CD19 / CD38 multispecific antibodies

Abstract

Described herein are multispecific binding antibodies having a reduced isoelectric focus point.

Description

Cross Reference to Related Applications

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

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on March 18, 2024, is named 51527-716601_SEQ.xml and is 19,873 bytes in size. BACKGROUND

[0003] Immunosuppressive B cell populations (i.e., regulatory B cells or Bregs) are those populations of B cells that suppress an individual’s anti-tumor immune response. Therapeutic agents that effectively and specifically target immunosuppressive B cells can thus be used to prevent immunosuppression and / or to eliminate immunosuppression, thereby providing effective treatment for different types of cancer. SUMMARY SUMMARY

[0004] In addition to binding to the intended target molecule, antibody drugs must also meet certain criteria related to manufacturability, storage stability, and the absence of off-target binding. This set of characteristics is often referred to as “developability.” Unfortunately, there is no single parameter (e.g., a measurable property) that can decisively predict developability and developability challenges from a range of antibodies that enter the clinic.

[0005] Exemplified and provided herein are multispecific antibodies with modifications that enable higher developability and manufacturability. In one aspect, provided herein is a multispecific antibody comprising: (a) a CD38 binding moiety comprising: (i) a first polypeptide comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region, 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 set forth in SEQ ID NO: 3; and (b) a CD19 binding moiety comprising: (i) a third polypeptide comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain constant region, 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 set forth 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 comprises a human IgGl or human IgG4 constant region. In certain embodiments, the polypeptide comprising a light chain variable region further comprises a light chain constant region. In certain embodiments, the second polypeptide, the fourth polypeptide, or both the second and fourth polypeptides comprise the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the CD38 binding moiety comprises one or more amino acid substitutions that inhibit homodimerization of the CD38 binding moiety. In certain embodiments, the CD38 binding moiety comprises a T366W substitution according to EU numbering or a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the CD19 binding moiety comprises one or more amino acid substitutions that inhibit homodimerization of the CD19 binding moiety. In certain embodiments, the CD19 binding moiety comprises 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 set forth in SEQ ID NO: 4. In certain embodiments, the third polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, a pharmaceutical composition comprises the multispecific antibody, a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, a nucleic acid encodes the multispecific antibody. Also described herein is a method of treating a cancer in an individual in need thereof, comprising administering to the individual the multispecific antibody. In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the solid tissue cancer comprises a breast cancer, a prostate cancer, a pancreatic cancer, a lung cancer, a kidney cancer, a gastric cancer, an esophageal cancer, a skin cancer, a colorectal cancer, or a head and neck cancer.In certain embodiments, the breast cancer is triple negative breast cancer, the lung cancer is non-small cell lung cancer, the head and neck cancer is head and neck squamous cell carcinoma, the kidney cancer is renal cell carcinoma, the brain cancer is glioblastoma multiforme, or the skin cancer is melanoma. 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's lymphoma. In certain embodiments, the hematological cancer is an aggressive B-cell lymphoma. In certain embodiments, the aggressive B-cell lymphoma comprises double hit lymphoma, double expressor lymphoma, or triple hit lymphoma. In certain embodiments, the hematological cancer is relapsed or refractory. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high immunosuppressive B-cells is a cancer or tumor comprising CD19-positive, CD38-high B-cell infiltration. In certain embodiments, the CD19-positive, CD38-high immunosuppressive B-cells express a B-cell activation marker. In certain embodiments, the B-cell activation marker comprises CD30. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high B-cells expresses PD-L1. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high B-cells is associated with CD20-low or CD20-negative B-cells. In certain embodiments, the CD38-high B-cells express at least about 30,000 CD38 proteins on the cell surface. In certain embodiments, the CD38-high B-cells express at least about 35,000 CD38 proteins on the cell surface. In certain embodiments, the CD38-high B-cells express at least about 40,000 CD38 proteins on the cell surface.

[0006] In one aspect, provided herein is a common light chain bispecific antibody comprising: an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, wherein the anti-CD38 heavy chain variable region comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9, and a negatively charged amino acid at heavy chain variable region position 1 according to Kabat numbering; an anti-CD19 heavy chain variable region and an anti-CD19 heavy chain constant region, wherein the anti-human CD19 heavy chain variable region comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12, and a negatively charged amino acid at heavy chain variable region position 1 according to Kabat numbering; a common light chain variable region comprising: (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ASS), and (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15, wherein: the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain constant region each lack 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 also has a hydrophobicity (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 comprises a serine at position 84 and / or a leucine at position 108 according to Kabat numbering. In certain embodiments, the common light chain variable region comprises a histidine at position 32 according to Kabat numbering. In certain embodiments, the negatively charged amino acid is a glutamic acid. In certain embodiments, the pi is between 8 and 9. In certain embodiments, a pharmaceutical composition comprises the common light chain bispecific antibody, a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, a nucleic acid encodes the bispecific antibody. Also provided herein is a method of treating a cancer in an individual in need thereof, comprising administering to the individual the common light chain bispecific antibody.In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the solid tissue cancer comprises a breast cancer, a prostate cancer, a pancreatic cancer, a lung cancer, a kidney cancer, a gastric cancer, an esophageal cancer, a skin cancer, a colorectal cancer, or a head and neck cancer. In certain embodiments, the breast cancer is a triple negative breast cancer, the lung cancer is a non-small cell lung cancer, the head and neck cancer is a head and neck squamous cell carcinoma, the kidney cancer is a renal cell carcinoma, the brain cancer is a glioblastoma multiforme, or the skin cancer is a melanoma. In certain embodiments, the cancer or tumor is a hematological cancer. In certain embodiments, the hematological cancer is a diffuse large B-cell lymphoma. In certain embodiments, the hematological cancer is a myeloma. In certain embodiments, the hematological cancer is a Burkitt’s lymphoma. In certain embodiments, the hematological cancer is an aggressive B-cell lymphoma. In certain embodiments, the aggressive B-cell lymphoma comprises a double hit lymphoma, a double expressor lymphoma, or a triple hit lymphoma. In certain embodiments, the hematological cancer is relapsed or refractory. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high immunosuppressive B-cells is a cancer or tumor comprising CD19-positive, CD38-high B-cell infiltration. In certain embodiments, the CD19-positive, CD38-high immunosuppressive B-cells express a B-cell activation marker. In certain embodiments, the B-cell activation marker comprises CD30. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high B-cells expresses PD-L1. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high B-cells is associated with CD20-low or CD20-negative B-cells. In certain embodiments, the CD38-high B-cells express at least about 30,000 CD38 proteins on the cell surface. In certain embodiments, the CD38-high B-cells express at least about 35,000 CD38 proteins on the cell surface. In certain embodiments, the CD38-high B-cells express at least about 40,000 CD38 proteins on the cell surface.

[0007] In another aspect, provided herein is a method of reducing the experimental isoelectric point of an antibody, the method comprising: (a) mutating a glutamine at heavy chain variable region position 1 according to Kabat numbering to a negatively charged amino acid; and (b) removing a lysine at the C-terminal position of the heavy chain constant region. In certain embodiments, the negatively charged amino acid is a glutamic acid. In certain embodiments, the antibody is a common light chain bispecific antibody comprising: an anti-CD38 heavy chain variable region, wherein the anti-CD38 heavy chain variable region comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, and (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-CD19 heavy chain variable region, wherein the anti-human CD19 heavy chain variable region comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, and (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12; a common light chain variable region comprising: (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ASS), and (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0008] Also provided herein is a method of making an antibody, the method comprising harvesting a bispecific antibody of the disclosure from a supernatant of a cell line, the supernatant comprising nucleic acid encoding the antibody, and subjecting the supernatant to one or more purification steps. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1A , Figure 1B and Figure 1C shows the polyreactivity profile of BSM-001F (BSM-001.1).

