C19 C38 dual-specific antibody

Abstract

Targeting immunosuppressive B cell populations using bispecific or multivalent targeting molecules presents a potential pathway for therapeutic interventions that effectively modulate antitumor immune responses and improve therapeutic outcomes. Therefore, we provide engineered antibody-based therapeutics that can effectively and selectively target immunosuppressive B cell populations for the treatment of cancer. [Solution] A multispecific antibody is provided 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 an antigen-binding fragment thereof, and the CD38 antigen-binding component comprises an antibody or an antigen-binding fragment thereof.

Description

[Technical Field]

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

[0002] While antibody therapies have been successfully used to treat a variety of diseases, their applications may be limited in terms of clinical efficacy in complex diseases such as cancer. Manipulating antibody-based therapies to alter target binding affinity and valency offers a potential pathway to achieving increased efficacy and improving treatment outcomes. Bispecific or polyvalent antibodies, therefore, offer a potential approach to addressing the 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 targeted agents for the treatment of numerous diseases. [Overview of the project]

[0003] The dynamic relationship between cancer biology and the immune system is a factor linked to 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 crucial role in the antitumor immune response, where immunosuppression is generally associated with tumor growth and invasion and correlates with negative outcomes. While B cells are known to positively modulate the immune response, a population of immunosuppressive B cells functions to suppress the antitumor immune response 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 presents a pathway for therapeutic interventions in cancer that effectively modulate the antitumor immune response and improve treatment outcomes (as opposed to selective depletion of, for example, epithelial cancer cell populations). The binding molecules provided herein may include bispecific antibodies that bind to B cell lineage surface markers (e.g., CD19, CD138, IgA, and / or CD20) as well as to 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 antibody binds to CD19 and CD38, and therefore has selectivity for a specific immunosuppressive B cell population.

[0005] As provided and described herein, bispecific antibodies that bind to CD19 and CD38 offer advantages in the selective binding of 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 hemagglutination, particularly compared to monospecific CD19 or CD38 antibodies. Thus, severe side effects seen with monospecific CD19 or CD38 antibodies, such as anemia, 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 compared to monospecific controls. Furthermore, bispecific antibodies that bind to CD19 and CD38 offer further advantages over simply using two independent 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 lower side effects seen with other B cell-targeting monoclonal antibodies (e.g., rituximab), such as lymphopenia.

[0006] Described herein are complex-binding molecules comprising 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 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 component comprises a polypeptide. In some embodiments, the first or second binding component comprises a polypeptide. In some embodiments, the first and second binding components comprise a polypeptide. In some embodiments, the polypeptide of the first or second binding component comprises an amino acid sequence of at least 100 amino acid residues. In some embodiments, the polypeptide of the first and second binding component comprises an amino acid sequence of at least 100 amino acid residues.

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

[0008] In some embodiments, the first or second binding component is an immunoglobulin heavy-chain and light-chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, or a variable region fragment (V) from an immunoglobulin novel antigen receptor. NAR ), or variable region derived from heavy chain antibodies (V HH) is included. In some embodiments, the first and second binding components are immunoglobulin heavy and light chain pairs, scFv, F(ab), F(ab')2, single-domain antibodies, and variable region fragments (V) from immunoglobulin novel antigen receptors. NAR ), or variable region derived from heavy chain antibodies (V H Includes 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 the HCDR1 amino acid sequence described in any one of SEQ ID NOs. 71-75, the HCDR2 amino acid sequence described in any one of SEQ ID NOs. 81-85 or 150-155, and the HCDR3 amino acid sequence described in any one of SEQ ID NOs. 91-95, and the immunoglobulin light chain comprises the LCDR1 amino acid sequence described in any one of SEQ ID NOs. 41-45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs. 51-55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs. 61-65. In some embodiments, the immunoglobulin heavy chain contains an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 3, and the immunoglobulin light chain contains an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 2. In some embodiments, the immunoglobulin heavy chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 3, and the immunoglobulin light chain contains the same amino acid sequence as the amino acid sequence described 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 includes scFv. In some embodiments, the first and second binding components include scFv. In some embodiments, the complex-binding molecule is a bispecific antibody or its biantigen-binding fragment.

[0011] In some embodiments, the dispecific antibody is selected from one of the following formats: common light chain dispecific IgG, Fab-Fc:scFv-Fc dispecific IgG, Fab-Fc-Fab:Fc dispecific IgG, Fab-Fc-scFv:Fab-Fc-scFv dispecific IgG, Fab-Fc-scFv:Fc dispecific IgG, Fab-Fc-Fab:Fab-Fc dispecific IgG, scFv-Fab-Fc:scFv-Fab-Fc dispecific IgG, Fab-Fab-Fc:Fab-Fab-Fc dispecific IgG, Fab-Fc-Fab:Fab-Fc-Fab dispecific IgG, scFv-Fab-Fc:Fc dispecific IgG, and Fab-Fc-scFv:Fab-Fc dispecific IgG. In some embodiments, the dispecific antibody is Fab-Fc:scFv-Fc dispecific IgG. In some embodiments, the dispecific antibody is Fab-Fc-scFv:Fab-Fc-scFv dispecific IgG. In some embodiments, the bispecific antibody is scFv-Fab-Fc:Fc bispecific IgG. In some embodiments, the complex-binding molecule includes an Fc region containing an amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate. In some embodiments, the amino acid residue modified with a native carbohydrate or a 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: 11-15, 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.

[0013] In some embodiments, the first binding component comprises an amino acid sequence that has at least about 90%, 95%, 97%, 99% identity to, or is 100% identical to, the amino acid sequence set forth in any one of SEQ ID NOs: 1 and SEQ ID NOs: 2.

[0014] In some embodiments, the first binding component comprises an amino acid sequence identical to the amino acid sequences set forth in SEQ ID NOs: 1 and SEQ ID NOs: 2.

[0015] In some embodiments, the second binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, 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, 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.

[0016] In some embodiments, the second binding component comprises an amino acid sequence that has at least about 90%, 95%, 97%, 99% identity to, or is 100% identical to, the amino acid sequence set forth in any one of SEQ ID NOs: 3 and SEQ ID NOs: 4. In some embodiments, the second binding component comprises an amino acid sequence identical to the amino acid sequences set forth in SEQ ID NOs: 3 and SEQ ID NOs: 4.

[0017] In some embodiments, the complex-binding molecule binds to CD19+ and 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 eukaryotic regulatory sequences. In some embodiments, the cells include prokaryotic cells. In some embodiments, the prokaryotic cells are Escherichia coli cells. In some embodiments, the cells include eukaryotic cells. In some embodiments, the eukaryotic cells are Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or human PER.C6 cells.

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

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

[0022] Provided are complex-binding molecules for use in methods of reducing immunosuppressive B cells located in, adjacent to, or around a tumor in an individual, or immunosuppressive B cells distal to the tumor site that affect the individual's anti-tumor immune response. Provided are complex-binding molecules for use in methods of reducing immunosuppressive B cells located in, adjacent to, or around a tumor in an individual. In some embodiments, tumor-infiltrating B cells or immunosuppressive B cells include CD19+ CD38+ B cells. Further provided are complex-binding molecules for use in methods of reducing or inhibiting the function of immunosuppressive B cells located in, adjacent to, or around a tumor in an individual and / or immunosuppressive B cells distal to the tumor site that affect the individual's anti-tumor immune response. In some embodiments, the function of immunosuppressive B cells includes the release of anti-inflammatory or immunosuppressive cytokines, e.g., IL-10, IL-35, TGF-beta, or combinations thereof.

[0023] Disclosed is a method for treating an individual affected by cancer or a tumor, comprising administering a complex-binding molecule to the individual affected by 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 lymphoblastic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin 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 (for example, cancer cells express CD19 and CD38).

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

[0025] Disclosed is a method for reducing immunosuppressive B cells that affect the antitumor immune response to a tumor in an individual with a tumor or cancer, comprising administering a complex-binding molecule to the individual with a tumor or cancer to reduce the immunosuppressive B cells that affect the antitumor immune response. Further disclosed is a method for reducing immunosuppressive B cells that are in, adjacent to, or around a tumor in an individual with a tumor or cancer, comprising administering a complex-binding molecule to the individual with a tumor or cancer to reduce the immunosuppressive B cells that are in, adjacent to, or around a tumor. In some embodiments, tumor-infiltrating B cells or immunosuppressive B cells include CD19+, CD38+ B cells.

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

[0027] Also disclosed is a method for producing a complex-binding molecule, comprising incubating a cell containing an expression vector comprising a nucleic acid sequence encoding the complex-binding molecule 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. Such isolation and purification may involve steps of contacting the cell culture medium or the cell culture medium subjected to one or more purification steps with a resin or column containing protein, 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 the cell culture medium subjected to one or more purification steps.

[0028] Provided herein are complex-binding molecules 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 its antigen-binding fragment, and the CD38-binding component comprises an antibody or its antigen-binding fragment. In some embodiments, provided are complex-binding molecules of any of the preceding embodiments in which the CD19 and / or CD38-binding components comprise an immunoglobulin heavy-chain and light-chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, a variable region fragment (VNAR) from an immunoglobulin neoantigen receptor, or a variable region (VHH) derived from a heavy-chain antibody. In some embodiments, provided are complex-binding molecules of any of the preceding embodiments in which the CD19 or CD38-binding component comprises an immunoglobulin heavy-chain and light-chain pair. In some embodiments, provided are complex-binding molecules of any of the preceding embodiments in which the CD19 and CD38-binding components comprise an immunoglobulin heavy-chain and light-chain pair.

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

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

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

[0032] Furthermore, the complex-binding molecule comprises a CD38 antigen-binding component that binds to CD38, which includes 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, which includes 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) containing an amino acid sequence described in any one of SEQ ID NOs. 71-75, and b) a component described in any one of SEQ ID NOs. 81-85 or 150-155. It is a complex-binding molecule comprising: c) a heavy chain complementarity determination region 2 (HCDR2) containing an amino acid sequence; c) a heavy chain complementarity determination region 3 (HCDR3) containing an amino acid sequence described in any one of SEQ ID NOs. 91 to 95; d) a light chain complementarity determination region 1 (LCDR1) containing an amino acid sequence described in any one of SEQ ID NOs. 101 to 105; e) a light chain complementarity determination region 2 (LCDR2) containing an amino acid sequence described in any one of SEQ ID NOs. 111 to 115; and / or f) a light chain complementarity determination region 3 (LCDR3) containing an amino acid sequence described in any one of SEQ ID NOs. 121 to 125.

[0033] In some embodiments, the provided material is a 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) containing an amino acid sequence described in any one of SEQ ID NOs: 11-15; h) a heavy chain complementarity-determining region 2 (HCDR2) containing an amino acid sequence described in any one of SEQ ID NOs: 21-25; i) a heavy chain complementarity-determining region 3 (HCDR3) containing an amino acid sequence described in any one of SEQ ID NOs: 31-35; j) a light chain complementarity-determining region 1 (LCDR1) containing an amino acid sequence described in any one of SEQ ID NOs: 101-105; k) a light chain complementarity-determining region 2 (LCDR2) containing an amino acid sequence described in any one of SEQ ID NOs: 111-115; and / or l) a light chain complementarity-determining region 3 (LCDR3) containing an amino acid sequence described in any one of SEQ ID NOs: 121-125.

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

[0035] In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD19 antigen-binding component comprises an immunoglobulin heavy chain variable region having the same amino acid sequence as SEQ ID NO: 1 or 6, and the immunoglobulin light chain variable region having the same amino acid sequence as SEQ ID NO: 4. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises a first immunoglobulin heavy chain constant region. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD38 immunoglobulin light chain variable region further comprises an immunoglobulin light chain constant region. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD19 immunoglobulin heavy chain variable region further comprises a second immunoglobulin heavy chain constant region. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, 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 are detrimental to the homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote the heterodimerization of the first and second heavy chain constant regions. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, 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 regions comprises a T366S / L368A / Y407V substitution (EU numbering), and as a result, 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 regions. In some embodiments, the provided molecule is a complex-binding molecule of any of the preceding embodiments, wherein a single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component.

[0036] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0037] Novel features of the present invention are precisely described in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0038] [Figure 1] This diagram illustrates the structure of common light chain bispecific IgG. [Figure 2] This diagram illustrates the structure of Fab-Fc:scFv-Fc bispecific IgG. [Figure 3] This diagram illustrates the structure of Fab-Fc-Fab:Fc bispecific IgG. [Figure 4] This diagram illustrates the structure of Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG. [Figure 5] This is a diagram illustrating the structure of Fab-Fc-scFv:Fc bispecific IgG. [Figure 6] This diagram illustrates the structure of Fab-Fc-Fab:Fab-Fc bispecific IgG. [Figure 7] This diagram illustrates the structure of scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG. [Figure 8] This diagram illustrates the structure of Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG. [Figure 9] This diagram illustrates the structure of Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG. [Figure 10] This is a diagram illustrating the structure of Fab-Fc-scFv:Fab-Fc bispecific IgG. [Figure 11]This diagram illustrates the structure of scFv-Fab-Fc:Fc bispecific IgG. [Figure 12A] This figure shows the binding data for CD19 and CD38 antibodies. Figure 12A shows the cell surface expression of CD19 and CD38. Figures 12B and 12C show the binding profiles of CD19 and CD38 antibodies. Figures 12D and 12E show the binding of CD19 and CD38 controls. Figure 12F shows the binding profile of cells that do not express CD19 and CD38. [Figure 12B] This figure shows the binding data for CD19 and CD38 antibodies. Figure 12A shows the cell surface expression of CD19 and CD38. Figures 12B and 12C show the binding profiles of CD19 and CD38 antibodies. Figures 12D and 12E show the binding of CD19 and CD38 controls. Figure 12F shows the binding profile of cells that do not express CD19 and CD38. [Figure 12C] This figure shows the binding data for CD19 and CD38 antibodies. Figure 12A shows the cell surface expression of CD19 and CD38. Figures 12B and 12C show the binding profiles of CD19 and CD38 antibodies. Figures 12D and 12E show the binding of CD19 and CD38 controls. Figure 12F shows the binding profile of cells that do not express CD19 and CD38. [Figure 12D] This figure shows the binding data for CD19 and CD38 antibodies. Figure 12A shows the cell surface expression of CD19 and CD38. Figures 12B and 12C show the binding profiles of CD19 and CD38 antibodies. Figures 12D and 12E show the binding of CD19 and CD38 controls. Figure 12F shows the binding profile of cells that do not express CD19 and CD38. [Figure 12E] This figure shows the binding data for CD19 and CD38 antibodies. Figure 12A shows the cell surface expression of CD19 and CD38. Figures 12B and 12C show the binding profiles of CD19 and CD38 antibodies. Figures 12D and 12E show the binding of CD19 and CD38 controls. Figure 12F shows the binding profile of cells that do not express CD19 and CD38. [Figure 12F]This figure shows the binding data for CD19 and CD38 antibodies. Figure 12A shows the cell surface expression of CD19 and CD38. Figures 12B and 12C show the binding profiles of CD19 and CD38 antibodies. Figures 12D and 12E show the binding of CD19 and CD38 controls. Figure 12F shows the binding profile of cells that do not express CD19 and CD38. [Figure 13A] This figure shows the binding data of antibodies against Daudi cells. [Figure 13B] This figure shows the binding data of antibodies against Daudi cells. [Figure 14A] This figure shows the binding data of antibodies against REH cells. [Figure 14B] This figure shows the binding data of antibodies against REH cells. [Figure 15A] This figure shows antibody binding data against CD19-transfected HEK293 cells. [Figure 15B] This figure shows antibody binding data against CD19-transfected HEK293 cells. [Figure 16A] This figure shows antibody binding data against CD38-transfected HEK293 cells. [Figure 16B] This figure shows antibody binding data against CD38-transfected HEK293 cells. [Figure 17A] This figure shows antibody binding data against non-transfected CHO cells. [Figure 17B] This figure shows antibody binding data against non-transfected CHO cells. [Figure 18A] This figure shows data on direct apoptosis in Daudi cells for the antibody test product. [Figure 18B] This figure shows data on direct apoptosis in Daudi cells for the antibody test product. [Figure 19A] This figure shows data on cross-linking-induced apoptosis in Daudi cells for the antibody test product. [Figure 19B] This figure shows data on cross-linking-induced apoptosis in Daudi cells for the antibody test product. [Figure 20A] This figure shows ADCC data for three donors across antibody test samples. [Figure 20B] This figure shows ADCC data for three donors across antibody test samples. [Figure 20C] This figure shows ADCC data for three donors across antibody test samples. [Figure 21A] This figure shows ADCC data for three donors across antibody test samples. [Figure 21B] This figure shows ADCC data for three donors across antibody test samples. [Figure 21C] This figure shows ADCC data for three donors across antibody test samples. [Figure 22A] This figure shows the CDC profiles across the test samples. [Figure 22B] This figure shows the CDC profiles across the test samples. [Figure 23] This figure shows ADCP data across antibody test samples. [Figure 24] This figure shows RBC binding data for antibody test samples. [Figure 25A] This figure shows the hemagglutination profile for the antibody test product. [Figure 25B] This figure shows the hemagglutination profile for the antibody test product. [Figure 26] This figure shows hemolysis data for antibody test samples. [Modes for carrying out the invention]

[0039] Immunosuppressive B cell populations that suppress antitumor immune responses can generally be defined by the presence of one or more cell surface biomarkers. Therefore, therapeutic agents that effectively and specifically target immunosuppressive B cells can be used to prevent and / or eliminate immunosuppression within, adjacent to, or around a tumor, or within the tumor environment. Provided herein are complex-binding molecules that target immunosuppressive B cells. Furthermore, provided are complex-binding molecules comprising 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 and the second target comprises a suppressive B cell surface marker. Disclosed herein are multivalent antibodies that specifically bind to B cell populations associated with negative modulation of antitumor responses or immunosuppression. Immunosuppressive B cells may comprise, or may 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 may also be used to treat immunosuppressive B cells and / or oncogenic conditions and / or cancers associated with immune dysfunction.

