Antibodies specific for CD38 and uses thereof

Abstract

CD38 is also expressed in a variety of hematological malignancies, including multiple myeloma. In the present invention, the inventors obtained a new antibody against CD38 that can be used to produce bispecific antibodies and CAR T cell populations. In particular, the inventors reported the development of Bi38-3, a new bispecific T cell engager that targets CD38 on MM cells and forms cytotoxic T cells through CD3ε. Bi38-3 lacks the Fc region of natural mAbs, which contributes to the resistance process, but triggers T cell proliferation, cytokine release, and lysis of CD38-positive MM cells in vitro. Similarly, Bi38-3 induces autologous T cells to eliminate tumor plasma cells isolated from MM patients at diagnosis and relapse. The cytotoxicity triggered by Bi38-3 is limited to cells expressing high levels of CD38 and maintains the integrity of T, B, and NK lymphocytes in vitro. Importantly, Bi38-3 rapidly reduced tumor cells in the MM1.S xenograft mouse model of human MM. In summary, the results show that the antibody of the present invention is an effective agent for specifically eliminating CD38-positive malignant cells without significantly affecting CD38-low-expressing cells, and is a promising new immunotherapy tool for treating malignant blood diseases, especially multiple myeloma.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, in particular to the field of oncology. Background Art

[0002] CD38 is a type II transmembrane glycoprotein. The functions of CD38 include receptor mediation in adhesion and signaling events as well as enzymatic activity. CD38 is commonly found in hematopoietic cells and solid tissues. In hematopoietic cells, most medullary thymocytes are CD38 + , resting and circulating T cells and B cells are CD38-, activated cells are CD38 + . CD38 is also expressed on approximately 80% of resting NK cells and monocytes, as well as lymphoblasts in the germinal centers of lymph nodes, plasma B cells, and some intrafollicular cells. CD38 can also be expressed by dendritic cells. A large proportion of normal bone marrow cells, especially precursor cells, express CD38. In addition, 50-80% of umbilical cord blood cells are CD38+ and remain present in human blood for the first two to three years of life. In addition to lymphoid precursor cells, CD38 is also expressed on red blood cells and platelets. For solid tissues, CD38 is expressed by epithelial cells and lamina propria lymphocytes in the intestine, by Purkinje cells and neurofibrillary tangles in the brain, by epithelial cells in the prostate, by β cells in the pancreas, by osteoclasts in the bones, by retinal cells in the eyes, and on the sarcolemma of smooth and striated muscles.

[0003] CD38 is also expressed in a variety of hematological malignancies, including multiple myeloma, B-cell chronic lymphocytic leukemia, B-cell acute lymphocytic leukemia, and Waldenstrom's macroglobulinemia. The leukemias include primary systemic amyloidosis, mantle cell lymphoma, pro-lymphocytic / myelocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, NK-cell leukemia, and plasma-cell leukemia. For example, multiple myeloma (MM) is a heterogeneous hematologic malignancy characterized by the accumulation of monoclonal immunoglobulin-secreting neoplastic plasma cells in the patient's bone marrow, as well as osteolytic lesions. 1 Current treatments have increased median overall survival to approximately 6 years, and recently developed monoclonal antibodies (mAbs), such as elotuzumab (anti-SLAMF7) and daratumumab (anti-CD38), have further improved outcomes. 2-4 However, overall survival for patients with relapsed disease after treatment with proteasome inhibitors (PIs), immunomodulatory agents (IMIDs), and mAbs remains extremely low, and MM remains an incurable disease. Therefore, new therapeutic strategies are needed to improve patient care and ultimately develop curative therapies.

[0004] Several anti-CD38 antibodies are described in the literature, for example, Lande R et al., Cellular Immunology 220(1), 30-8 (2002); Ausiello CM et al., Tissue Antigens 56(6), 539-47 (2000); and Cotner T et al., Int J Immunopharmacol 3(3), 255-68 (1981). For example, WO2006099875 describes several human anti-CD38 antibodies. Summary of the Invention Summary of the invention:

[0006] As defined in the claims, the present invention relates to antibodies specific for CD38 and uses thereof. Detailed description of the invention:

[0008] The inventors have developed a novel anti-CD38 / CD3 bispecific T cell-engaging antibody that triggers specific T cell-mediated lysis of CD38-positive MM cells in vitro, ex vivo, and in vivo. T cell killing of MM cells mediated by this novel anti-CD38 / CD3 bispecific T cell-engaging antibody, Bi38-3, is not affected by resistance mechanisms associated with anti-CD38 mAbs (e.g., daratumumab, an anti-CD38 monoclonal antibody approved for the treatment of MM), which is associated with binding of therapeutic antibodies to FcγRs. The inventors demonstrated that Bi38-3 mediates autologous T cell-mediated killing of tumor plasma cells from patients at diagnosis and relapse with similar efficiency. Furthermore, they demonstrated that Bi38-3 had no significant effects on T, B, and NK cells in vitro, readily induced T cell-mediated killing of MM cells, and simultaneously protected B cells from T cell cytotoxic activity. They showed that Bi38-3 could induce a 6-fold reduction in tumor burden in vivo in just 3 days. Thus, the inventors demonstrate that Bi38-3 is a selective and highly effective compound for the treatment of MM that can be used either in the first-line setting or at relapse, and support further evaluation in MM patients.

[0009] Main definitions:

[0010] As used herein, the term "CD38" has its ordinary meaning in the art and refers to ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1. An exemplary amino acid sequence of CD38 is represented by SEQ ID NO: 1. The extracellular domain of CD38 ranges from amino acid residue 43 to amino acid residue 300 in SEQ ID NO: 1.

[0011] SEQ ID NO: 1> sp|P28907|CD38_HUMAN ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1

[0012] OS=Homo sapiens OX=9606 GN=CD38 PE=1 SV=2

[0013]

[0014] As used herein, the term "CD3" has its ordinary meaning in the art and refers to the CD3 (cluster of differentiation 3) T cell co-receptor that helps activate cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). It consists of a protein complex composed of four different chains. In mammals, the complex contains one CD3γ chain, one CD3δ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) and the ζ chain (zeta chain) to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecules together constitute the TCR complex. An exemplary amino acid sequence of CD3ε is represented by SEQ ID NO: 2. The extracellular domain of CD3ε ranges from amino acid residue 23 to amino acid residue 207 in SEQ ID NO: 2.

[0015] SEQ ID NO: 2> sp|P07766|CD3E_HUMAN T cell surface glycoprotein CD3ε chain OS=Homosapiens

[0016] OX=9606 GN=CD3E PF=1 SV=

[0017]

[0018] Thus, the term "antibody" as used herein is used to refer to any antibody-like molecule having an antigen-binding region, and the term includes antibody fragments consisting of an antigen-binding domain, such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimers, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, diabodies, triabodies (scFv-Fab fusion, bispecific or trispecific, respectively); sc-diabodies; kappa (lambda) bodies (scFv-CL fusion); BiTEs (bispecific T cell engagers, scFv-scFv tandem to attract T cells); DVD-Ig (dual variable domain antibodies, bispecific format); SIPs (small immune proteins, a minibody); SMIPs ("small modular immunopharmaceuticals" scFv-Fc dimers); DARTs (ds-stabilized diabodies "dual affinity retargeting"); small antibody mimetics comprising one or more CDRs, etc. Techniques for preparing and using various antibody-based structures and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). In particular, diabodies are further described in EP 404,097 and WO 93 / 11161; while linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be produced by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques, or can be synthesized chemically. Techniques for producing antibody fragments are well known and described in the art. For example, Beckman et al., 2006; Holliger and Hudson, 2005; Le Gall et al., 2004; Reft and Heard, 2001; Reiter et al., 1996; and Young et al., 1995 each further describe and enable the production of efficient antibody fragments.

[0019] In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, λ (λ) and κ (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain consists of different sequence domains. The light chain consists of two domains, the variable domain (VL) and the constant domain (CL). The heavy chain consists of four (α, δ, γ) to five (μ, ε) domains, one variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4 collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity for the antigen. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental migration, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of the variable portion of one light chain and one heavy chain. Antibody specificity stems from the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may contribute to the antibody binding site or influence the overall domain structure, thereby affecting the binding site. CDRs are amino acid sequences that collectively define the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. An immunoglobulin light chain and heavy chain each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically includes six CDRs, including one set of CDRs from each of the heavy and light chain V regions. Framework regions (FRs) refer to the amino acid sequences intervening between the CDRs. Residues in the antibody variable domain are typically numbered according to the system devised by Kabat et al. This system is proposed in Kabat et al., 1987, Data in Immunologically Significant Protein Sequences, U.S. Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used in this specification. The Kabat residue names do not always directly correspond to the linear numbering of the amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or more amino acids than in the strict Kabat numbering, corresponding to shortening or insertion of structural components of the basic variable domain, whether framework or complementarity determining region (CDR). The correct Kabat numbering of the residues can be achieved by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35B (VH-CDR1), residues 50-65 (VH-CDR2) and residues 95-102 (VH-CDR3).According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (VL-CDR1), residues 50-56 (VL-CDR2), and residues 89-97 (VL-CDR3).

[0020] As used herein, the term "BB51 antibody" refers to a murine antibody characterized by a heavy chain variable domain as set forth in SEQ ID NO:3 and a light chain variable domain as set forth in SEQ ID NO:4.

[0021] SEQ ID NO: 3>IgH VH1.87-D1.1-J1:

[0022]

[0023] SEQ ID NO: 4>Igk Vk12.44-Jk5:

[0024]

[0025] As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked, for example, by a synthetic linker, such as a short, flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, an scFv can have the VL and VH variable regions in any order. For example, an scFv can comprise VL-linker-VH or VH-linker-VL, relative to the N- and C-termini of the polypeptide. As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity that has variable and constant regions derived from human germline immunoglobulin sequences.

[0026] As used herein, the term "chimeric antibody" refers to an antibody comprising the VH and VL domains of a non-human antibody, and the CH and CL domains of a human antibody. In some embodiments, a "chimeric antibody" is an antibody molecule in which (a) the constant region (i.e., heavy chain and / or light chain) or a portion thereof is changed, replaced, or exchanged so that the antigen binding site (variable region) is connected to a constant region of a different or changed class, effector function, and / or species, or a completely different molecule that gives the chimeric antibody new properties, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is changed, replaced, or exchanged by a variable region with a different or changed antigenic specificity. Chimeric antibodies also include primate antibodies, particularly humanized antibodies. In addition, chimeric antibodies may include residues that are not found in the recipient antibody or the donor antibody. These modifications are to further improve antibody performance. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). (See U.S. Patent No. 4,816,567; and Morrison et al., Proceedings of the National Academy of Sciences USA, 81:6851-6855 (1984)).

[0027] As used herein, the term "humanized antibody" refers to an antibody with a variable region framework and constant region from a human antibody, but retains the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains the minimum sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody and its antibody fragment can be a human immunoglobulin (receptor antibody or antibody fragment), wherein the residues from the complementary determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody), such as mice, rats or rabbits with desired specificity, affinity and ability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies / antibody fragments can include residues that are neither found in the receptor antibody nor in the CDR or framework sequences imported. Such antibodies are designed to maintain the binding specificity of the non-human antibodies derived from the binding region, but avoid immune responses directed against non-human antibodies. These modifications can further refine and optimize the performance of the antibody or antibody fragment. In general, a humanized antibody or antibody fragment thereof will include at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin, and all or a substantial portion of the FR regions are regions of a human immunoglobulin sequence. A humanized antibody or antibody fragment may also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0028] As used herein, the term "binding" in the context of binding of an antibody to a predetermined antigen or epitope is generally binding with low affinity, as measured by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte, with a K of D About 10 -6 m. (GE Healthcare, Piscaaway, NJ) is one of several surface plasmon resonance assay formats that are commonly used for epitope bin panels of monoclonal antibodies. Typically, the affinity of an antibody for its binding to a predetermined antigen corresponds to the K D Compared with its K binding to nonspecific antigens (such as BSA, casein) DThe antibody K is at least ten times lower, such as at least 100 times lower, such as at least 1000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, and the non-specific antigen is not identical to or closely related to the predetermined antigen. D When the antibody has a high affinity, its K value for binding to the antigen is very low. D Usually higher than the K of nonspecific antigens D An antibody is said to have substantially no binding to an antigen or epitope if its binding to the antigen or epitope is undetectable (e.g., using plasma resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100-fold, 500-fold, 1000-fold, or more than 1000-fold less than the binding detected for the antibody to an antigen or epitope having a different chemical structure or amino acid sequence.

[0029] As used herein, the term "bispecific antibody" has its ordinary meaning in the art and refers to an artificial hybrid antibody with two pairs of different heavy and light chains and two different antigen-binding sites.

[0030] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a bispecific antibody, which is a recombinant protein construct consisting of two flexibly linked single-chain antibodies (scFv). One of the scFv antibodies specifically binds to a selected tumor antigen expressed by a target cell, and the second specifically binds to another molecule, such as CD3, which is a subunit of the T cell receptor complex on T cells. In some embodiments, the BiTE antibody is able to transiently bind T cells to target cells while activating the cytolytic activity of the T cells. BiTE-mediated T cell activation does not require either a specific T cell receptor on the T cell or an MHC I molecule, peptide antigen, or co-stimulatory molecule on the target cell.

[0031] As used herein, the term "CAR-T cell" refers to a T lymphocyte that has been genetically modified to express a CAR. The definition of CAR T cells encompasses all classes and subclasses of T lymphocytes, including CD4+, CD8+ T cells, gamma delta T cells, and effector T cells, memory T cells, regulatory T cells, and the like. Genetically modified T lymphocytes can be "derived" or "obtained" from a subject who will receive therapy using genetically modified T cells, or they can be "derived" or "obtained" from a different subject.

