CD19 / CD38 multispecific antibody

Abstract

Multispecific antibodies with reduced isoelectric focusing points are described herein.

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Patent Application No. 63 / 491,441, filed on 21 March 2023, and U.S. Provisional Patent Application No. 63 / 590,886, filed on 17 October 2023, each of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing, which has been filed electronically in XML format and is incorporated herein by reference in its entirety. The above XML copy, created on 18 March 2024, is named 51527-716601_SEQ.xml and has a size of 19,873 bytes. [Background technology]

[0003] Immunosuppressive B cell populations (i.e., regulatory B cells or Bregs) are B cell populations that suppress an individual's anti-tumor immune response. Therefore, therapeutic agents that effectively and specifically target immunosuppressive B cells may be used to prevent and / or eliminate immunosuppression, thereby providing effective treatment for different types of cancer. [Overview of the Initiative]

[0004] Apart from binding to the intended target molecule, antibody drugs must also meet certain criteria regarding manufacturability, storage stability, and the absence of off-target binding. This set of characteristics is generally referred to as "developability." Unfortunately, there is no single parameter (e.g., assayable properties) that can clearly predict developability and its challenges based on the area of ​​antibody entering clinical practice.

[0005] Multispecific antibodies having modifications that enable better development potential and manufacturability are exemplified and provided herein. In one embodiment, a multispecific antibody is provided herein, which comprises: (a) a CD38 binding moiety comprising a CD38 binding moiety comprising (i) a first polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region comprising the amino acid sequence described in SEQ ID NO: 1, wherein the heavy chain constant region lacks a C-terminal lysine residue; and (ii) a second polypeptide comprising a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 3; and (b) a CD19 binding moiety comprising a CD19 binding moiety comprising (i) a third polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region comprising the amino acid sequence described in SEQ ID NO: 2, wherein the heavy chain constant region lacks a C-terminal lysine residue; and (ii) a fourth polypeptide comprising a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 3. In certain embodiments, the heavy chain constant region of the first polypeptide, the third polypeptide, or both the first and third polypeptides includes a human IgG1 or human IgG4 constant region. In certain embodiments, a polypeptide including a light chain variable region further includes a light chain constant region. In certain embodiments, the second polypeptide, the fourth polypeptide, or both the second and fourth polypeptides includes the amino acid sequence described in SEQ ID NO: 6. In certain embodiments, the CD38 binding moiety includes one or more amino acid substitutions that inhibit homodimerization of the CD38 binding moiety. In certain embodiments, the CD38 binding moiety includes a T366W substitution according to EU numbering, or a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the CD19 binding moiety includes one or more amino acid substitutions that inhibit homodimerization of the CD19 binding moiety. In certain embodiments, the CD19 binding moiety includes a T366W substitution according to EU numbering, or a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the first polypeptide consists of the amino acid sequence described in SEQ ID NO: 4. In certain embodiments, the third polypeptide consists of the amino acid sequence described in SEQ ID NO: 5.In certain embodiments, the pharmaceutical composition comprises a multispecific antibody, a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, the nucleic acid encodes a multispecific antibody. Methods for treating cancer in an individual requiring treatment, comprising the step of administering a multispecific antibody to the individual, are also described herein. In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the solid tissue cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, gastric cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In certain 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. In certain embodiments, the cancer or tumor is a hematological cancer. In certain embodiments, the hematological cancer is diffuse large B-cell lymphoma. In certain embodiments, the hematological cancer is myeloma. In certain embodiments, the hematological cancer is Burkitt lymphoma. In certain embodiments, hematological malignancies are aggressive B-cell lymphomas. In certain embodiments, aggressive B-cell lymphomas include double-hit lymphomas, double-expressor lymphomas, or triple-hit lymphomas. In certain embodiments, hematological malignancies are recurrent or refractory. In certain embodiments, cancers or tumors associated with CD19-positive, high-CD38, immunosuppressive B cells are cancers or tumors that include CD19-positive, high-CD38, B-cell infiltration. In certain embodiments, CD19-positive, high-CD38, immunosuppressive B cells express B-cell activation markers. In certain embodiments, the B-cell activation markers include CD30. In certain embodiments, cancers or tumors associated with CD19-positive, high-CD38 B cells express PD-L1. In certain embodiments, cancers or tumors associated with CD19-positive, high-CD38 B cells are associated with low-CD20 or CD20-negative B cells. In certain embodiments, CD38-high B cells express at least approximately 30,000 CD38 proteins on their cell surface. In certain embodiments, CD38-high B cells express at least approximately 35,000 CD38 proteins on their cell surface. In certain embodiments, CD38-high B cells express at least approximately 40,000 CD38 proteins on their cell surface.

[0006] In one embodiment, a bispecific antibody for a common light chain is provided herein, the bispecific antibody for a common light chain comprising an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, an anti-CD38 heavy chain constant region and an anti-CD19 heavy chain constant region, and a variable region of the common light chain, wherein the anti-CD38 heavy chain variable region comprises (a) a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, (b) a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, (c) a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering, and the anti-human CD19 heavy chain variable region comprises (a) a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, and (b) SEQ ID NO: 11 The common light chain variable region comprises (a) a light chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in (c) SEQ ID NO: 12, and a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering. The common light chain variable region comprises (a) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, (b) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence (AAS) described in SEQ ID NO: 14, and (c) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 15. Each of the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain variable region lacks a C-terminal lysine residue (e.g., K447 according to EU numbering), and the common light chain bispecific antibody has an experimental isoelectric point (pI) of less than 9. In certain embodiments, the common light chain bispecific antibody further comprises a hydrophobic (HIC) retention time of less than about 10 minutes. In certain embodiments, the terminal lysine residue is K447 according to EU numbering. In certain embodiments, the anti-CD19 heavy chain variable region contains serine at position 84 and / or leucine at position 108 according to Kabat numbering. In certain embodiments, the bispecific antibody of the common light chain contains histidine at position 32 according to Kabat numbering. In certain embodiments, the negatively charged amino acid is glutamic acid. In certain embodiments, the pI is 8-9.In certain embodiments, the pharmaceutical composition comprises a common light chain bispecific antibody, a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, the nucleic acid encodes the bispecific antibody. Methods for treating cancer in an individual requiring treatment are also provided herein, comprising the step of administering the individual with a common light chain bispecific antibody. In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the solid tissue cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, gastric cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In certain 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. In certain embodiments, the cancer or tumor is a hematological cancer. In certain embodiments, the hematological cancer is diffuse large B-cell lymphoma. In certain embodiments, the hematological cancer is myeloma. In certain embodiments, hematological malignancies are Burkitt lymphoma. In certain embodiments, hematological malignancies are aggressive B-cell lymphoma. In certain embodiments, aggressive B-cell lymphoma includes double-hit lymphoma, double-expressor lymphoma, or triple-hit lymphoma. In certain embodiments, hematological malignancies are recurrent or refractory. In certain embodiments, cancers or tumors associated with CD19-positive, CD38-high, immunosuppressive B cells are cancers or tumors that include CD19-positive, CD38-high, B-cell infiltration. In certain embodiments, CD19-positive, CD38-high, immunosuppressive B cells express a B-cell activation marker. In certain embodiments, the B-cell activation marker includes CD30. In certain embodiments, cancers or tumors associated with CD19-positive, CD38-high B cells express PD-L1. In certain embodiments, cancers or tumors associated with CD19-positive, CD38-high B cells are associated with CD20-low or CD20-negative B cells. In certain embodiments, CD38-high B cells express at least approximately 30,000 CD38 proteins on their cell surface. In certain embodiments, CD38-high B cells express at least approximately 35,000 CD38 proteins on their cell surface. In certain embodiments, CD38-high B cells express at least approximately 40,000 CD38 proteins on their cell surface.