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

[0041] As used herein, the term “cancer” can refer to or describe a physiological condition in mammals typically characterized by unregulated cell proliferation. Cancer may also include, but is not limited to, hematological malignancies and / or solid tumors. Cancer can refer to diseases of the blood, bones, organs, skin tissues and vascular system, including, but not limited to, diseases of the bladder, blood, bones, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lungs, lymph nodes, mouth, cervix, ovaries, pancreas, prostate, rectum, kidneys, skin, stomach, testes, 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 pre-lymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's giant spheroid tumor)). B-cell lymphoma, proteinemia or painless lymphoma, splenic marginal zone lymphoma, plasmacytosis myeloma, plasmacytosis, plasmacytoma, peri-implant immunoglobulin deposition, heavy chain disease, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodular marginal zone B-cell lymphoma (NMZL), gastrointestinal tumors (e.g., gastrointestinal stromal tumors (GIST)), follicular lymphoma, mantle cell lymphoma / leukemia, diffuse B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, and Burkitt's lymphoma (Burkitt lymphoma), mature T-cell and natural killer (NK) tumors (prolymphocytic leukemia, T-cell large lymphocytic leukemia). T-cell lymphoma, invasive NK cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides (Sézary syndrome), primary skin degenerative large cell lymphoma, lymphomatoid papular dysplasia, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma, and degenerative large cell lymphoma.Lymphoma), Hodgkin lymphoma (nodular sclerosis type, mixed cell type, lymphocyte-rich type, lymphocyte-depleted or non-reduced type, nodular lymphocyte type), Myeloma (multiple myeloma, inactive myeloma (inert) Myeloma, smoldering myeloma, chronic myeloproliferative disorders, spinal dysplasia / 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 bone disease, osteosarcoma, breast cancer (hormone-dependent, non-hormone-dependent), gynecological cancers (pediatric cervix, endometrium, fallopian tube, gestational trophoblast disease, ovary, peritoneum, uterus, vagina and vulva), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma, protuberous dermatofibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, hairy cell astrocytoma, embryonic hair growth neuroepithelial neoplasia Neoplasty, oligodendroglioma, ependymoma, glioblastoma multiforme, mixed glioma, oligodendroglioma / astrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, embryonic tissue tumor, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastrointestinal-pancreatic or gastrointestinal-pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, knot rectal cancer, invasive neuroendocrine tumor, leiomyosarcoma, mucinous adenocarcinoma, signet ring cell adenocarcinoma, hepatocellular carcinoma, hepatobiliary liver cancer Examples include, but are not limited to, cancer, 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, 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 "differentiation cluster 19" (also known as B4, T cell surface antigen Leu-12, and CVID3) refers to a B cell lineage surface biomarker or transmembrane protein encoded by the gene CD19 in humans. CD19 can function as a co-receptor for B cell antigen receptor complexes (BCRs) on B lymphocytes, for the activation of downstream signaling pathways and for lowering the threshold for triggering B cell responses to antigens. Structurally, the CD19 amino acid sequence has at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences of, for example, GenBank accession numbers NM_001178098.2→NP_001171569.1 or NM_001770.6→NP_001761.3, over a sequence length of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids, or over the entire length of the polypeptide. Structurally, the CD19 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of, for example, GenBank accession number NG_007275.1 or NCBI Gene ID 930, across at least 300, 500, 750, 1000, 1250, or 1500 nucleic acid sequences, or across the entire length of the polynucleotide. Sequence alignment can be performed using any alignment algorithm known in the art, such as BLAST, ALIGN set to default settings.

[0043] The term "CD38" or "differentiation cluster 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 that leads to cell activation and proliferation. Structurally, the CD38 amino acid sequence has at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of, for example, GenBank accession number NM_001775.4→NP_001766.2, across sequence lengths of at least 50, 100, 150, 200, 250 amino acids, or across the full length of the polypeptide. Structurally, the CD38 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of, for example, GenBank accession number NC_000004.12 or NCBI Gene ID 952, across the sequence lengths of at least 300, 500, or 750 nucleic acids, or across the entire length of the polynucleotide. Sequence alignment can be performed using any alignment algorithm known in the art, such as BLAST, ALIGN set to default settings.

[0044] The term “antibody” as used herein is used in its broadest sense and includes polyvalent or bispecific antibodies and monoclonal antibodies, e.g., intact antibodies and their functional (antigen-binding) antibody fragments, e.g., fragments, antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, e.g., single-chain variable fragments (sFv or scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody) fragments. The term also includes genetically engineered and / or otherwise modified forms of immunoglobulins, e.g., intrabody, peptidebody, chimeric antibody, fully human antibody, humanized antibody, and heteroconjugate antibody, polyspecific antibodies, e.g., bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to include its functional antibody fragments. The term also encompasses intact or full-length antibodies, for example, antibodies of any class or subclass, such as IgG and its subclasses, IgM, IgE, IgA, and IgD. An antibody may contain the human IgG1 constant region. An antibody may contain the human IgG4 constant region.

[0045] The antibodies offered include polyspecific or polyvalent antibodies (e.g., bispecific and polyreactive antibodies) and their antibody fragments. Antibodies also include antibody conjugates and antibody-containing molecules, such as chimeric molecules. Therefore, in addition to full-length and native antibodies, antibodies include fragments and parts that retain their binding specificity, such as any specific binding portion, such as any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), as well as biologically relevant (antigen-binding) fragments or their specific binding portions, including but not limited to Fab, F(ab')2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally found in substantially homogeneous antibody compositions, and therefore any individual antibodies contained within a monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies may contain the human IgG1 constant region or the human IgG4 constant region.

[0046] The terms "complementarity-determining region" (CDR), synonymous with "hypervariable region" or "HVR," and "CDR" are known in the art and refer to non-contiguous sequences of amino acids within the 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 are described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997), JMB 273, pp. 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262: pp. 732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, pp. 732-745 ("Contact" numbering scheme); and 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. It can be easily determined using one of many well-known schemes, including those described in Jan;27(1):55-77 ("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 ("Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0047] The boundaries of a given CDR or FR can 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. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions being applied by insertion characters, such as "30a", and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0048] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of 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, and each domain contains 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)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds a particular antigen can use the V H or V L domains from an antibody that binds the antigen to respectively complement the V L or V HDomains may be isolated by screening libraries (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 the intact antibody that contains the portion of the intact antibody that binds to the antigen to which the intact antibody binds. 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 polyspecific 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 a variety of techniques, including, but not limited to, production by recombinant host cells, in addition to proteolytic digestion of intact antibodies. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment containing a non-naturally occurring structure, e.g., a synthetic linker, e.g., having two or more antibody regions or chains joined by a polypeptide linker, and / or not produced by enzymatic digestion of naturally occurring intact antibodies.

[0050] In this specification, molecules, peptides, polypeptides, antibodies, or antibody fragments may be referred to as “dual-specific” or “bispecific,” including grammatical equivalents. A dual-specific molecule has the ability to specifically bind to at least two structurally distinct targets. Specific binding may include, but is not limited to, two distinct binding moieties that are structurally distinct at the molecular level, or high affinity (e.g., about 1 x 10⁻¹⁶). -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 1.25. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as “multispecific” refers to a molecule that has the ability to specifically bind to at least three structurally distinct targets. A “bispecific antibody,” including its grammatical equivalent, refers to a bispecific molecule that preserves at least one fragment of an antibody capable of specifically binding to a target, e.g., a variable region, heavy or light chain, or one or more complementarity-determining regions from the antibody molecule. A “multispecific antibody,” including its grammatical equivalent, refers to a multispecific molecule that preserves at least one fragment of an antibody capable of specifically binding to a target, e.g., a variable region, heavy or light chain, or a complementarity-determining region from the antibody molecule.

[0051] In this specification, “linker” is also referred to as “linker sequence,” “spacer,” “tethering sequence,” or their grammatical equivalents. “Linker” as used herein refers to two distinct molecules that are themselves target-binding, catalytically active, or naturally expressed and assembled as separate polypeptides, e.g., two distinct binding moieties or heavy / light chain pairs. Numerous strategies can be used to covalently link molecules. These include, but are not limited to, polypeptide linking between the N-terminus and C-terminus of a protein or protein domain, linking via disulfide bonds, and linking via chemical crosslinking reagents. In one embodiment of this specification, the linker is a peptide bond produced by recombinant technology or peptide synthesis. The linker peptide may primarily consist of the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be long enough to link the two molecules so that they adopt a relative conformation to each other and, as a result, retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids long or about 1 to 30 amino acids long. In one embodiment, a linker 1 to 20 amino acids long may be used. Useful linkers include glycine-serine polymers, e.g., (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n (where n is at least an integer of 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Exemplary linkers for linking antibody fragments or single-chain variable fragments may include AAEPKSS, AAEPKSDKTHTCPPCP, GGGG, or GGGGDKTHTCPPCP. Alternatively, various non-proteinoid polymers, not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol may have applications as linkers.

[0052] The “fragment-based” bispecific antibody or the bispecific antibody comprising a “single-chain variable fragment” or “scFv” in this disclosure may refer to a single-chain antibody or 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 inhibit the binding of either targeting moiety. Examples of fragment-based bispecific antibody formats include Tandem V. HH Examples include antibodies, tandem scFv, scFv-Fab, F(ab)2, and biaffinity retargeting antibodies (DART). Such fragment-based antibodies can be further manipulated with additional binding moieties specific to a given target, such as A2:B1, A1:B2, or A2:B2, or with fragments of Fc regions to improve pharmacokinetics or promote ADCC, ADCP, or CDC.

[0053] The “binding moiety” refers to the portion of a molecule, peptide, polypeptide, antibody, or antibody fragment that mediates specific binding to the described target, antigen, or epitope. For example, the binding moiety of an antibody may include a heavy / light chain variable region pair or one or more complementarity-determining regions (CDRs).

[0054] As used herein, “target” refers to a molecule, peptide, polypeptide, antibody, or a portion of a molecule involved with the binding portion of an antibody fragment. A target may include an amino acid sequence and / or carbohydrates, lipids, or other chemical entities. “Antigen” is a target comprising 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 polyspecific molecule refers to the number of targets to which the described molecule, peptide, polypeptide, antibody, or antibody fragment can bind. For example, a monovalent molecule can bind to one specific target molecule, a divalent molecule can bind to two molecules, and a tetravalent molecule can bind to four targets. For example, a bispecific divalent molecule is a molecule that can bind to two targets and two structurally different targets. For example, a bispecific divalent molecule, when brought into contact with a solution containing target A and target B, may bind to A2, B2, or A:B.

[0056] A “humanized” antibody is one 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. Humanized antibodies may optionally contain at least a portion of the 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 against humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0057] Among the antibodies offered are human antibodies. “Human antibodies” are antibodies having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or by a human antibody repertoire including a human antibody library, or by a non-human source utilizing other human antibody coding sequences. The term excludes humanized non-human antibodies that include non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. Human antibodies may also be prepared by administering an immunogen to a transgenic animal modified 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 human immunoglobulin loci that replace endogenous immunoglobulin loci, or are extrachromosomal, or randomly incorporated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are generally inactivated. Human antibodies may also originate from human antibody libraries, including phage displays and cell-free libraries containing antibody coding sequences derived 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 correlate with binding to FcγRIIIa, and increased binding to FcγRIIIa leads to increased ADCC activity. "ADCP" or "antibody-dependent cell-mediated phagocytosis," as used herein, may 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 polymers of amino acid residues, not limited to a minimum length. Polypeptides, including antibodies and antibody chains provided, as well as other peptides, e.g., linkers and binding peptides, may contain amino acid residues, including native and / or non-native amino acid residues. The term also includes post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may contain modifications to the native or natural sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0060] The sequence identity percentage (%) relative to the reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps where necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms required to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is 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, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is released by Genentech, Inc., South San Francisco, Calif., or can be compiled from 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 should not be changed.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 or against a given amino acid sequence B (which can also be expressed as a given amino acid sequence A having or containing a particular amino acid sequence identity % to 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 that the sequence alignment program ALIGN-2 scores as identical matches in its alignment of A and B, and Y is the total number of amino acid residues in B). If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it is recognized that the amino acid sequence identity % of A to B is not equal to 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 preceding paragraph.

[0061] Amino acid sequence variants of antibodies provided herein can be envisioned and recalled. Variants typically differ from polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the polypeptide sequences of the present invention and evaluating the biological activity of one or more polypeptides as described herein and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody, and amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and / or substitutions can be used to arrive at the final construct, as long as the final construct has the desired characteristics, e.g., antigen-binding properties. Antibody variants having one or more amino acid substitutions can be provided. Sites of interest for mutagenesis by substitution include CDRs and FRs. By introducing amino acid substitutions into an antibody of interest, the product can be screened for desired activities, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC.

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

[0063] Antibodies described herein may be encoded by nucleic acids. A nucleic acid is a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to transfer a polynucleotide encoding a polypeptide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule that has the ability to transport another nucleic acid ligated to it. One type of vector is a genome-integrated vector, or “integrated vector,” which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an “episome” vector, e.g., a nucleic acid with the ability to replicate outside of chromosomes. A vector that has the ability to direct the expression of a functionally linked gene is referred to herein as an “expression vector.” Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and viral vectors, among others. In expression vectors, regulatory elements such as promoters, enhancers, and polyadenylation signals for use in the control of transcription may be derived from mammalian, microorganism, viral, or insect genes. Additional selective genes may be incorporated to facilitate the ability to replicate in the host and to promote the recognition of transformants, which are usually conferred by the origin of replication. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses, may be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors may contain sequences that instruct site-specific integration (e.g., AttP-AttB recombination) into defined locations or a limited set of sites in the genome. Additionally, vectors may contain sequences derived from transposing elements.

[0064] As used herein, the terms “homonymous,” “homonymy,” or “homonymy percentage,” used herein to describe an amino acid sequence or nucleic acid sequence in comparison to a reference sequence, may be determined using formulas described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: pp. 2264-2268, 1990; modified in Proc. Natl. Acad. Sci. USA 90: pp. 5873-5877, 1993). Such formulas are incorporated into the basic local alignment search tool (BLAST) program by Altschul et al. (J. Mol. Biol. 215: pp. 403-410, 1990). The homology percentage of a sequence may be determined using the latest version of BLAST available as of the filing date of this application.

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

[0066] Where 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 treatment. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cattle, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0067] As used herein, the term “approximately” when used to modify a specific number refers to that number plus or minus 10%. When used to modify a range, the term “approximately” refers to a range from minus 10% of its minimum value to plus 10% of its maximum value.

[0068] Where used herein, the terms “treatment” or “to treat” are used in reference to a medical or other intervention regimen used to obtain beneficial or desired outcomes in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic and / or preventive benefits. A therapeutic benefit may refer to the eradication or remission of symptoms or the underlying disorder being treated. A therapeutic benefit may also be achieved with the eradication or remission of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, even though the subject may still be suffering from the underlying disorder. A preventive 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 a preventive benefit, a subject at risk of developing a particular disease, or a subject reporting one or more physiological symptoms of a disease, may receive treatment even if a diagnosis of the disease has not been made. Those skilled in the art will recognize that not all individuals in a given population of potential individuals for treatment will respond to the treatment, or will respond equally. Such individuals are considered to have been treated.

[0069] Section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described herein.

[0070] bispecific molecule Provided herein are bispecific, polyvalent, or complex-binding molecules comprising 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 and the second target comprises an inhibitory B cell surface marker. Immunosuppressive B cells or B cell populations may include B cell lineage surface biomarkers and inhibitory B cell surface biomarkers. B cell lineage surface markers may include CD19, CD138, IgA, or CD45. Immunosuppressive B cell surface markers may include 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 comprises CD19. In some embodiments, the inhibitory B cell surface marker includes 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] A polyvalent, bispecific, or complex-binding molecule has the ability to specifically bind to at least two structurally distinct targets. Specific binding may result from two structurally distinct binding moieties at the molecular level, including but not limited to distinct non-identical amino acid sequences, or from a single binding moiety capable of specifically binding to two structurally distinct targets. Molecules, peptides, polypeptides, antibodies, or antibody fragments referred to as "polyspecific," "polyvalent," or "bispecific" may refer to molecules having the ability to specifically bind 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 polypeptides. In certain embodiments, the first and second binding components consist of polypeptides. In certain embodiments, the polypeptide of the first or second binding component comprises an amino acid sequence with a length of at least 100 amino acid residues. In a particular embodiment, the polypeptides of the first and second binding components include an amino acid sequence having a length of at least 100 amino acid residues.