[0010] Figure 2 shows the hydrophobic interaction chromatography (HIC) profile of BSM-001F (BSM-001.1).

[0011] Figure 3 shows the B cell binding data of BSM-001F (BSM-001.1).

[0012] Figure 4 ADCC cell killing data showing BSM-001F (BSM-001.1).

[0013] Figure 5 Apoptotic cell killing data showing BSM-001F (BSM-001.1). DETAILED DESCRIPTION

[0014] As used herein, the term "immunosuppression" or "immunodepression" or "negative immune regulation" or "modulation" in reference to a particular cell population refers to a process or cell responsible for a decrease or inhibition of immune system function. Immunosuppression generally denotes a state in which immune system function is reduced or absent with respect to one or more functions such as cellular immune, antibody-based immune, or innate immune function. In certain instances, immunosuppression generally denotes a state in which immune system function is reduced or absent with respect to a tumor or within, surrounding, or adjacent to a tumor microenvironment. The entire immune response can be inhibited, the immune response within a local or specific region can be reduced, or a specific population of immune active lymphocytes can be selectively affected. Antigen-specific immunosuppression can be the result of a lack or inhibition of a specific antigen-specific cell population or the result of an enhanced regulation of the immune response by antigen-specific suppressor cells. Reference to immunosuppressive B cells refers to B cells or B cell populations that exert negative regulation on the immune response and can be identified by specific surface markers associated with such populations, such as CD38. In certain instances, immunosuppression can be identified by the presence or release of IL-10, IL-35, TGF-β, or a combination thereof. In certain instances, immunosuppression can be identified by the presence or release of IL-10, IL-35, TGF-β, or a combination thereof by B cells.

[0015] As used herein, the term "cancer" can refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer can also include solid tumors. Cancer can refer to diseases of the blood, bone, organs, skin tissue, and vascular system, including but not limited to diseases of the bladder, blood, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lung, lymph node, mouth, neck, ovary, pancreas, prostate, rectum, skin, stomach, testicle, throat, and uterus. Specific cancers include, but are not limited to, gastrointestinal tumors (e.g., gastrointestinal stromal tumor (GIST)), follicular lymphoma, mantle cell lymphoma / leukemia, Diffuse B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, and Burkitt's lymphoma, mature T-cell and natural killer cell (NK) neoplasms (pre-lymphocytic leukemia, T-cell large lymphocytic leukemia, invasive NK cell leukemia, adult T-cell leukemia / lymphoma, Extranodal NK / T-cell lymphoma, enteropathic T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sézary syndrome), primary Skin degenerative large cell lymphoma, lymphomatoid papulosis, anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and anaplastic large cell lymphoma.Papulosis, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma, degenerative large cell lymphoma, Hodgkin's lymphoma (nodular sclerosis, mixed cell type, lymphocyte-rich type, lymphocyte-depleted or unreduced type, nodular lymphocyte type), myeloma (multiple myeloma, inert myeloma, smoldering myeloma), chronic myeloproliferative diseases, myelodysplasia / myeloproliferative disorders. Diseases, myelodysplastic syndromes, lymphoproliferative disorders associated with immunodeficiency, histiocytic and dendritic cell tumors, hypercytosis, chondrosarcoma, Ewing sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent and non-hormone-dependent), gynecological cancers (cervix, endometrium, fallopian tubes, gestational trophoblastic disease, ovary, peritoneum, uterus, vagina, and vulva), basal cell carcinoma.carcinoma, BCC), squamous cell carcinoma (SCC), malignant melanoma, protuberous cutaneous fibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, hair cell astrocytoma, embryonic hair growth neuroepithelial neoplasia, oligodendroglioma, Ependymoma, glioblastoma multiforme, mixed glioma, oligodendrocyte astrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, embryonal tissue tumor, teratoma, Malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastric-intestinal-pancreatic or gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, knot Rectal cancer, invasive neuroendocrine tumor, leiomyosarcoma, mucinousadenocarcinoma), signet ring cell adenocarcinoma, hepatocellular carcinoma, hepatobiliary liver cancer, hepatic blastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung cancer (NSCLC) (squamous cell lung cancer, adenocarcinoma, large cell lung cancer), small cell lung cancer, thyroid cancer, prostate cancer (hormone refractory, non-androgen dependent, androgen dependent, hormone insensitive), renal cell carcinoma, and soft tissue sarcoma (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraskeletal osteosarcoma).

[0016] The term“CD19” or“cluster of differentiation 19” (also known as B4, T cell surface antigen Leu-12, and CVID3) refers to a B cell lineage surface biomarker or transmembrane protein encoded by the gene CD19 in humans. CD19 can serve as a co-receptor for the B cell antigen receptor complex (BCR) on B lymphocytes, which decreases the activation threshold of downstream signaling pathways and triggers a B cell’s response to an antigen. Structurally, the CD19 amino acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, over a sequence length of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids, or over the full length of the polypeptide, to the amino acid sequence of, e.g., GenBank Accession No. NM_001178098.2 → NP_001171569.1 or NM_001770.6 → NP_001761.3. Structurally, the CD19 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, over a sequence length of at least 300, 500, 750, 1000, 1250, 1500 nucleic acids, or over the full length of the polynucleotide, to the nucleic acid sequence of, e.g., GenBank Accession No. NG_007275.1 or NCBI Gene ID 930. Sequence alignments can be performed using any alignment algorithm known in the art, e.g., BLAST, ALIGN, set to default settings.

[0017] The term“CD38” or“cluster of differentiation 38” (also known as ADPRC1) refers to a B-cell surface biomarker or transmembrane protein encoded by the gene CD38 in humans. CD38 can play a role in B-cell signaling that leads to cell activation and proliferation. Structurally, the CD38 amino acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, over a sequence length of at least 50, 100, 150, 200, 250 amino acids, or over the full length of the polypeptide, to the amino acid sequence of, e.g., GenBank Accession No. NM_001775.4→ NP_001766.2. A second isoform of CD38, which has a premature stop codon, can be expressed at low levels in some cells. Structurally, the CD19 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, over a sequence length of at least 300, 500, 750 nucleic acids, or over the full length of the polynucleotide, to the nucleic acid sequence of, e.g., GenBank Accession No. NC_000004.12 or NCBI Gene ID 952. Sequence alignments can be performed using any alignment algorithm known in the art, e.g., BLAST, ALIGN, set to default settings.

[0018] The term“CD20” or“cluster of differentiation 20” (also known as B-lymphocyte surface antigen Bl) 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 about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, over a sequence length of at least 50, 100, 150, 200, 250 amino acids, or over the full length of the polypeptide, to the amino acid sequence of, e.g., Uniprot entry P11836.

[0019] “Binding,” as described herein in relation to binding molecules such as antibodies and bispecific antibodies, refers to the specific interaction of a target antigen with one or more amino acid residues of the variable region of a complementarity determining region. Such specific binding will typically result in a dissociation constant of less than about 1 x 10 -6 M, such affinities can be determined by one skilled in the art using techniques known in the art, such as by surface plasmon resonance.