[0032] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a group of polypeptides, typically two in the simplest embodiment, which, when in immune effector cells, provide cells with specificity for target cells, typically cancer cells, and generate intracellular signals. In some embodiments, CAR includes at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule defined below. In some aspects, the group of polypeptides is adjacent to each other. In some embodiments, the group of polypeptides includes a dimerization switch that couples polypeptides to each other when a dimerization molecule is present, for example, an antigen binding domain can be coupled to an intracellular signaling domain. In some embodiments, the stimulatory molecule is a zeta chain associated with a T cell receptor complex. In some embodiments, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule defined below. In some embodiments, the costimulatory molecules are selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and / or CD28. In some embodiments, CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR includes a chimeric fusion protein including an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain includes at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR includes an optional guide sequence at the amino terminus (N-ter) of the CAR fusion protein. In some embodiments, CAR further includes a guide sequence at the N-terminus of the extracellular antigen binding domain, wherein during the cell processing and positioning of CAR, the guide sequence is optionally cracked from the antigen binding domain (e.g., scFv). In particular aspects, CAR includes a single-chain variable fragment (scFv) fused to a monoclonal antibody, fused to a CD3-ζ (CD3-zeta) transmembrane domain and an inner domain.In some embodiments, CAR includes a domain for additional costimulatory signaling, such as CD3-ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40. In some embodiments, molecules can be co-expressed with CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate T cells after the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0033] As used herein, the term "T cell" has its general meaning in the art and represents an important component of the immune system that plays a central role in cell-mediated immunity. T cells are called conventional lymphocytes because they recognize antigens with their TCRs (T cell receptors for antigens) and present or restrict them through complex major histocompatibility molecules. There are several subpopulations of T cells, each with different functions, such as CD8+ T cells, CD4+ T cells, and γδ T cells.

[0034] As used herein, the term "CD8+ T cells" has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8+ T cells" are also referred to as cytotoxic T lymphocytes (CTLs), T killer cells, cytolytic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is an associative recognition element in the major histocompatibility complex class I restricted interaction. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to a patient's CD8+ T cell pool that has left the bloodstream and migrated into a tumor.

[0035] As used herein, the term "CD4+T cells" (also referred to as T helper cells or TH cells) refers to T cells that express CD4 glycoprotein on their surface and assist other white blood cells in the immune process, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. CD4+T cells are activated when peptide antigens are presented by MHC class II molecules, which are expressed on the surface of antigen presenting cells (APCs). Once activated, they rapidly divide and secrete cytokines that regulate or assist active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, TFH or Treg, which secrete different cytokines to promote different types of immune responses. Signals from APCs guide T cells into specific subtypes. In addition to CD4, TH cell surface biomarkers known in the art include CXCR3 (Th1), CCR4, Crth2 (Th2), CCR6 (Th17), CXCR5 (Tfh), and subtype-specifically expressed cytokines and transcription factors, including T-bet, GATA3, EOMES, RORγT, BCL6, and FoxP3.

[0036] As used herein, the term "γδT cells" has its general meaning in the art. γδT cells typically account for 1% to 5% of peripheral blood lymphocytes in healthy individuals (humans, monkeys). They are involved in initiating protective immune responses, and have been shown to recognize their antigen ligands through direct interaction with antigens without any presentation by MHC molecules of antigen-presenting cells. γ9δ2T cells (sometimes also referred to as γ2δ2T cells) are γδT cells with TCR receptors that have variable domains Vγ9 and Vδ2. They constitute the majority of γδT cells in human blood. When activated, γδT cells exert powerful, non-MHC-restricted cytotoxic activity, and are particularly effective in killing various types of cells, especially pathogenic cells. These may be cells infected by viruses ((Poccia et al., J. Leukocyte Biology, 1997, 62: 1-5) or other intracellular parasites, such as mycobacteria (Constant et al., Infection and Immunity, December 1995, vol. 63, no. 12: 4628-4633) or protozoa (Behr et al., Infection and Immunity, 1996, vol. 64, no. 8: 2892-2896). They may also be cancer cells (Poccia et al., J. Immunol., 159: 6009-6015; Fournie and Bonneville, Res. Immunol., 66th Immunol. Forum, 147: 338-347). Therefore, the possibility of modulating the activity of such cells in vitro, ex vivo or in vivo would provide novel and effective therapeutic approaches for the treatment of various diseases, such as infectious diseases (especially viral or parasitic), cancer, allergies and even autoimmune and / or inflammatory diseases.

[0037] As used herein, the term "treatment method" or "treatment" refers to preventative or prophylactic treatment, and also refers to curative or disease-modifying treatment, including treatment of patients at risk for or suspected of contracting a disease, as well as patients who are ill or diagnosed with a disease or medical condition, and includes suppressing clinical relapse. The treatment method can be used for patients who have a medical disease or may eventually acquire the disease to prevent, cure, delay the onset of the disease, reduce the severity of the disease, or improve one or more symptoms of the disease or recurrent disease, or to prolong the patient's life span beyond what would be expected in the absence of such treatment. The so-called "treatment regimen" refers to the treatment mode of the disease, such as the dosage mode used during treatment. The treatment regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide the patient with high levels of drug in the initial stage of the treatment regimen. The induction regimen may employ (part or all) a "loading regimen," which may include a larger dose of the drug than the doctor uses during the maintenance regimen, more frequent administration of the drug than the doctor uses during the maintenance regimen, or both. The phrases "maintenance regimen" or "maintenance period" refer to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during treatment for a disease, e.g., to keep the patient in remission for an extended period of time (e.g., months or years). A maintenance regimen can employ continuous therapy (e.g., dosing at regular intervals, e.g., weekly, monthly, annually, etc.) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, recurring treatment, or treatment upon reaching specific predetermined criteria (e.g., pain, disease manifestations, etc.)).

[0038] As used herein, the term "cancer" has its ordinary meaning in the art and refers to abnormal cells with the ability to grow autonomously, i.e., an abnormal state or condition characterized by rapid cell proliferation with the potential to invade or spread to other parts of the body. The term is intended to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of their histopathological type or stage of invasion. The term "cancer" includes, but is not limited to, malignant tumors of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic, gastrointestinal, and genitourinary tracts, as well as malignant tumors including most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, glioblastomas, non-small cell lung cancers, small intestine cancers, and esophageal cancers, including adenocarcinomas. The term "cancer" also includes, but is not limited to, solid tumors and blood-borne tumors.

[0039] The term "solid cancer" has its ordinary meaning in the art and refers to a cancer selected from the group consisting of, but not limited to, head and neck squamous cell carcinoma (HNSCC), adrenocortical carcinoma, anal cancer, bile duct cancer (e.g., pericytoma, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g., osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, multiple myeloma), brain and central nervous system cancer. Cancer (e.g., meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germ cell tumor, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer (e.g., endometrial adenocarcinoma, adenocarcinoma, papillary serous adenocarcinoma, clear cell carcinoma), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoids (e.g., choriocarcinoma, chorioadenoma), Kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma), laryngeal and hypopharyngeal cancer, liver cancer (e.g., hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g., esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, Pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), stomach cancer, testicular cancer (e.g., seminoma, non-seminoma germ cell cancer), thymic cancer, thyroid cancer (e.g., follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid cancer), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma).

[0040] The term "blood-borne cancer" or leukemia has its ordinary meaning in the art and refers to cancers of the blood cells. They begin in the bone marrow, the soft tissue in the center of the bones that makes blood cells. With leukemia, the bone marrow begins making abnormal cells that crowd out normal blood cells.

[0041] In some embodiments, the cancer is a CD38-positive hematological malignancy.

[0042] As used herein, the term "CD38-positive hematological malignancies" refers to hematological malignancies characterized by the presence of tumor cells expressing CD38, including leukemias, lymphomas, and myelomas. Examples of such CD38-positive hematologic malignancies include precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin lymphoma; acute promyelocytic leukemia, acute lymphoblastic leukemia, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT-type, nodal, and splenic), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasmacytoma, multiple myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, macroglobulinemia, plasma cell leukemia, and anaplastic large cell lymphoma (ALCL).

[0043] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma.

[0044] As used herein, the term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic effect within the necessary dosage and time period. The therapeutically effective amount of an active agent can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the active agent to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody portion are offset by the beneficial effects of the treatment. The effective dosage and dosing regimen of the active agent depend on the disease or condition to be treated and can be determined by one skilled in the art. A physician with ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician may start the dosage of the active agent used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, a suitable dose of a composition of the present invention is the amount of the compound that is the lowest dose that produces an effective therapeutic effect according to a particular dosing regimen. This effective dose generally depends on the factors mentioned above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize disease progression. Typically, the ability of a compound to inhibit cancer can be evaluated in an animal model system that is predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise improve the patient's symptoms. One of ordinary skill in the art will be able to determine the amount based on factors such as the patient's size, the severity of the patient's symptoms, and the specific composition or route of administration selected. An exemplary non-limiting range for a therapeutically effective amount of an inhibitor of the present invention is about 0.1-100 mg / kg, for example, about 0.1-50 mg / kg, for example, about 0.1-20 mg / kg, for example, about 0.1-10 mg / kg, for example, about 0.5, for example, about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. An exemplary non-limiting range for a therapeutically effective amount of an inhibitor of the present invention is 0.02-100 mg / kg, for example, about 0.02-30 mg / kg, for example, about 0.05-10 mg / kg or 0.1-3 mg / kg, for example, about 0.5-2 mg / kg. The route of administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and, for example, administered near the target site. The dosage regimen in the above-mentioned treatment and use methods is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over a period of time, or the dose can be proportionally reduced or increased according to the urgency of the treatment situation. In some embodiments, the efficacy of the treatment is monitored, for example, at predetermined time points during treatment. In some embodiments, the efficacy of the treatment can be monitored by visualization of the disease area, or by other diagnostic methods further described herein, for example, by performing one or more PET-CT scans, for example, using a labeled inhibitor of the present invention, a fragment derived from an inhibitor of the present invention, or a mini-antibody.If desired, the daily effective dose of the pharmaceutical composition can be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, the human monoclonal antibodies of the present invention are administered by slow continuous infusion for a long time, for example, over 24 hours, to minimize any unwanted side effects. The effective dose of the inhibitor of the present invention can also be administered using a weekly, biweekly or three-week dosing period. The dosing cycle can be limited to 8 weeks, 12 weeks or until clinical progression is determined. As a non-limiting example, treatment according to the present invention can be provided as a daily dose of an inhibitor of the invention in an amount of about 0.1-100 mg / kg, for example 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th or 15th day. 6, 4, or 2 hours, or any combination thereof, after initiation of treatment, or alternatively, on at least one of days 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, on at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, after initiation of treatment, or alternatively, on at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, using a single or divided dose.

[0045] Antibodies of the present invention:

[0046] The first object of the present invention relates to a monoclonal antibody having binding specificity to the extracellular domain of CD38, comprising:

[0047] A heavy chain comprising i) H-CDR1 shown in SEQ ID NO: 5, ii) H-CDR2 shown in SEQ ID NO: 6, and iii) H-CDR3 shown in SEQ ID NO: 7, and

[0048] A light chain comprising i) L-CDR1 represented by SEQ ID NO: 8, ii) L-CDR2 represented by SEQ ID NO: 9, and iii) L-CDR3 represented by SEQ ID NO: 10.

[0049] SEQ ID NO: 5(H-CDR1): GYTFTSYW

[0050] SEQ ID NO: 6(H-CDR2): IYPGDGDT

[0051] SEQ ID NO: 7(H-CDR3): ARERTTGAPRYFDV

[0052] SEQ ID NO: 8 (L-CDR1): ENIYSF

[0053] SEQ ID NO: 9 (L-CDR2): NTK

[0054] SEQ ID NO: 10 (L-CDR3): QHHYGIPLT

[0055] In some embodiments, the monoclonal antibody of the invention comprises a VH domain that is at least 70% identical to the amino acid sequence shown in SEQ ID NO:3.

[0056] In some embodiments, the monoclonal antibody of the invention comprises a VL domain that is at least 70% identical to the amino acid sequence shown in SEQ ID NO:4.

[0057] According to the present invention, a first amino acid sequence having at least 70% identity with a second amino acid sequence means that the first sequence has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the second amino acid sequence. According to the present invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence has 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the second amino acid sequence. Sequence identity is typically measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proceedings of the National Academy of Sciences of the United States of America, USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al., Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994; Altschul et al., Nat. Genet., 6:119-129, 1994, describe sequence alignment methods and homology calculations in detail. For example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) can be used to perform sequence comparisons (Internet 1996, W.R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences to each other, while LFASTA compares regions of local similarity. For example, these alignment tools and their respective tutorials are available on the Internet, such as the NCSA website. Alternatively, for comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function can be used, using the default BLOSUM62 matrix, set to default parameters (gap existence cost of 11, per residue gap cost of 1). When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used, using the PAM30 matrix set to default parameters (open gap 9, extended gap 1). For example, the BLAST sequence comparison system can be obtained from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al., Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang and Madden, Genome Res., 7:649-656, 1997.

[0058] Thus, the present invention provides antibodies comprising one or more functional variants of the VL region, VH region, or CDRs of the BB51 antibody. The functional variants of the VL, VH, or CDRs used in the context of the monoclonal antibodies of the present invention still allow the antibody to retain a significant portion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / hardness and / or specificity / selectivity of the parent antibody (i.e., BB51 antibody), and in some cases, such monoclonal antibodies of the present invention may have greater affinity, selectivity, and / or specificity than the parent Ab. Such functional variants generally retain significant sequence identity with the parent Ab. The sequence of the CDR variant may differ from the CDR sequence of the parent antibody sequence, mainly through conservative substitutions; for example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are substitutions of conservative amino acid residues. The sequence of the CDR variant may differ from the CDRs sequence of the parent antibody sequence, mainly through conservative substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions in the variant are substitutions of conservative amino acid residues. In the context of the present invention, conservative substitutions can be defined as substitutions within the following amino acid categories:

[0059] Aliphatic residues I, L, V and M

[0060] Cycloalkenyl-related residues F, H, W, and Y

[0061] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y

[0062] Negatively charged residues D and E

[0063] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0064] Positively charged residues H, K, and R

[0065] Small residues A, C, D, G, N, P, S, T, and V

[0066] Very small residues A, G, and S

[0067] Involving A, C, D, E, G, H, K, N, Q, R, S, P and T-forming residues in sequence

[0068] Flexible residues Q, T, K, S, G, P, D, E, and R.