[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, e.g., a variable region, heavy or light chain, or one or more complementarity-determining regions from the antibody molecule. In some embodiments, the complex-binding molecule described herein is a bispecific antibody and / or its biantigen-binding fragment. A bispecific antibody has 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, where 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0073] Immunosuppressive B cells or immunosuppressive B cell populations may contain cell surface biomarkers CD19 and CD38. Further disclosed herein are bispecific antibodies that target CD19 and CD38. In some embodiments, the CD19-binding component includes a variable heavy chain (VH) containing SEQ ID NO: 1. In certain embodiments, the CD19-binding component includes a VH CDR1 region containing 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 includes a VH CDR2 region containing 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 includes a VH CDR3 region containing 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 includes a variable light chain (VL) containing SEQ ID NO: 2. In certain embodiments, the CD19-binding component includes a VL CDR1 region containing 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 includes a VL CDR2 region containing 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 includes a VL CDR3 region containing 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, the first binding component comprising the HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11-15, the HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21-25, the HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31-35, the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 41-45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 51-55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 61-65.

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

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

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

[0079] In some embodiments, the CD19-binding component includes variable heavy and light chains or CDRs corresponding to or derived from ricebilizumab, tafacitamab, tapritumomab, obexerimab, blinatumomab, coltuximab, denintuzumab, or roncasutuximab, MOR208, MEDI-551, XmAb 5871, MDX-1342, or AFM11.

[0080] In some embodiments, the CD38 binding component includes a variable heavy chain (VH) containing SEQ ID NO: 3. In certain embodiments, the CD19 binding component includes a VH CDR1 region containing 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 includes a VH CDR2 region containing 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 includes a VH CDR3 region containing 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 includes a variable light chain (VL) containing SEQ ID NO: 4. In certain embodiments, the CD19-binding component includes a VL CDR1 region containing 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 includes a VL CDR2 region containing 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 includes a VL CDR3 region containing 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, the CD38-binding component comprising the HCDR1 amino acid sequence described in SEQ ID NO: 71, the HCDR2 amino acid sequence described in SEQ ID NO: 81, the HCDR3 amino acid sequence described in SEQ ID NO: 91, the LCDR1 amino acid sequence described in SEQ ID NO: 101, the LCDR2 amino acid sequence described in SEQ ID NO: 111, and / or the LCDR3 amino acid sequence described in SEQ ID NO: 121.

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

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

[0085] In some embodiments (for example, any prior embodiments), the CD38-binding component CDR-H2 comprises the amino acid residue P(X1)LG(X2)A, where X1 and X2 allow 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, the hydrophobic amino acids 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, the CD38-binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 3, and the VL sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 4, and the CD19-binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 1, and the VL sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 2.

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

[0088] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, the CD38-binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NOs. 3, 215, or 218-223, and the VL sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NOs. 4 or 223, and the CD19-binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NOs. 1, 201, or 216-217, and the VL sequence comprises at least about 90%, 95%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NOs. 2. In some embodiments, the CD19-binding component includes a VH amino acid sequence with substitutions at A84 and A108. In some embodiments, the substitutions include A84S and A108L.

[0089] In some embodiments, the bispecific antibody comprises a CD38-binding component and a CD19-binding component, the CD38-binding component comprising a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprising the same amino acid sequence as SEQ ID NOs. 3, 215, or 218-223, and the VL sequence comprising the same amino acid sequence as SEQ ID NOs. 4 or 223, and the CD19-binding component comprising a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprising the same amino acid sequence as SEQ ID NOs. 1, 201, 216-217, and the VL sequence comprising the same amino acid sequence as SEQ ID NOs. 2. In some embodiments, the CD19-binding component comprises a VH amino acid sequence with substitutions at A84 and A108. In some embodiments, the substitutions include 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 described in SEQ ID NO: 71, the HCDR2 amino acid sequence described in SEQ ID NO: 81, the HCDR3 amino acid sequence described in SEQ ID NO: 91, the LCDR1 amino acid sequence described in SEQ ID NO: 101, the LCDR2 amino acid sequence described in SEQ ID NO: 111, and / or the LCDR3 amino acid sequence described in SEQ ID NO: 121, and the CD19-binding component comprises the HCDR1 amino acid sequence described in SEQ ID NO: 11, the HCDR2 amino acid sequence described in SEQ ID NO: 21, the HCDR3 amino acid sequence described in SEQ ID NO: 31, the LCDR1 amino acid sequence described in SEQ ID NO: 41, the LCDR2 amino acid sequence described in SEQ ID NO: 51, and / or the LCDR3 amino acid sequence described 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 described in SEQ ID NO: 72, the HCDR2 amino acid sequence described in SEQ ID NO: 82, the HCDR3 amino acid sequence described in SEQ ID NO: 92, the LCDR1 amino acid sequence described in SEQ ID NO: 102, the LCDR2 amino acid sequence described in SEQ ID NO: 112, and / or the LCDR3 amino acid sequence described in SEQ ID NO: 122, and the CD19-binding component comprises the HCDR1 amino acid sequence described in SEQ ID NO: 12, the HCDR2 amino acid sequence described in SEQ ID NO: 22, the HCDR3 amino acid sequence described in SEQ ID NO: 32, the LCDR1 amino acid sequence described in SEQ ID NO: 42, the LCDR2 amino acid sequence described in SEQ ID NO: 52, and / or the LCDR3 amino acid sequence described in SEQ ID NO: 62.

[0092] In some embodiments, if the bispecific substance includes other structures that require a light chain constant region for Fab or bispecific format, the VL contains at least about 90%, 95%, 97%, 98%, 99%, or identical amino acid sequences to either SEQ ID NO: 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 described in SEQ ID NO: 75, the HCDR2 amino acid sequence described in SEQ ID NO: 85, the HCDR3 amino acid sequence described in SEQ ID NO: 95, the LCDR1 amino acid sequence described in SEQ ID NO: 105, the LCDR2 amino acid sequence described in SEQ ID NO: 115, and / or the LCDR3 amino acid sequence described in SEQ ID NO: 125, and the CD19-binding component comprises the HCDR1 amino acid sequence described in SEQ ID NO: 15, the HCDR2 amino acid sequence described in SEQ ID NO: 25, the HCDR3 amino acid sequence described in SEQ ID NO: 35, the LCDR1 amino acid sequence described in SEQ ID NO: 45, the LCDR2 amino acid sequence described in SEQ ID NO: 55, and / or the LCDR3 amino acid sequence described in SEQ ID NO: 65.

[0094] In some embodiments, the CD38-binding component includes 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 such 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 within the CDR. Such modifications may be made outside the CDR "hotspot." Variant V H and V LIn some embodiments of the sequences, each CDR remains unchanged. Amino acid sequence insertions and deletions include single or multiple amino acid insertions and deletions, as well as amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N or C terminus of an antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody. An example of an intra-sequence insertion variant of an antibody molecule is the insertion of three amino acids in the light chain. An example of a terminal deletion is an antibody with seven or fewer amino acid deletions at the end of the light chain.

[0096] Modifications (e.g., substitutions) may be made in the CDR, for example, to improve antibody affinity. Such modifications may be made in CDRs encoding codons that have a high mutagenesis rate during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: pp. 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-specific mutagenesis) may be used to improve antibody affinity (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178: pp. 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: pp. 1081-1085 (1989)). CDR-H3 and CDR-L3 are particularly often targeted. Alternatively, or additionally, the crystalline structure of the antigen-antibody complex is used to identify contact sites between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0097] Antibodies can be modified to increase or decrease their glycosylation (for example, 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 modified. Native antibodies from mammalian cells typically have the Asn of the CH2 domain in the Fc region. 297The antibody contains branched, bifurcated oligosaccharides attached by N-linking (see, for example, Wright et al., TIBTECH 15: pp. 26-32 (1997)). The oligosaccharides can be various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose attached to GlcNAc in the base of the bifurcated oligosaccharide structure. Modification of the oligosaccharide in the antibody may be done, for example, to create antibody variants having certain enhanced properties. Antibody glycosylated variants may have enhanced ADCC and / or CDC function. In some embodiments, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is the sum of all glycan structures attached to Asn297. 297 This is determined by calculating the average amount of fucose within the sugar chain (see, for example, International Publication No. 08 / 077546). Asn 297 This refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues) (see, for example, Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85). However, Asn 297Furthermore, due to minor sequence variations in the antibody, the fucosylated variant may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants can possess enhanced ADCC function (see, for example, Okazaki et al., J. Mol. Biol. 336: pp. 1239-1249 (2004); and Yamane-Ohnuki et al., Biotech. Bioeng. 87: p. 614 (2004)). Cell lines, such as knockout cell lines and methods of using them, can be used to produce defucosylated antibodies, such as Lec13 CHO cells lacking 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 glycosylated variants are also included (see, e.g., U.S. Patent No. 6,602,684).

[0098] In some embodiments, the complex-binding molecules provided herein are available in concentrations of approximately 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 less than 0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M D ) has. The antibody target can be a CD19 target, a CD38 target, or a target containing both CD19 and CD38. K DKD can be measured by any suitable assay. In certain embodiments, KD can be measured using a surface plasmon resonance assay (e.g., using 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 the antibody provided herein, thereby generating an Fc region variant. The Fc region is, as defined herein, the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. Examples of Fc regions include native sequence Fc regions and variant Fc regions. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0100] In some cases, the Fc region of immunoglobulins is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), which result in the killing of target cells by different mechanisms. Therefore, in some embodiments, the antibodies described herein include a variable domain of the present invention combined with a constant domain containing 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 contains an Fc region having a unique profile for binding to one or more Fcγ receptors (activating receptor FcγRI(CD64), FcγRIIA, FcγRIIC(CD32), FcγRIIIA and FcγRIIIB(CD16), and inhibitory receptor FcγRIIB) and for the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors, while IgG2 binds to FcγRIIA H131, and FcγRIIA with lower affinity R131 FcγRIIIA V158 IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 The inhibitory receptor FcγRIIB binds to IgG1, IgG2, and IgG3, and has lower affinity for them than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIIB does not bind to either IgG2 or IgG4. Ibid. In general, with respect to ADCC activity, human IgG1 ≥ IgG3 >> IgG4 ≥ IgG2.

[0101] In certain embodiments, the anti-CD19 or anti-CD38 variable region described herein is linked to an Fc that binds to one or more activated 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 region described herein is linked to a human IgG1 or IgG3 Fc, i.e., the antibody is an antibody of either the IgG1 or IgG3 isotype. In some embodiments, modifications in the Fc region produce an Fc variant having (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 the Fc receptor, compared to the parent Fc. In some embodiments, the Fc region variant includes 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 include, for example, two, three, four, or five substitutions of specific Fc region positions identified herein.

[0102] In some embodiments, ADCC activity can be increased by modifying the Fc region. With respect to ADCC activity, in some cases, human IgG1 and IgG3 show increased ADCC activity compared to IgG4 and IgG2, so the constant domains of IgG1 or IgG3, rather than IgG2 or IgG4, are selected for use in antibodies when ADCC is desired. In some embodiments, IgG3 is selected for activation of 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 activity compared to IgG2 or IgG4. Alternatively, in some embodiments, the Fc region is located at 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, 2 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, Modifications are made to increase affinity for antibody-dependent cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-mediated cytotoxicity (CDC), C1q, and / or Fcγ receptors by altering one or more amino acids in 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 378, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 (Kabat numbering). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods may be used (e.g., ACTI® and CytoTox 96® non-radioactive cytotoxicity assays).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0103] Antibodies may have an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, for example, U.S. Patent Application Publication No. 2005 / 0014934). Such antibodies may contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn, and may include an Fc region having substitutions in 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, for example, U.S. Patent No. 7,371,826). Other examples of Fc region variants are also conceivable (see, for example, Duncan & Winter, Nature 322:738–40 (1988), U.S. Patent Nos. 5,648,260 and 5,624,821, and International Publication No. 94 / 29351).

[0104] In some embodiments, it may be desirable to produce a cysteine-manipulated antibody, e.g., "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residue is located in an accessible site of the antibody. The reactive thiol group may be positioned at a site for conjugation to another part, e.g., a drug part or a linker drug part, in order to create an immunoconjugate. 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 include additional known and available non-proteinoid moieties. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be the same or different molecules.

[0106] Complex-binding molecules or bispecific antibodies can differ based on the binding portion associated with these molecules, and there are also several different formats applicable and assumed herein. Complex-binding molecules or bispecific antibodies may include antibody fragments, substantially intact antibodies, or combinations thereof. In some embodiments, the first or second binding component includes an immunoglobulin heavy-and-light chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, a variable region fragment (VNAR) from an immunoglobulin neoantigen receptor, or a variable region (VHH) derived from a heavy-chain antibody. In certain embodiments, the first and second binding components include an immunoglobulin heavy-and-light chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, a variable region fragment (VNAR) from an immunoglobulin neoantigen receptor, or a variable region (VHH) derived from a heavy-chain antibody. In some embodiments, the first or second binding component includes an immunoglobulin heavy-and-light chain pair. In certain embodiments, the first and second binding components include immunoglobulin heavy chain and light chain pairs. In some embodiments, the first or second binding component includes scFv. In certain embodiments, the first and second binding components include scFv.

[0107] The bispecific antibodies according to this disclosure comprise an intact antibody molecule or a substantially completely intact antibody molecule, and may be asymmetric or symmetric.

[0108] Asymmetric bispecific antibodies generally consist of a heavy / light chain (HC / LC) pair from an antibody specific to target A and an HC / LC pair from an antibody specific to target B, creating heterodimer antibodies. Such heterodimer antibodies face the problem of unproductive molecular formation during production. While HC / LC-A:HC / LC-B is desirable, it is usually thermodynamically or statistically undesirable from all possible combinations. Several schemes have been introduced to circumvent this problem. In some cases, the HC / LC pair from an antibody specific to A and the HC / LC pair from an antibody specific to B further include 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 manipulating structural features that promote heterodimer formation between HC-A and HC-B, e.g., a "knob" to the FC region of HC-A and a "hole" to HC-B, or vice versa. Another scheme for promoting HC-A:HC-B heterodimers is to manipulate amino acid residues in the FC portions of HC-A and HC-B to include charge pairs that facilitate electrostatic interactions between HC-B and HC-A. Another scheme for addressing the chain association problem is to modify the variable region of one of the HC / LC pairs to include a single-chain binding molecule (e.g., V HHThis involves replacing (or scFv) with the other. As a result, half of the molecule contains a classical HC / LC pair, and the other contains an HC constant region fused to or attached to a single-chain binding molecule. Further modifications are possible to facilitate the formation of appropriate HC / LC pairs, including manipulating mutations for either HC and LC of A or B to promote the formation of appropriate HC / LC pairs, and CrossMab technology that utilizes the exchange of corresponding constant regions of HC / LC pairs. Symmetrical bispecific antibodies avoid the problem of chain association by not relying on the formation of heterobifunctional molecules. Examples of such molecules include bivariable domain molecules containing stacked variable regions of different specificities, IgG-scFv molecules containing scFv of different specificities fused to the c-terminus of the heavy chain of a classical antibody molecule, (scFV)4-FC (where the Fc dimerizes to create a bispecific tetravalent molecule), DART-Fc, and two-in-one, which contain two scFv linked by the Fc region of Ig.

