosis (compare Figure 12A and Figure 7A). The increase in the level of apoptosis when cross-linking the anti-CD19 parental antibodies 851A and 851B was smaller for the CD38 antibody, likely due to the lower levels of CD19 compared to CD38 on Daudi cells (see Table 9). Cross-linking of the combination of anti-CD19 851A or 851B with anti-CD38 851D did not increase the level of apoptosis compared to 851D alone.

[0280] Figure 19B shows cross-linking-induced apoptosis in Daudi cells for bispecific antibodies BS1, BS2, BS4, (38K-VZVH + 19H-VZVK), daratumumab, and the IgG1 isotype control. When cross-linked, the BS1 and BS2 formats exhibited a level of apoptosis comparable to that of daratumumab. Notably, the bispecific format BS4 exhibited a level of cross-linking-induced apoptosis comparable to that of BS1, BS2, and daratumumab; without cross-linking, BS4 exhibited no apoptosis (see Figure 6B). The combination of the two control antibodies, 38K-VZVH and 19H-VZVK, exhibited significant apoptosis, but less than either of the bispecific formats, demonstrating the advantage of including anti-CD19 and anti-CD38 binding sites in a single antibody over separate antibodies.

[0281] Example 5: Cytotoxicity Daudi target cells were treated with a dose response of test article and incubated for 15 minutes at 37°C / 5% CO2. In addition to a 0 nM control, test articles were tested at a final top concentration of 133 nM, followed by a seven-point, 5-fold dilution series. Daratumumab and an IgG1 isotype control were used as positive and negative controls.

[0282] Pretreated target cells were co-cultured with human PBMCs from n=3 donors (E:T 25:1). PBMCs were "primed" overnight with 100 U / mL IL-2. PBMCs were labeled with ViaFluor 405. Samples were incubated for 4 hours at 37°C / 5% CO2 before flow cytometry analysis for cytotoxicity. For cytotoxicity analysis, cells were stained with propidium iodide (PI) and analyzed by high-throughput flow cytometry. The percentage of PI+ cells within the VF405- population was analyzed as an index of target cell cytotoxicity.

[0283] Figures 20A, 20B, and 20C show antibody-dependent cellular cytotoxicity (ADCC) for three donors. Results were similar for all three donors. The three bispecific formats, BS1, BS2, and BS4, and daratumumab exhibited similar levels of ADCC. The anti-CD19 bispecific control 19H-VZVK did not induce ADCC and was comparable to the IgG1 control antibody, likely due to the low levels of CD19 on the target Daudi cells (see Table 9). In contrast, the anti-CD38 bispecific control 38K-VZVH exhibited ADCC comparable to the bispecifics and daratumumab, likely due to the much higher levels of CD38 on Daudi cells compared to CD19.

[0284] Figures 21A-C show the ADCC for three donors. Results were similar for all three donors. The three bispecific formats, BS1, BS2, and BS4, exhibited similar levels of ADCC. The non-fucosylated versions of BS1, BS2, and BS4 showed approximately 10-fold increased ADCC compared to the fucosylated versions.

[0285] Complement-dependent cytotoxicity (CDC) assays were also performed. Target cells were treated with the following test articles: BS1, BS2, 38K-VZVH, 19H-VZVH, and the 38K-VZVH / 19H-VZVH combination, as well as dose-response controls: Darazalex, anti-CD20, WT IgG1 tafasitamab, and a human IgG1 isotype control. All were tested at a top concentration of 133 nM, followed by a 5-fold dilution series for a total of seven points, in addition to an untreated control. After a 15-minute incubation at 37°C and 5% CO2, complement was added to the treated cells at a final concentration of 25%. Cells were then incubated with complement for an additional 2 hours at 37°C and 5% CO2. After complement incubation, cells were washed, resuspended with 5 μg / mL of the viability dye propidium iodide (PI), and acquired via high-throughput flow cytometry.

[0286] Figures 22A and 22B show the results of a complement-dependent cytotoxicity (CDC) assay. The positive technical control, anti-CD20, induced robust, dose-dependent CDC activity. 38K-VZVH and 19H-VZVH (either alone or in combination), anti-CD19 tafasitamab (wt IgG1), and a human IgG1 isotype control did not induce any CDC activity. Darzalex, BS1, and BS2 all exhibited CDC activity (although not to the same magnitude as anti-CD20, as expected from the literature). The maximum cytotoxicity of Darzalex was higher than that of both BS1 and BS2.