[0069] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Compared with the CDR of BB51 antibody, the conservativeness of the variant CDR in terms of hydrophilicity / hydrophilicity and residue weight / size is also substantially retained. The importance of the hydrophilic amino acid index in conferring biological function on protein interactions is generally understood in the art. It is recognized that the relative hydrophilicity of amino acids helps to generate the secondary structure of proteins, which in turn determines the interaction of proteins with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge properties: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by using a BLAST program (e.g., BLAST 2.2.8 available through NCBI using standard settings BLOSUM62, Open Gap = 11, and Extended Gap = 1). Suitable variants typically exhibit at least about 70% identity to the parent polypeptide.

[0070] In some embodiments, the monoclonal antibodies of the present invention are chimeric antibodies. In some embodiments, the monoclonal antibodies of the present invention are chimeric antibodies having a heavy chain as set forth in SEQ ID NO: 3. In some embodiments, the monoclonal antibodies of the present invention are chimeric antibodies having a light chain as set forth in SEQ ID NO: 4. In some embodiments, the monoclonal antibodies of the present invention are chimeric antibodies having a heavy chain as set forth in SEQ ID NO: 3 and a light chain as set forth in SEQ ID NO: 4.

[0071] In some embodiments, the monoclonal antibodies of the invention are humanized antibodies.

[0072] The monoclonal antibodies of the present invention may be characterized by one or more functional or structural features of the aspects described above, or by any combination of selected functional and structural features.

[0073] The antibodies of the present invention can be of any isotype. The choice of isotype is generally guided by the desired effector function, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Any of the human light chain constant regions κ or λ can be used. If desired, the class of the monoclonal antibodies of the present invention can be converted by known methods. Typical class switching techniques can be used to convert one IgG subclass to another, such as from IgG1 to IgG2. Therefore, the effector function of the human monoclonal antibodies of the present invention can be changed by converting the isotype to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies for various therapeutic uses. In some embodiments, the antibodies of the present invention are full-length antibodies. In some embodiments, the full-length antibodies are IgG1 antibodies. In some embodiments, the full-length antibodies are IgG4 antibodies. In some embodiments, the IgG4 antibodies are stabilized IgG4 antibodies. Examples of suitable stabilized IgG4 antibodies are antibodies in which the arginine at position 409 of the heavy chain constant region of human IgG4 is substituted with lysine, threonine, methionine or leucine as shown in the EU index as described by Kabat et al., preferably lysine (described in WO2006033386) and / or in which the hinge region comprises a Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO2008145142, which is incorporated herein by reference in its entirety. In some embodiments, the monoclonal antibody of the present invention is an antibody of a non-IgG4 type, such as an IgG1, IgG2 or IgG3 that has been mutated so that the ability to mediate effector functions (e.g., ADCC) is reduced or even eliminated. For example, Dall'Acqua WF et al. describe such mutations, J Immunol 177(2):1129-1138 (2006) and Hezareh M, J Virol. 75(24):12161-12168 (2001).

[0074] In addition to modifications or substitutions made within the framework or CDR regions, the antibodies of the present invention can be designed to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, the monoclonal antibodies of the present invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to further alter one or more functional properties of the antibody. For example, it will be appreciated that the affinity of the antibodies provided herein can be altered using any suitable method known in the art. Thus, the present invention also relates to variants of the antibody molecules of the present invention having improved affinity for CD38. Such variants can be obtained by a variety of affinity maturation schemes, including mutating CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain replacement (Marks et al., Bio / Technology, 10, 779-783, 1992), use of E. coli mutants (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA replacement (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). These affinity maturation methods are discussed in Vaughan et al. (supra).

[0075] In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues, thereby altering the affinity of the antibody for an effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is further described in U.S. Patents 5,624,821 and 5,648,260, both authored by Winter et al.

[0076] In some embodiments, one or more amino acids selected from amino acid residues can be replaced by different amino acid residues, so that the antibody has a changed C1q combination and / or reduced or eliminated complement dependent cytotoxicity (CDC). This method is further described in detail by the people such as Idusogie in U.S. Patent number 6194551.

[0077] In some embodiments, one or more amino acid residues are altered to alter the ability of the antibody to fix complement. This approach is further described by Bodmer et al. in PCT publication WO 94 / 29351. In some embodiments, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fc receptors by modifying one or more amino acids. This approach is further described by Presta in PCT publication WO 00 / 42072. In addition, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields, RL et al., 2001 J. Biol. Chen. 276: 6591-6604, WO2010106180).

[0078] In some embodiments, the glycosylation of the antibody is modified. For example, a non-glycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. Glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. This carbohydrate modification can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. This glycosylation can increase the affinity of the antibody for the antigen. Such methods are further described in detail by Co et al. in U.S. Patents 5,714,350 and 6,350,861. Additionally or alternatively, antibodies with altered types of glycosylation can be prepared, such as low-fucosylated or non-fucosylated antibodies, in which the number of fucosyl residues is reduced or absent, or antibodies with increased bisecting GlcNac structures. This altered glycosylation pattern has been shown to increase the ADCC ability of the antibody. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express the recombinant antibodies of the present invention to produce antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes cell lines with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation or lack fucosyl residues. Thus, in some embodiments, the human monoclonal antibodies of the present invention can be produced by recombinant expression in cell lines that exhibit a hypofucosylation or afucosylation pattern, for example, a mammalian cell line deficient in expression of the FUT8 gene encoding the fucosyltransferase. PCT publication WO 03 / 035835 published by Presta describes a variant CHO cell line, Lec13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in reduced fucosylation of antibodies expressed in the host cells (see also Shields, RL et al., 2002 J. Biol. Chem. 277: 26733-26740). PCT publication WO 99 / 54342 by Umana et al. describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, thereby resulting in increased ADCC activity of the antibodies (see Umana et al., 1999 Nat. Biotech. 17: 176-180).Eureka Therapeutics further describes genetically engineered CHO mammalian cells that are capable of producing antibodies with altered mammalian glycosylation patterns that lack fucose residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human monoclonal antibodies of the present invention can be produced in yeast or filamentous fungi that are engineered for mammalian-like glycosylation patterns and are capable of producing antibodies that lack fucose as a glycosylation pattern (see, for example, EP1297172B1).

[0079] In some embodiments, the antibody is an antigen-binding fragment. Antibody fragments can be obtained by conventional techniques, such as by fragmentation of full-length antibodies or by expressing nucleic acids encoding antibody fragments in recombinant cells (see, for example, Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). The properties of the fragments can then be tested or screened in the same manner as for the full-length antibodies described herein.

[0080] In some embodiments, the monoclonal antibody of the present invention is a scFv fragment comprising the VH and VL domains of the antibody of the present invention. In some embodiments, the scFv fragment of the present invention consists of the amino acid sequence shown in SEQ ID NO:11.

[0081] SEQ ID NO: 11>scFv antibody

[0082]

[0083] Nucleic acid molecules and their use in producing antibodies of the present invention:

[0084] The monoclonal antibodies of the present invention can be produced by any technology known in the art, for example, not limited to any chemical, biological, genetic or enzymatic technology, no matter alone or in combination.For example, the amino acid sequence of the desired sequence is known, and those skilled in the art can easily produce the antibody by standard techniques for producing polypeptides.For example, they can be synthesized using well-known solid phase methods, preferably using commercially available peptide synthesis instruments (for example, instruments manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies of the present invention can be synthesized by recombinant DNA technology well known in the art. For example, after the DNA sequence encoding the antibody is incorporated into an expression vector and such vectors are introduced into a suitable eukaryotic or prokaryotic host, the DNA expression product of the antibody can be obtained, and these hosts will express the desired antibody, and subsequently known techniques can be used to isolate the desired antibody from these hosts.

[0085] Therefore, another object of the present invention relates to a nucleic acid sequence encoding a monoclonal antibody of the present invention. In some embodiments, the nucleic acid sequence encodes a heavy chain and / or a light chain of a monoclonal antibody of the present invention.

[0086] Generally, the nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector. As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which additional DNA fragments can be connected. Another type of vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origin). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of the gene effectively connected thereto. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article. Generally, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this manual, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which function equivalently.

[0087] Therefore, another object of the present invention relates to a vector comprising the nucleic acid according to the invention.

[0088] Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or guide the expression of the antibody after being applied to a subject. Examples of promoters and enhancers used in the expression vectors of animal cells include early promoters and enhancers of SV40 (Mizukami T. et al., 1987), LTR promoters and enhancers of Moloney murine leukemia virus (Kuwana Y et al., 1987), promoters (Mason JO et al., 1985) and enhancers (Gillies SD et al., 1983) of immunoglobulin H chains, etc. As long as the gene encoding human antibody C region can be inserted and expressed, any animal cell expression vector may be used. Examples of suitable vectors include pAGE107 (Miyaji H et al., 1990), pAGE103 (Mizukami T et al., 1987), pHSG274 (Brady G et al., 1984), pKCR (O'Hare K et al., 1981), pSG1β d2-4- (Miyaji H et al., 1990), and the like. Other examples of plasmids include replicating plasmids containing a replication origin, or integrating plasmids, such as pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and the like. Detailed protocols for the production of such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 98 / 1.

[0089] Another object of the present invention relates to a host cell which has been transfected, infected or transformed with the nucleic acid and / or vector of the present invention.

[0090] The term "transformation" refers to the introduction of a "foreign" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0091] The nucleic acid of the present invention can be used to produce the monoclonal antibody of the present invention in a suitable expression system.The term "expression system" refers to a host cell and a compatible vector under suitable conditions, for example, for expressing a protein encoded by the exogenous DNA carried and introduced into the host cell by a vector. Common expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors (Baculo virus vectors) and mammalian host cells and vectors. Other examples of host cells include but are not limited to prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces (Kluyveromyces) or yeast (Saccharomyces yeasts), mammalian cell lines (for example, Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established mammalian cell cultures (for example, produced by lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al., 1980), rat YB2 / 3HL.P2.g11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc.

[0092] The present invention also relates to a method for producing a recombinant host cell expressing an antibody of the present invention, comprising the steps of: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or in vitro, (ii) culturing the obtained recombinant host cell in vitro or in vitro, and (iii) optionally, selecting a host cell capable of expressing and / or secreting the antibody. Such a recombinant host cell can be used to produce the antibody of the present invention.

[0093] The multispecific antibodies of the present invention:

[0094] Another object of the present invention relates to a multispecific antibody comprising a first antigen-binding site and at least a second antigen-binding site derived from a monoclonal antibody of the present invention.

[0095] According to the present invention, the multispecific antibody of the present invention binds to the extracellular domain of CD38 and the extracellular domain of another antigen of interest.

[0096] In some embodiments, the second antigen binding site is used to mount a killing mechanism, for example by binding an antigen on a human effector cell or by binding a cytotoxic agent or a second therapeutic agent.

[0097] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include cells derived from bone marrow or lymphocytes, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, mast cells, and granulocytes, such as neutrophils, eosinophils, and basophils. Some effector cells express specific Fc receptors (FcRs) and perform specific immune functions. In some embodiments, effector cells can induce ADCC, such as natural killer cells. For example, monocytes and macrophages express FcRs, participate in specific killing of target cells, and present antigens to other components of the immune system. In some embodiments, effector cells can engulf target antigens or target cells. The expression of specific FcRs on effector cells may be regulated by humoral factors such as cytokines. Effector cells can engulf target antigens or engulf or lyse target cells. Suitable cytotoxic and secondary therapeutic agents, including toxins (eg, radiolabeled peptides), chemotherapeutic agents, and prodrugs, are exemplified below.

[0098] In some embodiments, the second antigen binding site is used to form T cells.In some embodiments, the second antigen binding site is specific for the extracellular domain of CD3ε.

[0099] In some embodiments, a multispecific antibody of the invention comprises an antigen binding domain that comprises, consists of, or consists essentially of a single-chain variable fragment (scFv) of the invention.

[0100] In some embodiments, the antigen binding domain comprises a connecting peptide, which can be located between the light chain variable region and the heavy chain variable region.

[0101] Exemplary formats of multispecific antibody molecules of the invention include, but are not limited to: (i) two antibodies, one specific for CD38 and the other specific for another antigen, such as CD3ε, that are chemically cross-linked; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, such as two scFvs linked in tandem by an additional linker peptide; (iv) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain comprises two variable domains linked by a short peptide molecule (Wu et al., Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) molecules, in: Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) chemically linked bispecific (Fab′)2 fragments; (vi) Tandab, which is a fusion of two single-chain diabodies to form a tetravalent bispecific antibody with two binding sites for each target antigen; (vii) flexible antibodies, which are a combination of scFvs and diabodies to produce multivalent molecules; (viii) so-called “dock and lock” molecules, based on the “dimerization and docking domain” in protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (ix) so-called Scorpion molecules, comprising, for example, two scFvs fused to the two ends of a human Fab arm; and (x) diabodies.

[0102] Another exemplary form of bispecific antibodies is an IgG-like molecule with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known techniques, such as those known as Triomab / Quadroma (Trion Pharmaceuticals / Fresenius Biotechnologies), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatic matching (Amgen), LUZ-Y (Genentech), SEEDbody (EMDSerono), Biclonic (Merus) and DuoBody (Genmab A / S).