[0109] The structures of complex-binding molecules or bispecific antibodies may be conceived and designed to alter the functionality or binding properties of the complex-binding molecules or bispecific antibodies (see, for example, "Bispecific antibodies: a mechanistic review of the pipeline," Nat Rev Drug Discovery. 2019 Aug;18(8):585-608) (see, for example, "The making of bispecific antibodies," MAbs. 2017 Feb-Mar; 9(2):182-212). For example, a 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 dispecific IgG A bispecific antibody having a common light chain bispecific IgG structure can be used in the present invention. Figure 1 illustrates a bispecific antibody having a common light chain bispecific IgG structure. The structure comprises a first and a second IgG heavy chain. Each heavy chain contains VH, CH1, CH2, and CH3 domains. The first heavy chain contains VH 102, CH1 104, CH2 106, and CH3 108. The second heavy chain contains VH 112, CH1 114, CH2 116, and CH3 118. The common light chain bispecific IgG structure also includes a light chain containing VL domain 120 and CL domain 122. Generally, the first heavy chain contains a sequence derived from the heavy chain of an antibody having first specificity, and the second heavy chain contains a heavy chain from an antibody having second specificity. The light chains paired with the first and second heavy chains are identical and can originate from the light chains of antibodies having either specificity or separate specificities. The heavy chains can be covalently linked to the light chain molecules via covalent bonds (e.g., disulfide bond 130). The heavy chains can be linked to other heavy chains via one or more covalent bonds (e.g., disulfide bonds 134 and / or 136). The common light chain two-specificity IgG structure may include first and second heavy chain molecules further containing mutations within the CH3 domain that promote the linkage of the first and second heavy chains and / or prevent the 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 the linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric interference, "knob" into "hole") or biochemically (e.g., electrostatic interaction). Exemplary knob-into-hole mutations may include T366W (EU numbering) in one heavy chain and T366S / L368A / Y407V (EU numbering) in the second heavy chain. Exemplary mutations that promote linkage of the first and second heavy chain molecules are disclosed, for example, in International Publication No. 2009089004, U.S. Patent No. 8,642,745, U.S. Patent Application Publication No. 20140322756, and “The making of bispecific antibodies,” MAbs. 2017 Feb-Mar; 9(2): pp. 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 B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and repressive 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 first heavy chain is configured to bind to the B cell lineage surface marker, and the second heavy chain is configured to bind to the repressive B cell surface marker. In some embodiments, the B cell lineage surface marker includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the repressive B cell surface marker includes CD38. In certain embodiments, the repressive B cell surface marker consists of CD38.

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

[0113] The BS1 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 its antigen-binding fragment, and the CD38-binding component comprises an antibody or its antigen-binding fragment, 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 the CD19 antibody or antigen-binding fragment is anti-CD38 immunoglobulin The CD38 antibody or antigen-binding component includes: a) a heavy chain complementarity-determining region 1 (HCDR1) containing an amino acid sequence described in any one of SEQ ID NOs. 71-75; b) a heavy chain complementarity-determining region 2 (HCDR2) containing an amino acid sequence described in any one of SEQ ID NOs. 81-85 or 150-155; c) a heavy chain complementarity-determining region 3 (HCDR3) containing an amino acid sequence described in any one of SEQ ID NOs. 91-95; and d) a heavy chain complementarity-determining region described in any one of SEQ ID NOs. 101-105. The CD19 antigen-binding component comprises a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence described, e) a light chain complementarity-determining region 2 (LCDR2) containing the amino acid sequence described in any one of SEQ ID NOs. 111 to 115, and / or) a light chain complementarity-determining region 3 (LCDR3) containing the amino acid sequence described in any one of SEQ ID NOs. 121 to 125, and the CD19 antigen-binding component comprises a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence described in any one of SEQ ID NOs. 11 to 15, and h) an amino acid sequence described in any one of SEQ ID NOs. 21 to 25. It includes a heavy chain complementarity determination region 2 (HCDR2) containing a column, i) a heavy chain complementarity determination region 3 (HCDR3) containing an amino acid sequence described in any one of sequence numbers 31 to 35, j) a light chain complementarity determination region 1 (LCDR1) containing an amino acid sequence described in any one of sequence numbers 101 to 105, k) a light chain complementarity determination region 2 (LCDR2) containing an amino acid sequence described in any one of sequence numbers 111 to 115, and / or l) a light chain complementarity determination region 3 (LCDR3) containing an amino acid sequence described in any one of sequence numbers 121 to 125.In some embodiments, the CD38 antigen-binding component comprises an HCDR2 amino acid sequence containing 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 contains the A84S and / or A108L substitution. In some embodiments, the CD38 light chain contains the W32H substitution.

[0114] Fab-Fc: scFv-Fc bispecific IgG A bispecific antibody 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 containing a single-chain variable fragment. The first heavy chain contains VH 202, CH1 204, CH2 206, and CH3 208 from the N-terminus to the C-terminus, respectively. The modified second heavy chain contains single-chain variable fragments (scFv) 210, CH2 216, and CH3 218 from the N-terminus to the C-terminus, respectively. The single-chain variable fragment (scFv) may contain a first domain 212 or a fragment thereof corresponding to a variable light chain domain, a second domain 214 or a fragment thereof corresponding to a variable heavy chain, and a linker polypeptide 215. The Fab-Fc:scFv-Fc bispecific IgG structure also includes a light chain containing a VL domain 220 and a CL domain 222. The first heavy chain may be covalently linked to the light chain molecule via a covalent bond (e.g., a disulfide bond 230). The first heavy chain may be linked to a 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 may include first and modified second heavy chain molecules further containing mutations within the CH3 domain that promote the linkage of the first and second heavy chains and / or prevent the 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 the linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric interference) or biochemically (e.g., electrostatic interaction). Exemplary mutations that promote the 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): pp. 182–212. The Fab-Fc:scFv-Fc bispecific IgG structure may also include a linked carbohydrate molecule 240 or additional modifications thereof.

[0115] Bispecific antibodies having a Fab-Fc:scFv-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and repressive 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the repressive B cell surface marker includes CD38. In certain embodiments, the repressive B cell surface marker consists of CD38.

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

[0117] The Fab-Fc:scFv-Fc bispecific IgG structure can be engineered so 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 containing a CD38-binding component, and the second heavy chain comprises a single-chain variable fragment (scFv) sequence containing a CD19-binding component. In certain embodiments, the VL sequence contains a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence containing a CD19-binding component comprises a CD19-binding component corresponding to the antibody heavy chain and light chain variable sequences, or the 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 its antigen-binding fragment, and the CD38-binding component comprises an antibody or its antigen-binding fragment, the CD38-antigen-binding component comprises a Fab that binds to CD38, which includes 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, which includes an anti-CD19 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and the CD38-binding component comprises an HCDR1 amino acid sequence described in any one of SEQ ID NOs. 71-75, an HCDR2 amino acid sequence described in any one of SEQ ID NOs. 81-85, or 150-155, and an HCDR3 amino acid sequence described in any one of SEQ ID NOs. 91-95 The immunoglobulin heavy chain contains an acid sequence, and the immunoglobulin light chain contains the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 101-105, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 111-115, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 121-125, and the CD19 binding component contains the immunoglobulin heavy chain containing the HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11-15, the HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21-25, and the HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31-35, and the immunoglobulin light chain contains the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 41-45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 51-55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 61-65. In some embodiments, the CD38 antigen-binding component comprises an HCDR2 amino acid sequence containing 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 contains the A84S and / or A108L substitution.In some embodiments, the CD38 light chain includes a W32H substitution.

[0119] Fab-Fc-Fab:Fc bispecific IgG A modified bispecific antibody 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-terminus 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-terminus 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 may be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 330). The first heavy chain may 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 may be linked in such a way 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 the second light chain may be linked in such a way 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 first heavy chain may be linked to a 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 may contain first and modified second heavy chain molecules, further including mutations within the CH3 domain that promote the linkage of the first and second heavy chains and / or prevent the 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 the linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric interference) or biochemically (e.g., electrostatic interaction).Exemplary mutations that promote the 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): pp. 182–212. The Fab-Fc-Fab:Fc bispecific IgG structure may also include a carbohydrate molecule 340 linked thereto or additional modifications thereof.

[0120] Bispecific antibodies having a Fab-Fc-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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

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

[0122] The Fab-Fc-Fab:Fc bispecific IgG structure can also be engineered so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 302) and VL domain (e.g., 320) contain CD38-binding components, and the second VH domain (e.g., 312) and VL domain (e.g., 324) contain CD19-binding components.

[0123] Fab-Fc-scFv: Fab-Fc-scFv bispecific IgG An engineered bispecific antibody 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 contain, from N-terminus 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) may contain a first domain 414 or a fragment thereof corresponding to a variable light chain domain, a second domain 416 or a fragment thereof corresponding to a 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 containing a VL domain 420 and a CL domain 422. A heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 430). A heavy chain may 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 may also include a carbohydrate molecule 440 linked thereto or additional modifications thereof.

[0124] A bispecific antibody having the Fab-Fc-scFv 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0125] The Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure can be engineered so 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) contain CD19-binding components, and the single-chain variable fragment (scFv) (e.g., 412) sequence contains CD38-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD38-binding components contains CD38-binding components, or their CD38-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

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

[0127] Fab-Fc-scFv:Fc bispecific IgG An engineered bispecific antibody 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-terminus 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) may include a first domain 514 or a fragment thereof corresponding to a variable light chain domain, a second domain 516 or a fragment thereof corresponding to a variable heavy chain, and a second linker polypeptide 515. The Fab-Fc-scFv:Fc bispecific IgG structure also comprises a first light chain containing a VL domain 520 and a CL domain 522. The Fab-Fc-scFv:Fc bispecific IgG structure also includes a second light chain containing a VL domain 524 and a CL domain 526. A heavy chain may be covalently linked to the light chain molecule via a covalent bond (e.g., a disulfide bond 530). The heavy chain may 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 may include first and modified second heavy chain molecules further containing mutations within the CH3 domain that promote 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. Mutations can prevent linkage of two heavy chain molecules or two second heavy chain molecules physically (e.g., steric interference) or biochemically (e.g., electrostatic interaction). Exemplary mutations that facilitate the linking 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): pp. 182–212. The Fab-Fc-scFv:Fc bispecific IgG structure may also include a linked carbohydrate molecule 540 or additional modifications thereof.

[0128] A bispecific antibody having the Fab-Fc-scFv: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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0129] The Fab-Fc-scFv:Fc bispecific IgG structure can be engineered so 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) contain CD19-binding components, and the single-chain variable fragment (scFv) (e.g., 512) sequence contains CD38-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD38-binding components contains CD38-binding components, or their CD38-binding fragments, corresponding to antibody heavy chain and light chain variable sequences.

[0130] The Fab-Fc-scFv:Fc bispecific IgG structure can also be engineered so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 502) and VL domain (e.g., 520) contain CD38-binding components, and the single-chain variable fragment (scFv) (e.g., 512) sequence contains CD19-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD19-binding components contains CD19-binding components, or their CD19-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

[0131] Fab-Fc-Fab:Fab-Fc bispecific IgG An engineered bispecific antibody 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-terminus to C-terminus, VH domain 602, CH1 domain 604, CH2 domain 606, CH3 domain 608, linker 610, second VH domain 612, and second CH1 domain 614, respectively. The second heavy chain comprises, from N-terminus to C-terminus, VH domain 652, CH1 domain 654, CH2 domain 656, and CH3 domain 658, respectively, as in the first heavy chain. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure also comprises a first light chain containing VL domain 620 and CL domain 622. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure also includes a second light chain containing a VL domain 624 and a CL domain 626. The heavy chain may be covalently linked to the light chain molecule via a covalent bond (e.g., a disulfide bond 630). The first heavy chain and the first light chain may be linked in such a way 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 the second light chain may be linked in such a way 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 may contain first and second heavy chain molecules further containing mutations within the CH3 domain that promote the linkage of the first and second heavy chains and / or prevent the 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 the linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric interference) or biochemically (e.g., electrostatic interaction).Exemplary mutations that promote the 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): pp. 182–212. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure may also include a carbohydrate molecule linked thereto or additional modifications thereof.

[0132] A bispecific antibody 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0133] The Fab-Fc-Fab:Fab-Fc bispecific IgG structure can be engineered so that the first antigen-binding site targets CD19 and the second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 602) and VL domain (e.g., 620) contain CD19-binding components, and the second VH domain (e.g., 612) and VL domain (e.g., 624) contain CD38-binding components.

[0134] The Fab-Fc-Fab:Fab-Fc bispecific IgG structure can also be engineered so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 602) and VL domain (e.g., 620) contain CD38-binding components, and the second VH domain (e.g., 612) and VL domain (e.g., 624) contain CD19-binding components.

[0135] scFv-Fab-Fc: scFv-Fab-Fc bispecific IgG An engineered bispecific antibody having the scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can be used in the present invention. Figure 7 illustrates a bispecific antibody having the scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure. The structure comprises two first heavy chain molecules. The first heavy chain each contains 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, from N-terminus to C-terminus. The single-chain variable fragment (scFv) may contain a first domain 714 or a fragment thereof corresponding to a variable light chain domain, a second domain 716 or a fragment thereof corresponding to a 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 containing a VL domain 720 and a CL domain 722. A heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., a disulfide bond 730). A heavy chain may 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 may also include a carbohydrate molecule 740 linked thereto or additional modifications thereof.

[0136] A bispecific antibody having the scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and repressive 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the repressive B cell surface marker includes CD38. In certain embodiments, the repressive B cell surface marker consists of CD38.

[0137] The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can be engineered so 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) contain CD19-binding components, and the single-chain variable fragment (scFv) (e.g., 712) sequence contains CD38-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD38-binding components contains CD38-binding components, or their CD38-binding fragments, corresponding to antibody heavy chain and light chain variable sequences.

[0138] The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can also be manipulated so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 702) and VL domain (e.g., 720) contain CD38-binding components, and the single-chain variable fragment (scFv) (e.g., 712) sequence contains CD19-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD19-binding components contains CD19-binding components, or their CD19-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

[0139] Fab-Fab-Fc: Fab-Fab-Fc bispecific IgG An engineered bispecific antibody having a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure may 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. Each heavy chain contains an additional VH domain 812 from the N-terminus to the C-terminus, and 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 containing 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 containing a VL domain 824 and a CL domain 826. The heavy chain molecules may be covalently linked to the light chain molecules via a covalent bond (e.g., a disulfide bond 830). The heavy chain and the first light chain may be linked in such a way that the VH domain and CH1 domain of the heavy chain pair with the VL domain and CL domain of the first light chain. The heavy chain and the second light chain may be linked in such a way that the additional VH domain and additional CH1 domain of the heavy chain pair with the VL domain and CL domain of the second light chain. The heavy chain may 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 may also include a carbohydrate molecule 840 linked thereto or further modifications thereof.

[0140] Bispecific antibodies having the 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

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

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

[0143] Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG An engineered bispecific antibody 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 contain, from N-terminus 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 containing 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 containing 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 the first light chain can be linked in such a way that the VH domain and CH1 domain of the heavy chain pair with the VL domain and CL domain of the first light chain. The heavy chain and the second light chain can be linked in such a way that the second VH domain and second CH1 domain of the heavy chain pair with the VL domain and CL domain 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 to it or additional modifications thereof.

[0144] A bispecific antibody 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0145] The Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can be engineered so that the first antigen-binding site targets CD19 and the second antigen-binding site targets CD38. In some embodiments, the first VH domain (e.g., 902) and VL domain (e.g., 920) contain CD19-binding components, and the second VH domain (e.g., 912) and VL domain (e.g., 924) contain CD38-binding components.

[0146] The Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can also be engineered so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the VH domain (e.g., 902) and VL domain (e.g., 920) contain CD38-binding components, and the second VH domain (e.g., 912) and VL domain (e.g., 924) contain CD19-binding components.

[0147] Fab-Fc-scFv: Fab-Fc bispecific IgG An engineered bispecific antibody 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-terminus 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) may include a first domain 1014 or a fragment thereof corresponding to a variable light chain domain, a second domain 1016 or a fragment thereof corresponding to a variable heavy chain, and a second linker polypeptide 1015. The second heavy chain, as in the first heavy chain, contains VH domain 1002, CH1 domain 1004, CH2 domain 1004, and CH3 domain 1008, respectively, from the N-terminus to the C-terminus. The Fab-Fc-scFv:Fab-Fc bispecific IgG structure also contains a first light chain containing VL domain 1020 and CL domain 1022. The heavy chain may be covalently linked to the 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 may include first and second heavy chain molecules further comprising mutations within the CH3 domain that promote the linkage of the first and second heavy chains and / or prevent the 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 the linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric interference) or biochemically (e.g., electrostatic interaction). Exemplary mutations that promote the 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): pp. 182–212.The Fab-Fc-scFv:Fab-Fc bispecific IgG structure may also include a carbohydrate molecule 1040 linked to it or additional modifications thereof.

[0148] A bispecific antibody having the 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker includes CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0149] The Fab-Fc-scFv:Fab-Fc bispecific IgG structure can be engineered so 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) contain CD19-binding components, and the single-chain variable fragment (scFv) (e.g., 1012) sequence contains CD38-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD38-binding components contains CD38-binding components, or their CD38-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

[0150] The Fab-Fc-scFv:Fab-Fc bispecific IgG structure can also be engineered so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 1002) and VL domain (e.g., 1020) contain CD38-binding components, and the single-chain variable fragment (scFv) (e.g., 1012) sequence contains CD19-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD19-binding components contains CD19-binding components, or their CD19-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

[0151] scFv-Fab-Fc:Fc bispecific IgG An engineered bispecific antibody 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 comprises a first heavy chain molecule containing scFv, VH, and Fc regions, and a second heavy chain molecule containing Fc. The scFv-Fab-Fc:Fc bispecific IgG structure may further include first and second heavy chain molecules containing mutations within the CH3 domain that promote the linkage of the first and second heavy chains and / or prevent the linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutations can promote the association of the first heavy chain molecule to the second heavy chain molecule physically (e.g., knob-in-hole structure) or biochemically (e.g., electrostatic interaction). The scFv-Fab-Fc:Fc bispecific IgG structure comprises a light chain molecule associated with a first heavy chain molecule that creates a first antigen-binding site. The second antigen-binding site is provided by an scFv fragment ligated to the N-terminus of the first heavy chain. Exemplary mutations that facilitate the ligation 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): pp. 182–212. The scFv-Fab-Fc:Fc bispecific IgG structure may also include a carbohydrate molecule 1140 ligated thereto or additional modifications thereof.