[0007] In another embodiment, a method for lowering the experimental isoelectric point of an antibody is provided herein, which comprises (a) mutating glutamine to a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering, and (b) removing lysine at the C-terminal position of the heavy chain constant region. In a particular embodiment, the negatively charged amino acid is glutamic acid. In a particular embodiment, the antibody is a common light chain bispecific antibody, which comprises an anti-CD38 heavy chain variable region, an anti-CD19 heavy chain variable region, and a common light chain variable region, where the anti-CD38 heavy chain variable region comprises (a) a heavy chain complementarity determining region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, (b) a heavy chain complementarity determining region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, and (c) a heavy chain complementarity determining region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and the anti-human CD19 heavy chain variable region comprises (a) the amino acid sequence described in SEQ ID NO: 10 The common light chain comprises a heavy chain complementarity determination region 1 (HCDR1) containing an acid sequence, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in (b) SEQ ID NO: 11, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in (c) SEQ ID NO: 12. The variable region of the common light chain comprises a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in (a) SEQ ID NO: 13, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence (AAS) described in (b) SEQ ID NO: 14, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in (c) SEQ ID NO: 15.

[0008] A method for producing an antibody is also provided herein, comprising the steps of: collecting the bispecific antibody of the present disclosure from the supernatant of a cell line containing a nucleic acid encoding the antibody; and subjecting the supernatant to one or more purification steps. [Brief explanation of the drawing]

[0009] [Figure 1A] The multireactivity profile of BSM-001F (BSM-001.0.1) is shown. [Figure 1B]The multireactivity profile of BSM-001F (BSM-001.0.1) is shown. [Figure 1C] The multireactivity profile of BSM-001F (BSM-001.0.1) is shown. [Figure 2] The hydrophobic chromatography (HIC) profile of BSM-001F (BSM-001.1) is shown. [Figure 3] This shows B-cell binding data for BSM-001F (BSM-001.1). [Figure 4] This shows ADCC cell death data for BSM-001F (BSM-001.1). [Figure 5] This shows apoptotic cell death data for BSM-001F (BSM-001.1). [Modes for carrying out the invention]

[0010] The terms “immunosuppression,” “negative immunomodulation,” or “modulation” refer to processes or cells involved in the reduction or suppression of immune system function with respect to specific cell populations as used herein. Immunosuppression generally means a state in which immune system function is reduced or absent with respect to one or more functions, such as cellular immunity, antibody-based immunity, or innate immune function. In specific examples, immunosuppression generally means a state in which immune system function is reduced or absent with respect to a tumor, or within, around, or adjacent to the tumor microenvironment. The entire immune response may be suppressed, the immune response in a local or specific area may be reduced, or a specific population of immunologically active lymphocytes may be selectively affected. Antigen-specific immunosuppression may result from the deletion or suppression of a specific 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 to the immune response and can 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-β, or a combination thereof from B cells.

[0011] As used herein, the term “cancer” generally refers to or describes a physiological condition in mammals characterized by uncontrolled cell proliferation. Cancer may also include solid tumors. Cancer may 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, breasts, cervix, chest, colon, endometrium, esophagus, eyes, head, kidneys, liver, lungs, lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum, kidneys, skin, stomach, testes, throat, and uterus. Specific cancers include 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 lymphoma, mature T-cell and natural killer (NK) tumors (prolymphocytic leukemia, T-cell macrolymphocytic leukemia, invasive NK-cell leukemia). Adult T-cell leukemia / lymphoma, exogenous negative NK / T-cell lymphoma, enteric disease T-cell lymphoma, hepatosplenic T-cell lymphoma, blastogenic NK-cell lymphoma, fungal infections (Sézary syndrome), primary cutaneous degenerative large cell lymphoma, lymphomatous papilloma, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma and degenerative large cell lymphoma, Hodgkin lymphoma (tubular sclerosis, mixed cell type, lymphocyte-rich type, lymphocyte-depleted type or non-reducing type, nodular Lymphocyte type, myeloma (multiple myeloma, inactive myeloma, smoldering myeloma), chronic myeloproliferative disorders, myelodysplastic / myeloproliferative disorders, myelodysplastic syndromes, lymphoproliferative disorders associated with immunodeficiency, histiocytic and dendritic cell neoplasms, hypercytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent, non-hormone-dependent), gynecological cancers (cervical, endometrial, fallopian tube, gestational trophoblast disease) (ovaries, peritoneum, uterus, vagina and vulva), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), dermatofibrosarcoma of the prostate, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, hairy cell astrocytoma, embryonic hair growth neuroepithelial tumor, oligodendrocyte, ependymal cell tumor, glioblastoma pleomorphic, mixed glioma, oligodendrocyte astrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, embryonic tissue tumor, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastric tumor.Carcinoid tumors, pancreatic endocrine tumors (PET), colorectal adenocarcinoma, colorectal cancer, invasive neuroendocrine tumors, leiomyosarcoma, mucinous adenocarcinoma, synnlet ring cell adenocarcinoma, hepatocellular carcinoma, hepatobiliary tract cancer, hepatoblastoma, hemangioma, liver adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamatoma), non-small cell lung cancer (NSCLC) (squamous cell lung cancer, adenocarcinoma, large cell lung cancer), small cell lung cancer, thyroid cancer. Prostate cancer (hormone-resistant, non-androgen-dependent, androgen-dependent, hormone-insensitive), renal cell carcinoma, and soft tissue sarcomas (fibrosarcoma, malignant fibrous histocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraosseous osteosarcoma) are listed, but are not limited to these.

[0012] The term "CD19" or "differentiation antigen group 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 the B cell antigen receptor complex (BCR) on B lymphocytes, thereby lowering the activation threshold of downstream signaling pathways and inducing a B cell response 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 GenBank accession numbers NM_001178098.2→NP_001171569.1 or NM_001770.6→NP_001761.3 over sequence lengths 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 approximately 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, over nucleic acid sequence lengths of at least 300, 500, 750, 1000, 1250, or 1500 nucleotides, or over the full length of the polynucleotide. Sequence alignment can be performed using any alignment algorithm known in the art, such as BLAST or ALIGN, set to default settings.

[0013] The term "CD38" or "differentiation antigen group 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, which 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 GenBank accession number NM_001775.4→NP_001766.2 over sequence lengths of at least 50, 100, 150, 200, 250 amino acids, or over the full length of the polypeptide. A second isoform of CD38 exists that has an early stop codon and can be expressed at low levels in some cells. Structurally, the CD19 nucleic acid sequence has at least approximately 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, over at least 300, 500, or 750 nucleic acid sequence lengths, or over the entire length of the polynucleotide. Sequence alignment can be performed using any alignment algorithm known in the art, such as BLAST or ALIGN, set to default settings.

[0014] The term "CD20" or "differentiation antigen group 20" (also known as B lymphocyte surface antigen B1) refers to a B cell lineage surface biomarker or transmembrane protein encoded by the gene CD20 in humans. Structurally, the CD20 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 Uniprot entry name P11836, for example, over a sequence length of at least 50, 100, 150, 200, 250 amino acids, or over the entire length of the polypeptide.

[0015] As described herein with reference to binding molecules such as antibodies and bispecific antibodies, "binds" refers to a specific interaction between a target antigen and one or more amino acid residues of the variable region of a complementarity determining region. Such specific binding generally results in a dissociation constant of less than about 1×10 -6 M, and such affinity can be determined by one of ordinary skill in the art using techniques known in the art, for example, by surface plasmon resonance.

[0016] As used herein, the term "antibody" is used in the broadest sense and includes intact antibodies and their functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, polyvalent antibodies or bispecific antibodies including monoclonal antibodies, single-chain variable fragments (sFv or scFv), and single-chain antibody fragments including single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or other modified forms of immunoglobulins, including intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecificities such as bispecific antibodies, diabodies, triabodies, and tetra-bodies, tandem di-scFv, tandem tri-scFv, etc. Unless otherwise indicated, the term "antibody" should be understood to encompass its functional antibody fragments. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. An antibody can comprise a human IgG1 constant region. An antibody can comprise a human IgG4 constant region.