[0103] In some embodiments, DuoBody technology is generally used to obtain or obtain bispecific antibodies by controlled Fab arm exchange. WO2008119353 and WO2011131746 (both by Genmab A / S) describe in vitro methods for producing bispecific antibodies by controlled Fab arm exchange. In an exemplary method described in WO2008119353, bispecific antibodies are formed by incubating "Fab arms" or "half molecules" exchange (exchange of heavy chain and connected light chain) between two monospecific antibodies under reducing conditions, and both antibodies include IgG4-like CH3 regions. The resulting product is a bispecific antibody with two Fab arms that can include different sequences. In another exemplary method described in WO2011131746, the bispecific antibody of the invention is prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is an antibody of the invention: a) providing a first antibody comprising an immunoglobulin Fc region, the Fc region comprising a first CH3 region; b) providing a second antibody comprising an immunoglobulin Fc region, the Fc region comprising a second CH3 region; wherein the sequences of the first and second CH3 regions are different. Such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between the first and second CH3 regions; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining the bispecific antibody, wherein the first antibody is an antibody of the invention and the second antibody has a different binding specificity, or vice versa. For example, reducing conditions can be provided by adding a reducing agent, for example, selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) can further include, for example, restoring the conditions to make them non-reducing or less reducing, for example, by removing the reducing agent, for example, by desalting. Preferably, the first and second CH3 regions differ in sequence and contain only a few relatively conservative asymmetric mutations, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between each of the first and second CH3 regions. Further details on these interactions and how to achieve them are provided in WO2011131746, the entire contents of which are incorporated herein by reference. In some other embodiments, the bispecific antibodies of the present invention are symmetrical bispecific antibodies of the IgG4 class, comprising two heavy chains, each heavy chain comprising a variable domain, a CH1 domain, and a hinge region, wherein in each heavy chain: a cysteine ​​in the CH1 domain that forms an interchain disulfide bond with a cysteine ​​in the light chain is replaced by another amino acid; and optionally, one or more amino acids in the upper hinge region are replaced by cysteine, wherein the constant region sequences of each heavy chain are similar or identical, and the variable regions of each heavy chain are different. Such bispecific formats are described in International Patent Application WO2013124450.In some embodiments, the bispecific antibody of the present invention is an asymmetric antibody comprising two heavy chains or heavy chain fragments, each heavy chain or heavy chain fragment comprising at least one variable region, a hinge region and a CH1 domain, wherein the first heavy chain or fragment thereof is of the IgG4 class and has a) an interchain cysteine ​​at position 127 in the CH1 domain numbered according to the Kabat numbering system is substituted with another amino acid; and b) optionally, one or more amino acids located in the upper hinge region are substituted with cysteine, and wherein the second heavy chain or fragment thereof is characterized in that part or all of the chain has an amino acid sequence different from that of the first heavy chain at least in a region outside the variable region (e.g., constant region). The bispecific form of the antibody is described in International Patent Application WO2013124451.

[0104] In some embodiments, the multispecific antibody of the invention is a bispecific T cell engager (BiTE) antibody.

[0105] In some embodiments, the multispecific antibody of the invention is a Antibody.

[0106] In some embodiments, the multispecific antibody of the invention comprises the sequence set forth in SED IQ NO:12.

[0107] SEQ ID NO: 12>Bi38-3 sequence

[0108]

[0109] The present invention also provides a nucleic acid encoding the multispecific antibody of the present invention. In some embodiments, the nucleic acid is incorporated into a vector as described above.

[0110] Chimeric antigen receptors (CARs) and their use in producing host cells expressing said CARs:

[0111] The present invention also provides chimeric antigen receptors (CARs) comprising the antigen binding domain of the antibodies of the present invention.

[0112] As used herein, the term "chimeric antigen receptor" or "CAR" has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing an antigen binding domain of an antibody (e.g., scFv) connected to a T cell signaling domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity to selected targets in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies. In addition, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) α and β chains. Typically, the chimeric antigen receptor includes at least one VH and / or VL sequence of an antibody of the present invention. The chimeric antigen receptor of the present invention also includes an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0113] In some embodiments, the antigen binding domain comprises a connecting peptide, which can be located between the light chain variable region and the heavy chain variable region.

[0114] In some embodiments, the present invention provides CARs comprising an antigen binding domain that comprises, consists of, or consists essentially of a single-chain variable fragment (scFv) of an antibody of the present invention.

[0115] In some embodiments, the CAR of the present invention consists of the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0116] SEQ ID NO: 13>CAR CD38 1G

[0117]

[0118] SEQ ID NO: 14>CAR CD38 3G

[0119]

[0120] In some embodiments, CAR includes an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from the group consisting of CD28, 4-1BB, and CD3ζ intracellular domains. CD28 is a T cell marker that is important in T cell co-stimulation. 4-1BB delivers effective co-stimulatory signals to T cells, promoting the differentiation of T lymphocytes and improving long-term survival. CD3ζ binds to TCRs to generate signals and contains an immunoreceptor tyrosine-based activation motif (ITAM).

[0121] In some embodiments, the chimeric antigen receptors of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, disulfide bonds, or converted into acid addition salts and / or optionally dimerized or polymerized.

[0122] The present invention also provides a nucleic acid encoding a chimeric antigen receptor of the present invention. In some embodiments, the nucleic acid is incorporated into a vector as described above.

[0123] Therefore, another object of the present invention relates to a host cell modified to express the above-mentioned chimeric antigen receptor (CAR).

[0124] In some embodiments, the host cell is a cytotoxic lymphocyte.

[0125] As used herein, the term "cytotoxic lymphocyte" has its ordinary meaning in the art and refers to a lymphocyte that targets and destroys intracellular pathogens, such as viral agents, where a lethal attack on the infected target cell is desired to limit the spread of the infection. According to the present invention, "cytotoxic lymphocyte" includes cytotoxic T cells and natural killer cells.

[0126] In some embodiments, the host cell is a natural killer cell.

[0127] As used herein, the term "natural killer cell" has its ordinary meaning in the art and refers to a type of cytotoxic lymphocyte that is crucial to the innate immune system. NK cells function similarly to cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virally infected cells and respond to tumor formation.

[0128] In some embodiments, host cell is a T cell, for example, separated from peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). In some embodiments, T cell can be any T cell, for example, cultured T cell, for example primary T cell, or from cultured T cell line such as Jurkat, SupT1 etc. T cell, from the T cell obtained by mammals. If obtained from mammals, T cell can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph node, thymus or other tissues or liquids. T cell can also be enriched or purified. T cell can be any type of T cell and can be the T cell of any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th2 cells, CD8+ T cells (such as cytotoxic T cells), tumor infiltrating cells, memory T cells, immature T cells etc. T cell can be CD8+ T cell or CD4+ T cell.

[0129] Therefore, another object of the present invention is a CAR-T cell comprising the chimeric antigen receptor (CAR) of the present invention.

[0130] In some embodiments, the host cell is a pluripotent stem cell (PSC). PSCs can indeed be modified by CAR and can then be used to derive T cells (e.g., WO 2017100403). PSCs include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). iPSCs can generally be derived or produced by introducing a specific set of pluripotency-related genes or "reprogramming factors" into a given cell type. Reprogramming factors include but are not limited to OCT4 (also known as "POU5FL"), SOX2, cMYC, and KLF4, which are also known as Yamanaka factors, see Takahashi, K, Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." Cell 126(4): 663-76.

[0131] In some embodiments, the host cell is a hematopoietic stem cell. As used herein, the term "hematopoietic stem cell" or "HSC" refers to a blood cell that has the ability to self-renew and differentiate into blood cell precursors. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem cells exhibit a variety of phenotypes, such as Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin-CD34+CD38+IL-3aloCD45RA-, and Lin-CD34+CD38+CD10+ (Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors, Vol. 3: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). In the bone marrow microenvironment, stem cells self-renew and maintain the continuous production of hematopoietic stem cells, which give rise to all mature blood cells throughout life.In some embodiments, hematopoietic progenitor cells or hematopoietic stem cells are isolated from peripheral blood cells.

[0132] In some embodiments, if it is desired to optimize the safety and effectiveness of CAR therapy, CAR activity can be controlled. CAR activity can be controlled in a variety of ways. For example, using, for example, inducible apoptosis of caspases fused to a dimerization domain (see, for example, Di et al., N Egnl. J. Med. 2011 Nov. 3; 365 (18): 1673-1683), can be used as a safety switch in the CAR therapy of the present invention.

[0133] Treatment Methods and Pharmaceutical Compositions:

[0134] The antibodies, multispecific antibodies and CAR-T cells of the present invention are particularly suitable for therapy.

[0135] Therefore, another object of the present invention relates to a method for treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody of the present invention and / or the multispecific antibody of the present invention and / or the CAR-T cell population of the present invention.

[0136] In particular, the multispecific antibodies and CAR-T cells of the present invention are particularly suitable for treating cancer, and more particularly suitable for treating CD38-positive hematological malignancies.

[0137] Therefore, the present invention relates to the antibodies of the present invention and / or the multispecific antibodies of the present invention and / or the CAR-T cell populations of the present invention for use in treating cancer in a subject in need thereof.

[0138] In some embodiments, the cancer is a CD38-positive cancer.

[0139] In some embodiments, the CD38-positive cancer is a CD38-positive hematological malignancy.

[0140] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma.

[0141] In some embodiments, the cancer is one that does not express or expresses very low levels of B-cell maturation antigen (BCMA).

[0142] In some embodiments, the cancer is a CD38-positive cancer that does not express or expresses very low levels of B-cell maturation antigen (BCMA).

[0143] In some embodiments, the cancer is a CD38-positive hematological malignancy that does not express or expresses very low levels of B-cell maturation antigen (BCMA).

[0144] In some embodiments, the multispecific antibodies of the invention and / or the CAR-T cell populations of the invention trigger specific T cell-mediated lysis of CD38-positive cancers.

[0145] In some embodiments, the multispecific antibodies of the invention and / or the CAR-T cell populations of the invention trigger specific T cell-mediated lysis of CD38-positive cancers while protecting B cells and NK cells from T cell cytotoxicity.

[0146] As used herein, the term "subject" refers to any mammal, such as rodents, felines, canines, and primates. In particular, in the present invention, the subject is a human suffering from or susceptible to cancer, preferably a CD38-positive cancer, more preferably a CD38-positive hematological malignancy.

[0147] In some embodiments, the subject has a relapsed cancer. In some embodiments, the subject has a relapsed CD38-positive cancer. In some embodiments, the subject has a relapsed CD38-positive hematological malignancy.

[0148] In some embodiments, the subject is resistant to a monoclonal antibody targeting CD38 (eg, daratumumab).

[0149] In some embodiments, the subject has been treated with a monoclonal antibody targeting CD38 and has acquired resistance to the anti-CD38 monoclonal antibody.

[0150] In a specific embodiment, the antibodies of the present invention and / or the multispecific antibodies of the present invention and / or the CAR-T cell populations of the present invention can be used in combination with anti-cancer therapy.

[0151] Therefore, the present invention relates to a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention and / or a multispecific antibody of the present invention and / or a CAR-T cell population of the present invention, and ii) a classical method of treating cancer as a combined preparation.

[0152] As used herein, the term "anti-cancer therapy" has its ordinary meaning in the art and refers to any natural or synthetic compound used to treat cancer.

[0153] In a specific embodiment, the anti-cancer therapy is radiation therapy, antibody therapy, or chemotherapy.

[0154] As used herein, the term "chemotherapeutic agent" refers to a compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multikinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, uredopa, ethylenimines, and methylamines including octahinipine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide, and trimethylolomelamine; acetylcholine; ogenins (especially bullatacin and bullatacinone); carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including synthetic analogues of adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, mustard); nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994)); dynemicins including dinamycin A;an esperamicin and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine acid (6-diazo-5-oxo-L-norleucine), doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgen hormones such as calusterone and dromostanolone propionate. propionate, epitiostanol, mepitiostane, testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquon; elfornithine; elliptinium acetate acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansinoids and streptozotocin; maytansinoids; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; Razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromto1; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, N) and doxetaxel ( Rhone-Poulenc Rorer, Antony, France; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included within the definition are antihormonal drugs that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, the aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0155] As used herein, the term "radiotherapy" has its ordinary meaning in the art and refers to the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the treated area (target tissue) by destroying their genetic material, making it impossible for these cells to continue to grow. A commonly used radiotherapy involves photons, such as X-rays. Depending on the amount of energy they possess, these rays can be used to destroy cancer cells on the surface of the body or deeper. The higher the energy of the X-ray beam, the deeper the X-rays penetrate into the target tissue. The X-ray energy produced by linear accelerators and electron accelerators (betatrons) is getting higher and higher. Using a machine to focus radiation (such as X-rays) on the cancer site is called external beam radiation therapy. Gamma rays are another form of photons used for radiation therapy. Gamma rays are radiation that certain elements (such as radium, uranium and cobalt 60) spontaneously produce when they decompose or decay. In some embodiments, radiotherapy is external beam radiation therapy. Examples of external radiation therapy include, but are not limited to, traditional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams from different directions to closely follow the shape of the tumor; intensity-modulated radiation therapy (IMRT), such as helical tomotherapy, which shapes the radiation beam to closely follow the shape of the tumor and varies the radiation dose based on the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technology to provide real-time images of the tumor to guide radiation therapy; intraoperative radiation therapy (IORT), which delivers radiation directly to the tumor during surgery; stereotactic radiosurgery, which can deliver large, precise doses of radiation to small tumor areas in a single treatment session; hyperfractionated radiation therapy, such as continuous hyperfractionated accelerated radiation therapy (CHART), in which the subject is given more than one radiation therapy (fraction) per day; and hypofractionated radiation therapy, in which a larger dose of radiation therapy is given in each fraction, but in fewer fractions.

[0156] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that fully or partially reduces, inhibits, interferes with, or modulates one or more immune checkpoint proteins.

[0157] As used herein, the term "immune checkpoint protein" has its ordinary meaning in the art and refers to a molecule expressed by T cells that either increases signaling (a stimulatory checkpoint molecule) or decreases signaling (an inhibitory checkpoint molecule).

[0158] Examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, PD-L1, LAG-3, TIM-3, and VISTA.