[0020] The term "antibody" herein is used in the broadest sense and includes multivalent or bispecific antibodies and monoclonal antibodies, including intact antibodies and functional (antigen binding) antibody fragments, including the following fragments: antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments (including single-chain variable fragments (sFv or scFv)), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact antibodies or full-length antibodies, including antibodies of any class or subclass, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgGl constant region. The antibody can comprise a human IgG4 constant region.

[0021] Among the provided antibodies are multispecific or multivalent antibodies (e.g., bispecific antibodies and polyreactive antibodies) and antibody fragments thereof. Antibodies include antibody conjugates and molecules comprising antibodies, such as chimeric molecules. Thus, antibodies include, but are not limited to, full-length antibodies and natural antibodies and fragments and portions thereof that retain their binding specificity, such as any specific binding portion thereof, including having any number of immunoglobulin classes and / or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and 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). A monoclonal antibody is typically one within a composition of substantially homogenous antibodies; thus, any individual antibody contained within a monoclonal antibody composition is identical, except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies can comprise a human IgGl constant region or a human IgG4 constant region.

[0022] The terms "complementarity determining region" and "CDR" include amino acid positions / residues (contiguous or non-contiguous) that confer antigen specificity and / or binding affinity. Typically, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework regions" and "FRs" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in: 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. 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 ImmunolJanuary 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol , June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Whitelegg NR and Rees AR , “WAM: an improved algorithm for modeling antibodies the WEB,” Protein Eng. December 2000;13(12):819-24 (“AbM” numbering scheme). In some embodiments, the CDR of the antibody described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0023] 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 structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody region sequences, with insertions (adapted by the insertion letter, e.g., "30a") and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("insertion deletions") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways.

[0024] The terms "variable region" or "variable domain" refer to the structural domains of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V L They typically have similar structures, where each domain contains four conserved frame regions (FRs) and three CDRs (see, for example, Kindt et al., Kuby). Immunology , 6th edition., WH Freeman and Co., page 91 (2007). Single V H or V L The structural domain is sufficient to confer antigen-binding specificity. Furthermore, V-terminal molecules derived from antibodies that bind to specific antigens can be used. H or V LDomain-separation antibodies that bind the antigen are screened separately to identify complementary V L or V H libraries (see, e.g., Portolano et al, J. Immunol . 150:880-887 (1993); Clarkson et al, Nature 352:624-628 (1991)).

[0025] The term“constant region” can refer to a light chain or heavy chain constant region. There are two major isotypes of light chain constant regions: kappa and lambda (sometimes abbreviated CK or CL, respectively). Heavy chain constant regions can include any of the following 5 isotypes: IgA, IgD, IgG, IgE, or IgM. The IgG isotype further includes IgGl, IgG2, IgG3, IgG4 subclasses. The heavy chain constant region includes CHI, hinge, CH2, and / or CH3 domains. Residues of light and heavy chain constant regions can be numbered according to the EU numbering scheme (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)) or the Kabat numbering scheme (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Ed. - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662, 680, 689 (1991)). An“Fc region” as described herein generally refers to the CH2 and CH3 domains of a heavy chain constant region.

[0026] Among the provided antibodies are antibody fragments. An“antibody fragment” can refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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 particular embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as a scFv. Antibody fragments can be made by various techniques including but not limited to proteolytic digestion of an intact antibody and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising a non-naturally occurring arrangement, such as a fragment having two or more antibody regions or chains connected by a synthetic linker (e.g., a polypeptide linker), and / or a fragment not produced by enzymatic digestion of a naturally occurring intact antibody.

[0027] In the present text, a molecule, peptide, polypeptide, antibody, or antibody fragment can be referred to as "bispecific" or "dual specific," including grammatical equivalents. A bispecific molecule has the ability to specifically bind at least two structurally distinct targets. Specific binding can be the result of two different binding moieties that are structurally distinct at the molecular level, including but not limited to different non-identical amino acid sequences; or a single binding moiety that is capable of specifically binding two structurally distinct targets with high affinity (e.g., a KD of less than about 1 x 10 -6 A molecule, peptide, polypeptide, antibody, or antibody fragment that is referred to as "multispecific" refers to a molecule that has the ability to specifically bind at least two or more structurally distinct targets. A "bispecific antibody," including grammatical equivalents, refers to a bispecific molecule that retains at least one antibody fragment (e.g., variable region, heavy chain, or light chain) or one or more complementarity determining regions from an antibody molecule that is capable of specifically binding a target. A "multispecific antibody," including grammatical equivalents, refers to a multispecific molecule that retains at least one antibody fragment (e.g., variable region, heavy chain, or light chain) or complementarity determining region from an antibody molecule that is capable of specifically binding a target.

[0028] A "linker" herein is also referred to as a "linker sequence," a "spacer sequence," a "tethering sequence," or a grammatical equivalent thereof. A "linker" as referred to herein links two different molecules, which themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides. For example, two different binding moieties or heavy / light chain pairs. A variety of strategies can be used to covalently link molecules together. These strategies include, but are not limited to, polypeptide linkages between the N- and C-termini of proteins or protein domains, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide linkage generated by recombinant techniques or peptide synthesis. The linker peptide can include primarily the following amino acid residues: Gly, Ser, Ala, or Thr. The length of the linker peptide should be sufficient to allow the two molecules to assume the correct conformation relative to each other such that they retain the desired activity to link the two molecules. In one embodiment, the length of the linker is about 1 to 50 amino acids or about 1 to 30 amino acids in length. In one embodiment, a linker of 1 to 20 amino acids in length can be used. Useful linkers include glycine-serine polymers including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Exemplary linkers for linking antibody fragments or single chain variable fragments can include AAEPKSS, AAEPKSSDKTHTCPPCP (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), or GGGGDKTHTCPPCP (SEQ ID NO: 21). Alternatively, a variety of non-proteinaceous polymers can be used as linkers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyl or copolymers of polyethylene glycol and polypropylene glycol.

[0029] A "fragment-based" bispecific antibody of the present disclosure or a bispecific antibody comprising a "single chain variable fragment" or "scFv" can refer to a single chain antibody or fragment thereof comprising two binding moieties and a linker connecting the two binding moieties. The linker can be a polypeptide linker or other linker with suitable flexibility so as not to inhibit binding of either targeting moiety. Fragment-based bispecific antibody formats include tandem V HH Antibodies, tandem scFv, scFv-Fab, F(ab)2, dual affinity retargeting antibodies (DARTs). Such fragment-based antibodies can be further manipulated to comprise additional binding moieties specific for a given target (e.g., A2:B1, A1:B2, or A2:B2) or Fc region fragments that improve pharmacokinetics or promote ADCC, ADCP, or CDC.

[0030] A "binding moiety" refers to a portion of a molecule, peptide, polypeptide, antibody, or antibody fragment that mediates specific binding to the target or antigen or epitope. By way of example, a binding moiety of an antibody can comprise a heavy / light chain variable region pair or one or more complementarity determining regions (CDRs).

[0031] A "target" as referred to herein refers to a portion of a molecule that participates with a binding moiety of a molecule, peptide, polypeptide, antibody, or antibody fragment. A target can comprise an amino acid sequence and / or a carbohydrate, lipid, or other chemical entity. An "antigen" is a target that comprises a portion capable of being bound by an adaptive immune molecule, such as an antibody or antibody fragment, B cell receptor, or T cell receptor.