[0017] The antibodies provided include multispecific or multivalent antibodies (e.g., bispecific antibodies and polyreactive antibodies) and antibody fragments thereof. Antibodies include molecules that contain antibodies such as antibody-conjugates and chimeric molecules. Thus, antibodies include full-length and native antibodies, as well as fragments and portions thereof that retain their binding specificity, e.g., any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), any specific binding portion thereof including those having such, as well as biologically relevant (antigen-binding) fragments or specific binding portions thereof including, but not limited to, Fab, F(ab’)2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally those within a composition of antibodies that is substantially homogeneous, and thus any individual antibody contained within a monoclonal antibody composition is identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies can include the human IgG1 constant region or the human IgG4 constant region.

[0018] The terms “complementarity-determining region” and “CDR” refer to amino acid positions / residues (continuous or discontinuous) that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-11, CDR-12, CDR-13). It is known in the art that “framework region” and “FR” refer to the non-CDR portions of the heavy chain and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).The precise amino acid sequence boundaries of a given CDR or FR are as follows: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J.Mol.Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J.Mol.Biol.262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003. This can be easily determined using one of several well-known schemes, including those described by 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 modeling 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 methods selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0019] 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 a structural alignment, and 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 accommodated by inserted letters, e.g., "30a", and deletions appearing in some antibodies. The two schemes place specific 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 in many respects to the Chothia numbering scheme.

[0020] 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 ), respectively) generally have a similar structure, 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., page 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 be isolated using the V H or V L domain from an antibody that binds the antigen, and libraries of complementary V L or V H domains can be screened (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993), Clarkson et al., Nature 352:624-628 (1991)).

[0021] The term "constant region" can refer to the constant region of the light chain or heavy chain. The light chain constant region has two main isotypes, kappa and lambda (sometimes abbreviated as CK or CL, respectively). The heavy chain constant region may contain one of five isotypes: IgA, IgD, IgG, IgE, or IgM. The IgG isotype further includes the IgG1, IgG2, IgG3, and IgG4 subclasses. The heavy chain constant region contains the CH1, hinge, CH2, and / or CH3 domains. Residues in the light and heavy chain constant regions can be numbered according to the EU numbering scheme (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)) or the Kabat numbering scheme (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication no 91-3242, pp 662, 680, 689 (1991)). As used herein, the “Fc region” generally refers to the CH2 and CH3 domains of the heavy chain constant region.

[0022] The antibodies provided include antibody fragments. “Antibody fragment” can refer to molecules other than the intact antibody, including a 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 multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a fragment produced by recombination, e.g., having two or more antibody regions or chains linked by a synthetic linker, e.g., a polypeptide linker, and / or containing non-spontaneous sequences, such as those not produced by enzymatic digestion of spontaneously occurring intact antibodies.

[0023] In this specification, molecules, peptides, polypeptides, antibodies, or antibody fragments may be referred to as “bispecific” or “bispecific,” including their grammatical equivalents. A bispecific molecule has the ability to specifically bind to at least two structurally distinct targets. Specific binding involves two distinct binding sites that are structurally different at the molecular level, including but not limited to different non-identical amino acid sequences, or high affinity (e.g., about 1 × 10⁻¹⁶). -6This may result from a single binding site that can specifically bind to two structurally different targets (having a KD of less than 1). 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 two or more structurally different targets. A "bispecific antibody" including a grammatical equivalent refers to a bispecific molecule that preserves at least one fragment of an antibody that can specifically bind to a target, e.g., a variable region, heavy or light chain, or one or more complementarity-determining regions derived from the antibody molecule. A "multispecific antibody" including a grammatical equivalent refers to a multispecific molecule that preserves at least one fragment of an antibody that can specifically bind to a target, e.g., a variable region, heavy or light chain, or a complementarity-determining region derived from the antibody molecule.

[0024] In this specification, “linker” is also referred to as “linker sequence,” “spacer,” “tether sequence,” or their grammatical equivalents. A “linker” as used herein connects two distinct molecules that are themselves target-binding, catalytically active, or naturally expressed and assembled as distinct polypeptides. For example, two different binding sites or heavy / light chain pairs. Molecules can be covalently linked using several strategies. These include, but are not limited to, polypeptide linkages between the N-terminus and C-terminus of a protein or protein domain, linkages via disulfide bonds, and linkages 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 have a length appropriate to link two molecules so that they assume the correct conformations relative to each other, in order to retain the desired activity. In one embodiment, the linker is about 1–50 amino acid length or about 1–30 amino acid length. In one embodiment, linkers with a length of 1 to 20 amino acids may be used. Useful linkers include, for example, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, including (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n (where n is an integer of at least 1). Exemplary linkers for linking antibody fragments or single-chain variable fragments include AAEPKSS, AAEPKSDKTHTCPPCP (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), or GGGGDKTHTCPPCP (SEQ ID NO: 21). Alternatively, a variety of non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may be used as linkers.

[0025] 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 sites and a linker connecting the two binding sites. The linker may be a polypeptide linker or another suitable flexible linker that does not inhibit the binding of either target site. The fragment-based bispecific antibody format includes Tandem V HH These include antibodies, tandem scFv, scFv-Fab, F(ab)2, and biaffinity retargeting antibodies (DART). Such fragment-based antibodies can be further manipulated to include additional binding sites specific to a given target, e.g., A2:B1, A1:B2, or A2:B2, or to include fragments of Fc regions to improve pharmacokinetics or promote ADCC, ADCP, or CDC.

[0026] The “binding region” refers to a portion of a molecule, peptide, polypeptide, antibody, or antibody fragment that mediates specific binding to the listed target, antigen, or epitope. For example, the binding region of an antibody may include a heavy / light chain variable region pair or one or more complementarity-determining regions (CDRs).

[0027] As used herein, “target” refers to a portion of a molecule that is involved with a binding portion of a molecule, such as a peptide, polypeptide, antibody, or antibody fragment. A target may include an amino acid sequence and / or carbohydrates, lipids, or other chemical entities. “Antigen” is a target that includes a portion that can be bound by an adaptive immune molecule, such as an antibody or antibody fragment, a B cell receptor, or a T cell receptor.

[0028] In some embodiments, the bispecific antibodies provided herein have 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 with respect to the antibody target (for example, 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 may be a CD19 target, a CD38 target, or a target containing both CD19 and CD38. K D KD can be measured by any suitable assay. In certain embodiments, KD can be measured using a surface plasmon resonance assay (e.g., BIACORE®-2000 or BIACORE®-3000 or Octet).

[0029] 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 form” of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0030] The antibodies offered include human antibodies. “Human antibodies” are antibodies that have an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or a non-human source, utilizing a human antibody repertoire, including a human antibody library, or a sequence encoding another human antibody. This term excludes humanized forms of non-human antibodies that contain a non-human antigen-binding region, such as those in which all or substantially all CDRs are non-human. Human antibodies can be prepared by administering an immunogen to a modified animal that has been modified to produce an intact human antibody or an intact antibody with a human variable region in response to antigen attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is located outside the chromosome, or is randomly incorporated into the animal's chromosome. In such modified animals, the endogenous immunoglobulin locus is generally inactivated. Human antibodies may also originate from a human antibody library, including phage displays and cell-free libraries, that contain sequences encoding antibodies derived from the human repertoire.

[0031] As used herein, “ADCC” or “antibody-dependent cell-mediated injury” refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, subsequently causing lysis of the target cells. ADCC can be correlated with binding to FcγRIIIa, and increased binding to FcγRIIIa results in increased ADCC activity. As used herein, “ADCP” or “antibody-dependent cell-mediated phagocytosis” may refer to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, subsequently causing phagocytosis of the target cells.

[0032] The terms “polypeptide” and “protein” are used interchangeably and refer to polymers of amino acid residues, not limited to the minimum length. Polypeptides, including antibodies and antibody chains and other peptides, e.g., linkers and binding peptides, may contain amino acid residues, including native and / or non-native amino acid residues. These terms also include post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, polypeptides may contain modifications to the native (native or natural) sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host producing the protein or errors in PCR amplification.

