Antibodies specific for CD38 and uses thereof

Abstract

In this study, we developed a novel antibody against CD38 that may be suitable for the generation of bispecific antibodies and CAR-T cells. In particular, we report the development of Bi38-3, a novel bispecific T cell engager that targets CD38 on MM cells and recruits cytotoxic T cells via CD3ε. Bi38-3 lacks the Fc region of natural mAbs, which contributes to the resistance process, yet it induced T cells to proliferate, release cytokines, and lyse CD38-positive MM cells in vitro. Similarly, Bi38-3 induced autologous T cells to eliminate tumor plasma cells isolated from MM patients both at diagnosis and at relapse. The cytotoxicity induced by Bi38-3 was restricted to cells expressing high levels of CD38 and maintained the integrity of T, B, and NK lymphocytes in vitro.

Description

[Technical Field]

[0001] The present invention is in the field of medicine, and in particular in the field of oncology. [Background technology]

[0002] CD38 is a type II transmembrane glycoprotein. CD38 functions include both receptor-mediated adhesion and signaling events and enzymatic activity. CD38 is normally found on hematopoietic cells and solid tissues. On hematopoietic cells, the majority of thymic medullary cells express CD38. + and resting and circulating T and B cells express CD38 - and activated cells are CD38 + CD38 is also expressed on approximately 80% of resting NK cells and monocytes, and on lymphoblasts in the germinal centers of lymph nodes, plasma B cells, and some intrafollicular cells. CD38 can also be expressed on dendritic cells. A high proportion of normal bone marrow cells, especially progenitor cells, express CD38. Furthermore, 50-80% of umbilical cord blood cells express CD38. + and is expressed in human blood during the first 2-3 years of life. CD38 is also expressed on erythrocytes and platelets, as well as on lymphoid progenitor cells. In solid tissues, CD38 is expressed in the intestine by intraepithelial cells and lymphocytes of the lamina propria, in the brain by Purkinje cells and neurofibrillary tangles, in the prostate by epithelial cells, in the pancreas by beta cells, in bone by osteoclasts, in the eye by retinal cells, and on the sarcolemma of smooth and striated muscle.

[0003] CD38 is also expressed in a variety of hematological malignancies, including multiple myeloma, B-cell chronic lymphocytic leukemia, B-cell acute lymphocytic leukemia, Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic / 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 hematological malignancy characterized by the accumulation of monoclonal immunoglobulin-secreting neoplastic plasma cells in the bone marrow of patients and osteolytic lesions. 1 Current treatments have increased median overall survival to approximately 6 years, and the recent development of monoclonal antibodies (mAbs), such as elotuzumab (anti-SLAMF7) and daratumumab (anti-CD38), has further improved prognosis. 2-4 However, overall survival for patients whose disease recurs after proteasome inhibitor (PI), immunomodulatory agent (IMID), and mAb treatment remains extremely poor, making MM an incurable disease. Thus, new therapeutic strategies are needed to improve patient care and ultimately develop curative treatments.

[0004] Several anti-CD38 antibodies have been described in the literature, for example, in Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3. For example, Patent Document 1 describes several human anti-CD38 antibodies. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2006 / 099875 [Non-patent literature]

[0006] [Non-Patent Document 1] Lande R et al., Cell Immunol.220(1), 30-8(2002) [Non-patent document 2] Ausiello CM et al., Tissue Antigens.56(6), 539-47(2000), [Non-patent document 3] Cotner T et al., Int J Immunopharmacol.3(3), 255-68(1981) Summary of the Invention [Means for solving the problem]