[0152] A bispecific antibody having the scFv-Fab-Fc:Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and repressive 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 includes CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the repressive B cell surface marker includes CD38. In certain embodiments, the repressive B cell surface marker consists of CD38.

[0153] The scFv-Fab-Fc:Fc bispecific IgG structure can be engineered so 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 contain CD19-binding components, and the single-chain variable fragment (scFv) sequence contains CD38-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence contains CD38-binding components, or their CD38-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

[0154] The scFv-Fab-Fc:Fc bispecific IgG structure can also be engineered so that the first antigen-binding site targets CD38 and the second antigen-binding site targets CD19. In some embodiments, the heavy chain VH and VL domains contain CD38-binding components, and the single-chain variable fragment (scFv) sequence contains CD19-binding components. In certain embodiments, the single-chain variable fragment (scFv) sequence containing CD19-binding components contains CD19-binding components, or their CD19-binding fragments, corresponding to the antibody heavy chain and light chain variable sequences.

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

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

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

[0158] Framework domain Mutations or reverse mutations to germline sequences made within the heavy and light chain framework regions may be advantageous for improving the pharmacokinetic and pharmacodynamic properties of the CD19 and CD38 binding molecules described herein. In certain cases, mutations or reverse mutations 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 reverse mutations 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). Therefore, in some embodiments, the heavy and / or light chain framework regions include 1, 2, 3, 4, 5, 8, or 10 mutations or reverse mutations returning to the germline sequence. In some embodiments, the heavy and / or light chain framework regions include from 1 mutation or reverse mutation returning to the germline sequence to 10 mutations or reverse mutations returning to the germline sequence. In some embodiments, the framework region of the heavy chain and / or light chain includes at least one mutation or reversion mutation that returns to the germline sequence. In some embodiments, the framework region of the heavy chain and / or light chain includes up to 10 mutations or reversion mutations that return to the germline sequence. In some embodiments, the framework region of the heavy chain and / or light chain includes from one mutation or reversion mutation that returns to the germline sequence to two mutations or reversion mutations that return to the germline sequence, from one mutation or reversion mutation that returns to the germline sequence to three mutations or reversion mutations that return to the germline sequence, from one mutation or reversion mutation that returns to the germline sequence to four mutations or reversion mutations that return to the germline sequence, from one mutation or reversion mutation that returns to the germline sequence to five mutations or reversion mutations that return to the germline sequence, from one mutation or reversion mutation that returns to the germline sequence to eight mutations or reversion mutations that return to the germline sequence, from one mutation or reversion mutation that returns to the germline sequence to ten mutations or reversion mutations that return to the germline sequence,From 2 mutations or revert mutations returning to the germline sequence to 3 mutations or revert mutations returning to the germline sequence, from 2 mutations or revert mutations returning to the germline sequence to 4 mutations or revert mutations returning to the germline sequence, from 2 mutations or revert mutations returning to the germline sequence to 5 mutations or revert mutations returning to the germline sequence, from 2 mutations or revert mutations returning to the germline sequence to 8 mutations or revert mutations returning to the germline sequence, from 2 mutations or revert mutations returning to the germline sequence to 10 mutations or revert mutations returning to the germline sequence, from 3 mutations or revert mutations returning to the germline sequence to 4 mutations or revert mutations returning to the germline sequence, from 3 mutations or revert mutations returning to the germline sequence to 5 mutations or revert mutations returning to the germline sequence, from 3 mutations or revert mutations returning to the germline sequence to germline Up to 8 mutations or revert mutations returning to the column, from 3 mutations or revert mutations returning to the germline sequence up to 10 mutations or revert mutations returning to the germline sequence, from 4 mutations or revert mutations returning to the germline sequence up to 5 mutations or revert mutations returning to the germline sequence, from 4 mutations or revert mutations returning to the germline sequence up to 8 mutations or revert mutations returning to the germline sequence, from 4 mutations or revert mutations returning to the germline sequence up to 10 mutations or revert mutations returning to the germline sequence, from 5 mutations or revert mutations returning to the germline sequence up to 8 mutations or revert mutations returning to the germline sequence, from 5 mutations or revert mutations returning to the germline sequence up to 10 mutations or revert mutations returning to the germline sequence, or from 8 mutations or revert mutations returning to the germline sequence up to 10 mutations or revert mutations returning to the germline sequence. In some embodiments, the framework regions of the heavy chain and / or light chain include 1 mutation or revert mutation returning to the germline sequence, 2 mutations or revert mutations returning to the germline sequence, 3 mutations or revert mutations returning to the germline sequence,The molecule includes four mutations or revertant mutations that return to the germline sequence, five mutations or revertant mutations that return to the germline sequence, eight mutations or revertant mutations that return to the germline sequence, or ten mutations or revertant mutations that return to the germline sequence. In some embodiments, the CD38 binding moiety includes the heavy chain framework region described in SEQ ID NO: 5. In some embodiments, the CD19 binding moiety includes the heavy chain framework region described in SEQ ID NO: 6 or 7.

[0159] Medicinally acceptable excipients, carriers, and diluents A composition comprising the complex-binding molecule of this disclosure is included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibody of this disclosure is administered suspended in a sterile and / or isotonic solution. In certain embodiments, the solution contains about 0.9% NaCl. In certain embodiments, the solution contains about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of the following: buffers, e.g., acetic acid (salt), citric acid (salt), histidine, succinic acid (salt), phosphoric acid (salt), bicarbonate (salt), and hydroxymethylaminomethane (Tris); surfactants, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyols / disaccharides / polysaccharides, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, e.g., glycine or arginine; antioxidants, e.g., ascorbic acid, methionine; or chelating agents, e.g., EDTA or EGTA.

[0160] Subcutaneous formulations for antibody administration may contain one or more of the following: buffers, e.g., acetate(salt), citrate(salt), histidine, succinic acid(salt), phosphoric acid(salt), bicarbonate(salt), and hydroxymethylaminomethane(Tris); surfactants, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyols / disaccharides / polysaccharides, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, e.g., glycine or arginine; antioxidants, e.g., ascorbic acid, methionine; or chelating agents, e.g., EDTA or EGTA. Additionally, compounds or molecules to relieve pain at the injection site, e.g., hyaluronidase at concentrations of approximately 2,000 U / ml to approximately 12,000 U / ml, may be included.

[0161] In certain embodiments, the complex-binding molecules of this disclosure are lyophilized for transport / storage and reconstituted before administration. In certain embodiments, the lyophilized antibody formulation comprises a filler, e.g., 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, may be buffered to a certain pH, generally below 7.0, whether or not it is reconstituted. 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, as well as one or more additional components selected from instructions for use, diluents, excipients, carriers, and devices for administration.

[0163] In certain embodiments, a method for preparing a cancer treatment is described herein, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with a complex-binding molecule of the Disclosure. In certain embodiments, a method for preparing a cancer treatment for storage or transport is described herein, comprising lyophilizing one or more antibodies of the Disclosure.

[0164] Manufacturing and production Nucleic acids encoding complex-binding molecules (e.g., bispecific antibodies) described herein may be used to enable the production of complex-binding molecules for commercial or therapeutic use by infecting, transfecting, transforming, or otherwise transgenicizing suitable cells with respect to the nucleic acids. Standard cell lines and methods for antibody production from large-scale cell cultures are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production," Mabs. 2010 Sep-Oct; 2(5): pp. 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 functionally linked to a eukaryotic regulatory sequence. In some embodiments, the cell contains the nucleic acid sequence.

[0166] In some embodiments, the cells include nucleic acids encoding complex-binding molecules disclosed herein. In certain embodiments, the cells include prokaryotic cells. In certain embodiments, the prokaryotic cells are Escherichia coli cells. In certain embodiments, the cells include eukaryotic cells. In certain embodiments, the eukaryotic cells are Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or human PER.C6 cells.

[0167] In a particular embodiment, the method described herein for producing a complex-binding molecule comprises culturing cells containing nucleic acids encoding the complex-binding molecule under conditions sufficient to enable the production and secretion of the complex-binding molecule in vitro.

[0168] In certain embodiments, a master cell bank is described herein, comprising (a) a mammalian cell line containing a nucleic acid encoding an antibody described herein, integrated at a genomic location, and (b) a cryoprotective agent. In certain embodiments, the cryoprotective agent comprises glycerol. In certain embodiments, the master cell bank comprises (a) a CHO cell line containing a nucleic acid encoding a complex-binding molecule, integrated at a genomic location, and (b) a cryoprotective agent. In certain embodiments, the cryoprotective agent comprises glycerol. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding 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 in cell culture medium under conditions sufficient to allow expression and secretion of the complex-binding molecules, and further collecting the complex-binding molecules from the cell culture medium. The collection may further include one or more purification steps to remove live cells, cell debris, non-complex-binding molecular proteins or polypeptides, undesirable salts, buffers, and medium components. In certain embodiments, the additional purification steps include centrifugation, ultracentrifugation, purification of protein A, protein G, protein A / G, or protein L, and / or ion-exchange chromatography.

[0170] How to use Suppression of the immune response by immunomodulatory cells can promote tumor growth, migration, and metastasis. Immunosuppression or negative immunomodulation 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 the body of the subject or patient. While B cells are primarily known as positive immunomodulators through the production of antibodies that promote the neutralization of pathogens, certain populations of B cells can function to suppress or negatively modulate the immune response. Such populations of B cells can be defined by the expression of one or more cell surface biomarkers. Immunosuppressive B cells or B cell populations can include B cell lineage surface biomarkers and suppressive B cell surface biomarkers. B cell lineage surface markers can include CD19, CD138, IgA, or CD45. B cell surface markers may 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 the immune response 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 the immune response by negatively modulating lymphoid structures and / or promoting the conversion of T cells to regulatory T cells. Disclosed herein are methods for targeting immunosuppressive B cell populations to effectively modulate the response.

[0171] Targeting immunosuppressive B cells or B cell populations can result in immune activation or positive modulation of the immune response against tumor or tumor-forming cells. Provided herein are methods for treating an individual with cancer or tumor, comprising administering to the individual with cancer or tumor a complex-binding molecule disclosed herein. Also provided herein are methods for reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual with tumor or cancer, comprising administering to the individual with tumor or cancer a complex-binding molecule disclosed herein, thereby reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor. Further disclosed are methods for contacting immunosuppressive B cells in a subject with a complex-binding molecule, comprising administering the complex-binding molecule to a subject. In certain embodiments, the subject has a tumor or cancer.

[0172] The type, subtype, or morphology of the tumor or cancer may be an important factor in the treatment strategy and method. 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 the antitumor immune response. In some embodiments, tumors or cancers include B cells containing B cell lineage surface biomarkers and suppressive B cell surface biomarkers. B cell lineage surface markers may include CD19, CD138, IgA, or CD45. B cell surface markers may include IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or TGFB. In some embodiments, B cell surface markers include CD19 (e.g., CD19+) and CD38 (e.g., CD38+). In some embodiments, tumor-infiltrating B cells or immunosuppressive B cells include 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 of attempting to improve or induce remission of the treated condition. With respect to cancer, treatment includes, but is not limited to, a reduction in tumor volume, a reduction in tumor volume growth, an increase in progression-free survival, or an increase in overall life expectancy. In certain embodiments, treatment affects the remission of the treated cancer. In certain embodiments, treatment encompasses use as a prophylactic or maintenance dose intended to prevent 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 the administered treatment, but nevertheless, these individuals are considered to be treated.

[0175] In certain embodiments, cancer or tumor is a solid cancer or tumor. In certain embodiments, cancer or tumor is a blood cancer or tumor. In certain embodiments, cancer or tumor includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, and liver. In certain embodiments, tumors that can be treated with the antibodies of the present invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, tumors / cancers include 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, and Swing's sarcoma. Sarcoma, focal nodular hyperplasia, gastrinoma, germline tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial hyperplasia, intraepithelial squamous cell hyperplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, nerve sheath, medulloblastoma, medullary epithelioma The following are selected from the group of cancers: mesothelioma, mucosal epithelial carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumors, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar cancer, VIPoma (VIPpoma), and Wilms' tumor.In certain embodiments, tumors / cancers treated with one or more antibodies of the present invention 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 ductal carcinoma, and / or breast cancer. In certain embodiments, cancers treated with 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 treated with one or more antibodies of this disclosure includes colon cancer. In certain embodiments, the cancer treated includes 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 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, cancers and / or tumors treated with the complex-binding molecules herein are mature B-cell neoplasms: chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, non-Hodgkin lymphoma (diffuse large B-cell lymphoma, follicular lymphoma), mucosa-associated lymphoid tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma and Waldenström macroglobulinemia, nodular marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, Burkitt lymphoma, or primary central nervous system lymphoma.

[0177] In certain embodiments, cancers and / or tumors treated with the complex-binding molecules described herein include T-cell neoplasms such as T-cell non-Hodgkin 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 target requiring it via any route suitable for administering the antibody-containing pharmaceutical composition, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral. 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 on 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 dose. In certain embodiments, the therapeutically acceptable dose is about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the therapeutically acceptable dose is about 1 mg / kg to about 40 mg / kg. In certain embodiments, the therapeutically acceptable dose is about 5 mg / kg to about 30 mg / kg. A therapeutically effective dose is an amount sufficient to relieve one or more symptoms associated with the disease or illness being treated.

[0179] Exemplary Embodiments Provided herein are complex-binding molecules 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 its antigen-binding fragment, and the CD38-binding component comprises an antibody or its antigen-binding fragment. In some embodiments, provided are complex-binding molecules of any of the preceding embodiments in which the CD19 and / or CD38-binding components comprise an immunoglobulin heavy-chain and light-chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, a variable region fragment (VNAR) from an immunoglobulin neoantigen receptor, or a variable region (VHH) derived from a heavy-chain antibody. In some embodiments, provided are complex-binding molecules of any of the preceding embodiments in which the CD19 or CD38-binding component comprises an immunoglobulin heavy-chain and light-chain pair. In some embodiments, provided are complex-binding molecules of any of the preceding embodiments in which the CD19 and CD38-binding components comprise an immunoglobulin heavy-chain and light-chain pair.

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

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

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

[0183] Furthermore, the complex-binding molecule comprises a CD38 antigen-binding component that binds to CD38, which includes 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, which includes 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) containing an amino acid sequence described in any one of SEQ ID NOs. 71-75, and b) a component described in any one of SEQ ID NOs. 81-85 or 150-155. It is a complex-binding molecule comprising: c) a heavy chain complementarity determination region 2 (HCDR2) containing an amino acid sequence; c) a heavy chain complementarity determination region 3 (HCDR3) containing an amino acid sequence described in any one of SEQ ID NOs. 91 to 95; d) a light chain complementarity determination region 1 (LCDR1) containing an amino acid sequence described in any one of SEQ ID NOs. 101 to 105; e) a light chain complementarity determination region 2 (LCDR2) containing an amino acid sequence described in any one of SEQ ID NOs. 111 to 115; and / or f) a light chain complementarity determination region 3 (LCDR3) containing an amino acid sequence described in any one of SEQ ID NOs. 121 to 125.

[0184] In some embodiments, the provided material is a 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) containing an amino acid sequence described in any one of SEQ ID NOs: 11-15; h) a heavy chain complementarity-determining region 2 (HCDR2) containing an amino acid sequence described in any one of SEQ ID NOs: 21-25; i) a heavy chain complementarity-determining region 3 (HCDR3) containing an amino acid sequence described in any one of SEQ ID NOs: 31-35; j) a light chain complementarity-determining region 1 (LCDR1) containing an amino acid sequence described in any one of SEQ ID NOs: 101-105; k) a light chain complementarity-determining region 2 (LCDR2) containing an amino acid sequence described in any one of SEQ ID NOs: 111-115; and / or l) a light chain complementarity-determining region 3 (LCDR3) containing an amino acid sequence described in any one of SEQ ID NOs: 121-125.

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

[0186] In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD19 antigen-binding component comprises an immunoglobulin heavy chain variable region having the same amino acid sequence as SEQ ID NO: 1 or 6, and the immunoglobulin light chain variable region having the same amino acid sequence as SEQ ID NO: 4. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises a first immunoglobulin heavy chain constant region. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD38 immunoglobulin light chain variable region further comprises an immunoglobulin light chain constant region. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, wherein the anti-CD19 immunoglobulin heavy chain variable region further comprises a second immunoglobulin heavy chain constant region. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, 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 are detrimental to the homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote the heterodimerization of the first and second heavy chain constant regions. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, 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 regions comprises a T366S / L368A / Y407V substitution (EU numbering), and as a result, 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 regions. In some embodiments, the provided molecule is a complex-binding molecule of any of the preceding embodiments, wherein a 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 that includes a light chain variable region and a heavy chain variable region that binds to CD38. In some embodiments, what is provided is a 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, what is provided is a 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, what is provided is a 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, the provided complex-binding molecule is one of the preceding embodiments, wherein the CD19-binding component comprises the HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11-15, the HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21-25, and the HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31-35, and the immunoglobulin light chain comprises the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 41-45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 51-55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 61-65.