[0159] As used herein, the terms "combination therapy," "combination therapy," or "therapeutic combination" refer to treatment with more than one drug. A combination therapy can be a dual therapy or dual therapy.

[0160] According to the present invention, the drugs used in the combination therapy are administered to the subject simultaneously, separately or sequentially.

[0161] As used herein, the term "simultaneous administration" refers to administration of two active ingredients by the same route and at the same time or substantially the same time. The term "separate administration" refers to administration of two active ingredients by different routes and at the same time or substantially the same time. The term "sequential administration" refers to administration of two active ingredients at different times, by the same or different routes.

[0162] Specifically, the CAR-T cell group prepared as described above can be used in the methods and compositions of adoptive immunotherapy according to known techniques or variants thereof that are obvious to those skilled in the art based on the present disclosure. For example, see U.S. Patent Application Publication No. 2003 / 0170238 of Grunberg et al.; See also U.S. Patent No. 4690915 of Rosenberg. At present, most adoptive immunotherapy is autologous lymphocyte therapy (ALT) for treatment using the patient's own immune cells. These therapies involve treating the patient's own lymphocytes. Typically, these therapies are accomplished by removing the patient's lymphocytes and converting the cells into CAR-T cell groups as described above. Once CAR-T cells are prepared with the CAR of the present invention, these ex vivo cells are reintegrated into the patient's body to enhance the immune system's ability to kill dead tumor cells. In some embodiments, the preparation method of the cell is to first harvest the cells from the culture medium and then wash and concentrate the cells in a culture medium and container system (a "pharmaceutically acceptable" carrier) suitable for a therapeutically effective amount. Suitable infusion media can be any isotonic formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition depends on the relative representation of T cells with the desired specificity, the age and weight of the recipient, the severity of the targeted condition, and the immunogenicity of the targeted Ags. These cell amounts can be as low as about 10 3 / kg, preferably 5×10 3 / kg; and up to 10 7 / kg, preferably 10 8 / kg. The number of cells will depend on the final use of the composition, as well as the cell types contained therein. For example, if specific cells for a particular Ag are desired, the population will contain greater than 70%, typically greater than 80%, 85%, and 90-95% of such cells. For the uses provided herein, the volume of cells is typically one liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. Clinically relevant numbers of immune cells can be distributed into multiple infusions that cumulatively equal or exceed the total amount of cells required.

[0163] For administration, the antibodies of the present invention are formulated into pharmaceutical compositions. Pharmaceutical compositions comprising the antibodies of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is mixed with a pharmaceutically acceptable carrier to form a mixture. If the administration of a composition can be tolerated by the recipient patient, the composition is referred to as a "pharmaceutically acceptable carrier." Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. (See, for example, Gennaro et al., Remington's Pharmaceutical Sciences (Mack Publishing Company, 9th edition, 1995)). The formulation may also include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the surface of the vial, etc. The form, route of administration, dosage, and course of treatment of the pharmaceutical composition naturally depend on the condition to be treated, the severity of the disease, the patient's age, weight, and sex, etc. The pharmaceutical composition of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc.

[0164] Typically, the pharmaceutical composition comprises a pharmaceutically acceptable carrier for an injectable formulation. These carriers may be, in particular, isotonic, sterile saline solutions (monosodium phosphate or disodium phosphate, sodium, potassium, calcium or magnesium, or mixtures of these salts), or dried, in particular freeze-dried, compositions, optionally containing sterile water or physiological saline, to form injectable solutions.

[0165] The dosage to be administered can be adjusted according to various parameters, in particular according to the mode of administration used, the pathology concerned or the desired duration of treatment.

[0166] To prepare a pharmaceutical composition, an effective amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0167] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or propylene glycol in water; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and must be fluid enough to permit easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0168] Solutions of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water and suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0169] The antibodies of the present invention can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed from free amino groups of proteins) and are formed from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0170] In some embodiments, the present invention provides the composition of the present invention.Carrier can also be a solvent or dispersion medium comprising for example water, ethanol, polyol (for example glycerol, propylene glycol and liquid polyethylene glycol etc.), its suitable mixture and vegetable oil.Suitable fluidity can be by using coating, for example lecithin, by maintaining required particle size and by using surfactant to keep in the case of dispersion.The effect of microorganisms can be prevented by various antibacterial and antifungal agents, for example parahydroxybenzoate, chlorobutanol, phenol, sorbic acid, thimerosal etc. In many cases, preferably include isotonic agent, for example sugar or sodium chloride.By using delayed absorption agent in the composition, for example aluminum monostearate and gelatin, the absorption time of injectable composition can be prolonged.

[0171] Sterile injectable solutions are prepared by mixing the required amount of the active compound with the various other ingredients listed above, as needed, in a suitable solvent, followed by sterile filtration. Dispersions are generally prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, whereby a powder of the active ingredient and any additional desired ingredients is extracted from a previously sterile-filtered solution.

[0172] Preparation of more or highly concentrated solutions for direct injection is also contemplated, where it is envisioned that the use of DMSO as a solvent would result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0173] When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is readily administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules and the like may also be used.

[0174] For parenteral administration in the aqueous solution, for example, the solution should be suitably buffered if necessary, and first the liquid diluent and enough saline or glucose are isotonic. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this respect, according to the disclosure, operable sterile aqueous medium will be known to those skilled in the art. For example, a dosage can be dissolved in 1ml isotonic NaCl solution, then added to 1000ml subcutaneous lysate or injected into the infusion site of suggestion (for example, referring to " Remington's Pharmaceutical Science ", the 15th edition, pages 1035-1038 and 1570-1580). According to the situation of the therapeutic object, some changes will inevitably occur in dosage. In any case, the person responsible for administration will determine the appropriate dosage for individual subjects.

[0175] The antibodies of the invention can be formulated in therapeutic mixtures to contain about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 or even about 10 mg of the antibody per dose. Multiple doses can also be administered.

[0176] In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; sustained-release capsules; and any other forms currently used.

[0177] In some embodiments, it is contemplated that liposomes and / or nanoparticles are used to introduce antibodies into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0178] Nanocapsules can typically capture compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, these ultrafine particles (approximately 0.1 μm in size) are typically designed using polymers that can degrade in vivo. The present invention contemplates the use of biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements and can be easily prepared.

[0179] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also known as multicellular vesicles (MLVs). MLVs typically have diameters ranging from 25 nm to 4 μm. Ultrasonication of MLVs results in the formation of vesicles with diameters ranging from 200 to Liposomes are small unilamellar vesicles (SUVs) in the range of 1.5 to 2.5 mm² / cm² that contain an aqueous solution in their core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0180] The present invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0181] Figure 1 : Bi38-3 dose-dependent autologous T cell-mediated lysis of patient MM tumor cells. CD138+ plasma cells were purified from the patient's bone marrow and co-cultured with autologous CD3+ T cells isolated from PBMC at an E:T cell ratio of 5:1 for 24 hours. The culture was analyzed by FACS to monitor the number of CD138+ cells that fell into the live gate. Shown are the averages of three experiments, indicating the percentage of live CD138+ cells in 4 different patients at diagnosis and 3 different patients at relapse (relative to the untreated case). The histogram shows the average effects of Bi38-3 alone, T cells alone, and T cells containing Bi38-3 (100 ng / mL) on tumor plasma cells in 5 patients at diagnosis (top) and 3 patients at relapse (bottom). Standard deviations are shown, and p values ​​were calculated using Student's t test (*p<0.05, **p<0.01, ***p<0.001).

[0182] Figure 2 : In vivo activity of Bi38-3 in the MM1.Slue xenograft mouse model. A. Treatment schedule. NSG mice were inoculated 5.10 6 MM1.SLue cells (iv) and treatment was started on day 13, when similar levels of luciferase-expressing MM cells were detected in all mice. Purified T cells (5.10 6cells / mouse) were injected intravenously with Bi38-3 or PBS (blue arrows). Bi38-3 (0.1 mg / Kg) was injected intravenously every day for 9 days (black arrows). Luciferase activity (red arrows) was measured using an IVIS imaging system 7, 11, 13, 15, 18 and 21 (or 22) days after tumor injection. B. Continuous bioluminescence imaging for evaluating myeloma progression / regression. Radiation was measured on the whole body of the mouse. The image on the left represents the luminescence 7 days after inoculation with MM.1S myeloma cells and before the start of treatment. The image on the right represents 18 days after inoculation with MM.1S cells and 4 days after treatment with Bi38-3 (upper panel) or vehicle (lower panel). The radiation color scale is indicated on the right. C. Longitudinal radiation levels of mice treated with vehicle (blue line) and Bi38-3 (red line). Representative groups of 9 mice were inoculated with T cells from 2 independent donors. The p value was calculated using Student's t-test on day 22 (***p<0.001).

[0183] Figure 3 : In vitro activity of anti-CD38 CAR-T cells. A. Schematic diagram of different chimeric antigen receptor (CAR) and costimulatory receptor (CCR) structures. First-generation (1G) CARs include the CD3ζ signaling domain, and third-generation CARs (3G) contain CD28, 4-1BB, and CD3z signaling domains. CCR includes CD28 and 4-1BB signaling domains but lacks the CD3ζ domain. CAR Mock does not have the anti-CD38 scFv region. B. Cytotoxic activity of different CAR-T pairs against CD38-expressing MM (MM.1S and RPMI8226) and CD38-negative fibroblast (HEK293) cell lines in vitro. Luciferase-expressing cells were cultured with the indicated CAR-T cells at different effector / target ratios (E:T) for 20 hours. Cytotoxic activity was determined by measuring the level of luciferase in the culture. Representative of 4 independent experiments.

[0184] Figure 4: Sensitivity of blood cells and bone marrow hematopoietic progenitor cells to Bi38-3. A. Bi38-3-mediated T cell lysis Treg versus MM1.S cells. In the presence of MM1.S cells, purified T cells from healthy donors (n=3) were co-cultured with increasing concentrations of Bi38-3 for 24 hours. B. Bi38-3-mediated T cell lysis CD34+ bone marrow hematopoietic progenitor cells versus MM1.S cells. In the presence of MM1.S cells, paired CD34+ hematopoietic progenitor cells and T cells purified from the bone marrow of healthy donors (hip surgery) (n=4) were co-cultured with increasing concentrations of Bi38-3 for 24 hours. The number of live CD20+ (B cells), FoxP3+ (Treg cells), CD34+ (hematopoietic progenitor cells) and CD138+ (MM1.S cells) were calculated by FACS using counting beads and expressed as ratios to the untreated control group. The histogram shows the ratios of B, Treg, CD34+ hematopoietic progenitor cells, and MM.1S cells at each Bi38-3 concentration, with error bars representing SD. Normality of the CD34+ population was established by the Shapiro-Wilk normality test, and p values ​​were determined by unpaired Student's t-test (*p<0.05, **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0185] Example 1: Novel CD38 / CD3 bispecific T cell engagers for the treatment of multiple myeloma.

[0186] method:

[0187] Construction and purification of Bi38-3.

[0188] Bi38-3 is produced by the fusion of two scFvs from mouse hybridomas producing antibodies against human CD38 and CD3ε (BB51 and OKT3, respectively), connected by a 15-amino acid glycine-serine (G4S1x3) spacer. The human CD8 leader peptide was genetically linked to the N-terminus of the fusion fragment, and Myc-tag and His-tag sequences were introduced at the C-terminus. The coding sequence of Bi38-3 was cloned into the pCDNA3 expression vector (ThermoFisher) and confirmed by sequencing. This vector was transiently transfected into HEK-293T cells, and the 55.6 Kd protein corresponding to Bi38-3 was purified from the supernatant using a HisTrap HP column (GE). The integrity of Bi38-3 was analyzed by Coomassie blue staining and western blot using an anti-Myc-tag antibody.

[0189] cell lines

[0190] MM1.S, NCI-H929, and KMS-11 MM cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin, 10 μg / mL streptomycin, and 2 mM L-glutamine. All cell lines were monitored for mycoplasma contamination. Luciferase-expressing MM1.S and KMS-11 cells (KMS111uc and MM1.Sluc) were generated by lentiviral transduction of a luciferase expression vector (Addgene, pLenti CMVPuro LUC (w168-1), a gift from Eric Campeau and Paul Kaufman). To generate CD38-deficient MM1.S cells, two pairs of RNA guides were designed to delete exons 2 and 3 of the CD38 gene. The annealed oligonucleotides were cloned into the pX458 vector (Addgene plasmid ID 48138, a gift from Dr. Feng Zhang) and verified by sequencing. For Cas9 deletion, 2 × 10 6 MM1.Sluc cells were nucleofected with 2 μg of each Cas9 vector and incubated in culture for 24 h. GFP-positive cells were sorted by FACS and cloned into 96-well plates. Subclones were analyzed for CD38 expression by flow cytometry, and CD38-negative clones were selected for further analysis.

[0191] Blood and bone marrow samples

[0192] Peripheral blood samples from healthy donors were obtained from the Etablissement Francais du Sang (EFS). Fresh tumor plasma cells were collected from the buffy coat of bone marrow aspirates from myeloma patients and further purified using anti-CD138-coated beads (Miltenyi). In all cases, informed consent was obtained from patients and volunteers in accordance with the Declaration of Helsinki and approved by the Internal Review Board of St. Louis Hospital.