[0287] Antibody-dependent cellular phagocytosis (ADCP) was further assayed using pHrodo Green AM (pHG)-labeled Raji cells treated with a dose-response of test article and incubated for 15 minutes at 37°C with 5% CO2. pHG is a pH-sensitive dye that is only weakly fluorescent at neutral pH but highly fluorescent at the low pH of mature macrophage phagosomes. pHG-labeled Raji target cells with anti-CD20 antibody and an IgG1 isotype control were used as positive and negative controls, with a top concentration of 133 nM, a seven-point 5-fold dilution series, and a 0 nM control. Pretreated target cells were cocultured with human macrophages (differentiated in vitro from monocytes) from three donors (E:T 1:2). Macrophages were labeled with Cell Trace Violet (CTV). Samples were incubated for 4 hours at 37°C with 5% CO2 before flow cytometry analysis of phagocytosis. The percentage of pHGhi / CTV+ cells was analyzed as an index of target cell phagocytosis. The percentage was plotted against the logarithm of the test article concentration on an XY chart, and the data was fitted to a four-parameter nonlinear regression curve from which the EC50 was calculated.

[0288] Figure 23 shows the results of an antibody-dependent cellular phagocytosis (ADCP) assay using Raji cells and donor macrophages as targets. The positive control, anti-CD20, demonstrated dose-dependent phagocytosis for all three donors after 4 hours (5-10% of maximum phagocytosis). The negative control, IgG1 isotype control, demonstrated no dose-dependent phagocytosis for all three donors after 4 hours. Darzalex demonstrated dose-dependent phagocytosis for all three donors after 4 hours (4-10% of maximum phagocytosis). BS-1, BS-2, nonfucosylated BS-1, and nonfucosylated BS-2 demonstrated minimal dose-dependent phagocytosis, with the nonfucosylated format resulting in an increase in ADCP.

[0289] Example 6: Interaction with RBCs Flow cytometry-based red blood cell (RBC) binding studies were performed to assess test article binding to red blood cells from n=3 cynomolgus monkeys and n=3 human donors. Whole blood was washed with 1X PBS and then diluted 20-fold with PBS before treatment with test articles. In addition to a 0 nM control, bispecifics (BS1, BS2), parental monoclonals (851A, 851D), and controls (anti-CD38 Darzalex, recombinant anti-CD19 Tafasitamab, IgG1 isotype control, anti-CD47 conjugated to Alexa Fluor 647) were tested in triplicate at a top final concentration of 133 nM, followed by a total of seven 5-fold serial dilutions. Single-arm controls (38K-VZVH, 19H-VZVK) were tested in combination, both at a top concentration of 133 nM and with the same dose response.

[0290] After 30 minutes of incubation on ice with the primary antibody, the cells were washed and stained with 5 μg / mL of secondary antibody (Alexa Fluor 647-labeled goat anti-human Fcγ F(ab')2) to detect binding of the test article on red blood cells. No secondary antibody was used for anti-CD47-A647 stained cells. After an additional 30 minutes of incubation on ice with the secondary antibody, the stained cells were washed, diluted, and acquired by high-throughput flow cytometry. The Alexa Fluor 647 geometric mean fluorescence intensity (MFI) of the single cell population was calculated. The MFI of AF647 was plotted on an XY chart, and the MFI was graphed against the logarithm of the concentration. The data was fitted to a nonlinear regression curve from which the EC50 was calculated.

[0291] Figure 24 shows that AF647-conjugated anti-CD47 demonstrated dose-response binding curves using red blood cells from all three human donors. Darzalex also demonstrated a dose-dependent increase in binding with all three donors, although the maximum MFI was an order of magnitude lower than that of anti-CD47. Anti-CD38 851D demonstrated the next highest maximum MFI after Darzalex, followed by BS1, BS2, the 38K-VZVH & 19H-VZVK combination, and anti-CD19 tafasitamab. Finally, anti-CD19 851A and the IgG1 isotype demonstrated only a slight increase in MFI at the highest concentrations.