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

[0189] In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, 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 are detrimental to the homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote the heterodimerization of the first and second heavy chain constant regions. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided, 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 regions comprises a T366S / L368A / Y407V substitution (EU numbering), and as a result, the heterodimerization of the first and second immunoglobulin heavy chain constant regions is promoted more than the homodimerization of the first or second immunoglobulin heavy chain constant region. In some embodiments, the provided molecule is a complex-binding molecule of any of the preceding embodiments, wherein a single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component.

[0190] In some embodiments, the provided molecule is a complex-binding molecule of any of the preceding embodiments, which is a bispecific antibody or a biantigen-binding fragment thereof. In some embodiments, the provided molecule is a complex-binding molecule of any of the preceding embodiments, which comprises an Fc region containing an amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate. In some embodiments, the provided molecule is a complex-binding molecule of any of the preceding embodiments, in which 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, a complex-binding molecule of any of the preceding embodiments is provided that binds to CD19+, CD38+ B cells. In some embodiments, a complex-binding molecule of any of the preceding embodiments is provided that exhibits reduced hemagglutination compared to a CD19 or CD38 monospecific antibody containing an Fc region.

[0192] Provided is a nucleic acid comprising a polynucleotide sequence encoding one of the complex-binding molecules of the prior embodiments. Embodiment 49: The nucleic acid of Embodiment 47, wherein the polynucleotide sequence encoding the complex-binding molecule is functionally linked to a eukaryotic regulatory sequence. In some embodiments, a cell comprising the nucleic acid described in one of the prior embodiments is provided. In some embodiments, a cell of any of the prior embodiments, comprising a prokaryotic cell, is provided. In some embodiments, a cell of any of the prior embodiments, wherein the prokaryotic cell is an E. coli cell, is provided. In some embodiments, a cell of any of the prior embodiments, comprising a eukaryotic cell, is provided. In some embodiments, a cell of any of the prior 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 are pharmaceutical compositions, for example, compositions comprising a complex-binding molecule described in any one of the prior 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 are complex-binding molecules or pharmaceutical compositions described in any one of the prior embodiments for use in methods of treating tumors or cancer in an individual. In some embodiments, the tumor is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In certain embodiments, the B-cell malignancy is a B-cell acute lymphoblastic leukemia. In certain embodiments, the B-cell malignancy is a chronic lymphocytic leukemia, a small lymphocytic lymphoma, a mantle cell lymphoma, or a non-Hodgkin lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological malignancy is a plasma malignancy. In certain embodiments, the plasma malignancy is multiple myeloma. In some embodiments of any of the prior embodiments, the hematological malignancy expresses CD19 and CD38 (for example, cancer cells express CD19 and CD38).

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

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

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

[0198] Further provided is a method for treating an individual affected by cancer or a tumor, comprising administering to the individual affected by cancer or a tumor a complex-binding molecule described in any one of the preceding embodiments or a pharmaceutical composition described in 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 lymphoblastic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin 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 (for example, cancer cells express CD19 and CD38).

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

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

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

[0202] Also provided herein are methods for producing a complex-binding molecule described in any one of the prior embodiments, comprising incubating the cells of the prior 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 are methods for preparing a cancer treatment for an individual, comprising mixing the complex-binding molecule described in any one of the prior embodiments with a pharmaceutically acceptable diluent, carrier, or excipient.

[0203] Therefore, provided herein are complex-binding molecules comprising 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 and the second target comprises an inhibitory B cell surface marker, and the first and second targets are not identical. In some embodiments, the first or second binding component comprises a polypeptide. In some specific embodiments, the first or second binding component comprises a polypeptide. In some embodiments, the first and second binding components comprise a polypeptide. In some specific embodiments, the first and second binding components comprise a polypeptide. In some embodiments, the polypeptide of the first or second binding component comprises an amino acid sequence with a length of at least 100 amino acid residues. In some embodiments, the polypeptides of the first and second binding components comprise an amino acid sequence with a length of at least 100 amino acid residues.

[0204] The B cell lineage surface marker may 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] The inhibitory B cell surface marker may 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 some specific embodiments, the inhibitory B cell surface marker is IgD. In some embodiments, the inhibitory B cell surface marker includes CD1. In some specific embodiments, the inhibitory B cell surface marker is CD1. In some embodiments, the inhibitory B cell surface marker includes CD5. In some specific embodiments, the inhibitory B cell surface marker is CD5. In some embodiments, the inhibitory B cell surface marker includes CD21. In some specific embodiments, the inhibitory B cell surface marker is CD21. In some embodiments, the inhibitory B cell surface marker includes CD24. In some specific 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 may include an antibody or a target-binding fragment thereof. In some embodiments, the first or second binding component includes an immunoglobulin heavy-chain and light-chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, a variable region fragment (VNAR) from an immunoglobulin neoantigen receptor, or a variable region (VHH) derived from a heavy-chain antibody. In some embodiments, the first and second binding components include an immunoglobulin heavy-chain and light-chain pair, scFv, F(ab), F(ab')2, a single-domain antibody, a variable region fragment (VNAR) from an immunoglobulin neoantigen receptor, or a variable region (VHH) derived from a heavy-chain antibody. In certain embodiments, the first or second binding component includes an immunoglobulin heavy-chain and light-chain pair. In certain embodiments, the first and second binding components include an immunoglobulin heavy-chain and light-chain pair. In certain embodiments, the first or second binding component includes scFv. In a particular embodiment, the first and second bonding components include scFv.

[0207] A complex-binding molecule as described herein, which is a bispecific antibody or a biantigen-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 the HCDR1 amino acid sequence described in any one of SEQ ID NOs. 71-75, the HCDR2 amino acid sequence described in any one of SEQ ID NOs. 81-85 or 150-155, and the HCDR3 amino acid sequence described in any one of SEQ ID NOs. 91-95, and the immunoglobulin light chain comprises the LCDR1 amino acid sequence described in any one of SEQ ID NOs. 41-45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs. 51-55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs. 61-65. In a particular embodiment, the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO. 3, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO. 2. In certain embodiments, the immunoglobulin heavy chain contains the same amino acid sequence as described in SEQ ID NO: 3, and the immunoglobulin light chain contains the same amino acid sequence as described 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 common light chain bispecific IgG. In certain embodiments, the bispecific antibody is Fab-Fc:scFv-Fc bispecific IgG. In certain embodiments, the dispecific antibody is Fab-Fc-Fab:Fc dispecific IgG. In certain embodiments, the dispecific antibody is Fab-Fc-scFv:Fab-Fc-scFv dispecific IgG. In certain embodiments, the dispecific antibody is Fab-Fc-scFv:Fc dispecific IgG. In certain embodiments, the dispecific antibody is Fab-Fc-Fab:Fab-Fc dispecific IgG. In certain embodiments, the dispecific antibody is scFv-Fab-Fc:scFv-Fab-Fc dispecific IgG. In certain embodiments, the dispecific antibody is Fab-Fab-Fc:Fab-Fab-Fc dispecific IgG. In certain embodiments, the dispecific antibody is Fab-Fc-Fab:Fab-Fc-Fab dispecific IgG. In certain embodiments, the dispecific antibody is IgG-scFv.

[0210] The complex-binding molecule may include post-translational modifications. In some embodiments, the complex-binding molecule includes an Fc region containing 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 includes the HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11-15, the HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21-25, the HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31-35, the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 41-45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 51-55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 61-65. In certain embodiments, the first binding component includes an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical to, or 100% identical to, the amino acid sequence described in either SEQ ID NOs: 1 or SEQ ID NOs: 2.

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

[0213] The complex-binding molecule can bind to a first target and a second target, the first target containing a B cell lineage surface marker, and the second target containing 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] Complex-binding molecules can be encoded by nucleic acid molecules. Disclosed herein are nucleic acids comprising polynucleotide sequences encoding the complex-binding molecules disclosed herein. In some embodiments, the polynucleotide sequences encoding the complex-binding molecules are functionally linked to eukaryotic regulatory sequences.

[0215] The cells may contain nucleic acids that encode complex-binding molecules. In some embodiments, the cells include prokaryotic cells. In certain embodiments, the prokaryotic cells are E. coli cells. In some embodiments, the cells include eukaryotic cells. In certain embodiments, the eukaryotic cells are Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or human PER.C6 cells.

[0216] Also disclosed herein are compositions comprising complex-binding molecules and pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, the compositions are formulated for intravenous administration. In some embodiments, the compositions are formulated for subcutaneous administration.

[0217] The complex-binding molecules disclosed herein can inhibit and / or reduce the number of immunosuppressive B cells that suppress the antitumor immune response. Therefore, the complex-binding molecules disclosed herein can be used in methods for treating tumors or cancer in an individual. 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 lymphoblastic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin 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 (for example, cancer cells express CD19 and CD38).

[0218] In some embodiments, cancer or tumor is solid tissue cancer. In some embodiments, 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, cancer is breast cancer. In some specific embodiments, breast cancer is triple-negative breast cancer. In some embodiments, cancer is lung cancer. In some specific embodiments, lung cancer is non-small cell lung cancer. In some embodiments, cancer is head and neck cancer. In some specific embodiments, head and neck cancer is head and neck squamous cell carcinoma. In some embodiments, cancer is kidney cancer. In some specific embodiments, kidney cancer is renal cell carcinoma. In some embodiments, cancer is brain cancer. In some embodiments, brain cancer is glioblastoma multiforme. In some embodiments, cancer is skin cancer. In some specific embodiments, skin cancer is melanoma.

[0219] The complex-binding molecules described herein may be used in methods to reduce tumor-infiltrating B cells and / or immunosuppressive B cells that suppress the antitumor immune response against tumors in an individual. The complex-binding molecules described herein may be used in methods to inhibit the function of tumor-infiltrating B cells and / or immunosuppressive B cells that suppress the antitumor immune response against tumors in an individual. The complex-binding molecules may be used in methods to reduce suppressive B cells that are in, adjacent to, or around tumors in an individual. In some embodiments, tumor-infiltrating B cells or immunosuppressive B cells include CD19+, CD38+ B cells.

[0220] Further disclosed herein are methods for treating an individual affected by cancer or a tumor, comprising administering a complex-binding molecule disclosed herein to the individual affected by 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 lymphoblastic leukemia. In certain embodiments, the B-cell malignancy is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin 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 (for example, cancer cells express CD19 and CD38).

[0221] In some embodiments, cancer or tumor is solid tissue cancer. In some embodiments, 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, cancer is breast cancer. In some specific embodiments, breast cancer is triple-negative breast cancer. In some embodiments, cancer is lung cancer. In some specific embodiments, lung cancer is non-small cell lung cancer. In some embodiments, cancer is head and neck cancer. In some specific embodiments, head and neck cancer is head and neck squamous cell carcinoma. In some embodiments, cancer is kidney cancer. In some specific embodiments, kidney cancer is renal cell carcinoma. In some embodiments, cancer is brain cancer. In some embodiments, brain cancer is glioblastoma multiforme. In some embodiments, cancer is skin cancer. In some specific embodiments, skin cancer is melanoma.

[0222] Also disclosed is a method for reducing tumor-infiltrating B cells in, adjacent to, or surrounding a tumor in an individual affected by a tumor or cancer, comprising administering a complex-binding molecule disclosed herein to an individual affected by a tumor or cancer, thereby reducing tumor-infiltrating B cells in the tumor. Also disclosed is a method for reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual affected by a tumor or cancer, comprising administering a complex-binding molecule disclosed herein to an individual affected by a tumor or cancer, thereby reducing immunosuppressive B cells in the tumor. In some embodiments, tumor-infiltrating B cells or immunosuppressive B cells include CD19+, CD38+ B cells. In some embodiments, reducing tumor-infiltrating B cells includes 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 includes reducing and / or blocking and / or preventing and / or inhibiting intercellular contact-induced immunosuppression mediated by immunosuppressive B cells. In some embodiments, reducing tumor-infiltrating B cells includes reducing and / or blocking and / or preventing and / or inhibiting the differentiation of immunosuppressive B cells.

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

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

[0225] The following examples are provided 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 Illustrating the disclosures herein, a complex-binding molecule comprising 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 and the second target comprises an inhibitory B cell surface marker, was tested for binding to cells expressing CD19 and CD38. Binding characteristics of antibodies containing CD19 and CD38 light and heavy chains to CD19 and CD38-expressing Raji cells. Figure 12A shows cell surface expression data for CD19 and CD38 in Raji cells. Raji cells expressing CD19 and CD38 were incubated with antibodies containing CD19 and CD38 light and heavy chains. Cells were incubated with 30 μg / mL of antibody at 11 different concentrations to generate binding profiles for each sample. CD19 and CD38 expression was validated using commercially available antibodies. The tested samples were: (A) Matched CD19 heavy and light chains, where the CD19 heavy chain contains SEQ ID NO: 1 and the CD19 light chain contains SEQ ID NO: 2; (B) Swapped CD19 heavy and CD38 light chains, where the CD19 heavy chain contains SEQ ID NO: 1 and the CD38 light chain contains SEQ ID NO: 4; (C) Swapped CD38 heavy and CD19 light chains, where the CD38 heavy chain contains SEQ ID NO: 3 and the CD19 light chain contains SEQ ID NO: 2; (D) Matched CD38 heavy and light chains, where the CD38 heavy chain contains SEQ ID NO: 3 and the CD38 light chain contains SEQ ID NO: 4; (E) CD19 single-chain variable fragments (scFv) containing SEQ ID NOs: 1-2; (F) CD38 single-chain variable fragments (scFv) containing SEQ ID NOs: 3-4; Darzalex (CD38 control); Anti-CD19 PE (CD19 control); Anti-CD38 Includes PE (CD38 control) and IgG1 isotype control.

[0227] Figures 12B and 12C show the binding profiles of samples A-F, Darzalex, and IgG1 isotype controls. Tables 1 and 2 show the EC of samples A-F, Darzalex, and IgG1 isotype controls. 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 among samples. Figures 12D and 12E show the 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 that do not express CD19 and CD38.

[0228] [Table 1]

[0229] [Table 2]

[0230] Example 2: Octet-bound data The binding affinity of the parental and bispecific antibodies was determined using biolayer interferometry. Binding experiments were performed on an Octet Red96 at 25°C using assay buffer consisting of 0.1% BSA, 1XPBS, 0.02% Tween-20, and 0.05% NaN3. The antibody was loaded onto an anti-hIgG Fc Capture biosensor for 300 seconds. The ligand-loaded sensor was immersed in serial dilutions of the antigen (starting at 300 nM: 2-fold serial dilution for CD19, 3-fold serial dilution 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 sample 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) containing 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 approximately a 5-fold reduction in binding to CD19. As expected, parental antibodies (851D / 851F) containing anti-CD38 003 VH and VL did not bind to CD19.

[0239] Table 3 shows the binding data. The two parent antibodies (851D / 851F) with anti-CD38 003 VH and VL bound to CD38 with similar KD. Substitution of anti-CD38 003 VL with anti-CD19 VL (851C) resulted in a significant reduction in binding to CD38. As expected, the parent antibodies (851A / 851E) with anti-CD19 VH and VL did not bind to CD38, and neither did 851B. 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] The bispecific antibody (format) test kit included the following:

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

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

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

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

[0246] BS3 = 3C10scFv-003Fab-FcKnob:FcHole:003LC)(scFv-Fab-Fc:Fc bispecific IgG1) in a ratio of 1:1:1

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

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

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

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

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

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

[0253] Table 4 shows binding data for bispecificity tests in single-antigen format. The bispecific antibodies BS1 / BS2 / BS4 bound to both target antigens with a KD of no more than 4 times that of the parent antibody (indicated by 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. The 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 a 500 nM primary antigen for 500 seconds, followed by a 300 nM secondary antigen for 240 seconds. Rate constants were calculated using a monovalent (1:1) binding model. Table 5 shows that the bispecific antibodies BS1 / BS2 / BS4 were able to simultaneously bind to both target antigens with a ka (1 / Ms) no more than twice that of the parental antibodies (851B, 851D, and 851E). Similar to the one-antigen format, BS3 bound only to CD19 and not to CD38.

[0256] [Table 5]

[0257] Variants were further tested for their ability to bind to CD19 and / or CD38. Binding experiments were performed on an Octet Red at 25 °C. Antibodies were loaded onto an anti-hIgG Fc Capture (AHC) biosensor for 300 seconds. The sensors loaded with the ligand were immersed in a two-fold serial dilution of the antigens (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. Kinetic 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 Study Cell Binding Study Protocol: Five cell lines (HEK293-CD19, HEK293-CD38, HEK293-CD19 / CD38, Daudi, and REH), in addition to untreated controls, were incubated in triplicate with the test article at 133 nM, followed by a three-fold dilution series (a total of 7 points). Transient transfection into the HEK293 cell line was performed.