[0007] As defined by the claims, the present invention relates to antibodies having specificity for CD38 and uses thereof. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows the dose-dependent autologous T cell-mediated lysis of Bi38-3 in a patient's MM tumor cells. CD138+ plasma cells were purified from the patient's bone marrow and cocultured with autologous CD3+ T cells isolated from PBMCs at a 5:1 E:T cell ratio for 24 hours. Cultures were analyzed by FACS to monitor the number of CD138+ cells entering the viability gate. The average of triplicate experiments showing the percentage of viable CD138+ cells (relative to the untreated condition) in four different patients at diagnosis and three different patients at relapse is shown. Histograms show the average effect of Bi38-3 alone, T cells alone, and Bi38-3 (100 ng / mL) plus T cells on tumor plasma cells from five identical patients at diagnosis (top) and three identical patients at relapse (bottom). Standard deviations are shown, and p values ​​were calculated by Student's t-test (*p<0.05; **p<0.01; ***p<0.001). [Figure 2]Figure 2 shows the in vivo activity of Bi38-3 in the MM1.Sluc xenograft mouse model. A. Treatment schedule. NSG mice were inoculated (intravenously) with 5.10 MM1.SLuc cells, and treatment began on day 13, when similar levels of luciferase-expressing MM cells were detected in all mice. Purified T cells (5.10 cells / mouse) were intravenously injected with Bi38-3 or PBS (blue arrow). Intravenous injections of Bi38-3 (0.1 mg / kg) were repeated daily for 9 days (black arrow). Luciferase activity was measured using an IVIS imaging system at 7, 11, 13, 15, 18, and 21 (or 22) days after tumor injection (red arrow). B. Serial bioluminescence imaging to assess myeloma progression / regression. Radiation intensity was measured throughout the mouse body. The image on the left shows luminescence 7 days after inoculation of MM.1S myeloma cells and before the start of treatment. The image on the right shows 18 days after inoculation of MM.1S cells and 4 days after treatment with Bi38-3 (upper panel) or vehicle (lower panel). The radiation color scale is shown on the right. C. Longitudinal radiation levels of mice treated with vehicle (blue line) and Bi38-3 (red line). Nine mice per group are shown, inoculated with T cells from two separate donors. p-values ​​were calculated by Student's t-test at day 22 (***p<0.001). [Figure 3]Figure 3 shows the in vitro activity of anti-CD38 CAR-T cells. A. Schematic diagram of the structures of various chimeric antigen receptors (CARs) and costimulatory receptors (CCRs). First-generation (1G) CARs contain the CD3ζ signaling domain, while third-generation CARs (3G) contain the CD28, 4-1BB, and CD3z signaling domains. CCRs contain the CD28 and 4-1BB signaling domains but lack the CD3ζ domain. CAR Mock lacks the anti-CD38 scFv region. B. In vitro cytotoxic activity of various CAR-Ts in CD38-expressing MM (MM.1S and RPMI8226) and CD38-negative fibroblast (HEK293) cell lines. Luciferase-expressing cells were cultured with the above-mentioned CAR-T cells at various effector / target (E:T) ratios for 20 hours. Cytotoxic activity was determined by measuring luciferase levels in the cultures. Four independent experiments are shown. [Figure 4] Figure 4 shows the sensitivity of blood cells and bone marrow hematopoietic progenitor cells to Bi38-3. A. Relative Bi38-3-mediated T cell lysis in Tregs versus MM1.S cells. Purified T cells (n=3) from a healthy donor were co-cultured for 24 hours with increasing concentrations of Bi38-3 in the presence of MM1.S cells. B. Relative Bi38-3-mediated T cell lysis in CD34+ bone marrow hematopoietic progenitor cells versus MM1.S cells. Paired CD34+ hematopoietic progenitor cells and T cells (n=4) purified from the bone marrow of a healthy donor (hip surgery) were co-cultured for 24 hours with increasing concentrations of Bi38-3 in the presence of MM1.S cells. The numbers of viable 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 a ratio to the untreated control. Histograms show the ratios of B cells, Treg cells, CD34+ hematopoietic progenitor cells, and MM1.S cells at each Bi38-3 concentration, with error bars indicating SD. Normality of CD34+ populations was determined by the Shapiro-Wilk normalization test, and p values ​​were determined by unpaired Student's t-test (*p<0.05; **p<0.01; ***p<0.001). DETAILED DESCRIPTION OF THE INVENTION

[0009] The present inventors have developed a novel anti-CD38 / CD3 bispecific T cell engager antibody that induces 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 engager antibody, Bi38-3, appears to be unaffected by mechanisms of resistance to anti-CD38 mAbs (such as daratumumab, an anti-CD38 mAb approved for the treatment of MM) that involve FcγR binding of therapeutic antibodies. The present inventors have shown that Bi38-3 mediates autologous T cell-mediated killing of patients' tumor plasma cells at diagnosis and relapse with similar efficacy. Furthermore, the inventors have shown that Bi38-3 has no significant effects on T cells, B cells, or NK cells in vitro, rapidly inducing T cell-mediated killing of MM cells while protecting B cells from T cell cytotoxic activity. We have shown that Bi38-3 can induce a six-fold reduction in tumor burden in vivo in just three days. Thus, we have demonstrated that Bi38-3 is a selective and effective compound for the treatment of MM, which may be used in both frontline and relapse settings and may support further evaluation in MM patients.