[0193] Flow cytometry and cytotoxicity

[0194] To determine lysis of KMS-11luc or MM1Sluc MM cell lines, purified peripheral T cells (effectors) were incubated in culture medium (RPMI, 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin, 10 μg / mL streptomycin, and 2 mM L-glutamine) with luciferase-expressing MM1.S or KMS-11 cells (targets) at a 1:5 effector to target ratio in flat-bottom 96-well plates with varying concentrations of Bi38-3. The cells were cultured according to the manufacturer's instructions (Bright-Glo TM Luciferase assay system (Promega), within 20 minutes after adding firefly luciferase substrate, the luciferase signal produced by surviving MM cells 24 hours later was measured using a CLARIOstar Plus luminometer plate reader (BMG LABTECH GmbH, Ortenberg, Germany). Under similar co-culture conditions, T cell cytotoxicity against primary cells was determined by flow cytometry. Effector T cells were co-cultured with purified target MM cells and serial dilutions of Bi38-3. After incubation, anti-human CD138-allophycocyanin (APC) (clone 44F9-Miltenyi Biotec), anti-CD20-Brilliant Violet (BV) 605 (clone 2H7-BioLegend), anti-CD4-APC / Cyanine 7 (clone RPA-T4-BioLegend), and anti-CD8-BV421 (clone RPA-T8-BioLegend) antibodies were added to the cells to distinguish target cells from effector cells, and the number of viable cells was determined by flow cytometry using Brightcount beads (ThermoFisher). All FACS acquisitions were performed on a Canto II (Beckon Dickinson). Analysis and calculation of the proliferation index were performed using FlowJo.

[0195] T cell activation and proliferation assays

[0196] To detect activation, effector cells (T cells) and target cells (MM1.S) were co-cultured at a 5:1 ratio for 24 hours, stained with anti-human CD4-APC-Cyanocyanine 7 (clone RPA-T4), anti-CD8-BV421 (clone RPA-T8), anti-CD25-Phycoerythrin (Pe) / Cyanocyanine 5 (clone BC96), and anti-CD69-BV711 (clone FN50) antibodies (all from Biolegend), and analyzed by flow cytometry. To analyze proliferation, T cells were labeled with CellTrace Violet dye (ThermoFisher) and stimulated with MM1.S cells or MM1.S-CD38KO with or without Bi38-3 (10 ng / ml) for 96 hours. Cells were stained with anti-CD4-APC / Cy7 (clone RPA-T4) and anti-CD8-BV421 (clone RPA-T8) antibodies and analyzed by flow cytometry.

[0197] Quantification of cytokines in cell culture supernatants

[0198] Using BD TM The concentration of cytokines in supernatants from cytotoxicity assays was analyzed using the CBA Human Th1 / Th2 Cytokine Kit II (Beckon Dickinson). Data were acquired on a Canto II and analyzed using an FCAP Array. TM Analyses were performed using the ELISA software (Beckon Dickinson).

[0199] Mouse systemic tumor models

[0200] We used 6- to 12-week-old NOD / SCID / IL-2Rγnull mice (The Jackson Laboratory) according to a protocol approved by the Institutional Animal Care and Use Committee (Comité d'éthique Paris-Nord). Mice were inoculated with 5 × 10 6 MM1.Sluc cells were then transfected 14 days later with 5 × 10 6Purified human T cells (purified using the Pan T cell isolation kit from Miltenyi Biotec) contained (or did not contain) 0.08 mg / kg of Bi38-3. Bi38-3 (or PBS for the control group) was injected into the tail vein repeatedly every day for 9 days. No randomization or blinding was used. Bioluminescence imaging was performed every 3 or 4 days. Mice were injected intraperitoneally with 240 μL of D-luciferin (15 mg / mL) (XenoLight D-luciferin potassium salt, Perkin Elmer), and 15 minutes later, images were collected using the IVIS imaging system (PerkinElmer) and Living Image software (PerkinElmer) in a 25 cm field of view at a medium bin level and different exposure times. All mice were sacrificed 22 days after inoculation of MM cells.

[0201] result:

[0202] Construction, production and binding properties of Bi38-3

[0203] Bi38-3 consists of two scFvs derived from mouse hybridomas, raised against human CD38 and CD3ε (BB51 and OKT3, respectively), linked by a 15-amino acid glycine-serine (G4S1x3) spacer (not shown). The amino acid sequences of the anti-CD38 heavy and light chain variable domains corresponding to the anti-CD38 scFv and Bi38-3 are described in Table 1.

[0204] Table 1: Amino acid sequences of immunoglobulins (IgH and Igk) from BB515 hybridoma (anti-CD38), the corresponding scFvs, and Bi38-3.

[0205]

[0206]

[0207] The anti-CD38 scFv is located at the N-terminus, the anti-CD3ε scFv is located at the C-terminus, followed by Myc-Tag and Hisx6-Tag sequences (not shown). Western blot analysis of HEK-293 cells transiently transfected with the Bi38-3 expression vector showed a unique protein recognized by the anti-Myc-Tag antibody with an expected size of 55.6 Kd (data not shown). Bi38-3 was purified from the culture supernatant of transiently transfected HEK-293 cells using a HisTrap HP column (GE). The purity of the monomeric Bi38-3 protein was confirmed by gel electrophoresis and Coomassie blue staining (data not shown). The binding of Bi38-3 to MM1.S, KMS11, and NCI-H939 MM cells expressing CD38 was analyzed by flow cytometry using an anti-Fab antibody that recognizes the scFv domain. We observed that Bi38-3 was detected on the surface of MM cell lines, with less intense staining of KMS11 cells, which express low levels of CD38, and stronger signals for MM1.S and NCI-H929 cells, which display higher levels of CD38 (data not shown). To verify the specificity of Bi38-3 for CD38, we used the CRISPR / Cas9 method to inactivate the CD38 gene in MM1.S cells (MM1.S-KO) (data not shown). FACS analysis showed that Bi38-3 could not be detected on the surface of CD38-negative MM1.S-KO cells (data not shown). Therefore, purified Bi38-3 effectively and specifically recognizes CD38 on MM cells.

[0208] Bi38-3 induces T cell activation and proliferation in response to MM cells in vitro

[0209] We next studied the reaction of T cells to MM cells triggered by Bi38-3. First, we performed FACS analysis to measure the proliferation index of T cells stained with purple fluorescence. In the presence of MM1.S target cells (T), stimulating donor effector T cells (E) with Bi38-3 resulted in strong proliferation, with an average of 5 cell divisions (amplification index) after 4 days, slightly higher than the level induced by treatment with anti-CD3 / CD28 beads (data not shown). Proliferation requires expression of CD38 on target cells because T lymphocytes cultured with CD38-deficient MM1.S cells and Bi38-3 do not proliferate. In addition, independent Bi38-3 culture and independent MM1.S cell culture did not induce significant T cell expansion.

[0210] Secondly, we analyzed the expression of early activation markers CD69 and CD25 on donor T cells. After overnight co-culture with MM1.S cells, CD4 and CD8 T cells readily upregulated both markers in a Bi38-3 dose-dependent manner, with up to 80% CD69-positive T cells detected at the highest concentration (data not shown). In contrast, we observed weaker percentages of T cells expressing CD25 and CD69 upon stimulation with 100 ng / mL Bi38-3 alone (15% and 30%, respectively). In addition, co-culture with MM1.S target cells alone did not induce the expression of activation markers (data not shown). Consistent with this, co-culture with MM1.SKO cells and Bi38-3 triggered lower CD69 and CD25 induction compared to co-culture with wild-type MM1.S cells (data not shown), demonstrating that CD38 expression on target cells enhances the upregulation of activation markers.

[0211] Finally, we monitored the production of cytokines triggered by Bi38-3. Co-culture of donor T cells with MM1.S triggered the production of interferon-γ (IFNg), tumor necrosis factor-α (TNFa), interleukin-2 (IL-2), IL-4, and IL-10 in a Bi38-3 dose-dependent manner (data not shown). In contrast, stimulation with Bi38-3 alone or co-culture with MM1.S alone failed to induce T cells to secrete any of these cytokines (not shown). Together, these results indicate that Bi38-3 directs T cell proliferation, activation, and cytokine release in response to CD38-expressing MM cells in vitro.

[0212] Bi38-3 induces CD38-dependent T cell-mediated killing of MM cells in vitro

[0213] To evaluate the functionality of Bi38-3, we performed co-culture assays to measure the cytotoxic activity of effector T cells isolated from PBMCs of healthy donors against firefly luciferase expressing target KMS11 and MM1.S MM cell lines. Luciferase levels, indicating the number of remaining viable MM target cells, were compared to those observed in untreated controls to determine the percentage of killing in the presence of varying concentrations of Bi38-3. T cells readily killed KMS11 target cells in a Bi38-3 dose-dependent manner, with half-maximal effective concentrations (EC 50 ) was approximately 5 ng / mL, equivalent to 0.09 nM of the 55.6 Kd protein (data not shown). Bi38-3-mediated T cell cytotoxic activity was also observed in co-culture with MM1.S cells. However, in this cell line expressing higher levels of CD38, EC 50The concentration of Bi38-3 in the co-culture was ten times lower (0.5 ng / mL), indicating that Bi38-3 is more efficient. In contrast, the viability of MM1.S or KMS11MM cells was not affected by co-culture with T cells or Bi38-3 (data not shown). In addition, Bi38-3 induced poor T cell-mediated killing of MM1.S-KO cells. Even at the highest dose of Bi38-3 (1 μg / mL), about half of the CD38-deficient MM1.S cells survived in co-culture (data not shown). Therefore, Bi38-3 has effective T cell cytotoxic activity against MM cells expressing CD38.

[0214] Bi38-3 induces autologous T cell-mediated tumor plasma cell killing in vitro

[0215] We next analyzed the potential of Bi38-3 to induce MM cell lysis by autologous T cells. Target tumor plasma cells isolated from patients at diagnosis were incubated with purified autologous effector T cells at an E:T ratio of 1:5 in the presence of varying concentrations of Bi38-3. FACS analysis of overnight co-cultures revealed a Bi38-3 dose-dependent decrease in the number of viable CD138-positive MM cells, with EC 50 Range is 0.5 to 1 ng / mL, depending on the patient ( Figure 1 Importantly, Bi38-3 showed no cytotoxicity against fresh primary MM cells in the absence of T cells. Bi38-3-induced cytotoxicity of autologous T cells was further investigated in tumor plasma cells from relapsed MM patients and demonstrated similar efficacy, EC 50 Range 0.2 to 1 ng / mL ( Figure 1 ). Thus, in these in vitro experiments, Bi38-3 triggered autologous T cell-mediated tumor plasma cell killing at diagnosis and relapse.

[0216] In vitro specific activity of Bi38-3 against MM cells with high CD38 expression

[0217] Although CD38 is highly expressed on plasma cells, it is also expressed on various cell types, including hematopoietic cell subsets. To investigate the effects of Bi38-3 on blood cells, PBMCs from donors were treated with various concentrations of Bi38-3 for 24 hours, and the different cell populations were analyzed by FACS (data not shown). We observed that the percentage of CD14-expressing monocytes that fell into the live gate was significantly reduced in a Bi38-3 dose-dependent manner (data not shown). In contrast, the percentages of CD4 and CD8 T lymphocytes, which together represent approximately 60% of the PBMC population, increased slightly in response to Bi38-3 as the percentage of CD14-positive cells decreased. Similarly, even at high concentrations of Bi38-3 (100 ng / mL), B (CD19+) and NK (CD56+) cell populations were slightly elevated or remained at similar levels (approximately 10% and 5%, respectively) (data not shown). Next, we investigated whether CD38 expression on the surface of blood cells was affected by Bi38-3. FACS analysis showed that the mean fluorescence intensity (MIF) of CD38 on T, B, and NK cells remained similar in cultures containing increasing doses of Bi38-3 (data not shown). Consistent with this, CD38 expression on CD14+ bone marrow cells did not decrease significantly, although analysis could not be performed at higher doses of Bi38-3 (1 and 100 ng / mL) because no or too few cells could be detected. To compare the activity of Bi38-3 in CD38 high (CD38hi) MM and CD38 intermediate (CD38int) cells, we performed co-culture assays with MM1.S expressing high levels of CD38 (data not shown), freshly isolated B cells, expressing moderate amounts of CD38 (data not shown), and autologous T cells. After overnight culture, the percentage of live CD20-positive B cells and CD138-positive MM1.S cells was analyzed by flow cytometry. We observed a decrease in the percentage of MM1.S cells at a Bi38-3 concentration of 0.1 ng / mL, and this decrease was more pronounced at higher doses (data not shown). In contrast, the percentage of surviving CD20-positive B cells remained unchanged even at high Bi38-3 concentrations compared to untreated conditions (data not shown).

[0218] We developed a similar autologous triple culture assay to investigate the potential cytotoxic effects of Bi38-3 on CD34+ bone marrow hematopoietic progenitor cells and regulatory T cells (Tregs), both of which express low levels of CD38. Although Bi38-3 readily induced MM cell death at low concentrations (10-2 ng / mL and above), we found that it did not trigger significant T cell-mediated cytotoxicity against Foxp3+ Tregs ( Figure 2Similarly, at concentrations below 10 ng / mL, there was no significant toxicity to CD34+ hematopoietic progenitor cells (>40% survival rate), and at the highest concentration, there was moderate toxicity ( Figure 2 B). In summary, our results indicate that Bi38-3 does not impair CD38 surface expression and triggers T cell-mediated killing only of cells expressing high levels of CD38, while exhibiting no or limited toxicity towards cells expressing intermediate levels of CD38, such as hematopoietic progenitors, B, T, or NK cells.

[0219] In summary, our results indicate that Bi38-3 does not impair CD38 surface expression and triggers T cell-mediated killing of CD38hi cells, while having no significant activity against CD38int cells.

[0220] Bi38-3 controls the expansion of MM cells in vivo

[0221] The in vivo anti-tumor activity of Bi38-3 was evaluated using a human MM xenograft mouse model. MM1.Sluc cells were injected into the tail vein of NSG mice, and luciferase levels were measured every 4 days using an IVIS imaging system. 14 days after MM1.S injection, purified human T cells were transplanted intravenously with or without Bi38-3 (0.08 mg / kg). Treatment with Bi38-3 or vehicle was repeated daily for 7 days ( Figure 2 A). Eleven days after tumor cell injection, all mice showed similar levels of radiance (luciferase), indicating that MM cells had effectively engrafted into the host animals prior to Bi38-3 treatment ( Figure 2 B). While control mice showed rapid tumor progression, all Bi38-3 treated animals showed a five-fold reduction in tumor growth within the first 4 days of Bi38-3 treatment ( Figure 2 C). After 7 days, the level of luciferase-expressing MM cells in Bi38-3-treated mice was only one-tenth of the initial level and 50-fold lower than that in the untreated control group. These results indicate that Bi38-3 can effectively control MM tumor progression in vivo.