[0262] We conducted studies to evaluate the cell surface expression of CD19 and CD38 on Daudi, Raji, and REH cell lines. Cells were stained in triplicates using commercially available PE-conjugated antibodies, washed, and acquired via flow cytometry. To quantify molecular expression on the cell surface, we used the Quantum Simply Cellular anti-mouse IgG kit (catalog #815-A) from Bangs Laboratories to generate standard curves for interpolating MFI against molecule count / cell values ​​(Table 9).

[0263] [Table 9]

[0264] Figure 13A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (each having one arm against CD19 or CD38 and the other arm against varicella-zoster virus) to Daudi cells. Considering that Daudi cells have approximately 1 million copies of CD38 on their surface but only about 200,000 copies of CD19, Figure 13A shows that binding of anti-CD38 antibodies 851D and 38K-VZVH is efficient, while binding of anti-CD19 antibodies 851A, 851B, and 19H-VZVK is only moderate. Notably, 851D, which has two CD38-binding Fabs, binds approximately 5 times better than 38K-VZVH, which has only one CD38-binding Fab.

[0265] Figure 13B shows the binding of bispecific antibodies BS1, BS2, and BS4 to Daudi cells. The avidity of the bispecific antibodies that bind to both CD38 and CD19 is revealed by comparing their binding to that of 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 having one arm against CD19 or CD38 and the other arm against varicella-zoster virus) to REH cells. Considering that REH cells have approximately 300,000 copies of CD38 on their surface but only about 50,000 copies of CD19, Figure 14A shows that binding of anti-CD38 851D and 38K-VZVH is efficient, while binding of anti-CD19 851A, 851B, and 19H-VZVK is only moderate. The scale of MFI is significantly lower compared to Daudi cells, due to the lower expression levels of both CD38 and CD19 on REH cells (Figures 2A, 2B). Furthermore, 851D, which has two CD38-binding Fabs, binds approximately five times better to CD38 than 38K-VZVH, which has only one CD38-binding Fab.

[0267] Figure 14B shows the binding of bispecific antibodies BS1, BS2, and BS4 to REH cells. The avidity of bispecific antibodies that bind to both CD38 and CD19 is revealed 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 did not bind to these cells. Notably, 851A and 851B, each possessing two CD19-binding Fabs, bound significantly better than 19H-VZVK, which possessed only one CD19-binding Fab.

[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 to CD19 approximately 10 times 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. Notably, 851D, which has two CD38-binding Fabs, binds better than 38K-VZVH, which has only one CD38-binding Fab.

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

[0272] Cell Binding Study Protocol - Non-Specific Background Binding: Studies were 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. Two cell lines were stained with viability dye and then incubated in triplicate with the untreated control, untreated and unsecondary controls, as well as the highest concentration of 1,250 nM, followed by a 5-fold dilution series (4 samples in total) of the test samples.

[0273] Figure 17A shows the binding of parental antibodies (851A, 851B, 851D) to untransfected CHO-S cells. Nonspecific binding was observed 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 untransfected Expi293T cells. Nonspecific binding was observed for all three parental antibodies, starting at 250 nM, and was more pronounced with anti-CD38 851D.

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

[0276] For direct evaluation of apoptosis, each test was tested in three consecutive sequences at a final peak concentration of 33 nM, followed by a 7-point 5-fold dilution series, in addition to an untreated control. For cross-linking-induced apoptosis, each individual test (BS1, BS2, BS4, 851A, 851B, and 851D) and combinations of test (851A and 851D, 851B and 851D, and 38K-VZVH and 19H-VZVK) was tested in three consecutive sequences at a final peak concentration of 33 nM, followed by a 7-point 5-fold dilution series, in addition to an untreated control, in addition to daratumumab and IgG1 isotype controls. 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 bispecific control antibodies (38K-VZVH, 19H-VZVK), daratumumab, and IgG1 isotype controls. 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 show recognizable direct apoptosis.

[0278] Figure 18B shows direct apoptosis in Daudi cells for the bispecific antibodies BS1, BS2, BS4, daratumumab, and IgG1 isotype controls. The BS1 and BS2 formats showed significantly higher levels of direct apoptosis compared to daratumumab. The bispecific format BS4 showed levels of direct apoptosis comparable to the parental anti-CD19 851A / 851B antibody (comparison in Figure 12A), which may be due to the BS4 format being unable to bring CD19 and CD38 into proximity to initiate apoptosis.

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

[0280] Figure 19B shows crosslinking-induced apoptosis in Daudi cells for the bispecific antibodies BS1, BS2, BS4, (38K-VZVH+19H-VZVK), daratumumab, and IgG1 isotype controls. When crosslinked, the BS1 and BS2 formats showed levels of apoptosis comparable to daratumumab. Notably, the bispecific format BS4 showed levels of crosslinking-induced apoptosis comparable to BS1, BS2, and daratumumab, while without crosslinking, BS4 showed no apoptosis (see Figure 6B). The combination of the two control antibodies, 38K-VZVH and 19H-VZVK, showed significant apoptosis, but lower than either of the bispecific formats, indicating that including anti-CD19 and anti-CD38 binding sites in a single antibody is advantageous over using independent antibodies.

[0281] Example 5: Cytotoxic Daudi target cells were treated with the test product in dose-response mode and incubated at 37°C / 5% CO2 for 15 minutes. In addition to a 0 nM control, the test product was tested at a final peak concentration of 133 nM, followed by a 7-point 5-fold dilution series. Daratumumab and IgG1 isotype controls were used as positive and negative controls.

[0282] Pre-treated target cells were co-cultured with human PBMCs from n=3 donors (E:T 25:1). The PBMCs were primed overnight with 100 U / mL IL-2. The PBMCs were labeled with ViaFluor 405. After incubation of the samples at 37C / 5% CO2 for 4 hours, flow cytometry analysis was performed to assess 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 indicator of target cell cytotoxicity.

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

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

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

[0286] Figures 22A and 22B show the results of the 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 tafacitamab (wt IgG1), and the human IgG1 isotype control did not induce any CDC activity. Darzalex, BS1, and BS2 all showed CDC activity (though not to the same extent 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 cell phagocytosis (ADCP) was further assayed using pHrodo Green AM (pHG)-labeled Raji cells treated with the test sample in a dose-response manner and incubated for 15 minutes at 37°C and 5% CO2. pHG is a pH-sensitive dye that exhibits only weak fluorescence at neutral pH but is highly fluorescent at low pH in the mature phagosomes of macrophages. pHG-labeled Raji target cells with anti-CD20 antibody and IgG1 isotype controls were used as positive and negative controls, with a maximum concentration of 133 nM, a 7-point 5-fold dilution series, and a 0 nM control. Pre-treated target cells were co-cultured with human macrophages (differentiated in vitro from monocytes) from n=3 donors (E:T 1:2). Macrophages were labeled with Cell Trace Violet (CTV). After incubation of samples at 37°C and 5% CO2 for 4 hours, flow cytometry analysis was performed for phagocytosis. The percentage of pHGhi / CTV+ cells was analyzed as an indicator of target cell phagocytosis. The percentages were plotted against the logarithm of the test substance concentration on an XY chart, and the data were fitted to a four-parameter nonlinear regression curve for which EC50 was calculated.

[0288] Figure 23 shows the results of antibody-dependent cell phagocytosis (ADCP) assays using Raji cells and donor macrophages as targets. The positive control, anti-CD20, demonstrated dose-dependent phagocytosis in all three donors after 4 hours (maximum phagocytosis of 5–10%). The negative control, IgG1 isotype control, did not demonstrate dose-dependent phagocytosis in all three donors after 4 hours. Darzalex demonstrated dose-dependent phagocytosis in all three donors after 4 hours (maximum phagocytosis of 4–10%). BS-1, BS-2, non-fucosylated BS-1, and non-fucosylated BS-2 showed slight dose-dependent phagocytosis, with the non-fucosylated format resulting in increased ADCP.

[0289] Example 6: Interaction with RBCs Flow cytometry-based erythrocyte (RBC) binding studies were performed to evaluate the binding of the test substance to erythrocytes from n=3 cynomolgus monkeys and n=3 human donors. Whole blood was washed with 1X PBS, then diluted 20-fold with PBS, and treated with the test substance. In addition to a 0 nM control, two specific substances (BS1, BS2), parental monoclonals (851A, 851D), and controls (anti-CD38 darazalex, recombinant anti-CD19 tafacitamab, IgG1 isotype control, anti-CD47 conjugated to Alexa Fluor 647) were tested in triplicate at a peak 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 peak concentration of 133 nM and the same dose-response.

[0290] After 30 minutes of incubation on ice with the primary antibody, cells were washed and stained with 5 ug / mL of secondary antibody (Alexa Fluor 647-labeled goat anti-human Fcγ F(ab')2) to detect binding of the test substance to erythrocytes. The secondary antibody was not used for anti-CD47-A647 stained cells. After a further 30 minutes of incubation on ice with the secondary antibody, stained cells were washed, diluted, and acquired by high-throughput flow cytometry. The geometric mean fluorescence intensity (MFI) of AlexaFluor 647 for single cell populations was calculated. The MFI of AF647 was plotted on an XY chart, the MFI was graphed against the logarithm of concentration, and the data were fitted to a nonlinear regression curve for which EC50 was calculated.

[0291] Figure 24 shows that AF647-conjugated anti-CD47 exhibited dose-response binding curves using erythrocytes from all three human donors. Darzalex also showed a dose-dependent increase in binding to all three donors, but its maximum MFI was orders of magnitude lower than that of anti-CD47. Anti-CD38 851D showed the second highest maximum MFI after darzalex, followed by BS1, BS2, the 38K-VZVH & 19H-VZVK combination, and anti-CD19 tafacitamab. Finally, anti-CD19 851A and the IgG1 isotype showed only a slight increase in MFI at the highest concentrations.

[0292] In vitro hemagglutination assays were performed on erythrocytes 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 coagulation. The blood was then washed with PBS and diluted 1:50 to obtain "whole blood substrate". The whole blood substrate was plated in 96-well round-bottom plates and treated in triplicate with a 0 nM control, followed by a maximum final concentration of 133 nM, and then six 5-fold serial dilutions in PBS of test products (BS1, BS2, 38K-VZVH+19H-VZVK, 851A, and 851D), controls (tafacitamab with wild-type IgG1), darazalex, and human IgG1 isotype control), or a positive technical control (IGM-55.5). After 1 hour of incubation at 37°C and 5% CO2, plates were photographed to check the level of hemagglutination. Using the photographs as reference, each well was scored on a specific hemagglutination scale from 0 to 5. The specific manifestation 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 in all three human donors, starting at 0.04–1.1 nM. BS1, BS2, 38K-VZVH+19H-VZVK, darazalex, tafacitamab, and human IgG1 isotype controls all did not induce hemagglutination at any concentration in all three donors. Both monoclonal antibodies 851A (anti-CD19) and 851D (anti-CD38) induced hemagglutination in all three donors, starting at 0.2 or 1.1 nM respectively, and the response was similar in scale to the technical control (anti-CD47). In contrast to the parental monoclonal antibodies, BS1 and BS2 did not induce any hemagglutination at any concentration.

[0294] Figure 25B shows the results of the hemagglutination assay for cynoquizal donor 3. The positive control, IGM-55.5 (anti-little i antigen IgM antibody), induced hemagglutination in all three cynoquizal donors, starting at 0.04 or 0.2 nM. BS1, BS2, 38K-VZVH+19H-VZVK, Darzalex, Tafacitamab, and the human IgG1 isotype control all did not induce hemagglutination at any concentration in any of the three donors. Both monoclonal antibodies 851A (anti-CD19) and 851D (anti-CD38) induced hemagglutination in all three donors, starting at 1.1 nM each. In contrast to the parental monoclonal antibodies, BS1 and BS2 did not induce any hemagglutination at any concentration.

[0295] In vitro hemolysis assays were also performed on erythrocytes from 3 healthy cynomolgus monkeys (cyno) and 3 healthy human donors (n=3). Whole blood was obtained on the day of the study and checked for coagulation. The blood was washed with PBS and diluted 1:10 to obtain "whole blood substrate". The whole blood substrate was treated with the test samples and controls in PBS. In addition to the 0 nM control, two specific substances (BS1, BS2), parental monoclonals (851A, 851D) and controls (anti-CD38 darazalex, recombinant anti-CD19 tafacitamab, IgG1 isotype control) were tested in triplicate at a maximum final concentration of 133 nM, followed by a total of 7 5-fold serial dilutions. Single-arm controls (38K-VZVH, 19H-VZVK) were tested in combination, both at a maximum concentration of 133 nM and the same dose response. Saponins were tested at a maximum concentration of 0.1% in seven 3-fold serial dilutions. After incubation at 37°C and 5% CO2 for 1 hour, the plates were centrifuged and the supernatant was collected. The supernatant was analyzed for optical density (OD) at 540 nm via a plate reader. As a positive control, saponins induced dose-dependent hemolysis in all species and donors, starting at 0.001% and increasing to 0.10%. None of the test samples induced any hemolysis at any of the concentrations tested.

[0296] Figure 26 shows that none of the test samples induced any hemolysis at any of the concentrations tested. In the positive control, saponins induced dose-dependent hemolysis in all species and donors, starting at 0.001% and increasing to 0.10%.

[0297] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Numerous variations, modifications, and substitutions will now come to mind to those skilled in the art without deviating from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the invention. The following claims are intended to define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are thereby covered.

[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. 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 an antigen-binding fragment thereof, and the CD38-binding component comprises an antibody or an antigen-binding fragment thereof.

2. The CD19-binding component and / or the CD38-binding component are immunoglobulin heavy chain and light chain pairs, scFv, F(ab), F(ab') ２ , single-domain antibodies, variable region fragments from immunoglobulin novel antigen receptors (V ＮＡＲ ), or a variable region derived from a heavy chain antibody (V Ｈ A complex-binding molecule according to claim 1, comprising H).

3. The complex-binding molecule according to claim 2, wherein the CD19-binding component or the CD38-binding component comprises an immunoglobulin heavy chain and light chain pair.

4. The complex-binding molecule according to claim 3, wherein the CD19-binding component and the CD38-binding component include an immunoglobulin heavy chain and light chain pair.

5. The CD38-binding component comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence described in any one of SEQ ID NOs. 71 to 75, an HCDR2 amino acid sequence described in any one of SEQ ID NOs. 81 to 85 or 150 to 155, and an HCDR3 amino acid sequence described in any one of SEQ ID NOs. 91 to 95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence described in any one of SEQ ID NOs. 101 to 105, an LCDR2 amino acid sequence described in any one of SEQ ID NOs. 111 to 115, and / or an LCDR3 amino acid sequence described in any one of SEQ ID NOs. 121 to 125, and the CD19 A complex-binding molecule according to any one of claims 1 to 4, wherein the binding component comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprises an HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11 to 15, an HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21 to 25, and an HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31 to 35, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence described in any one of SEQ ID NOs: 101 to 105, an LCDR2 amino acid sequence described in any one of SEQ ID NOs: 111 to 115, and / or an LCDR3 amino acid sequence described in any one of SEQ ID NOs: 121 to 125.

6. The complex-binding molecule according to claim 5, wherein the CD38-binding component comprises an immunoglobulin heavy chain having an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 3 or 5, and an immunoglobulin light chain having an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 4, and / or the CD19-binding component comprises an immunoglobulin heavy chain having an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 1 or 6, and an immunoglobulin light chain having an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO:

4.

7. The complex-binding molecule according to claim 6, wherein the immunoglobulin heavy chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 3 or 5, and the immunoglobulin light chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 4, and / or the immunoglobulin heavy chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1 or 6, and the immunoglobulin light chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO:

4.

8. A complex-binding molecule according to any one of claims 1 to 7, wherein the common light chain is a bispecific IgG.

9. The CD38-binding component comprises an immunoglobulin heavy chain containing the HCDR1 amino acid sequence described in any one of SEQ ID NOs: 71-75, the HCDR2 amino acid sequence described in any one of SEQ ID NOs: 81-85 or 150-155, and the HCDR3 amino acid sequence described in any one of SEQ ID NOs: 91-95, and the immunoglobulin light chain comprises the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 101-105, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 111-115, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 121-125, and the C A complex-binding molecule according to any one of claims 1 to 3, wherein the D19-binding component comprises an immunoglobulin heavy chain containing the HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11 to 15, the HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21 to 25, and the HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31 to 35, and the immunoglobulin light chain comprises the LCDR1 amino acid sequence described in any one of SEQ ID NOs: 41 to 45, the LCDR2 amino acid sequence described in any one of SEQ ID NOs: 51 to 55, and / or the LCDR3 amino acid sequence described in any one of SEQ ID NOs: 61 to 65.