[0010] (Main definitions) As used herein, the term "CD38" has its general meaning in the art and refers to ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1. An exemplary amino acid sequence of CD38 is set forth in 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. SEQ ID NO: 1 >sp|P28907|CD38_HUMAN ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1 OS=Homo sapiens OX=9606 GN=CD38 PE=1 SV=2 MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLL GYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI

[0011] As used herein, the term "CD3" has its general meaning in the art and refers to the CD3 (cluster of differentiation 3) T-cell coreceptor, which mediates the activation of both cytotoxic T cells (CD8+ naive T cells) and also helper T cells. It consists of a protein complex, composed of four specific chains. In mammals, this complex includes the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T-cell receptor (TCR) and the ζ chain (zeta chain) to generate activation signals for T lymphocytes. The TCR, ζ chain, and CD3 molecule together comprise the TCR complex. An exemplary amino acid sequence of CD3ε is set forth in SEQ ID NO:2. The extracellular domain of CD3ε spans from amino acid residue 23 to amino acid residue 207 in SEQ ID NO:2. SEQ ID NO:2 >sp|P07766|CD3E_HUMAN T-cell surface glycoprotein CD3 epsilon chain OS=Homo sapiens OX=9606 GN=CD3E PE=1 SV=2 MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0012] As used herein, the term "antibody" is thus used to refer to any antibody-like molecule having an antigen-binding region, and this term includes antibody fragments comprising an antigen-binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DAB), TandAb dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (bispecific or trispecific scFv-Fs, respectively). These include antibody-based fragments (e.g., scFv-ab fusions), sc-diabodies, kappa (lambda) bodies (scFv-CL fusions), BiTEs (bispecific T cell engagers, scFv-scFv tandems that attract T cells), DVD-Igs (dual variable domain antibodies, bispecific formats), SIPs (small molecule immune proteins, a type of minibody), SMIPs ("small molecule modular immunopharmaceuticals" scFv-Fc dimers), DARTs (ds-stabilized diabodies "dual affinity retargeting"), and small molecule antibody mimics containing one or more CDRs. Techniques for preparing and using various antibody-based constructs and fragments are known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, inter alia, are further described in EP 404,097 and WO 93 / 11161, and linear antibodies are further described by Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to generate Fab' fragments. Fab fragments can be formed by papain digestion. Fab, Fab', and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, ds-scFv, dimeric, minibody, diabody, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or chemically. Techniques for producing antibody fragments are known and described in the art.For example, Beckman et al., 2006; Holliger and Hudson, 2005; Le Gall et al., 2004; Reff and Heard, 2001; Reiter et al., 1996; and Young et al., 1995 each further describe and enable the generation of effective antibody fragments.

[0013] In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to one light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (K). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains a specific sequence domain. Light chains contain two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four (α, δ, γ) to five (μ, ε) domains: a variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4, collectively referred to as CH). The variable regions of both the light chain (VL) and heavy chain (VH) determine binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, placental transport, complement fixation, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is primarily composed of residues from hypervariable regions or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) may participate in the antibody-combining site or affect the overall domain structure, i.e., the binding site. CDRs together refer to the amino acid sequences that define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3. Thus, an antigen-binding site typically contains six CDRs, including each set of CDRs in the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequence located between the CDRs. Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al.This system is set forth in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues in the sequence of SEQ ID NO: 1. The actual linear amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering, corresponding to truncations of, or insertions into, structural elements, whether in the framework regions or complementarity-determining regions (CDRs) of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain, according to the Kabat numbering system, are located at residues 31-35B (VH-CDR1), residues 50-65 (VH-CDR2), and residues 95-102 (VH-CDR3). 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) according to the Kabat numbering system.