[0032] In some embodiments, the dissociation constant (KD) of a bispecific antibody pair provided herein for an antibody target is about 10 μΜ, 1 μΜ, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 D M or less, e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). The antibody target can be a CD19 target, a CD38 target, or a target comprising both CD19 and CD38. K D The KD can be measured by any suitable assay. In certain embodiments, the KD can be measured using a surface plasmon resonance assay (e.g., using a BIACORE®-2000 or BIACORE®-3000 or Octet).

[0033] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally can include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that typically has been humanized, i.e., changed to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0034] Among the provided antibodies are human antibodies. A "human antibody" is one which possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including a human antibody phage library. The term "human antibody" does not include antibodies that contain non-human antigen binding sites, such as those in which all or a substantial portion of the CDRs are non-human. Human antibodies can be produced by a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to an antigenic challenge. Such an animal typically contains all or a portion of the human immunoglobulin loci, which replace the

[0035] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" means a cell-mediated reaction in which nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC can be associated with binding to FcγRIIIa, where increased binding to FcγRIIIa results in an increase in ADCC activity. As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis can mean a cell-mediated reaction in which nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0036] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains as well as other peptides (e.g., linkers and binding peptides), can comprise amino acid residues, including natural and / or non-natural amino acid residues. The terms also encompass post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, a polypeptide can contain modifications relative to the original or native sequence, so long as the protein maintains a desired activity. These modifications can be intentional, as by site-directed mutagenesis, or they can be accidental, such as by mutation of the host that produces the protein or by errors due to PCR amplification.

[0037] The percent (%) sequence identity with reference to a polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.) or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not variable. In the case of amino acid sequence comparisons using ALIGN-2, the amino acid sequence identity of a given amino acid sequence A to, with, or versus a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A having or comprising a certain amino acid sequence identity to, with, or versus a given amino acid sequence B) is calculated as follows: 100 X fraction X / Y, where X is the number of amino acid residues in the alignment of sequences A and B scored by the sequence alignment program ALIGN-2 as identical matches, and where Y is the total number of amino acid residues in B. It will be appreciated that, where the length of amino acid sequence A does not equal the length of amino acid sequence B, the amino acid sequence identity of A to B will not equal the amino acid sequence identity of B to A. All amino acid sequence identity percentage values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph, unless otherwise specifically indicated.

[0038] Amino acid sequence variants of the antibodies provided herein can be considered and envisioned. Variants will typically differ in one or more substitutions, deletions, additions, and / or insertions from the polypeptides specifically disclosed herein. Such variants can be naturally occurring or can be synthetically generated, e.g., by modifying one or more of the above polypeptide sequences of the application and evaluating one or more biological activities of the polypeptides as described herein and / or using any of a variety of known techniques. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody, amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion(s), insertion(s), and substitution(s) can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics {e.g., antigen binding). Antibody variants having one or more amino acid substitutions can be provided. Sites of interest for mutagenesis by substitution include the CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest and the products screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0039] The present disclosure also provides for "immunoconjugates" or "antibody conjugates" or "antibody-drug conjugates," which refer to conjugates of an antibody with one or more heterologous molecules. For example, an immunoconjugate can include a conjugate of an antibody with one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, protein domains, toxins such as enzymatically active toxins of

[0040] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are a class of polynucleotides comprising two or more nucleotide bases. In certain embodiments, nucleic acids are components of vectors that can be used to transfer polypeptide-encoding polynucleotides into cells. As used herein, the term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrating vector, or "integrating vector," which can integrate into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, i.e., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In an expression vector, regulatory elements such as promoters, enhancers, polyadenylation signals used in controlling transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. Viral-derived vectors can be employed, such as lentivirus, retrovirus, adenovirus, adeno-associated virus, and the like. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors can comprise sequences that direct site-specific integration into a determined location in the genome or into a set of restriction sites (e.g., AttP-AttB recombination). Additionally, vectors can comprise sequences derived from transposable elements.

[0041] As used herein, the term "homologous," "homology," or "percent homology" when used in reference to an amino acid sequence or nucleic acid sequence relative to a reference sequence can be determined using the formula 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). This formula is incorporated into the basic local alignment search tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). The percent homology of sequences can be determined using the most recent version of BLAST as of the filing date of this application.

[0042] The nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform suitable cells, or otherwise genetically modify suitable cells to target said nucleic acids, thereby achieving the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures are known in the art. See, for example, Li et al., “Cell culture processes for monoclonal antibody production.” Mabs September-October 2010; 2(5): 466-477. In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the mammalian cells are cell lines that can be used to produce antibodies, such as Chinese hamster ovary cells (CHO) cells, NSO mouse myeloma cells, or PER.C6® cells. In some embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of the cell that can be used to produce the antibody. In some embodiments, a method for preparing an antibody is described herein, the method comprising culturing cells containing nucleic acids encoding the antibody under in vitro conditions sufficient to allow the production and secretion of said antibody.

[0043] As used herein, the terms "individual," "patient," or "object" refer to an individual diagnosed with, suspected of having, or at risk of having at least one disease that can be treated with the described compositions and methods. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0044] As used herein, the term "about" to modify a specific number means the number plus or minus 10%. The term "about" to modify a range means the range minus 10% of its lowest value and plus 10% of its highest value.

[0045] As used herein, the term "treatment" or "treating" is used in reference to a pharmaceutical or other intervention regimen used for obtaining beneficial or desired results for a subject. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or its symptoms. In addition, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, notwithstanding the fact that the subject can still be afflicted with the underlying disorder. Prophylactic benefit includes delaying, preventing or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease or condition, or subject reporting one or more of the physiological symptoms associated with the disease or condition, can be treated, even though the subject can not yet be diagnosed with the disease or condition. The skilled artisan will recognize that not all subjects treated with a therapeutic will respond to treatment or have equivalent responses. Such individuals are considered to be treated.

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] Also described herein are methods of making the antibodies described herein. Such methods include incubating a cell or cell line comprising a nucleic acid encoding the antibody in cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further harvesting the antibody from the cell culture medium. Harvesting can also include one or more purification steps to remove live cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and media components. In certain embodiments, one or more additional purification steps include centrifugation, ultracentrifugation, protein A purification, protein G purification, protein A / G purification, or protein L purification and / or ion exchange chromatography.

[0048] As used herein, "treat," "treatment," or "treating" refers to the intentional intervention of a physiological condition, for example, to reduce the severity of a disease or condition; to reduce the duration of a condition process; to improve or eliminate one or more symptoms associated with a disease or condition; or to provide a beneficial effect to a subject with a disease or condition. Treatment does not require that the underlying disease or condition be cured.

[0049] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes the regression of a disease (as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of damage or disability due to disease affliction). The ability of a therapeutic agent to promote the regression of a disease can be assessed using a variety of methods known to the skilled practitioner, for example, in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by determining the activity of the agent in in vitro assays.

[0050] As used herein, "pharmaceutically acceptable" in relation to a "carrier," "excipient," or "diluent" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., the antibody) can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0051] The antibodies, bispecific antibodies, and multispecific antibodies described herein can include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt (see, e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19) that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids (such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like), as well as those derived from nontoxic organic acids (such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Base addition salts include those derived from alkaline, earth- alkaline metals (such as sodium, potassium, magnesium, calcium, and the like), as well as from nontoxic organic amines (such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like).