[0033] Percent (%) sequence identity with respect to a 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 as necessary to achieve maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various known ways 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 necessary to achieve 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 publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program must 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 remain unchanged.In situations where the ALIGN-2 program is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or, to put it another way, a given amino acid sequence A that has or contains a particular % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: fraction X / Y multiplied by 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be 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.

[0034] The amino acid sequence variants of antibodies provided herein are intended and conceived. The variants differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may occur spontaneously or may be produced synthetically by, for example, modifying one or more of the polypeptide sequences of the present invention described herein, evaluating the biological activity of one or more of the polypeptides described herein, and / or using any of several known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of antibody amino acid sequence variants of antibodies that can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions is performed to reach the final construct, provided that the final construct has the desired properties, e.g., antigen binding. Antibody variants having one or more amino acid substitutions may be provided. The target sites for mutagenesis by substitution include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest, and the product is screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC.

[0035] This disclosure also provides “immune complexes,” “antibody complexes,” or “antibody-drug complexes,” which refer to antibodies conjugated to one or more heterologous molecules. For example, an immune complex may include an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitor, a protein domain, a toxin (e.g., a protein toxin, an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants, or animals), or a radioisotope. In some embodiments, an immune complex may include the complex conjugation molecules or fragments thereof of this disclosure (e.g., scFv).

[0036] The 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 polypeptide encoding a polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of carrying another ligated nucleic acid. One type of vector is a genomic integration vector or “integration vector” that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an “episomal” vector, such as a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of a functionally ligated gene is referred to herein as an “expression vector.” Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and viral vectors. In expression vectors, regulatory elements such as promoters, enhancers, and polyadenylation signals for use in regulating transcription may be derived from mammalian, microbial, viral, or insect genes. Selective genes to facilitate the ability to replicate in the host and the recognition of transformants, usually conferred by the origin of replication, may be further incorporated. 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 sites. Vectors may contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into defined locations or a limited set of sites in the genome. Furthermore, vectors may contain sequences derived from transposition factors.

[0037] As used herein, the terms “homologous,” “homologousness,” or “percent homology” may be used herein to describe an amino acid sequence or nucleic acid sequence in comparison to a reference sequence, and may be determined using the formulas described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 1564-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). These formulas are incorporated into the basic local alignment search tool (BLAST) program by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST as of the filing date of this application.

[0038] The nucleic acids encoding antibodies described herein can be used to transgenicize cells suitable for the nucleic acid by infection, transfect, transformation, or other means, thus enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large cell media are known in the art. See, for example, Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. 2010 Sep-Oct;2(5):466-477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells that are useful cell lines for antibody production are Chinese hamster ovary cells (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 a cell useful for antibody production. In certain embodiments, a method for producing an antibody is described herein, the method comprising the step of culturing cells containing the nucleic acid encoding the antibody under in vitro conditions sufficient to enable the production and secretion of the antibody.

[0039] As used herein, the terms “individual,” “patient,” or “subject” refer to an individual who is diagnosed with, suspected of having, or at risk of developing, at least one disease for which the described compositions and methods are useful in 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.

[0040] As used herein, the term “approximately” used to modify a particular number refers to a number that is plus or minus 10% of that number. The term “approximately” used to modify a range refers to a range of minus 10% of the lowest value and plus 10% of the highest value.

[0041] As used herein, the terms “treatment” or “to treat” refer to a pharmaceutical or other intervention regimen used to obtain a beneficial or desired outcome in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits and / or preventive benefits. Therapeutic benefits may refer to the elimination or improvement of symptoms or the underlying disorder being treated. Therapeutic benefits may also be achieved by the elimination or improvement of one or more physiological symptoms associated with the underlying disorder, such that improvement in the subject is observed despite the subject still being affected by the underlying disorder. Preventive benefits include delaying, preventing, or eliminating the onset of a disease or illness; delaying or eliminating the onset of symptoms of a disease or illness; slowing, stopping, or reversing the progression of a disease or illness; or any combination thereof. With respect to preventive benefits, subjects at risk of progression of a particular disease, or 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, when considering a potential population of individuals for treatment, not all will respond to the treatment or respond equally well. Such individuals are considered treated.

[0042] The paragraph headings used in this specification are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0043] Methods for producing antibodies as described herein are also described herein. Such methods include the steps of incubating cells or cell lines containing nucleic acids encoding antibodies in a cell medium under conditions sufficient to enable antibody expression and secretion, and further the steps of collecting the antibodies from the cell medium. The collection step may further include one or more purification steps to remove live cells, cell fragments, non-antibody proteins or polypeptides, unwanted salts, buffers, and culture medium components. In certain embodiments, additional purification steps may include centrifugation, ultracentrifugation, purification of protein A, protein G, protein A / G, or protein L, and / or ion exchange chromatography.

[0044] As used herein, “to treat,” “to treat,” or “to treat” means, for example, intentional intervention of a physiological condition that results in a reduction of the severity of a disease or illness, shortening the duration of the course of a disease, improving or eliminating one or more symptoms associated with a disease or illness, or providing a beneficial effect to a person having a disease or illness. Treatment does not require a cure of the underlying disease or illness.

[0045] The “therapeutic effective dose,” “effective dose,” or “effective amount” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with other therapeutic agents, protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional or physical impairment resulting from the distress of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as by assaying the activity of the drug in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or in in vitro assays.

[0046] As used herein, “pharmaceutically acceptable” includes any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., with respect to “carriers,” “excipients,” or “diluents.” In some embodiments, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, may be coated with a material to protect the compound from acids or other natural conditions that can inactivate the compound.

[0047] The antibodies, bispecific antibodies, and multispecific antibodies described herein may contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” is defined as a salt that retains the desired biological activity of the parent compound and does not impart any unwanted toxic effects (see, for example, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanoates, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, and calcium, as well as non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0048] In one embodiment, a multispecific antibody is described herein, which comprises: (a) a CD38 binding moiety comprising: a first polypeptide comprising (i) an immunoglobulin heavy chain variable region and a heavy chain constant region comprising the amino acids described in SEQ ID NO: 1, wherein the heavy chain constant region lacks a C-terminal lysine residue; and (ii) a second polypeptide comprising a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 3; and (b) a CD19 binding moiety comprising: a third polypeptide comprising (i) an immunoglobulin heavy chain variable region and a heavy chain constant region comprising the amino acids described in SEQ ID NO: 2, wherein the heavy chain constant region lacks a C-terminal lysine residue; and (ii) a fourth polypeptide comprising a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 3. In certain embodiments, the heavy chain constant regions of the first polypeptide, the third polypeptide, or both the first polypeptide and the third polypeptide comprise a human IgG1 or human IgG4 constant region. In certain embodiments, the polypeptide containing a light chain variable region further contains a light chain constant region. In certain embodiments, the second polypeptide, the fourth polypeptide, or both the second and fourth polypeptides contain the amino acid sequence described in SEQ ID NO: 6. In certain embodiments, the CD38 binding moiety contains one or more amino acid substitutions that inhibit homodimerization of the CD38 binding moiety. In certain embodiments, the CD38 binding moiety contains a T366W substitution according to EU numbering, or a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the CD19 binding moiety contains one or more amino acid substitutions that inhibit homodimerization of the CD19 binding moiety. In certain embodiments, the CD19 binding moiety contains a T366W substitution according to EU numbering, or a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the first polypeptide consists of the amino acid sequence described in SEQ ID NO: 4. In certain embodiments, the third polypeptide consists of the amino acid sequence described in SEQ ID NO: 5. In certain embodiments, the pharmaceutical composition comprises a multispecific antibody, a pharmaceutically acceptable excipient, a diluent, or a carrier. In certain embodiments, the nucleic acid encodes a multispecific antibody.