[0014] 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. SEQ ID NO: 3 > IgH VH1.87-D1.1-J1: QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTVTVSS SEQ ID NO: 4 >Igk Vk12.44-Jk5: DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQHHYGIPLTFGAGTKLELK

[0015] As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked, e.g., by a synthetic linker, e.g., a small flexible polypeptide linker, and can be expressed as a single polypeptide chain, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified otherwise, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., relative to the N- and C-termini of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL. 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 antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences.

[0016] 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 (a) an antibody molecule in which the constant regions (i.e., heavy and / or light chains) or portions thereof have been altered, substituted, or replaced so that the antigen-binding site (variable region) binds to constant regions of a different or altered class, effector function, and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, or drug, that confers new properties to the chimeric antibody; or (b) an antibody molecule in which the variable region, or portions thereof, have been altered, substituted, or replaced with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized antibodies and, particularly, humanized antibodies. Furthermore, chimeric antibodies can contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody function. For further 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. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0017] As used herein, the term "humanized antibody" refers to an antibody having variable and constant regions from a human antibody but retaining the CDRs of the original non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from a non-human immunoglobulin. Often, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibodies or antibody fragments) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced with residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, residues from the Fv framework regions (FRs) of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to retain the binding specificity of the non-human antibody from which the binding regions are derived, but to prevent an immune response against the non-human antibody. These modifications can further refine and optimize the function of the antibody or antibody fragment. Generally, a humanized antibody or antibody fragment thereof can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or most of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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.

[0018] As used herein, the term "binding" in the context of an antibody binding to a given antigen or epitope typically refers to a binding affinity of about 10, as measured, for example, by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and a soluble form of the antibody as the analyte. -6 K of M D BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats routinely used for epitope binning of monoclonal antibodies. Typically, antibodies exhibit a K of 0.05 for binding to nonspecific antibodies (e.g., BSA, casein) that are not identical to or closely related to the given antigen. D at least 10 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, D The antibody binds to a given antigen with an affinity corresponding to the K D is very low (i.e., the antibody has high affinity), the K D However, typically, K D An antibody is said to not substantially bind an antigen or epitope if such binding is undetectable (e.g., using the antigen as the ligand and a soluble form of the antibody as the analyte in a BIAcore3000 instrument using surface plasmon resonance (SPR) technology) or is 100-fold, 500-fold, 1000-fold, or more than 1000-fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

[0019] As used herein, the term "bispecific antibody" has its general meaning in the art and refers to an artificial hybrid antibody having two different pairs of heavy and light chains and also two different antigen-binding sites.

[0020] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a bispecific antibody, a recombinant protein construct composed of two movably linked single-chain antibodies (scFvs). One of the scFv antibodies specifically binds to a selected target cell-expressed tumor antigen, and the second specifically binds to another molecule, such as CD3, a subunit of the T cell receptor complex on T cells. In some embodiments, the BiTE antibody can transiently bind T cells to target cells and simultaneously activate the cytolytic activity of the T cell. BiTE-mediated activation of T cells does not require a specific T cell receptor on the T cell, nor an MHC I molecule, peptide antigen, or costimulatory molecule on the target cell.

[0021] As used herein, the term "CAR-T cells" refers to T lymphocytes that have been genetically engineered to express a CAR. The definition of CAR-T cells encompasses all classes and subclasses of T lymphocytes, including CD4+, CD8+ T cells, γδ T cells, and effector T cells, memory T cells, regulatory T cells, etc. Genetically modified T lymphocytes can be "derived" or "obtained" from the subject to be treated using the genetically modified T cells, or can be "derived" or "obtained" from a different subject.

[0022] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that in an immune effector cell, confer specificity for a target cell, typically a cancer cell, and intracellular signal generation to the cell. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some aspects, the polypeptides of the set are in close proximity to each other. In some embodiments, the polypeptides of the set comprise a dimerization switch that can link the polypeptides to each other in the presence of a dimerization molecule, e.g., link the antigen-binding domain to the 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 comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is chosen from costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In some embodiments, the CAR comprises 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, the CAR comprises 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, the CAR comprises 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, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising 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, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) upon cellular processing and localization of the CAR to the cell membrane. In certain aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane domain and the intracellular domain CD3-zeta. In some embodiments, the CAR comprises an additional costimulatory signaling domain, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with the CAR.