[0052] In one aspect, described herein is a multispecific antibody comprising: (a) a CD38 binding moiety comprising: (i) a first polypeptide comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region, 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 set forth in SEQ ID NO: 3; and (b) a CD19 binding moiety comprising: (i) a third polypeptide comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain constant region, 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 set forth in SEQ ID NO: 3. In certain embodiments, the heavy chain constant region of the first polypeptide, the third polypeptide, or both the first polypeptide and the third polypeptide comprises a human IgGl or human IgG4 constant region. In certain embodiments, the polypeptide comprising a light chain variable region further comprises a light chain constant region. In certain embodiments, the second polypeptide, the fourth polypeptide, or both the second polypeptide and the fourth polypeptide comprise the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the CD38 binding moiety comprises one or more amino acid substitutions that inhibit homodimerization of the CD38 binding moiety. In certain embodiments, the CD38 binding moiety comprises a T366W substitution according to EU numbering or a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the CD19 binding moiety comprises one or more amino acid substitutions that inhibit homodimerization of the CD19 binding moiety. In certain embodiments, the CD19 binding moiety comprises 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 set forth in SEQ ID NO: 4. In certain embodiments, the third polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the pharmaceutical composition comprises the multispecific antibody, a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, the nucleic acid encodes the multispecific antibody.

[0053] The bispecific antibodies according to the present disclosure comprise intact antibody molecules or substantially fully intact antibody molecules and can be asymmetric or symmetric.

[0054] Asymmetric bispecific antibodies typically comprise a heavy chain / light chain (HC / LC) pair from an antibody specific for target A and a HC / LC pair from an antibody specific for target B, resulting in a hetero-bifunctional antibody. Hetero-bifunctional antibodies such as these suffer from problems of non-productive formation of molecules upon production. HC / LC-A:HC / LC-B is desirable, but from all possible possible combinations, it is typically thermodynamically or statistically unfavorable. Multiple solutions have been introduced to circumvent this problem. In some cases, the HC / LC pair from an antibody specific for A and the HC / LC pair from an antibody specific for B also comprise 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 engineering structural features such as a "knob" into the FC region of HC-A and a "hole" into HC-B or vice versa, facilitating heterodimer formation between HC-A and HC-B. Another solution to facilitate HC-A:HC-B heterodimers is to engineer amino acid residues in the FC portion of HC-A and HC-B to comprise charge pairs that favor electrostatic interactions between HC-B and HC-A. Another solution to the chain association problem is to replace the variable region of one of the HC / LC pairs with a single chain binding molecule (e.g., a V HH or scFv). One half of the molecule comprises a classic HC / LC pair, while the other half comprises an HC constant region fused or otherwise linked to a single chain binding molecule. Additional modifications can be made to facilitate correct HC / LC pairing, and the modifications include engineered mutations to the HC and LC of A or B to favor formation of the correct HC / LC pair; CrossMab technology, which requires swapping of the corresponding constant regions of the HC / LC pairs. Symmetric bispecific antibodies circumvent the chain association problem by not relying on formation of a hetero-bifunctional molecule. Examples of such include: dual variable domain molecules, which comprise stacked variable regions of different specificity; IgG-scFv molecules, which comprise an scFv of different specificity fused to the C-terminus of a classic antibody molecule heavy chain; (scFV)4-FC, which comprises two scFv linked by an Ig Fc region (Fc dimerization results in a bispecific tetravalent molecule); DART-Fc and two-in-one, among others.

[0055] Structures of multispecific or bispecific antibodies can be envisioned and designed to alter the functional or binding properties of the complexed binding molecule or bispecific antibody (see, e.g., “Bispecific antibodies: a mechanistic review of the pipeline.” Nat Rev Drug Discovery. 2019 Aug; 18(8): 585-608) (see, e.g., “The making of bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 112-212). For example, a bispecific antibody can be selected from one of the following formats: common light chain bispecific IgG, Fab-Fc:scFv-Fc bispecific IgG, Fab-Fc-Fab:Fc bispecific IgG, Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG, Fab-Fc-scFv:Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc bispecific IgG, scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG, Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and Fab-Fc-scFv:Fab-Fc bispecific IgG.

[0056] Exemplary knobs-in-holes mutations for the antibodies described herein can comprise T366W (EU numbering) in one heavy chain and T366S / L368A / Y407V (EU numbering) in the second heavy chain. Exemplary mutations that facilitate coupling of a first heavy chain molecule and a second heavy chain molecule are disclosed, e.g., in WO2009089004, US 8,642,745, US PG-PUB: US20140322756, and “The making of bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 112-212.

[0057] In some embodiments, provided herein are common light chain bispecific antibodies comprising: (1) an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, wherein the anti-CD38 heavy chain variable region comprises: (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, (iii) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9, and (iv) a Q1E heavy chain variable region mutation according to Kabat numbering, (2) an anti-CD19 heavy chain variable region and an anti-CD19 heavy chain constant region, wherein the anti-human CD19 heavy chain variable region comprises: (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, (iii) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12, and (iv) a Q1E heavy chain variable region mutation according to Kabat numbering, (3) a common light chain variable region comprising: (i) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, (ii) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (AAS), and (iii) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15, wherein: the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain constant region each lack a C-terminal lysine residue (e.g., K478 according to Kabat numbering); and the common light chain bispecific antibody has an experimental isoelectric point (pi) of less than about 9. In certain embodiments, the anti-CD38 heavy chain variable region can comprise a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8 or 16-18.

[0058] In certain embodiments, the common light chain bispecific antibody also has a hydrophobic (HIC) retention time of less than about 10 minutes. In certain embodiments, the anti-CD19 heavy chain variable region comprises a serine at position 84 and / or a leucine at position 108 according to Kabat numbering. In certain embodiments, the common light chain variable region comprises a histidine at position 32 according to Kabat numbering. In certain embodiments, the pi is less than 9. In certain embodiments, the pi is between 8 and 9. In certain embodiments, the pi is between 8.5 and 9.0. In certain embodiments, the pi is between 8.7 and 9.0. In certain embodiments, the pi is between 8.8 and 9.0. In certain embodiments, the pi is 8.9. Methods of treatment

[0059] In certain embodiments, disclosed herein are antibodies useful in the treatment of cancer or a tumor. Treatment refers to a method that seeks to improve or alleviate the condition being treated. In the case of cancer, treatment includes, but is not limited to, reducing tumor volume, reducing growth of tumor volume, increasing progression-free survival or overall expected lifespan. In certain embodiments, treatment will achieve remission of the cancer being treated. In certain embodiments, treatment encompasses use as a prophylactic or maintenance dose aimed at preventing recurrence or progression of a previously treated cancer or tumor. It will be understood by one skilled in the art that not all individuals will respond equally or at all to an administered treatment, but that these individuals are considered to have been treated.