[0292] An in vitro hemagglutination assay was performed on red blood cells from a total of three healthy (n=3) cynomolgus monkey (Cyno) donors and three healthy (n=3) human donors. Whole blood was obtained on the day of the study and checked for clotting. The blood was then washed with PBS and diluted 1:50 to obtain "whole blood substrate." The whole blood substrate was plated in a 96-well round-bottom plate and treated in triplicate with test articles (BS1, BS2, 38K-VZVH+19H-VZVK, 851A, and 851D), controls (tafasitamab with wild-type IgG1), Darazalex, and a human IgG1 isotype control), or a positive technical control (IGM-55.5) in PBS at a top final concentration of 133 nM, followed by six 5-fold serial dilutions, in addition to a 0 nM control. After 1 hour of incubation at 37°C, 5% CO2, the plates were photographed to confirm the level of hemagglutination. Using the photograph as a reference, each well was scored on a unique hemagglutination scale of 0 to 5. The unique appearance of each score is somewhat relative to the individual donor.

[0293] Figure 25A shows the results of the hemagglutination assay for human donor 3. The positive control, anti-CD47, induced hemagglutination for all three human donors, starting at 0.04–1.1 nM. BS1, BS2, 38K-VZVH+19H-VZVK, Darazalex, tafasitamab, and the human IgG1 isotype control all showed no induction of hemagglutination at any concentration for all three donors. Both monoclonal antibodies 851A (anti-CD19) and 851D (anti-CD38), starting at 0.2 or 1.1 nM each, induced hemagglutination for all three donors, with responses similar in magnitude to the technical control (anti-CD47). In contrast to the parent monoclonal antibodies, BS1 and BS2 did not show any induction of hemagglutination at any concentration.

[0294] Figure 25B shows the results of the hemagglutination assay for cynomolgus donor 3. The positive control, IGM-55.5 (anti-little i antigen IgM antibody), induced hemagglutination in all three cynomolgus donors starting at 0.04 or 0.2 nM. BS1, BS2, 38K-VZVH+19H-VZVK, Darazalex, tafasitamab, and the human IgG1 isotype control all showed no induction of hemagglutination at any concentration in all three donors. Both monoclonal antibodies 851A (anti-CD19) and 851D (anti-CD38), starting at 1.1 nM each, induced hemagglutination in all three donors. In contrast to the parent monoclonal antibodies, BS1 and BS2 did not show any induction of hemagglutination at any concentration.

[0295] An in vitro hemolysis assay was also performed on red blood cells from three (n=3) healthy cynomolgus monkeys (cyno) and three (n=3) healthy human donors. Whole blood was obtained on the day of the study and checked for clotting. Blood was washed with PBS and diluted 1:10 to obtain the "whole blood matrix." The whole blood matrix was treated with test articles and controls in PBS. In addition to a 0 nM control, bispecifics (BS1, BS2), parental monoclonals (851A, 851D), and controls (anti-CD38 Darazalex, recombinant anti-CD19 Tafasitamab, IgG1 isotype control) were tested in triplicate at a top final concentration of 133 nM, followed by a total of seven 5-fold serial dilutions. Single-arm controls (38K-VZVH, 19H-VZVK) were tested in combination, both at a top concentration of 133 nM and with the same dose response. Saponin was tested at a top concentration of 0.1%, with a total of seven three-fold serial dilutions. After 1 hour of incubation at 37°C, 5% CO2, the plates were centrifuged and the supernatants were collected. The supernatants were analyzed for optical density (OD) at 540 nm via a plate reader. The positive control, saponin, induced dose-dependent hemolysis starting at 0.001% up to 0.10% for all species and donors. None of the test articles induced any hemolysis at any of the concentrations tested.

[0296] Figure 26 shows that none of the test articles induced any hemolysis at any concentration tested. The positive control, saponin, induced dose-dependent hemolysis starting at 0.001% up to 0.10% for all species and donors.