10. The complex-binding molecule according to claim 9, wherein the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 3 or 5, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 4, and / or the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 1 or 7, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO:

2.

11. The complex-binding molecule according to claim 10, wherein the immunoglobulin heavy chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 3 or 5, and the immunoglobulin light chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 4, and the immunoglobulin heavy chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1 or 7, and the immunoglobulin light chain contains the same amino acid sequence as the amino acid sequence described in SEQ ID NO:

2.

12. The complex-binding molecule according to any one of claims 9 to 11, wherein the CD19-binding component or the CD38-binding component contains scFv.

13. The complex-binding molecule according to claim 12, wherein the CD19-binding component includes scFv.

14. The complex-binding molecule according to claim 12, wherein the CD38-binding component includes scFv.

15. The complex-binding molecule according to any one of claims 9 to 14, wherein the CD19-binding component or the CD38-binding component comprises an immunoglobulin heavy chain / light chain pair.

16. The complex-binding molecule according to claim 15, wherein the CD19-binding component includes an immunoglobulin heavy chain / light chain pair.

17. The complex-binding molecule according to claim 15, wherein the CD38-binding component comprises an immunoglobulin heavy chain / light chain pair.

18. The complex-binding molecule according to any one of claims 1 to 17, wherein the CD38-binding component comprises an HCDR2 amino acid sequence including the sequence P-X1-LG-X2-A, and X1 and X2 are each selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E.

19. The complex-binding molecule according to claim 18, wherein X1 is H and X2 is T.

20. A complex-binding molecule according to any one of claims 1 to 19, wherein the heavy chain variable region that binds to CD19 includes A84S and A108L modifications according to Kabat numbering.

21. A complex-binding molecule according to any one of claims 1 to 20, wherein the light chain variable region bound to CD38 includes a W32H modification according to Kabat numbering.

22. The complex-binding molecule comprises a CD38 antigen-binding component that binds to CD38, which includes 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, which includes an anti-CD19 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, wherein the CD38 antigen-binding component is a) Heavy chain complementarity determination region 1 (HCDR1) containing an amino acid sequence described in any one of SEQ ID NOs. 71 to 75, b) Heavy chain complementarity determination region 2 (HCDR2) containing an amino acid sequence described in any one of sequence numbers 81-85 or 150-155, c) Heavy chain complementarity determination region 3 (HCDR3) containing an amino acid sequence described in any one of sequence numbers 91 to 95, d) Light chain complementarity determination region 1 (LCDR1) containing an amino acid sequence described in any one of sequence numbers 101 to 105, e) Light chain complementarity determination region 2 (LCDR2) containing an amino acid sequence described in any one of sequence numbers 111 to 115, and / or f) Light chain complementarity determination region 3 (LCDR3) containing an amino acid sequence described in any one of sequence numbers 121 to 125. Includes, The aforementioned CD19 antigen-binding component is g) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in any one of sequence numbers 11 to 15, h) Heavy chain complementarity determination region 2 (HCDR2) containing an amino acid sequence described in any one of sequence numbers 21 to 25, i) Heavy chain complementarity determination region 3 (HCDR3) containing an amino acid sequence described in any one of sequence numbers 31 to 35, j) Light chain complementarity determination region 1 (LCDR1) containing an amino acid sequence described in any one of sequence numbers 101 to 105, k) Light chain complementarity determination region 2 (LCDR2) containing an amino acid sequence described in any one of sequence numbers 111 to 115, and / or l) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in any one of sequence numbers 121 to 125. including, Complex-binding molecules.

23. The complex-binding molecule according to claim 22, wherein the CD38 antigen-binding component comprises an HCDR2 amino acid sequence containing the sequence P-X1-L-G-X2-A, and X1 and X2 are each selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E.

24. The complex-binding molecule according to claim 23, wherein X1 is H and X2 is T.

25. The complex-binding molecule according to any one of claims 22 to 24, wherein the CD38 antigen-binding component comprises an immunoglobulin heavy chain variable region having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 3 or 5, and an immunoglobulin light chain variable region having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO:

4.

26. The complex-binding molecule according to claim 25, wherein the CD38 antigen-binding component includes an immunoglobulin heavy chain variable region having the same amino acid sequence as SEQ ID NO: 3 or 5, and the immunoglobulin light chain variable region has the same amino acid sequence as SEQ ID NO:

4.

27. The complex-binding molecule according to any one of claims 22 to 26, wherein the CD19 antigen-binding component comprises an anti-CD19 immunoglobulin heavy chain variable region having an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 1 or 6, and an immunoglobulin light chain variable region having an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO:

4.

28. The complex-binding molecule according to claim 27, wherein the anti-CD19 antigen-binding component includes an immunoglobulin heavy chain variable region having the same amino acid sequence as SEQ ID NO: 1 or 6, and the immunoglobulin light chain variable region has the same amino acid sequence as SEQ ID NO:

4.

29. The complex-binding molecule according to any one of claims 22 to 28, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises a first immunoglobulin heavy chain constant region.

30. The complex-binding molecule according to any one of claims 22 to 29, wherein the anti-CD38 immunoglobulin light chain variable region further comprises an immunoglobulin light chain constant region.

31. The complex-binding molecule according to any one of claims 22 to 30, wherein the anti-CD19 immunoglobulin heavy chain variable region further comprises a second immunoglobulin heavy chain constant region.

32. A complex-binding molecule according to any one of claims 22 to 31, 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 detrimental to the homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote the heterodimerization of the first and second heavy chain constant regions.

33. The complex-binding molecule according to claim 32, 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 regions comprises a T366S / L368A / Y407V substitution (EU numbering), and as a result, 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 regions.

34. The complex-binding molecule according to any one of claims 22 to 33, wherein the CD19 antigen-binding component comprises a heavy chain immunoglobulin sequence described in SEQ ID NO: 201 and a light chain immunoglobulin sequence described in SEQ ID NO: 213, and the CD38-binding component comprises a heavy chain immunoglobulin sequence described in SEQ ID NO: 202 and a light chain immunoglobulin sequence described in SEQ ID NO:

213.

35. A complex-binding molecule according to any one of claims 22 to 34, wherein the heavy chain variable region that binds to CD19 includes A84S and A108L modifications according to Kabat numbering.

36. A complex-binding molecule according to any one of claims 22 to 35, wherein the light chain variable region bound to CD38 includes a W32H modification according to Kabat numbering.

37. A complex-binding molecule according to any one of claims 22 to 36, wherein a single bispecific binding molecule is formed from the CD38 antigen-binding component and the CD19 antigen-binding component.

38. 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 includes an scFV that binds to CD19, and the CD38-binding component includes a Fab region that includes a light chain variable region and a heavy chain variable region that binds to CD38.

39. The complex-binding molecule according to claim 38, wherein the scFv that binds to CD19 is linked to the constant region of the first immunoglobulin heavy chain.

40. The complex-binding molecule according to claim 38 or 39, wherein the heavy chain variable region of the CD38-binding component further comprises a second immunoglobulin heavy chain constant region.

41. The complex-binding molecule according to any one of claims 38 to 40, wherein the light chain variable region of the CD38-binding component further comprises an immunoglobulin light chain constant region.

42. The complex-binding molecule according to any one of claims 38 to 41, wherein the CD19-binding component comprises an HCDR1 amino acid sequence described in any one of SEQ ID NOs: 11 to 15, an HCDR2 amino acid sequence described in any one of SEQ ID NOs: 21 to 25, and an HCDR3 amino acid sequence described in any one of SEQ ID NOs: 31 to 35, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence described in any one of SEQ ID NOs: 41 to 45, an LCDR2 amino acid sequence described in any one of SEQ ID NOs: 51 to 55, and / or an LCDR3 amino acid sequence described in any one of SEQ ID NOs: 61 to 65.

43. The complex-binding molecule according to any one of claims 38 to 42, wherein the CD38-binding component comprises an HCDR1 amino acid sequence described in any one of SEQ ID NOs: 71 to 75, an HCDR2 amino acid sequence described in any one of SEQ ID NOs: 81 to 85 or 150 to 155, and an HCDR3 amino acid sequence described in any one of SEQ ID NOs: 91 to 95, and the immunoglobulin light chain comprises an LCDR1 amino acid sequence described in any one of SEQ ID NOs: 101 to 105, an LCDR2 amino acid sequence described in any one of SEQ ID NOs: 111 to 115, and / or an LCDR3 amino acid sequence described in any one of SEQ ID NOs: 121 to 125.

44. The complex-binding molecule according to claim 43, wherein the CD38-binding component comprises an HCDR2 amino acid sequence containing the sequence P-X1-L-G-X2-A, and X1 and X2 are each selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E.

45. The complex-binding molecule according to claim 44, wherein X1 is H and X2 is T.

46. The complex-binding molecule according to any one of claims 38 to 45, wherein the CD19-binding component comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 1 or 7, and the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO:

2.

47. The complex-binding molecule according to any one of claims 38 to 45, wherein the CD38-binding component comprises an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO: 3 or 5, and an amino acid sequence having at least about 90%, 95%, 97%, and 99% identity with SEQ ID NO:

4.

48. The complex-binding molecule according to any one of claims 38 to 47, wherein the CD19-binding component includes an amino acid sequence identical to the amino acid sequence described in SEQ ID NO: 1 or 7, and an amino acid sequence identical to the amino acid sequence described in SEQ ID NO:

2.

49. The complex-binding molecule according to any one of claims 38 to 48, wherein the CD38-binding component includes the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 3 or 5, and the same amino acid sequence as the amino acid sequence described in SEQ ID NO:

4.

50. The complex-binding molecule according to any one of claims 38 to 49, 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 unfavor the homodimerization of the anti-CD38 immunoglobulin heavy chain constant region and / or promote the heterodimerization of the first heavy chain constant region and the second heavy chain constant region.

51. The complex-binding molecule according to claim 50, 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 regions comprises a T366S / L368A / Y407V substitution (EU numbering), and as a result, heterodimerization of the first and second immunoglobulin heavy chain constant regions is promoted more than homodimerization of the first or second immunoglobulin heavy chain constant regions.

52. The complex-binding molecule according to any one of claims 38 to 51, wherein the CD19-binding component includes the sequence described in any one of the sequence numbers, and the CD38-binding component includes the light chain and heavy chain described in sequence numbers 213 and 202, respectively.

53. A complex-binding molecule according to any one of claims 38 to 52, wherein the heavy chain variable region that binds to CD19 includes A84S and A108L modifications according to Kabat numbering.

54. A complex-binding molecule according to any one of claims 38 to 53, wherein the light chain variable region bound to CD38 includes a W32H modification according to Kabat numbering.

55. A complex-binding molecule according to any one of claims 38 to 54, wherein a single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component.

56. A complex-binding molecule according to any one of claims 1 to 55, which is a bispecific antibody or a biantigen-binding fragment thereof.

57. A complex-binding molecule according to any one of claims 1 to 55, comprising an Fc region containing an amino acid residue modified with a native carbohydrate or a non-fucosylated carbohydrate.

58. The complex-binding molecule according to claim 57, wherein the amino acid residue modified with the native carbohydrate or the non-fucosylated carbohydrate corresponds to asparagine 297 according to EU numbering.

59. A complex-binding molecule according to any one of claims 1 to 58, which binds to CD19+ and CD38+ B cells.

60. A complex-binding molecule according to any one of claims 1 to 59, which exhibits reduced hemagglutination compared to a CD19 or CD38 monospecific antibody containing an Fc region.

61. A nucleic acid or a plurality of nucleic acids comprising a polynucleotide sequence encoding a complex-binding molecule according to any one of claims 1 to 60.

62. The nucleic acid according to claim 61, wherein the polynucleotide sequence encoding the complex-binding molecule is functionally linked to a eukaryotic regulatory sequence.

63. A cell comprising the nucleic acid according to claim 61 or 62.

64. The cell according to claim 63, which includes a prokaryotic cell.

65. The prokaryotic cell according to claim 64, which is an E. coli cell.

66. The cell according to claim 63, which includes a eukaryotic cell.

67. The eukaryotic cells according to claim 66, which are Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or human PER. C6 cells.

68. A composition comprising a complex-binding molecule according to any one of claims 1 to 60 and a pharmaceutically acceptable diluent, carrier, or excipient.

69. The composition according to claim 68, formulated for intravenous administration.

70. The composition according to claim 68, formulated for subcutaneous administration.

71. A complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70 for use in a method of treating a tumor or cancer in an individual.

72. The use according to claim 71, wherein the cancer or tumor is a blood cancer.

73. The use according to claim 72, wherein the blood cancer is a B-cell malignant tumor.

74. The use according to claim 73, wherein the B-cell malignant tumor is B-cell acute lymphoblastic leukemia.

75. The use according to claim 74, wherein the B-cell malignant tumor is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin lymphoma (diffuse large B-cell lymphoma, follicular lymphoma).

76. The use according to claim 72, wherein the blood cancer is a plasma malignant tumor.

77. The use according to claim 76, wherein the plasma malignant tumor is multiple myeloma.

78. The use according to any one of claims 72 to 77, wherein the hematological cancer expresses CD19 and CD38.

79. The use according to claim 71, wherein the cancer or tumor is a solid tissue cancer.

80. The use according to claim 79, wherein 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.

81. The use according to claim 80, wherein 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.

82. A complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70 for use in a method of reducing and / or modulating the function of tumor-infiltrating B cells and / or immunosuppressive B cells located in, adjacent to, or surrounding a tumor in an individual.

83. A complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70 for use in a method of reducing and / or modulating the function of immunosuppressive B cells located in, adjacent to, or surrounding a tumor in an individual.

84. The use according to claim 82 or 83, wherein the function of immunosuppressive B cells includes the release of IL-10, IL-35, TGF-beta, or a combination thereof.

85. A complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70, for use in a method for reducing immunosuppression by immunosuppressive B cells.

86. The tumor-infiltrating B cells or the immunosuppressive B cells are CD19-positive B cells, CD38 ＋ The use according to any one of claims 82 to 85, comprising positive B cells, CD19, CD38 double-positive B cells, or a combination thereof.

87. A method for treating an individual afflicted with cancer or a tumor, comprising administering to the individual afflicted with cancer or the tumor a complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70, thereby treating the cancer or tumor.

88. The method according to claim 87, wherein the cancer or tumor is a blood cancer.

89. The method according to claim 88, wherein the blood cancer is a B-cell malignant tumor.

90. The method according to claim 89, wherein the B-cell malignant tumor is B-cell acute lymphoblastic leukemia.

91. The method according to claim 89, wherein the B-cell malignant tumor is chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, or non-Hodgkin lymphoma (diffuse large B-cell lymphoma, follicular lymphoma).

92. The use of method 88, wherein the aforementioned blood cancer is a plasma malignancy.

93. The use of method 92, wherein the plasma malignant tumor is multiple myeloma.

94. The method according to any one of claims 88 to 93, wherein the hematological cancer expresses CD19 and CD38.

95. The method according to claim 87, wherein the cancer or tumor is a solid tissue cancer.

96. The method according to claim 95, wherein 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.

97. The method according to claim 96, wherein 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.

98. A method for reducing tumor-infiltrating B cells in, adjacent to, or surrounding a tumor in an individual affected by a tumor or cancer, comprising administering to the individual affected by the tumor or cancer a complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70, thereby reducing tumor-infiltrating B cells in the tumor.

99. A method for reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual affected by a tumor or cancer, comprising administering to the individual affected by the tumor or cancer a complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70, thereby reducing immunosuppressive B cells in the tumor.

100. A method for inhibiting the function of immunosuppressive B cells located in, adjacent to, or surrounding a tumor in an individual affected by a tumor or cancer, comprising administering to the individual affected by the tumor or cancer a complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70, thereby reducing immunosuppression by immunosuppressive B cells in the tumor.

101. A method for inhibiting the function of immunosuppressive B cells located in, adjacent to, or surrounding a tumor, comprising contacting the immunosuppressive B cells with a complex-binding molecule according to any one of claims 1 to 60 or a composition according to any one of claims 68 to 70, thereby reducing immunosuppression by the immunosuppressive B cells in the tumor.

102. The method according to claim 100 or 101, wherein the function of immunosuppressive B cells includes the release of IL-10, IL-35, TGF-beta, or a combination thereof.

103. The tumor-infiltrating B cells or the immunosuppressive B cells are CD19-positive B cells, CD38 ＋ The method according to any one of claims 98 to 102, comprising positive B cells, CD19, CD38 double-positive B cells, or a combination thereof.

104. A method for producing a complex-binding molecule according to any one of claims 1 to 60, comprising incubating the cells according to claim 66 or 67 in a cell culture medium under conditions sufficient to enable the expression, assembly, and secretion of the complex-binding molecule into the cell culture medium.

105. The method according to claim 104, comprising isolating and purifying the molecule from the cell culture medium.

106. A method for preparing a cancer treatment for an individual, comprising mixing a complex-binding molecule according to any one of claims 1 to 60 with a pharmaceutically acceptable diluent, carrier, or excipient.

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