[0049] The bispecific antibodies described herein comprise intact antibody molecules or substantially completely intact antibody molecules, and may be asymmetric or symmetric.

[0050] 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 a heterodimer antibody. Such heterodimer antibodies face the problem of unproductive molecular formation when produced. While HC / LC-A:HC / LC-B is desirable, it is usually thermodynamically or statistically undesirable from all possible combinations. Several methods have been introduced to circumvent this problem. In some examples, 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 likelihood of forming an antibody with HC / LC-A:HC / LC-B. This can be achieved by manipulating structural features such as a "knob" to the FC region of HC-A and a "hole" to HC-B, or vice versa, which promote the formation of a heterodimer between HC-A and HC-B. Another scheme to promote HC-A:HC-B heterodimers is to manipulate the amino acid residues in the FC portions of HC-A and HC-B to include charge pairs favorable to electrostatic interactions between HC-B and HC-A. Another scheme to address the chain association problem is to make one variable region of the HC / LC pair a single-chain binding molecule (e.g., V HHThis involves replacing with (or scFv). Thus, half of the molecule contains a classical HC / LC pair, and the other half contains an HC constant region fused to or otherwise linked to a single-chain binding molecule. Further modifications can be made to facilitate proper HC / LC pairing, including manipulating mutations for HC and LC in either A or B to favor the formation of a proper HC / LC pair, and CrossMab technology involves the exchange of the corresponding constant regions of the HC / LC pair. Symmetric bispecific antibodies avoid the problem of chain association by not relying on the formation of heterobifunctional molecules. Such examples 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 containing two scFVs linked by the FC region of Ig (Fes dimerizes to produce a bispecific tetravalent molecule), DART-Fc, and two-in-one, among others.

[0051] Multispecific or bispecific antibodies can be conceived and designed to alter the combined functionality or binding properties of 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):112-212). 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, aFab-Fc-scFv:Fab-Fc-scFv bispecific IgG, aFab-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.

[0052] Exemplary knob-into-hole mutations for use with the antibodies described herein may include T366W (EU numbering) in one heavy chain and T366S / L368A / Y407V (EU numbering) in the second heavy chain. Exemplary mutations that facilitate coupling of the first and second heavy chain molecules are disclosed, for example, in WO2009089004, US 8,642,745, U.S. Patent Publication: US20140322756, and “The making of bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 112-212.

[0053] In some embodiments, a bispecific antibody for a common light chain is provided herein, the bispecific antibody for a common light chain comprises (1) an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, (2) an anti-CD38 heavy chain constant region and an anti-CD19 heavy chain constant region, and (3) a variable region for a common light chain, wherein the anti-CD38 heavy chain variable region comprises (i) a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, (ii) a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, (iii) a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and (iv) a Kabat-numbered Q1E heavy chain variable region mutation, and the anti-human CD19 heavy chain variable region comprises (i) a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, and (ii) The common light chain variable region includes (iii) a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 11, (iii) a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 12, and (iv) a Kabat-numbered Q1E heavy chain variable region mutation. The common light chain variable region includes (i) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, (ii) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence (AAS) described in SEQ ID NO: 14, and (iii) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 15. Each of the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain variable region lacks a C-terminal lysine residue (e.g., Kabat-numbered K478), and the bispecific antibody of the common light chain has an experimental isoelectric point (pI) of less than approximately 9. In certain cases, the anti-CD38 heavy chain variable region may include a heavy chain complementarity-determining region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NOs. 8 or 16-18.

[0054] In certain embodiments, the bispecific antibody of the common light chain further includes a hydrophobic (HIC) retention time of less than approximately 10 minutes. In certain embodiments, the anti-CD19 heavy chain variable region includes serine at position 84 and / or leucine at position 108 according to Kabat numbering. In certain embodiments, the bispecific antibody of the common light chain includes histidine at position 32 according to Kabat numbering. In certain embodiments, the pI is less than 9. In certain embodiments, the pI is 8 to 9. In certain embodiments, the pI is 8.5 to 9.0. In certain embodiments, the pI is 8.7 to 9.0. In certain embodiments, the pI is 8.8 to 9.0. In certain embodiments, the pI is 8.9.

[0055] Treatment method In certain embodiments, antibodies useful for treating cancer or tumors are disclosed herein. This refers to a method that seeks to improve or ameliorate the disease being treated. With respect to cancer, treatment includes, but is not limited to, a reduction in tumor volume, a reduction in tumor volume growth, or an increase in progression-free survival or overall life expectancy. In certain embodiments, the treatment would result in remission of the treated cancer. In certain embodiments, the treatment includes use as a prophylactic or maintenance dose intended to prevent recurrence or progression of a previously treated cancer or tumor. Those skilled in the art will understand that not all individuals will respond equally to, or not at all to, the treatment administered; nevertheless, these individuals are considered treated.

[0056] Methods for treating cancer in an individual requiring treatment, comprising the step of administering a multispecific antibody to the individual, are also described herein. In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the solid tissue cancer includes breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, gastric cancer, esophageal cancer, skin cancer, colorectal cancer, or head and neck cancer. In certain 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. In certain embodiments, the cancer or tumor is a hematological cancer. In certain embodiments, the hematological cancer is diffuse large B-cell lymphoma. In certain embodiments, the hematological cancer is myeloma. In certain embodiments, the hematological cancer is Burkitt lymphoma. In certain embodiments, the hematological cancer is aggressive B-cell lymphoma. In certain embodiments, aggressive B-cell lymphomas include double-hit lymphomas (e.g., defined by mutations and / or rearrangements in either the MYC gene and the BCL2 gene or the BCL6 gene), double-expressor lymphomas (e.g., defined by the co-expression of c-MYC and BCL-2), or triple-hit lymphomas (e.g., defined as having morphological, biological, and cytogenetic characteristics similar to both diffuse large B-cell lymphoma and Burkitt lymphoma, but with three gene rearrangements instead of two: the c-MYC gene, the BCL-2 gene, and the BCL-6 gene). In certain embodiments, hematological malignancies are recurrent (e.g., the cancer recurs after a period of remission) or refractory (e.g., the cancer has become unresponsive to previous treatments with different therapies / treatments). In certain embodiments, CD19-positive, CD38-high, immunosuppressive B-cell associated cancers or tumors are CD19-positive, CD38-high, B-cell infiltrate-containing cancers or tumors. In certain embodiments, CD19-positive, CD38-hyposuppressive B cells express a B cell activation marker. In certain embodiments, the B cell activation marker includes CD30. In certain embodiments, cancer or tumors associated with CD19-positive, CD38-hyposuppressive B cells express PD-L1.In certain embodiments, cancers or tumors associated with CD19-positive, CD38-high B cells are associated with CD20-low or CD20-negative B cells. In certain embodiments, CD38-high B cells express at least about 30,000 CD38 proteins on their cell surface. In certain embodiments, CD38-high B cells express at least about 35,000 CD38 proteins on their cell surface. In certain embodiments, CD38-high B cells express at least about 40,000 CD38 proteins on their cell surface. In a particular embodiment, the multispecific antibody comprises (a) a CD38 binding moiety comprising (i) a first polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region containing the amino acids described in SEQ ID NO: 1, wherein the heavy chain constant region lacks a C-terminal lysine residue, and (ii) a second polypeptide comprising a light chain variable region containing the amino acid sequence described in SEQ ID NO: 3; and (b) a CD19 binding moiety comprising (i) a third polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region containing the amino acids described in SEQ ID NO: 2, wherein the heavy chain constant region lacks a C-terminal lysine residue, and (ii) a fourth polypeptide comprising a light chain variable region containing the amino acid sequence described in SEQ ID NO: 3.