[0222] Past Discussion:

[0223] Here we report the development of Bi38-3, a novel anti-CD38 / CD3 bispecific T cell-engaging antibody that triggers specific T cell-mediated lysis of CD38-positive MM cells in vitro, ex vivo, and in vivo.

[0224] Monoclonal antibodies (Mab) targeting CD38 are effective in the treatment of MM. 13 Daratumumab is an anti-CD38 Mab approved for MM, whether used alone or in combination with 14Or in combination with normal standard of care 2，15 Both showed good therapeutic effects. These clinical data indicate that CD38, which is highly expressed on tumor plasma cells, is the preferred target for MM immunotherapy. However, despite significant improvements in survival, many patients treated with daratumumab eventually relapse due to resistance mechanisms, including FcγR-dependent downregulation of CD38 on tumor cells and inhibition of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis. 16 Bi38-3 lacks the Fc region found on natural immunoglobulins and mobilizes cytotoxic T cells through its anti-CD3 scFv without downregulating CD38 expression on target cells (data not shown). Therefore, Bi38-3-mediated T cell killing of MM cells is not affected by the resistance mechanism of anti-CD38 mAbs, such as daratumumab, which is related to the binding of therapeutic antibodies to FcγRs. Similarly, in relapse 17 The upregulation of complement inhibitors CD55 and CD59 on cytotoxic cells observed with MM and thought to contribute to drug resistance should not occur after Bi38-3 treatment. In addition, MM is characterized by a defective immune system, and the standard of care regimen that associates IMIDs with dexamethasone may limit the effectiveness of cytotoxic cells. However, our data suggest that Bi38-3 mediates autologous T cell-mediated killing of tumor plasma cells with similar efficiency at diagnosis and relapse ( Figure 1 Together, these data suggest that Bi38-3 can effectively eliminate MM cells in patients resistant to standard therapy, including those including daratumumab.

[0225] Because CD38 is expressed on the surface of blood cells, including T, B, and NK lymphocytes 18 , anti-CD38 mAbs may target them and impair their function. In fact, daratumumab has been shown to eliminate regulatory T cells 19 This process may be related to the increase in the number and activation of T cells at the beginning of treatment. 16 Furthermore, daratumumab treatment resulted in NK cell depletion. 20 , and may contribute to the patient's susceptibility to infection 21 Our data showed that Bi38-3 had no significant effects on T, B, and NK cells in vitro (data not shown). We also report that even at high doses (10 ng / mL), it readily induced T cell-mediated killing of MM cells while protecting B cells from T cell cytotoxic activity. Interestingly, these results contrast with the activity of AMG424, a recently described anti-CD38 BiTE, which triggered T cell cytotoxicity against B, T, and NK cells in vitro. 22Although additional experiments are needed to evaluate the toxicity of Bi38-3 in in vivo models, particularly against myeloid cells, our results suggest that Bi38-3 can effectively induce the elimination of MM cells without affecting cells expressing low levels of CD38.

[0226] Recently, bispecific antibodies against Fc receptor-like 5 (Fcrl5 or FcHR5) or B cell maturation antigen (BCMA) have been reported. 10，12,23 BI 836909, a BiTE targeting BCMA and CD3ε, was shown to eliminate MM cells at a dose of 0.5 mg / kg in the NCI-H929 mouse xenograft model. 23 Similarly, EM801, an asymmetric bispecific antibody containing a mutant Fc region, was shown to be effective at the same dose (0.5 mg / kg) 10 BCMA expression is restricted to germinal B cells, including memory B cells and normal and malignant plasma cells. 24 However, although the majority of MM patients express BCMA, 6–9% of cases are negative for this marker, and expression levels on neoplastic plasma cells are heterogeneous among patients. 25，26 Furthermore, in MM patients treated with T cells expressing anti-BCMA chimeric antigen receptors, BCMA was expressed in tumor plasma cells. 27 14,15,16 These findings suggest that the development of bispecific antibodies with enhanced potency and safety could improve the treatment of MM.

[0227] Our data showed that targeting CD38 with Bi38-3 at a dose of 0.1 mg / kg was effective in xenograft models ( Figure 2 ), which is significantly lower than the dose reported for BCMA bispecific antibodies in similar mouse models. 10,23 Therefore, Bi-38-3 may represent an attractive therapeutic option for MM cases expressing no or very low BCMA levels.

[0228] Although BiTEs have demonstrated efficacy in a variety of malignancies, their clinical development is hampered by their short half-life in patients, requiring continuous infusion via pumps. 9 The CD19 / CD3 BiTE Blinatumomab was recently approved for the treatment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Interestingly, early phase 2 clinical trials have shown that MRD negativity, which is associated with improved survival, can occur by the end of the first treatment cycle. 28，29While the optimal number of cycles of blinatumomab in the MRD setting remains to be further investigated in ALL, clinical data suggest that limited BiTE therapy may improve outcomes in MRD+ patients over time. In our study, we showed that Bi38-3 was able to reduce tumor burden in vivo by 6-fold in just 3 days ( Figure 2 C). Therefore, this rapid and significant activity against tumor plasma cells suggests that, like blinatumomab in ALL, Bi38-3 can eliminate MRD in MM patients after a limited number of cycles and improve outcomes after standard therapy.

[0229] In summary, the data presented in this manuscript demonstrate that Bi38-3 is a selective and highly effective compound for the treatment of MM, either in the first-line or relapse setting, and support further evaluation in MM patients.

[0230] Example 2: Production of CAR-T cells

[0231] method:

[0232] Production of transduced CAR-T cells

[0233] HEK293 cells were transfected with calcium phosphate using 10 μg of CAR constructs and auxiliary plasmids (psPAX2 and pMD2.G). 12 hours after transfection, complete culture medium (DMEM, 10% FVS) was updated, and 2 days after transfection, the cell-free supernatant containing retroviral particles was collected, concentrated by centrifugation and used for transduction. T cells (purified using the Pan T cell isolation kit from Miltenyi Biotec) were stimulated with CD3 / CD28 beads (ThermoFisher) in culture medium (RPMI1640, 10% FBS, penicillin; 100 U / mL, streptomycin; 100 mg / mL). After 16 hours, the cells were transferred to a 6-well plate (Falcon) coated with retronectin-coated (15 mg / mL) of reverse transcriptase binding protein (Takara) and transduced overnight with the specified lentiviral particles. 72 hours after transduction, GFP and CAR expression were measured by flow cytometry to determine transduction efficiency. The transduced CAR-T cells accounted for more than 80% of the total cells used for in vitro experiments.

[0234] result:

[0235] Anti-CD38 CAR-T cells triggered MM cell lysis in vitro.

[0236] Since Bi38-3-induced T cells mediated lysis of MM cells (see Example 1), we investigated whether its anti-CD38 scFv could trigger direct cell killing by transgenic T cells in the context of a chimeric antigen receptor (CAR). We developed a first-generation anti-CD38 CAR construct (CAR CD38 1G) containing a BB51-derived anti-CD38 scFv, the hinge and transmembrane regions of human CD8, and the CD3ζ signaling domain ( Figure 3 A). Because the combination of the CD3ζ activation domain and the costimulatory signaling domain has been shown to enhance the activity of CAR, we also constructed a third-generation anti-CD38 CAR (CAR CD38 3G), which consists of an anti-CD38 scFv, a CD28 transmembrane region, and the signaling domains of CD28, CD137 (4-1BB), and CD3ζ in sequence ( Figure 3 A). As controls, we generated a CAR without the scFv domain (CAR Mock) and a costimulatory CAR (CCR CD38) that resembles CD38 3G but lacks the CD3ζ signaling domain. The protein sequences corresponding to these constructs are described in Table 2. The DNA sequence encoding each CAR was cloned into a lentiviral vector allowing for GFP co-expression to produce viral particles and transduce donor T cells. All CAR constructs expressed upon transduction of human T lymphocytes (CAR-T); however, the expression level of CAR CD38 3G was lower than that of the other constructs (data not shown). Despite this difference, all transduced T cells could be expanded in vitro for more than 2 weeks and stably expressed the CAR, indicating that the potential fratricidal effect of CD38-expressing T cells still allowed CAR-T culture (data not shown). To investigate the cytotoxic function of effector CAR-T cells (E), we performed co-culture experiments with different ratios of luciferase-expressing target cells (T). Compared with mock and CCR CD38 negative controls, MM1.S and RPMI8288MM cells expressing CD38 were readily lysed by CAR CD38 1G and 3G ( Figure 3 B). Even at low E / T ratios (<2.5), anti-CD38 CAR-T cells were able to kill MM cells, while CCR CD38 and Mock transduced T cells showed poor cytotoxicity. In contrast, CARCD38 1G and 3G did not induce or barely induced lysis of CD38-negative HEK293 cells ( Figure 3 B) Thus, the anti-CD38 scFv derived from the BB51 hybridoma can effectively drive T cell cytotoxicity against MM cells in the context of different CAR constructs.

[0237] Table 2:Amino acid sequences of the first- and third-generation anti-CD38 CARs (CAR CD38 1G and 3G, respectively) and the costimulatory CAR (CCR CD38).

[0238]

[0239]

[0240] References:

[0241] Throughout this application, various references describe prior art related to the present invention. The disclosures of these references are hereby incorporated into the present disclosure by reference.

[0242] 1Dimopoulos, M.A., Richardson, P.G., Moreau, P., & Anderson, K.C. Current treatment landscape for relapsed and / or refractory multiple myeloma. Journal of Oncology 12, 42–54, doi: 10.1038 / nrclinonc.2014.200 (2015).

[0243] Dimopoulos, M.A., et al. Daratumumab, lenalidomide, and dexamethasone in the treatment of multiple myeloma. New England Journal of Medicine 375, 1319–1331, doi: 10.1056 / NEJMoa1607751 (2016).

[0244] Facon, T. et al. Daratumumab plus lenalidomide and dexamethasone in previously untreated myeloma. New England Journal of Medicine 380, 2104–2115, doi: 10.1056 / NEJMoa1817249 (2019).

[0245] Moreau, P. et al. Bortezomib, thalidomide, and dexamethasone with or without daratumumab before and after autologous stem cell transplantation in newly diagnosed multiple myeloma (CASSIOPEIA): a randomized, open-label, phase 3 study. The Lancet, doi: 10.1016 / S0140-6736(19)31240-1 (2019).

[0246] 5Bannas, P., Hambach, J. & Koch-Nolte, F. Nanobodies and Nanobody-based Human Heavy Chain Antibodies as Antitumor Therapeutics. Front Immunol 8, 1603, doi:10.3389 / fimmu.2017.01603 (2017).

[0247] 6 Brischwein, K. et al. MT110: a novel bispecific single-chain antibody construct with high potency in eradicating established tumors. Molecular Immunity 43, 1129-1143, doi: 10.1016 / j.molimm.2005.07.034 (2006).

[0248] 7Foster, JB, and Maude, SL. New advances in immunotherapy for pediatric leukemia. Pediatrician 30, 25–29, doi: 10.1097 / MOP.0000000000000572 (2018).

[0249] 8 Zimmerman, Z., Maniar, T. & Nagorsen, D. Unleashing the clinical power of T cells: the CD19 / CD3 bispecific T cell engager (BiTE(R)) antibody construct blinatumomab as a potential therapy. International Immunology 27, 31-37, doi:10.1093 / intimm / dxu089 (2015).

[0250] 9 Velasquez, MP, Bonifant, CL & Gottschalk, S. Redirecting T cells to hematological malignancies with bispecific antibodies. Blood 131, 30–38, doi: 10.1182 / blood-2017-06-741058 (2018).

[0251] 10Seckinger, A. et al. Target expression, generation, preclinical activity, and pharmacokinetics of the BCMA-T cell bispecific antibody EM801 for the treatment of multiple myeloma. Cancer Cell 31, 396–410, doi: 10.1016 / j.ccell.2017.02.002 (2017).

[0252] 11Hipp, S. et al. A novel BCMA / CD3 bispecific T cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo. Leukemia 31, 1743–1751, doi: 10.1038 / leu.2016.388 (2017).

[0253] Li, J. et al. Membrane-proximal epitopes promote efficient T cell synapse formation by anti-FcRH5 / CD3 and are essential for killing myeloma cells. Cancer Cell 31, 383–395, doi: 10.1016 / j.ccell.2017.02.001 (2017).

[0254] 13 van de Donk, N., Richardson, P. G. & Malavasi, F. CD38 antibodies in multiple myeloma: back to the future. Blood 131, 13–29, doi:10.1182 / blood-2017-06-740944 (2018).

[0255] Usmani, S. Z., et al. Clinical efficacy of daratumumab monotherapy in heavily pretreated patients with relapsed or refractory multiple myeloma. Blood 128, 37–44, doi: 10.1182 / blood-2016-03-705210 (2016).

[0256] Palumbo, A. et al. Daratumumab, bortezomib, and dexamethasone in the treatment of multiple myeloma. New England Journal of Medicine 375, 754–766, doi: 10.1056 / NEJMoa1606038 (2016).

[0257] 16 van de Donk, N. & Usmani, S.Z. CD38 antibodies in multiple myeloma: mechanisms of action and resistance patterns. Front Immunol 9, 2134, doi:10.3389 / fimmu.2018.02134 (2018).

[0258] Nijhof, I.S., et al. CD38 expression and complement inhibitors influence response and resistance to daratumumab in myeloma. Blood 128, 959–970, doi:10.1182 / blood-2016-03-703439 (2016).

[0259] 18 Deaglio, S., Aydin, S., Vaisitti, T., Bergui, L. & Malavasi, F. CD38 lies at the interface between prognostic marker and therapeutic target. Trends in Molecular Medicine 14, 210-218, doi:10.1016 / j.molmed.2008.02.005 (2008).