[0297] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0298] [Table 10-1]

[0299] [Table 10-2]

[0300] [Table 10-3]

[0301] [Table 10-4]

[0302] [Table 10-5]

[0303] [Table 10-6]

[0304] Table 10-7

[0305] Table 10-8

Claims

1. 1. A common light chain bispecific antibody comprising a CD19 antigen-binding component configured to bind to CD19 and a CD38 antigen-binding component configured to bind to CD38, wherein the CD19 antigen-binding component comprises an antibody or antigen-binding fragment thereof, and the CD38 antigen-binding component comprises an antibody or antigen-binding fragment thereof.

2. the CD38 antigen-binding component a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; Including, the CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and l) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The common light chain bispecific antibody of claim 1 , comprising:

3. the CD38 antigen-binding component a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 151 to 155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; Including, the CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and l) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The common light chain bispecific antibody of claim 1 , comprising:

4. 3. The common light chain bispecific antibody of claim 2, wherein the CD38 antigen-binding component comprises an anti-CD38 immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 3 or 5, and an immunoglobulin light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

4.

5. 5. The common light chain bispecific antibody of claim 4, wherein the CD38 antigen-binding component comprises an anti-CD38 immunoglobulin heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 3 or 5, wherein the phenylalanine at position 54 of SEQ ID NO: 3 or position 54 of SEQ ID NO: 5 is substituted with glutamine, and an immunoglobulin light chain variable region comprising an amino acid sequence identical to SEQ ID NO:

4.

6. 6. The common light chain bispecific antibody of any one of claims 2 to 5, wherein the CD19 antigen-binding component comprises an anti-CD19 immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 6, and an immunoglobulin light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

4.

7. 7. The common light chain bispecific antibody of claim 6, wherein the CD19 antigen-binding component comprises the anti-CD19 immunoglobulin heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 1 or 6, and the immunoglobulin light chain variable region comprising an amino acid sequence identical to SEQ ID NO:

4.

8. 8. The common light chain bispecific antibody of claim 2, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises a first immunoglobulin heavy chain constant region and the anti-CD19 immunoglobulin heavy chain variable region further comprises a second immunoglobulin heavy chain constant region.

9. 9. The common light chain bispecific antibody of any one of claims 1 to 8, wherein the first immunoglobulin heavy chain constant region and / or the second immunoglobulin heavy chain constant region comprises one or more amino acid substitutions that disfavor homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote heterodimerization of the first heavy chain constant region and the second heavy chain constant region.

10. 10. The common light chain bispecific antibody of claim 9, wherein one of the first immunoglobulin heavy chain constant region or the second immunoglobulin heavy chain constant region comprises a T366W substitution (EU numbering), and the other of the first immunoglobulin heavy chain constant region or the second immunoglobulin heavy chain constant region comprises a T366S / L368A / Y407V substitution (EU numbering), such that heterodimerization of the first immunoglobulin heavy chain constant region and the second immunoglobulin heavy chain constant region is favored compared to homodimerization of the first immunoglobulin heavy chain constant region or the second immunoglobulin heavy chain constant region.

11. The common light chain bispecific antibody of any one of claims 1 to 10, wherein the heavy chain variable region that binds to CD19 comprises the A84S and A108L modifications according to the Kabat numbering.

12. The common light chain bispecific antibody according to any one of claims 1 to 11, wherein the light chain variable region that binds to CD38 comprises the W32H modification according to Kabat numbering.

13. 13. The common light chain bispecific antibody of any one of claims 1 to 12, wherein the common light chain bispecific antibody exhibits reduced hemagglutination compared to a CD19 or CD38 monospecific antibody comprising an Fc region.

14. A nucleic acid or a plurality of nucleic acids comprising a polynucleotide sequence encoding the common light chain bispecific antibody of any one of claims 1 to 13.

15. A composition comprising the common light chain bispecific antibody of any one of claims 1 to 13 and a pharmaceutically acceptable diluent, carrier, or excipient.

16. 18. Use of the common light chain bispecific antibody of any one of claims 1 to 13 or the composition of claim 15 in the manufacture of a medicament for use in a method of treating a tumor or cancer in an individual.

17. 17. The use of claim 16, wherein the cancer or tumor is a blood cancer.

18. 17. The use according to claim 16, wherein the cancer or tumor is a solid tissue cancer.

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