[0057] In certain embodiments, cancer or tumor is a solid cancer or solid tumor. In certain embodiments, cancer or tumor is a blood cancer or hematoma. 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 tumor, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatocytoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, tumors / cancers include melanoma ascites, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, Maltrin's adenocarcinoma, basal cell carcinoma, bronchial carcinoma, capillary carcinoma, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, swing sarcoma, focal nodular hyperplasia, gastoma, germline tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, and Nslinite, carcinoma in situ, squamous cell carcinoma in situ, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, medulloblastoma, medullary epithelioma, mesothelioma, mucosal epidermal 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. Selected from the group of undifferentiated carcinoma, uveal melanoma, leukoplakia, vaginal / vulvar cancer, 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, the cancer treated with the antibodies of this disclosure includes glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of this disclosure includes pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure includes ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure includes lung cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure includes 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 is 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.

[0058] In certain embodiments, the antibody can be administered to a target requiring administration via any route suitable for the administration of the antibody-containing pharmaceutical composition, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. 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 dosage schedule, such as weekly, twice weekly, monthly, twice monthly, every two weeks, every three weeks, or once a month. In certain embodiments, the antibody is administered every three weeks. The antibody is 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 1 mg / kg to about 20 mg / kg. In certain embodiments, the therapeutically acceptable dose is approximately 1 mg / kg to approximately 10 mg / kg. In certain embodiments, the therapeutically acceptable dose is approximately 5 mg / kg to approximately 30 mg / kg. In certain embodiments, the therapeutically acceptable dose is approximately 5 mg / kg to approximately 20 mg / kg. The therapeutically effective dose includes an amount sufficient to improve one or more symptoms associated with the disease or distress being treated.

[0059] Method for producing antibodies with reduced pI In some embodiments, methods for lowering the experimental isoelectric point of an antibody are provided herein, comprising the steps of (a) mutating glutamine to a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering, and (b) removing lysine at the C-terminal position of the heavy chain constant region. In some embodiments, the negatively charged amino acid is glutamic acid. In some embodiments, the negatively charged amino acid is aspartic acid. In some embodiments, the lysine at the C-terminal position of the heavy chain constant region is K447 according to EU numbering.

[0060] In some embodiments, the antibody is a common light chain bispecific antibody, which comprises (1) an anti-CD38 heavy chain variable region, (2) an anti-CD19 heavy chain variable region, and (3) a common light chain variable region, where the anti-CD38 heavy chain variable region comprises (i) a heavy chain complementarity determining region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, (ii) a heavy chain complementarity determining region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, and (iii) a heavy chain complementarity determining region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and the anti-CD19 heavy chain variable region comprises (i) the sequence described in SEQ ID NO: 10 The variable region of the common light chain includes (i) a light chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in (ii) SEQ ID NO: 11, and (iii) a light chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 12.

[0061] In certain embodiments, the bispecific antibody of the common light chain further includes a hydrophobic (HIC) retention time of less than approximately 10 minutes. In certain embodiments, the anti-CD19 heavy chain variable region includes serine at position 84 and / or leucine at position 108 according to Kabat numbering. In certain embodiments, the bispecific antibody of the common light chain includes histidine at position 32 according to Kabat numbering. In certain embodiments, the pI is less than 9. In certain embodiments, the pI is 8 to 9. In certain embodiments, the pI is 8.5 to 9.0. In certain embodiments, the pI is 8.7 to 9.0. In certain embodiments, the pI is 8.8 to 9.0. In certain embodiments, the pI is 8.9.

[0062] pharmaceutically acceptable excipients, carriers, and diluents In certain embodiments, the multispecific antibodies of the Disclosure are contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. The pharmaceutically acceptable excipients, carriers, and diluents may be included to increase the shelf life, stability, or administration of the antibody. Such compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibodies of the Disclosure are administered suspended in a sterile aqueous solution. In certain embodiments, the aqueous solution contains about 0.9% NaCl. In certain embodiments, the aqueous solution contains about 5.0% dextrose. In certain embodiments, the aqueous solution further comprises one or more of the following: buffers, e.g., acetates, citrates, histidines, succinates, phosphates, bicarbonates, and hydroxyketylaminomethane (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.

[0063] In certain embodiments, the antibodies of this disclosure may be shipped / stored, lyophilized, and reconstituted before administration. In certain embodiments, the lyophilized antibody formulation may contain an expander, e.g., mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized antibody formulation may be contained in a vial made of glass or other suitable non-reactive material. The formulated antibody may be buffered at a specific pH, generally below 7.0, whether or not it is reconstituted. In certain embodiments, the pH may be 4.5–7.0, 4.5–6.5, 4.5–6.0, 4.5–5.5, 4.5–5.0, or 5.0–6.0.

[0064] Kits comprising one or more antibodies described herein in a suitable container, along with one or more additional components selected from instructions for use, diluents, excipients, carriers, and devices for administration, are also described herein.

[0065] In certain embodiments, a method for preparing a cancer treatment is described herein, comprising the step of mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with the antibodies of the Disclosure. In certain embodiments, a method for preparing a cancer treatment for storage or shipment is described herein, comprising the step of lyophilizing one or more antibodies of the Disclosure.

[0066] Further exemplary embodiments Exemplary Embodiment 1. A common light chain bispecific antibody, wherein the common light chain bispecific antibody is Anti-CD38 heavy chain variable region and anti-CD38 heavy chain constant region, Anti-CD19 heavy chain variable region and anti-CD19 heavy chain constant region, Including the variable region of the common light chain, Here, the anti-CD38 heavy chain variable region includes heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering. The anti-human CD19 heavy chain variable region includes heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, and the amino acid sequence described in SEQ ID NO: 11. The common light chain includes a heavy chain complementarity determination region 2 (HCDR2) containing an amino acid sequence, a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 12, and a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering, and the variable region of the common light chain includes a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence described in SEQ ID NO: 14, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 15. Each of the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain variable region lacks a C-terminal lysine residue, and the common light chain bispecific antibody has an experimental isoelectric point (pI) of less than 9.0, preferably the common light chain bispecific antibody further has a hydrophobic (HIC) retention time of less than about 10 minutes (as assayed in Example 2).

[0067] Exemplary Embodiment 2: A bispecific antibody of a common light chain according to Embodiment 1, further comprising a hydrophobic (HIC) retention time of less than 10 minutes (as assayed in Example 2, for example).

[0068] Exemplary Embodiment 3. A bispecific antibody of the common light chain according to any one of Embodiments 1-2, wherein the terminal lysine residue is K447 according to EU numbering.

[0069] Exemplary Embodiment 4. A bispecific antibody of the common light chain according to any one of Embodiments 1 to 3, wherein the CD19 heavy chain variable region contains serine at position 84 and / or leucine at position 108 according to Kabat numbering.

[0070] Exemplary Embodiment 5. A bispecific antibody of the common light chain according to any one of Embodiments 1 to 4, wherein the variable region of the common light chain contains histidine at position 32 according to Kabat numbering.

[0071] Exemplary Embodiment 6. A common light chain bispecific antibody according to any one of Embodiments 1 to 5, wherein the negatively charged amino acid is glutamic acid.

[0072] Exemplary Embodiment 7. A common light chain bispecific antibody according to any one of Embodiments 1 to 6, wherein pI is 8.7 to 9.0.

[0073] Exemplary Embodiment 8. A common light chain bispecific antibody according to any one of Embodiments 1 to 7, wherein the anti-CD19 heavy chain constant region includes a T366W substitution by EU numbering or a T366S / L368A / Y407V substitution by EU numbering.

[0074] Exemplary Embodiment 9. A common light chain bispecific antibody according to any one of Embodiments 1 to 8, wherein the anti-CD38 heavy chain constant region includes a T366W substitution by EU numbering or a T366S / L368A / Y407V substitution by EU numbering.

[0075] Exemplary Embodiment 10. A bispecific antibody of a common light chain according to any one of Embodiments 1 to 9, further comprising a light chain constant region.

[0076] Exemplary embodiment 11. (i) The anti-CD38 heavy chain variable region and the anti-CD38 heavy chain constant region contain the amino acid sequence of SEQ ID NO: 4, (ii) The anti-CD19 heavy chain variable region and the anti-CD38 heavy chain constant region contain the amino acid sequence of SEQ ID NO: 5 (iii) The variable region and constant region of the common light chain include the amino acid sequence of SEQ ID NO: 6 A bispecific antibody of a common light chain as described in any one of Embodiments 1 to 10.