[0060] Also provided herein is a method of treating a cancer in an individual in need thereof, comprising administering to the individual the bispecific antibody. In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the solid tissue cancer comprises a breast cancer, a prostate cancer, a pancreatic cancer, a lung cancer, a kidney cancer, a gastric cancer, an esophageal cancer, a skin cancer, a colorectal cancer, or a head and neck cancer. In certain embodiments, the breast cancer is a triple negative breast cancer, the lung cancer is a non-small cell lung cancer, the head and neck cancer is a head and neck squamous cell carcinoma, the kidney cancer is a renal cell carcinoma, the brain cancer is a glioblastoma multiforme, or the skin cancer is a melanoma. In certain embodiments, the cancer or tumor is a hematological cancer. In certain embodiments, the hematological cancer is a diffuse large B-cell lymphoma. In certain embodiments, the hematological cancer is a myeloma. In certain embodiments, the hematological cancer is a Burkitt’s lymphoma. In certain embodiments, the hematological cancer is an aggressive B-cell lymphoma. In certain embodiments, the aggressive B-cell lymphoma comprises a double hit lymphoma (e.g., defined by mutations and / or rearrangements within the MYC gene and the BCL2 gene or the BCL6 gene), a double expressor lymphoma (e.g., defined by co-expression of c-MYC and BCL-2), or a triple hit lymphoma (e.g., defined as having morphologic, biological, and cytogenetic features similar to diffuse large B-cell lymphoma and Burkitt’s lymphoma, but with rearrangements of three instead of two genes: the c-MYC, BCL-2, and BCL-6 genes). In certain embodiments, the hematological cancer is relapsed (e.g., the cancer has relapsed after a remission period) or refractory (e.g., the cancer has stopped responding to prior treatment with a different therapeutic agent / therapy). In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high immunosuppressive B-cells is a cancer or tumor comprising CD19-positive, CD38-high B-cell infiltration. In certain embodiments, the CD19-positive, CD38-high immunosuppressive B-cells express a B-cell activation marker. In certain embodiments, the B-cell activation marker comprises CD30. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high B-cells expresses PD-L1. In certain embodiments, the cancer or tumor associated with CD19-positive, CD38-high B-cells is associated with CD20-low B-cells or CD20-negative B-cells. In certain embodiments, the CD38-high B-cells express at least about 30,000 CD38 proteins on the cell surface. In certain embodiments, the CD38-high B-cells express at least about 35,000 CD38 proteins on the cell surface. In certain embodiments, the CD38-high B-cells express at least about 40,000 CD38 proteins on the cell surface.In certain embodiments, the multispecific antibody comprises: (a) a CD38 binding moiety comprising: (i) a first polypeptide comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region, 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 set forth in SEQ ID NO: 3; and (b) a CD19 binding moiety comprising: (i) a third polypeptide comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain constant region, 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 set forth in SEQ ID NO: 3.

[0061] In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer or tumor. In certain embodiments, the cancer or tumor comprises a tumor of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicle, and liver. In certain embodiments, the tumor that can be treated by the antibodies of the application comprises an adenoma, adenocarcinoma, angiosarcoma, astrocytoma, carcinoma, germ cell tumor, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratocarcinoma. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, Bartholin gland carcinoma, basal cell carcinoma, bronchiolar adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, focal nodular hyperplasia, gastrinoma, germ line tumor, glioblastoma, glucagonoma, hemangio-blastoma, hemangioendothelioma, hemangioma, hepatoadenoma, hepatoadenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasm, intraepithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, neurinoma, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian carcinoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate carcinoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar carcinoma, vasoactive intestinal peptide tumor (VIPpoma), and Wilms' tumor.In certain embodiments, the tumor / cancer to be treated with one or more antibodies of the application includes brain cancer, head and neck cancer, colorectal cancer, acute myeloid leukemia, precursor 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 adenocarcinoma, and breast cancer (preferably breast ductal carcinoma and / or breast carcinoma). In certain embodiments, the cancer treated with an antibody of the disclosure includes glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of the disclosure includes pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of the disclosure includes ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of the disclosure includes lung cancer. In certain embodiments, the cancer treated with one or more antibodies of the disclosure includes prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of the disclosure includes colon cancer. In certain embodiments, the cancer treated is includes glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is otherwise refractory to treatment. In certain embodiments, the cancer treated is recurrent.

[0062] In certain embodiments, the antibody can be administered to a subject in need thereof by any route suitable to administer a pharmaceutical composition containing the antibody, e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally, etc. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered according to a suitable dosage regimen, e.g., once a week, twice a week, once a month, twice a month, once every two weeks, once every three weeks, or once a month, etc. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 20 mg / kg. A therapeutically effective amount includes an amount sufficient to improve one or more symptoms associated with the disease or illness being treated. Methods of making antibodies with reduced pi

[0063] In some embodiments, provided herein are methods of reducing the experimental isoelectric point of an antibody, the method comprising: (a) mutating a glutamine at heavy chain variable region position 1 according to Kabat numbering to a negatively charged amino acid; and (b) removing a lysine at the C-terminal position of the heavy chain constant region. In some embodiments, the negatively charged amino acid is a glutamic acid. In some embodiments, the negatively charged amino acid is an aspartic acid. In some embodiments, the lysine at the C-terminal position of the heavy chain constant region is K447 according to EU numbering.

[0064] In some embodiments, the antibody is a common light chain bispecific antibody comprising: (1) an anti-CD38 heavy chain variable region, wherein the anti-CD38 heavy chain variable region comprises: (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, and (iii) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9, (2) an anti-CD19 heavy chain variable region, wherein the anti-human CD19 heavy chain variable region comprises: (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, and (iii) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12, and (3) a common light chain variable region comprising: (i) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, (ii) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ASS), and (iii) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0065] In certain embodiments, the common light chain bispecific antibody also has a hydrophobic (HIC) retention time of less than about 10 minutes. In certain embodiments, the anti-CD19 heavy chain variable region comprises a serine at position 84 and / or a leucine at position 108 according to Kabat numbering. In certain embodiments, the common light chain variable region comprises a histidine at position 32 according to Kabat numbering. In certain embodiments, the pi is less than 9. In certain embodiments, the pi is between 8 and 9. In certain embodiments, the pi is between 8.5 and 9.0. In certain embodiments, the pi is between 8.7 and 9.0. In certain embodiments, the pi is between 8.8 and 9.0. In certain embodiments, the pi is 8.9. Pharmaceutically acceptable excipients, carriers and diluents

[0066] In certain embodiments, the multispecific antibodies of the present disclosure are included in a pharmaceutical composition containing one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents can be included to increase the shelf-life, stability, or administrability of the antibodies. Such compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibodies of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffer, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylamino methane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / di- / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine; or a chelator, such as EDTA or EGTA.

[0067] In certain embodiments, the antibodies of the present disclosure can be shipped / stored, lyophilized, and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation comprises a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation can be housed in a vial composed of glass or other suitable non-reactive material. When formulated, whether or not reconstitution is performed, the antibody can be buffered at a certain pH, typically less than 7.0. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

[0068] Also described herein are kits comprising one or more antibodies described herein and one or more additional components selected from the following: instructions for use; diluents, excipients, carriers, and administration devices, in suitable containers.

[0069] In certain embodiments, described herein is a method of preparing a cancer therapeutic comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein is a method of preparing a cancer therapeutic for storage or shipping comprising lyophilizing one or more antibodies of the present disclosure. Additional exemplary embodiments

[0070] Exemplary Embodiment 1. A common light chain bispecific antibody comprising: an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, wherein the anti-CD38 heavy chain variable region comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9, and a negatively charged amino acid at heavy chain variable region position 1 according to Kabat numbering; an anti-CD19 heavy chain variable region and an anti-CD19 heavy chain constant region, wherein the anti-human CD19 heavy chain variable region comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12, and a negatively charged amino acid at heavy chain variable region position 1 according to Kabat numbering; a common light chain variable region comprising: a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15, wherein: the anti-CD38 heavy chain constant region and the anti-CD19 heavy chain constant region each lack a C-terminal lysine residue; and the common light chain bispecific antibody has an experimental isoelectric point (pi) of less than 9.0, preferably wherein the common light chain bispecific antibody also has a hydrophobic (HIC) retention time of less than about 10 minutes (e.g., as determined in Example 2).

[0071] Exemplary Embodiment 2. The common light chain bispecific antibody of embodiment 1, wherein the common light chain bispecific antibody also has a hydrophobic (HIC) retention time of less than about 10 minutes (e.g., as determined in Example 2).