[0023] As used herein, the term "T cell" has its common meaning in the art and refers to an important component of the immune system that plays a central role in cell-mediated immunity. T cells are known as general lymphocytes because they recognize antigens with their TCR (T cell receptor for antigen) through presentation or restriction by molecules of the major histocompatibility complex. There are several subsets of T cells, including CD8+ T cells, CD4+ T cells, and γδ T cells, each with a specific function.

[0024] As used herein, the term "CD8+ T cells" has its common 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 called 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 the relevant recognition element in class I-restricted interactions of the major histocompatibility complex. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to a patient's pool of CD8+ T cells that have left the bloodstream and migrated to the tumor.

[0025] As used herein, the term "CD4+ T cells" (also called T helper cells or TH cells) refers to T cells that express the CD4 glycoprotein on their surface and assist other white blood cells in immune processes, including the maturation of B cells into plasma cells and memory B cells and the activation of cytotoxic T cells and macrophages. CD4+ T cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete cytokines that regulate or support active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, TGF-β, or Treg, which secrete various cytokines to promote various types of immune responses. Signaling from APCs directs T cells to 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-specific expression of cytokines and transcription factors including T-bet, GATA3, EOMES, RORγT, BCL6, and FoxP3.

[0026] As used herein, the term "γδ T cells" has its general meaning in the art. γδ T cells typically account for 1-5% of peripheral blood lymphocytes in healthy individuals (humans, monkeys). They have been shown to be involved in the initiation of protective immune responses and recognize antigenic ligands by directly interacting with the antigen without presentation by MHC molecules on antigen-presenting cells. γ9δ2 T cells (sometimes referred to as γ2δ2 T cells) are γδ T cells that possess TCR receptors with variable domains Vγ9 and Vδ2. They constitute the majority of γδ T cells in human blood. Upon activation, γδ T cells exhibit potent, non-MHC-restricted cytotoxic activity, which is effective in killing various types of cells, especially pathogen cells. It may be a cell infected with a virus (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), or it may be a cancer cell (Poccia et al., J. Immunol., 159:6009-6015; Fournie and Bonneville, Res. Immunol., 66th Forum in Immunology, 147:338-347). Thus, the possibility of modulating the activity of such cells in vitro, ex vivo, or in vivo may provide new and effective therapeutic approaches in the treatment of various pathologies such as infectious diseases (especially viral or parasitic), cancer, allergies, and even autoimmune and / or inflammatory disorders.

[0027] As used herein, the term "treatment" or "treating" refers to both preventative or preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, and patients who have been diagnosed with or are suffering from a disease or medical condition, including the prevention of clinical recurrence. Treatment can be administered to patients who have or may ultimately suffer from a medical disorder to prevent, cure, or delay the onset of the disorder or recurrent disorder, lessen the severity of the disorder or recurrent disorder, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to prolong the patient's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., a pattern of medication used during treatment. Therapeutic regimens can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used in the early treatment of a disease. The overall purpose of an induction regimen is to provide high levels of a drug to the patient during the initial period of the treatment regimen. An induction regimen can use (in part or in whole) a "loading regimen," which can involve administering a higher dose of a drug than a physician would use during a maintenance regimen, administering a drug more frequently than a physician would use during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in a state of remission for an extended period of time (months or years). A maintenance regimen can use continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., treatment with breaks, intermittent treatment, treatment at recurrence, or treatment upon meeting certain predetermined conditions (e.g., pain, symptoms of disease, etc.)).

[0028] As used herein, the term "cancer" has its general meaning in the art and refers to abnormal cells, characterized by rapidly proliferating cell proliferation capable of autonomous growth, i.e., the potential to invade or spread to other parts of the body. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignant cancerous cells, tissues, or organs, regardless of the type or stage of histopathological invasiveness. The term "cancer" includes, but is not limited to, malignant lesions of various organ systems, such as affected lung, breast, thyroid, lymphoid, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including malignant lesions such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, glioblastomas, non-small cell lung cancers, small intestine cancers, and esophageal cancers. The term "cancer" also includes, but is not limited to, solid tumors and blood-borne tumors.