[0077] Exemplary Embodiment 12. A composition comprising one or more nucleic acid molecules encoding a common light chain bispecific antibody as described in any one of Embodiments 1 to 11.

[0078] Exemplary Embodiment 13. A cell comprising one or more nucleic acid molecules encoding a common light chain bispecific antibody as described in any one of Embodiments 1 to 11.

[0079] Exemplary Embodiment 14. A method for treating cancer in an individual requiring treatment, comprising the step of administering to the individual a common light chain bispecific antibody described in any one of Embodiments 1 to 11.

[0080] Exemplary Embodiment 15. The method according to Embodiment 14, wherein the cancer includes a solid tumor.

[0081] Exemplary Embodiment 16. The method according to Embodiment 14, wherein the cancer is a hematological cancer.

[0082] Exemplary Embodiment 17. The method according to Embodiment 16, wherein the hematological cancer is a B-cell malignant tumor.

[0083] Exemplary Embodiment 18. A method for lowering the experimental isoelectric point of an antibody, comprising the steps of (a) mutating glutamine to a negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering, and (b) removing lysine at the C-terminal position of the heavy chain constant region, The antibody is a common light chain bispecific antibody, and common light chain bispecific antibodies include the following: An anti-CD38 heavy chain variable region comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and an anti-CD38 heavy chain constant region. An anti-CD19 heavy chain variable region comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 11, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 12, and an anti-CD19 heavy chain constant region.

[0084] A variable region of the common light chain, comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence described in SEQ ID NO: 14, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 15. [Examples]

[0085] Example 1 Polyspecificity was measured using ELISA (BVP, PSR HEK293, PSR CHO). Three plates were coated overnight at 4°C with baculovirus particles (BVP), HEK293, and CHO. Antibody concentrations of 150, 50, 16.7, and 5.6 μg / mL were tested in triplicates. The plates were blocked (blocking buffer: PBS + 2% BSA). Background signal was measured for coated wells containing only secondary (secondary) Ab. A 5x background signal cutoff was used. The positive control (PC) contained MEDNA Bio#H1308, and the negative control (NC) contained MEDNA Bio#H1314.

[0086] BSM-001F (BSM-001.1) is a common light chain antibody, and the common light chain antibody includes (1) an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, (2) an anti-CD19 heavy chain variable region and an anti-CD19 heavy chain constant region, and (3) a common light chain variable region, where the anti-CD38 heavy chain variable region includes (i) a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, (ii) a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, and (iii) a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 9, and the anti-CD19 heavy chain variable region is (i (ii) a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, (ii) a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 11, and (iii) a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 12. The variable region of the common light chain includes (i) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, (ii) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence (AAS) described in SEQ ID NO: 14, and (iii) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 15. BSM-001F includes a Kabat-numbered Q1E heavy chain variable region mutation, an anti-CD38 heavy chain constant region, and an anti-CD19 heavy chain constant region in each heavy chain, each lacking a C-terminal lysine residue (e.g., K447 according to EU numbering).

[0087] For data analysis, the BVP / HEK / CHO score was determined using the formula: BVP / HEK / CHO score = (average OD450 value of 3 consecutive samples) / (average OD450 value of background signal). Figures 1A-1C show the ELISA OD450 readings for guselkumab, BSM-001F (BSM-001.1), NC, and PC. BSM-001F (BSM-001.1) showed a substantial decrease in polyspecificity compared to guselkumab and PC.

[0088] Example 2 To determine the experimental isoelectric point, the sample was diluted in a matrix of methylcellulose, 4M urea, 3-10% Pharmalyte (4%), 5nM arginine, and pI markers (pI 4.22 and pI 10.10). The mixture was subjected to an iCE3 IEF analyzer (ProteinSimple), pre-focused at 1,500V, and then focused at 3,000V. The isoelectric point of each peak was calculated from the pre- and post-peak pI markers. The experimental isoelectric point of BSM-001F (BSM-001.1) was measured to be 8.9. Unmodified BSM-001, which (i) lacks the negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering, and (ii) has a heavy chain constant region containing a terminal lysine residue (e.g., K447 according to EU numbering), showed a pI of 9.1. The experimental isoelectric point of BSM-001F (BSM-001.1) decreased to less than 9 (8.9).

[0089] Curia hydrophobic interaction chromatography (HIC) (Curia) was also determined. For the HIC protocol, the sample was titrated with 1 M (NH4)2SO4 before injection. 10 μL of the sample was injected at a flow rate of 0.8 mL / min into a Sepax Proteomix HIC butyl-NP1.7, 4.6 × 35 mm column. The mobile phase started with 60% buffer C (1.8 M (NH4)2SO4), 0.1 M NaH2PO4, pH 6.5) and 40% buffer D (0.1 M NaH2PO4, pH 6.5), and was changed from 1 min to 16 min using a linear gradient to 0% buffer C and 100% buffer D. The signal was measured at 280 nm. A HIC retention time of 9.9 was observed. Figure 2 shows the HIC profile of BSM-001F (BSM-001.1).

[0090] Example 3 Cell binding to Daudi cells was measured. BSM-001F (BSM-001.1) showed comparable binding to Daudi cells compared to unmodified BSM-001 (e.g., lacking Q1E and terminal lysine modifications). Figure 3 shows the cell binding profile.

[0091] ADCC cell death assays were also performed using human donor PBMC samples. For direct evaluation of apoptosis, cells were treated with the test substance and incubated at 37°C / 5%CO2 for 48 hours. For evaluation of cross-linking-induced apoptosis, cells were incubated with the test substance on ice for 30 minutes, followed by the addition of rabbit anti-human Fc gamma-specific F(ab')2. The cells were then incubated at 37°C / 5%CO2. After incubation, the cells were washed, stained with Annexin V, and then resuspended in Annexin V buffer containing the survival dye (propidium iodide; PI) for flow cytometry. Early apoptotic cells were defined as Annexin V+ / PI- unicellular cells, and late apoptotic / necrotic cells were defined as Annexin V+ / PI+ unicellular cells. The sum of Annexin V+ / PI- and Annexin V+ / PI- 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. BSM-001F (BSM-001.1) showed comparable ADCC-mediated death of Daudi cells compared to unmodified BSM-001 (e.g., lacking Q1E and terminal lysine modifications). Figure 4 shows the ADCC profile.

[0092] For direct evaluation of apoptosis, cells were treated with the test substance and incubated at 37°C / 5%CO2 for 48 hours. For evaluation of cross-linking-induced apoptosis, cells were incubated with the test substance on ice for 30 minutes, followed by the addition of rabbit anti-human Fc gamma-specific F(ab')2. The cells were then incubated at 37°C / 5%CO2 for 48 hours. After incubation, cells were washed, stained with Annexin V, and then resuspended in Annexin V buffer containing the survival dye (propidium iodide; PI) for flow cytometry. Early apoptotic cells were defined as Annexin V+ / PI- unicellular cells, and late apoptotic / necrotic cells were defined as Annexin V+ / PI+ unicellular cells. The sum of Annexin V+ / PI- and Annexin V+ / PI- 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. BSM-001F (BSM-001.1) showed comparable apoptosis-mediated death of Daudi cells compared to unmodified BSM-001 (e.g., lacking Q1E and terminal lysine modifications). Figure 5 shows the apoptosis profile.

[0093] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternative forms to the embodiments of the present invention described herein may be employed when carrying out the present invention.

[0094] All publications, patent applications, issued patents, and other documents referenced herein are incorporated herein by reference in such a manner as each individual publication, patent application, issued patent, or other document is specifically and individually incorporated into the whole by reference. Definitions contained in the texts incorporated by reference are excluded to the extent that they do not contradict the definitions in this disclosure.