[0072] Exemplary Embodiment 3. The common light chain bispecific antibody of any one of embodiments 1-2, wherein the terminal lysine residue is K447 according to EU numbering.

[0073] Exemplary Embodiment 4. The common light chain bispecific antibody of any one of embodiments 1-3, wherein the anti-CD19 heavy chain variable region comprises a serine at position 84 and / or a leucine at position 108 according to Kabat numbering.

[0074] Exemplary Embodiment 5. The common light chain bispecific antibody of any one of embodiments 1-4, wherein the common light chain variable region comprises a histidine at position 32 according to Kabat numbering.

[0075] Exemplary Embodiment 6. The common light chain bispecific antibody of any one of embodiments 1-5, wherein the negatively charged amino acid is glutamic acid.

[0076] Exemplary Embodiment 7. The common light chain bispecific antibody of any one of embodiments 1-6, wherein the pi is between 8.7 and 9.0.

[0077] Exemplary Embodiment 8. The common light chain bispecific antibody of any one of embodiments 1-7, wherein the anti-CD19 heavy chain constant region comprises a T366W substitution according to EU numbering or a T366S / L368A / Y407V substitution according to EU numbering.

[0078] Exemplary Embodiment 9. The common light chain bispecific antibody of any one of embodiments 1-8, wherein the anti-CD38 heavy chain constant region comprises a T366W substitution according to EU numbering or a T366S / L368A / Y407V substitution according to EU numbering.

[0079] Exemplary Embodiment 10. The common light chain bispecific antibody of any one of embodiments 1-9, wherein common light chain bispecific antibody further comprises a light chain constant region.

[0080] Exemplary Embodiment 11. The common light chain bispecific antibody of any one of embodiments 1-10, wherein: (i) the anti-CD38 heavy chain variable region and the anti-CD38 heavy chain constant region comprise 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 comprise the amino acid sequence of SEQ ID NO: 5; (iii) the common light chain variable region and the light chain constant region comprise the amino acid sequence of SEQ ID NO: 6.

[0081] Exemplary Embodiment 12. A composition comprising one or more nucleic acid molecules encoding the common light chain bispecific antibody of any one of embodiments 1-11.

[0082] Exemplary Embodiment 13. A cell comprising one or more nucleic acid molecules encoding a common light chain bispecific antibody of any one of embodiments 1-11.

[0083] Exemplary Embodiment 14. A method of treating cancer in an individual in need thereof, comprising administering to the individual a common light chain bispecific antibody of any one of embodiments 1-11.

[0084] Exemplary Embodiment 15. The method of embodiment 14, wherein the cancer comprises a solid tumor.

[0085] Exemplary Embodiment 16. The method of embodiment 14, wherein the cancer is a hematological cancer.

[0086] Exemplary Embodiment 17. The method of embodiment 16, wherein the hematological cancer is a B-cell malignancy.

[0087] Exemplary Embodiment 18. A method of reducing the experimental isoelectric point of an antibody, the method comprising: (a) mutating a glutamine at heavy chain variable region position 1 according to Kabat numbering to a negatively charged amino acid; and (b) removing a lysine at the C-terminal position of the heavy chain constant region, wherein the antibody is a common light chain bispecific antibody comprising: an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, wherein the anti-CD38 heavy chain variable region comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, and a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9, an anti-CD19 heavy chain variable region and an anti-CD19 heavy chain constant region, wherein the anti-human CD19 heavy chain variable region comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12, a common light chain variable region comprising: a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15. Examples

[0088] Example 1 ELISA (BVP, PSR HEK293, PSR CHO) measurements for multispecific. Plates (3) were coated with baculovirus particles (BVP), HEK293, and CHO at 4°C overnight. Antibody concentrations of 150, 50, 16.7, and 5.6 ug / mL were tested in triplicate. Plates were blocked (blocking buffer: PBS + 2% BSA). Background signal was measured for cells coated with secondary antibody (2nd Ab) only. 5x background signal was used as a cutoff. Positive control (PC) included MEDNA Bio #H1308, and negative control (NC) included MEDNA Bio #H1314.

[0089] BSM-001F (BSM-001.1) is a common light chain antibody comprising: (1) an anti-CD38 heavy chain variable region and an anti-CD38 heavy chain constant region, wherein the anti-CD38 heavy chain variable region comprises (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 7, (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 8, (iii) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 9, (2) an anti-CD19 heavy chain variable region and an anti-CD19 heavy chain constant region, wherein the anti-human CD19 heavy chain variable region comprises (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10, (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11, and (iii) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 12, (3) a common light chain variable region comprising: (i) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13, (ii) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (AAS), and (iii) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15. BSM-001F comprises a Q1E heavy chain variable region mutation according to Kabat numbering in each heavy chain, the anti-CD38 heavy chain constant region and the CD19 heavy chain constant region each lack a C-terminal lysine residue (e.g., K447 according to Kabat numbering) To perform data analysis, BVP / HEK / CHO scores were determined, wherein the BVP / HEK / CHO score= (Average OD450 value of three samples) / (Average OD450 value of background signal). Figure 1A -C shows the ELISA OD450 readings for guselkumab, BSM-001F (BSM-001.1), NC, and PC. Compared to guselkumab and PC, BSM-001F (BSM-001.1) exhibited significantly reduced multireactivity.

[0090] Example 2 To determine the experimental isoelectric point, the sample was diluted in a matrix containing methylcellulose, 4 M urea, 3-10 Pharmalyte (4%), 5 mM arginine, and pI markers (at pI 4.22 and pI 10.10). The mixture was placed in an iCE3 IEF analyzer (ProteinSimple), pre-focused at 1,500 V, and then focused at 3,000 V. The isoelectric point of each peak was calculated based on the pI marker in parentheses. The experimental isoelectric point of BSM-001F (BSM-001.1) was measured to be 8.9. Unmodified BSM-001 exhibited a pI of 9.1, which (i) lacked the negatively charged amino acid at position 1 of the heavy chain variable region according to the Kabat number, and (ii) had a heavy chain constant region including a terminal lysine residue (e.g., K447 according to the EU number). The experimental isoelectric point of BSM-001F (BSM-001.1) was reduced to below 9 (8.9).

[0091] Curia hydrophobic interaction chromatography (HIC) was also determined. For the HIC protocol, the sample was titrated with 1M (NH4)2SO4 prior to injection. 10 μL of sample was injected into a Sepax Proteomix HIC butyl-NP1.7 4.6 x 35 mm column at a flow rate of 0.8 mL / min. 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 transitioned linearly to 0% buffer C and 100% buffer D from 1 min to 16 min. The signal was measured at 280 nm. A HIC retention time of 9.9 was observed. Figure 2 The HIC profile of BSM-001F (BSM-001.1) is displayed.

[0092] Example 3 Cellular binding to Daudi cells was measured. BSM-001F (BSM-001.1) showed equivalent binding to Daudi cells compared to unmodified BSM-001 (e.g., lacking Q1E, and terminal lysine modifications). Figure 3 Cellular binding profiles are shown.