[0260] 19 Krejcik, J. et al. Daratumumab depletes CD38+ immunoregulatory cells, promotes T cell expansion, and distorts the T cell repertoire in multiple myeloma. Blood 128, 384–394, doi: 10.1182 / blood-2015-12-687749 (2016).

[0261] 20Casneuf, T. et al. Effects of daratumumab on natural killer cells and clinical outcomes in relapsed or refractory multiple myeloma. Blood Adv 1, 2105–2114, doi: 10.1182 / bloodadvances.2017006866 (2017).

[0262] 21Nahi, H. et al. Infectious complications and NK cell depletion after daratumumab treatment in multiple myeloma. PLoS ONE 14, e0211927, doi:10.1371 / journal.pone.0211927 (2019).

[0263] 22 Zuch de Zafra, CL, et al. Targeting multiple myeloma with AMG 424, a novel anti-CD38 / CD3 bispecific T cell recruitment antibody optimized for cytotoxicity and cytokine release. Clinical Cancer Research, doi: 10.1158 / 1078-0432.CCR-18-2752 (2019).

[0264] 23 Hipp, S. et al. A novel BCMA / CD3 bispecific T cell receptor for the treatment of multiple myeloma induces selective lysis in vitro and in vivo. Leukemia 31, 2278, doi: 10.1038 / leu.2017.219 (2017).

[0265] 24 Darce, JR, Arendt, BK, Wu, X. & Jelinek, DF. Regulation of BAFF binding receptor expression during human B cell differentiation. J Immunol. 179, 7276-7286, doi: 10.4049 / jimmunol.179.11.7276 (2007).

[0266] 25 Lee, L. et al. Evaluation of B cell maturation antigen as a target for antibody-drug conjugate-mediated cytotoxicity in multiple myeloma. Journal of Hematology 174, 911–922, doi: 10.1111 / bjh.14145 (2016).

[0267] 26 Salem, D.A., et al. Quantification of B cell maturation antigens, targets for novel chimeric antigen receptor T cell therapy for myeloma. Leukemia Research 71, 106–111, doi: 10.1016 / j.leukres.2018.07.015 (2018).

[0268] 27Brudno, J.N., et al. Transgenic T cells expressing chimeric antigen receptors against B-cell maturation antigens lead to remission in relapsed multiple myeloma with a poor prognosis. Journal of Clinical Oncology 36, 2267–2280, doi: 10.1200 / jco.2018.77.8084 (2018).

[0269] 28 Gokbuget, N. et al. Blinatumomab for minimal residual disease in adult patients with B-cell precursor acute lymphoblastic leukemia. Blood 131, 1522–1531, doi: 10.1182 / blood-2017-08-798322 (2018).

[0270] 29 Topp, MS, et al. Targeted therapy with the T-cell engaging antibody blinatumomab in patients with chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia results in high response rates and prolonged leukemia-free survival. Journal of Clinical Oncology 29, 2493–2498, doi: 10.1200 / JCO.2010.32.7270 (2011). Sequence Listing <110> INSERM (French National Institute of Health and Medical Research) University of Paris <120> Antibodies specific for CD38 and uses thereof <130> 24020CAP <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 300 <212> PRT <213> Homo sapiens <400> 1 Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys 1 5 10 15 Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 25 30 Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40 45 Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 55 60 Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val 65 70 75 80 Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys 85 90 95 His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu 100 105 110 Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile 115 120 125 Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr 130 135 140 Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys 145 150 155 160 Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175 Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185 190 Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu 195 200 205 Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser 210 215 220 Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu Glu Ala 225 230 235 240 Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln Asp 245 250 255 Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln 260 265 270 Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285 Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295 300 <210> 2 <211> 207 <212> PRT <213> Homo sapiens <400> 2 Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120 125 Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140 Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys 145 150 155 160 Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170 175 Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg 180 185 190 Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195 200 205 <210> 3 <211> 121 <212> PRT <213> Mus musculus <400> 3 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Mus musculus <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 5 <211> 8 <212> PRT <213> Mus musculus <400> 5 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 6 <211> 8 <212> PRT <213> Mus musculus <400> 6 Ile Tyr Pro Gly Asp Gly Asp Thr 1 5 <210> 7 <211> 14 <212> PRT <213> Mus musculus <400> 7 Ala Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val 1 5 10 <210> 8 <211> 6 <212> PRT <213> Mus musculus <400> 8 Glu Asn Ile Tyr Ser Phe 1 5 <210> 9 <211> 3 <212> PRT <213> Mus musculus <400> 9 Asn Thr Lys 1 <210> 10 <211> 9 <212> PRT <213> Mus musculus <400> 10 Gln His His Tyr Gly Ile Pro Leu Thr 1 5 <210> 11 <211> 243 <212> PRT <213> Artificial <220> <223> scFV antibody <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly 165 170 175 Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys Gly Lys Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Asn Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Arg Thr Thr 210 215 220 Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser <210> 12 <211> 503 <212> PRT <213> Artificial <220> <223> Bi38-3 bispecific antibody <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly 165 170 175 Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys Gly Lys Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Asn Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Arg Thr Thr 210 215 220 Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro 260 265 270 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 275 280 285 Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 290 295 300 Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln 305 310 315 320 Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr 325 330 335 Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 340 345 350 Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly 355 360 365 Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 370 375 380 Gly Gly Ser Gly Gly Gly Gly Ser Val Asp Asp Ile Gln Leu Thr Gln 385 390 395 400 Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr 405 410 415 Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys 420 425 430 Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala 435 440 445 Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr 450 455 460 Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 465 470 475 480 Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys 485 490 495 Leu Glu Leu Lys Ala Ala Ala 500 <210> 13 <211> 442 <212> PRT <213> Artificial <220> <223> CAR CD38 1G <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly 165 170 175 Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys Gly Lys Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Asn Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Arg Thr Thr 210 215 220 Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser Leu Glu His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 245 250 255 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 260 265 270 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 275 280 285 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 290 295 300 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 305 310 315 320 Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Val Lys Phe Ser Arg Ser 325 330 335 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 340 345 350 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 355 360 365 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro 370 375 380 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 385 390 395 400 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 405 410 415 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 420 425 430 Ala Leu His Met Gln Ala Leu Pro Pro Arg 435 440 <210> 14 <211> 508 <212> PRT <213> Artificial <220> <223> CAR CD38 3G <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly 165 170 175 Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys Gly Lys Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Asn Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Arg Thr Thr 210 215 220 Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser Leu Glu Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp 245 250 255 Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu 260 265 270 Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu 275 280 285 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 290 295 300 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 305 310 315 320 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 325 330 335 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 340 345 350 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 355 360 365 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 370 375 380 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Ser Arg Val Lys Phe 385 390 395 400 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 405 410 415 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 420 425 430 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 435 440 445 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 450 455 460 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 465 470 475 480 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 485 490 495 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 <210> 15 <211> 545 <212> PRT <213> Artificial <220> <223> Bi38-3 + leader sequence <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 35 40 45 Asn Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 50 55 60 Pro Gln Leu Leu Val Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile 85 90 95 Asn Asn Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His 100 105 110 Tyr Gly Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 145 150 155 160 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 165 170 175 Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala 275 280 285 Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser 290 295 300 Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro 305 310 315 320 Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr 325 330 335 Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp 340 345 350 Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu 355 360 365 Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys 370 375 380 Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly 385 390 395 400 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Asp Asp 405 410 415 Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu 420 425 430 Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn 435 440 445 Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp 450 455 460 Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly 465 470 475 480 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp 485 490 495 Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe 500 505 510 Gly Ser Gly Thr Lys Leu Glu Leu Lys Ala Ala Ala Glu Gln Lys Leu 515 520 525 Ile Ser Glu Glu Asp Leu Asn Gly Ala Val Glu His His His His His 530 535 540 His 545 <210> 16 <211> 463 <212> PRT <213> Artificial <220> <223> CAR CD38 1G + leader sequence <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 35 40 45 Asn Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 50 55 60 Pro Gln Leu Leu Val Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile 85 90 95 Asn Asn Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His 100 105 110 Tyr Gly Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 145 150 155 160 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 165 170 175 Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ser Leu Glu His Phe Val Pro Val Phe 260 265 270 Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 275 280 285 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 290 295 300 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 305 310 315 320 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 325 330 335 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Val 340 345 350 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 355 360 365 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 370 375 380 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln 385 390 395 400 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 405 410 415 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 420 425 430 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 435 440 445 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 <210> 17 <211> 529 <212> PRT <213> Artificial <220> <223> CAR CD38 3G + leader sequence <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 35 40 45 Asn Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 50 55 60 Pro Gln Leu Leu Val Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile 85 90 95 Asn Asn Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His 100 105 110 Tyr Gly Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 145 150 155 160 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 165 170 175 Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ser Leu Glu Ile Glu Val Met Tyr Pro 260 265 270 Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val 275 280 285 Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys 290 295 300 Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 305 310 315 320 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 325 330 335 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 340 345 350 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 355 360 365 Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 370 375 380 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 385 390 395 400 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly 405 410 415 Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 420 425 430 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 435 440 445 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 450 455 460 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 465 470 475 480 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 485 490 495 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 500 505 510 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 515 520 525 Arg <210> 18 <211> 415 <212> PRT <213> Artificial <220> <223> CCR - CD38 <400> 18 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 35 40 45 Asn Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 50 55 60 Pro Gln Leu Leu Val Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile 85 90 95 Asn Asn Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His 100 105 110 Tyr Gly Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 145 150 155 160 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 165 170 175 Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ser Leu Glu Ile Glu Val Met Tyr Pro 260 265 270 Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val 275 280 285 Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys 290 295 300 Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 305 310 315 320 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 325 330 335 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 340 345 350 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 355 360 365 Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 370 375 380 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 385 390 395 400 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 405 410 415 <210> 19 <211> 21 <212> PRT <213> Artificial <220> <223> CD8a Leader <400> 19 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 20 <211> 264 <212> PRT <213> Artificial <220> <223> Anti-CD38 scFv (Table 1) <400> 20 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 35 40 45 Asn Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 50 55 60 Pro Gln Leu Leu Val Tyr Asn Thr Lys Thr Leu Thr Glu Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile 85 90 95 Asn Asn Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His 100 105 110 Tyr Gly Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 145 150 155 160 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 165 170 175 Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Glu Arg Thr Thr Gly Ala Pro Arg Tyr Phe Asp Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ser 260

Claims

1. A monoclonal antibody having binding specificity to the extracellular domain of CD38, comprising: a heavy chain comprising i) H-CDR1 set forth in SEQ ID NO: 5, ii) H-CDR2 set forth in SEQ ID NO: 6, and iii) H-CDR3 set forth in SEQ ID NO: 7; and A light chain comprising i) the L-CDR1 represented by SEQ ID NO: 8, ii) the L-CDR2 represented by SEQ ID NO: 9, and iii) the L-CDR3 represented by SEQ ID NO:

10.

2. The monoclonal antibody of claim 1, comprising a VH domain having at least 70% identity to the amino acid sequence shown in SEQ ID NO:

3.

3. The monoclonal antibody of claim 1, comprising a VL domain having at least 70% identity to the amino acid sequence shown in SEQ ID NO:

4.

4. The monoclonal antibody according to claim 1, which is a chimeric antibody having a heavy chain as shown in SEQ ID NO: 3 and / or a light chain as shown in SEQ ID NO:

4. The monoclonal antibody according to claim 1 , which is a humanized antibody. A single-chain variable fragment (scFv) comprising the VH and VL domains of the antibody of claim 1.

7. The scFv fragment according to claim 6, which consists of the amino acid sequence shown in SEQ ID NO:

11.

8. A bispecific antibody comprising a first antigen-binding site and a second antigen-binding site derived from the complementarity determining regions (CDRs) of the monoclonal antibody of claim 1; The second antigen binding site binds an antigen on a human effector cell selected from the group consisting of lymphocytes, killer cells, natural killer cells, macrophages, monocytes, mast cells, and granulocytes.

9. The bispecific antibody of claim 8, wherein the second antigen binding site is specific for the extracellular domain of CD3ε. 10 . The bispecific antibody according to claim 8 , wherein the first antigen-binding site comprises the single-chain variable fragment (scFv) according to claim 6 .

11. The bispecific antibody according to claim 8, which is a BITE ® Antibody.

12. The bispecific antibody according to claim 11, comprising the sequence as shown in SED IQ NO:

12.

13. A chimeric antigen receptor (CAR), comprising the antigen binding domain of the antibody of claim 1; the antigen binding domain comprises the complementarity determining region (CDR) of the antibody of claim 1.

14. The chimeric antigen receptor (CAR) of claim 13, wherein the antigen binding domain comprises the single-chain variable fragment (scFv) of claim 6.

15. The chimeric antigen receptor (CAR) of claim 14, comprising an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from the group consisting of CD28, 4-1BB, and CD3ζ intracellular domains.

16. The chimeric antigen receptor (CAR) according to claim 14, wherein the chimeric antigen receptor (CAR) consists of the amino acid sequence shown in SEQ ID NO: 13 or 14.

17. A nucleic acid encoding the monoclonal antibody of claim 1, the bispecific antibody of claim 8, or the chimeric antigen receptor (CAR) of claim 13. A nucleic acid, wherein the nucleic acid sequence encodes the heavy chain and light chain of the monoclonal antibody according to claim 1.

19. A vector comprising the nucleic acid of claim 17.

20. A host cell engineered to express the monoclonal antibody of claim 1, the bispecific antibody of claim 8, or the chimeric antigen receptor (CAR) of claim 13. The host cell according to claim 20 , which is a CAR-T cell.

22. Use of the antibody according to claim 1 and / or the bispecific antibody according to claim 8 and / or the CAR-T cell according to claim 21 in the preparation of a medicament for treating multiple myeloma.

23. A pharmaceutical composition comprising a certain amount of the antibody of claim 1 and / or the bispecific antibody of claim 8 and / or the CAR-T cell of claim 21.

Images