[0095] [Table 1]

[0096] Table 2

Claims

1. A multispecific antibody, wherein the multispecific antibody is a) CD38 bonding portion, i) A first polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region having the amino acid sequence described in Sequence ID No. 1, wherein the heavy chain constant region lacks a C-terminal lysine residue, and ii) A second polypeptide comprising a light chain variable region containing the amino acid sequence described in Sequence ID No.

3. The CD38 bonding portion includes, b) CD19 bonding portion, i) A third polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region having the amino acid sequence described in Sequence ID No. 2, wherein the heavy chain constant region lacks a C-terminal lysine residue, and ii) A fourth polypeptide comprising a light chain variable region containing the amino acid sequence described in SEQ ID NO: 3 CD19 bond portion including A multispecific antibody containing [specific antibody].

2. The multispecific antibody according to claim 1, wherein the heavy chain constant region of the first polypeptide, the third polypeptide, or both the first polypeptide and the third polypeptide comprises a human IgG1 or human IgG4 constant region.

3. The multispecific antibody according to claim 1, wherein the polypeptide containing a light chain variable region further comprises a light chain constant region.

4. The multispecific antibody according to any one of claims 1 to 3, wherein the second polypeptide, the fourth polypeptide, or both the second polypeptide and the fourth polypeptide contain the amino acid sequence described in SEQ ID NO:

6.

5. The multispecific antibody according to any one of claims 1 to 4, wherein the CD38 binding portion comprises one or more amino acid substitutions that inhibit heavy chain homodimerization of the CD38 binding portion.

6. The multispecific antibody according to claim 5, wherein the CD38 binding portion includes a T366W substitution by EU numbering, or a T366S / L368A / Y407V substitution to the heavy chain of the CD38 binding portion by EU numbering.

7. The multispecific antibody according to claim 1 or 2, wherein the CD19 binding portion comprises one or more amino acid substitutions that inhibit heavy chain homodimerization of the CD19 binding portion.

8. The multispecific antibody according to claim 7, wherein the CD19 binding portion includes a T366W substitution by EU numbering, or a T366S / L368A / Y407V substitution to the heavy chain of the CD19 binding portion by EU numbering.

9. The multispecific antibody according to any one of claims 1 to 8, wherein the first polypeptide comprises the amino acid sequence described in SEQ ID NO:

4.

10. The multispecific antibody according to any one of claims 1 to 8, wherein the third polypeptide comprises the amino acid sequence described in SEQ ID NO:

5.

11. A bispecific antibody of a common light chain, wherein the bispecific antibody of the common light chain is Anti-CD38 heavy chain variable region and anti-CD38 heavy chain steady region, Anti-human CD19 heavy chain variable region and anti-CD19 heavy chain constant region, Variable region of the common light chain Includes, Here, The aforementioned anti-CD38 heavy chain variable region is Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in Sequence ID No. 9, The negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering and Includes, The aforementioned anti-CD19 heavy chain variable region is Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 11, Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in Sequence ID No. 12, The negatively charged amino acid at position 1 of the heavy chain variable region according to Kabat numbering and Includes, The variable region of the common light chain is Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence (AAS) described in Sequence ID No. 14, Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in Sequence ID No. 15 and Includes, Each of the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain variable region lacks a C-terminal lysine residue (e.g., K447 according to EU numbering), The bispecific antibody of the common light chain contains an experimental isoelectric point (pI) of less than 9. A bispecific antibody of the common light chain.

12. The common light chain bispecific antibody according to claim 11, further comprising a hydrophobic (HIC) retention time of less than approximately 10 minutes.

13. A common light chain bispecific antibody according to any one of claims 11 to 12, wherein the terminal lysine residue is K447 according to EU numbering.

14. The common light chain bispecific antibody according to any one of claims 11 to 13, wherein the anti-CD19 heavy chain variable region contains serine at position 84 and / or leucine at position 108 according to Kabat numbering.

15. A bispecific antibody of a common light chain according to any one of claims 11 to 14, wherein the variable region of the common light chain contains histidine at position 32 according to Kabat numbering.

16. The common light chain bispecific antibody according to any one of claims 11 to 15, wherein the negatively charged amino acid is glutamic acid.

17. A common light chain bispecific antibody according to any one of claims 11 to 16, wherein the pI is 8.7 to 9.

18. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 10 or a common light chain bispecific antibody according to any one of claims 11 to 17, and a pharmaceutically acceptable excipient, diluent, or carrier.

19. A nucleic acid or a plurality of nucleic acids encoding a common light chain bispecific antibody according to any one of claims 1 to 10 or a common light chain bispecific antibody according to any one of claims 11 to 17.

20. A method for treating cancer in an individual requiring treatment, comprising the step of administering a common light chain bispecific antibody according to any one of claims 1 to 10 or a common light chain bispecific antibody according to any one of claims 11 to 17 to the individual, thereby treating the cancer in the individual requiring treatment.

21. The method according to claim 20, wherein the cancer or tumor is a solid tissue carcinoma.

22. The method according to claim 21, 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.

23. The method according to claim 22, 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.

24. The method according to claim 20, wherein the cancer or tumor is a blood cancer.

25. The method according to claim 24, wherein the hematological cancer is diffuse large B-cell lymphoma.

26. The method according to claim 24, wherein the blood cancer is myeloma.

27. The method according to claim 24, wherein the blood cancer is Burkitt lymphoma.

28. The method according to claim 24, wherein the blood cancer is B-cell lymphoma.

29. The method according to claim 28, wherein the B-cell lymphoma includes double-hit lymphoma, double-expressor lymphoma, or triple-hit lymphoma.

30. The method according to any one of claims 24 to 29, wherein the hematological cancer is recurrent or refractory to treatment.

31. The method according to any one of claims 20 to 30, wherein the cancer or tumor associated with CD19-positive, CD38-hyposuppressive B cells is a cancer or tumor comprising CD19-positive, CD38-hyposuppressive B cell infiltration.

32. The method according to claim 31, wherein the CD19-positive, CD38-hyposuppressive B cells express a B cell activation marker.

33. The method according to claim 32, wherein the B cell activation marker includes CD30.

34. The method according to any one of claims 20 to 33, wherein the cancer or tumor associated with CD19-positive, CD38-high B cells expresses PD-L1.

35. The method according to any one of claims 22 to 34, wherein the cancer or tumor associated with CD19-positive, CD38-high B cells is associated with CD20-low or CD20-negative B cells.

36. The method according to claim 35, wherein the CD38-enhanced B cells express at least about 30,000 CD38 proteins on their cell surface.

37. The method according to claim 35, wherein the CD38-enhanced B cells express at least about 35,000 CD38 proteins on their cell surface.

38. The method according to claim 35, wherein the CD38-enhanced B cells express at least about 40,000 CD38 proteins on their cell surface.

39. A method for lowering the electric point of an antibody during experiments, (a) A step of mutating glutamine to a negatively charged amino acid at position 1 of the heavy chain variable region by Kabat numbering, and (b) Step of removing lysine from the C-terminal position of the heavy chain constant region. Methods that include...

40. The method according to claim 39, wherein the negatively charged amino acid is glutamic acid.

41. The aforementioned antibody Anti-CD38 heavy chain variable region, Anti-CD19 heavy chain variable region, Variable region of the common light chain Includes, The aforementioned anti-CD38 heavy chain variable region is Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 7, Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 8, Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in Sequence ID No. 9 and Includes, The anti-human CD19 heavy chain variable region, Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 10, Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 11, Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 12 and Includes, The variable region of the common light chain is Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 13, Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence (AAS) described in Sequence ID No. 14, Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in Sequence ID No. 15 and The method according to any one of claims 39 to 40, including the method described in any one of claims 39 to 40.

42. A method for producing an antibody, comprising the steps of: collecting a bispecific antibody according to any one of claims 1 to 17 from the supernatant of a cell line containing a nucleic acid encoding the antibody; and subjecting the supernatant to one or more purification steps.

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