[0093] ADCC killing assays were also performed using human donor PBMC samples. To assess direct apoptosis, cells were treated with test articles and incubated at 37°C / 5% CO2for 48 hours. To assess cross-linking induced apoptosis, cells were incubated with test articles on ice for 30 minutes, then rabbit anti-human Fc gamma specific F(ab')2was added. Cells were then incubated at 37°C / 5% CO2for 48 hours. Following incubation, cells were washed and stained with Annexin V, then cells were resuspended in Annexin V buffer containing a viability dye (propidium iodide; PI) prior to flow cytometry acquisition. Early apoptotic cells were defined as Annexin V+ / PI single cells, while late apoptotic / necrotic cells were defined as Annexin V+ / PI+single cells. The sum of Annexin V+ / PI and Annexin V+ / PI was defined as total apoptotic / necrotic cells. Percent Annexin V+ / PI cells or Annexin V+ / PI+were plotted to compare various apoptosis conditions. BSM-001F (BSM-001.1) showed equivalent ADCC-mediated killing of Daudi cells compared to unmodified BSM-001 (e.g., lacking Q1E, and terminal lysine modifications). Figure 4 ADCC profiles are shown.

[0094] To assess direct apoptosis, cells were treated with test articles and incubated at 37°C / 5% CO2for 48 hours. To assess cross-linking induced apoptosis, cells were incubated with test articles on ice for 30 minutes, then rabbit anti-human Fc gamma specific F(ab')2was added. Cells were then incubated at 37°C / 5% CO2for 48 hours. Following incubation, cells were washed and stained with Annexin V, then cells were resuspended in Annexin V buffer containing a viability dye (propidium iodide; PI) prior to flow cytometry acquisition. Early apoptotic cells were defined as Annexin V+ / PI single cells, while late apoptotic / necrotic cells were defined as Annexin V+ / PI+single cells. The sum of Annexin V+ / PI and Annexin V+ / PI was defined as total apoptotic / necrotic cells. Percent Annexin V+ / PI cells or Annexin V+ / PI+were plotted to compare various apoptosis conditions. BSM-001F (BSM-001.1) showed equivalent ADCC-mediated killing of Daudi cells compared to unmodified BSM-001 (e.g., lacking Q1E, and terminal lysine modifications).Figure 5 Apoptosis profiles were shown.

[0095] While preferred embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that many changes, modifications, and alternatives can be made to the embodiments of the application described herein without departing from the application. It is to be understood that various alternatives to the embodiments of the application described herein can be employed in practicing the application.

[0096] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in the text of the document, including definitions that are contained in the specification and the claims, are hereby incorporated by reference. SEQUENCE LISTING

[0097]

[0098]

[0099]

Claims

1. A multispecific antibody comprising: a) The CD38 binding portion includes: i) A first polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown 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 shown in SEQ ID NO: 3; and b) The CD19 binding portion, which includes: i) A third polypeptide comprising an immunoglobulin heavy chain variable region and a heavy chain constant region, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, and 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 shown in SEQ ID NO:

3.

2. The multispecific antibody of 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 of claim 1, wherein the polypeptide comprising the light chain variable region further comprises the 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 comprise the amino acid sequence shown in SEQ ID NO:

6.

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

6. The multispecific antibody of claim 5, wherein the CD38 binding moiety comprises a T366W substitution according to EU number or a T366S / L368A / Y407V substitution according to EU number for the heavy chain of the CD38 binding moiety.

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

8. The multispecific antibody of claim 7, wherein the CD19 binding moiety comprises a T366W substitution according to EU designation or a T366S / L368A / Y407V substitution according to EU designation for the heavy chain of the CD19 binding moiety.

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

4.

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

5.

11. A common light chain bispecific antibody comprising: The anti-CD38 heavy chain variable region and the anti-CD38 heavy chain constant region, wherein the anti-CD38 heavy chain variable region comprises: Contains the heavy chain complementarity-determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:

7. Contains heavy chain complementarity-determining region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO:

8. Contains the heavy chain complementarity-determining region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 9, and Based on the Kabat number, the negatively charged amino acid at position 1 in the heavy chain variable region, Anti-CD19 heavy chain variable region and anti-CD19 heavy chain constant region, wherein the anti-human CD19 heavy chain variable region comprises: Contains the heavy chain complementarity-determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:

10. Contains heavy chain complementarity-determining region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO:

11. Contains the heavy chain complementarity-determining region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 12, and Based on the Kabat number, the negatively charged amino acid at position 1 in the heavy chain variable region, A common light chain variable region, which includes: Contains the light chain complementarity-determining region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO:

13. Contains the light chain complementarity-determining region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 14 (AAS), and Contains the light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO:

15. in: The anti-CD38 heavy chain constant region and the anti-CD19 heavy chain constant region each lack a C-terminal lysine residue (e.g., according to EU designation K447); and The common light chain bispecific antibody has an experimental isoelectric point (pI) of less than 9.

12. The common light chain bispecific antibody of claim 11, wherein the common light chain bispecific antibody also has a hydrophobic (HIC) retention time of less than about 10 minutes.

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

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

15. The common light chain bispecific antibody according to any one of claims 11-14, wherein the common light chain variable region comprises histidine at position 32 according to the Kabat number.

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

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

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

19. One or more nucleic acids encoding a common light chain bispecific antibody as described in any one of claims 1 to 10 or a common light chain bispecific antibody as described in any one of claims 11 to 17.

20. A method of treating cancer in an individual in need, comprising administering to the individual a common light chain bispecific antibody as claimed in any one of claims 1 to 10 or a common light chain bispecific antibody as claimed in any one of claims 11 to 17, thereby treating the cancer in the individual in need.

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

22. The method of 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 of 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 of claim 20, wherein the cancer or tumor is a blood cancer.

25. The method of claim 24, wherein the blood cancer is diffuse large B-cell lymphoma.

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

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

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

29. The method of claim 28, wherein the B-cell lymphoma includes double-hit lymphoma, dual-expressing lymphoma, or triple-hit lymphoma.

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

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

32. The method of claim 31, wherein the CD19-positive, CD38-hyperimmunosuppressive B cells express B cell activation markers.

33. The method of claim 32, wherein the B cell activation marker comprises CD30.

34. The method of 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 of 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 B cells or CD20-negative B cells.

36. The method of claim 35, wherein the CD38 high B cells express at least about 30,000 CD38 proteins on the cell surface.

37. The method of claim 35, wherein the CD38 high B cells express at least about 35,000 CD38 proteins on the cell surface.

38. The method of claim 35, wherein the CD38 high B cells express at least about 40,000 CD38 proteins on the cell surface.

39. A method for lowering the experimental isoelectric point of an antibody, the method comprising: (a) Mutate the glutamine at position 1 of the heavy chain variable region according to the Kabat number to a negatively charged amino acid; and (b) Remove lysine residues at the C-terminal position of the heavy chain constant region.

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

41. The method of any one of claims 39-40, wherein the antibody is a common light chain bispecific antibody comprising: Anti-CD38 heavy chain variable region, wherein the anti-CD38 heavy chain variable region comprises: Contains the heavy chain complementarity-determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:

7. Contains the heavy chain complementarity-determining region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 8, and Contains the heavy chain complementarity-determining region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO:

9. Anti-CD19 heavy chain variable region, wherein the anti-human CD19 heavy chain variable region comprises: Contains the heavy chain complementarity-determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:

10. Contains the heavy chain complementarity-determining region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 11, and Contains the heavy chain complementarity-determining region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO:

12. A common light chain variable region, which includes: Contains the light chain complementarity-determining region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO:

13. Contains the light chain complementarity-determining region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 14 (ASS), and Contains the light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO:

15.

42. A method for preparing an antibody, the method comprising harvesting a bispecific antibody as described in any one of claims 1 to 17 from a supernatant of a cell line, and subjecting the supernatant to one or more purification steps, the supernatant containing a nucleic acid encoding the antibody.

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