[0029] The term "solid tumor" has its common meaning in the art and includes head and neck squamous cell carcinoma (HNSCC), adrenocortical carcinoma, anal cancer, bile duct cancer (e.g., perihilar cancer, 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 (e.g., meningioma), , astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germinoma, 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, adenosquamous cell carcinoma, papillary serous adenocarcinoma, clear cell), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g., choriocarcinoma, destructive chorioadenocarcinoma), Kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma), pharyngeal 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 It refers to a solid tumor selected from the group consisting of, but not limited to, myosinoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminomatous germ cell carcinoma), thymic cancer, thyroid cancer (e.g., follicular carcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma).

[0030] The term "blood-borne cancer" or "leukemia" has its common meaning in the art and refers to a cancer of the blood cells. It begins in the bone marrow, the soft tissue in the center of bones where blood cells are made. In leukemia, the bone marrow begins to make abnormal cells that crowd out normal blood cells.

[0031] In some embodiments, the cancer is a CD38-positive hematological malignancy.

[0032] As used herein, the term "CD38-positive hematological malignancies" refers to hematological malignancies characterized by the presence of tumor cells expressing CD38, including leukemia, lymphoma, and myeloma. Examples of such CD38-positive hematological malignancies include precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin's 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 lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL). (including low-grade, intermediate-grade, and high-grade FL), cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT, nodal, and splenic types), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasmacytoma, multiple myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom macroglobulinemia, plasma cell leukemia, and anaplastic large cell lymphoma (ALCL).

[0033] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma.

[0034] As used herein, the term "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an active agent may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the active agent to induce a desired response in the individual. A therapeutically effective amount is one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The effective dosage and administration regimen of an active agent depend on the disease or condition being treated and can be determined by one of ordinary skill in the art. A skilled physician can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can 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, an appropriate dose of the composition of the present invention may be the amount of the compound that is the lowest dose effective to produce a therapeutic effect with a particular administration regimen. Such an effective dose may generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use may be determined by its ability to stabilize the progression of a disease. Typically, the ability of a compound to inhibit cancer can be evaluated in an animal model system that is predictive of efficacy in, for example, human tumors. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a patient. One of skill in the art can determine such amounts based on factors such as the size of the patient, the severity of the patient's symptoms, and the particular 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, e.g., about 0.1-50 mg / kg, e.g., about 0.1-20 mg / kg, e.g., about 0.1-10 mg / kg, e.g., about 0.5, e.g., 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, e.g., about 0.02-30 mg / kg, e.g., about 0.05-10 mg / kg, or 0.1-3 mg / kg, e.g., about 0.5-2 mg / kg. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and can be administered, for example, proximal to the target site.The dosing regimen in the above-described treatment methods and uses is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased if the exigencies of the therapeutic situation indicate. In some embodiments, treatment efficacy is monitored during treatment, e.g., at predetermined time points. In some embodiments, efficacy can be monitored by visualization of the diseased area or by other diagnostic methods further described herein, e.g., by performing one or more PET-CT scans using labeled inhibitors of the invention, fragments derived from inhibitors of the invention, or miniantibodies thereof. If desired, the effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six, or more divided doses administered separately at appropriate intervals throughout the day, optionally in a single dosage form. In some embodiments, the human monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time, e.g., more than 24 hours, to minimize any undesirable side effects. An effective dose of the inhibitor of the invention can also be administered using a weekly, biweekly, or triweekly dosing period. The duration of treatment can be limited, for example, to 8 weeks, 12 weeks, or until clinical progression is observed.As a non-limiting example, treatment herein can be administered by administering 20 mg of 20 ... A daily dose of an inhibitor of the invention in an amount of about 0.1 to 100 mg / kg, e.g., 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, can be provided using a single dose or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0035] (Antibody of the present invention) The first object of the present invention is to - a heavy chain comprising (i) an H-CDR1 as set forth in SEQ ID NO: 5, (ii) an H-CDR2 as set forth in SEQ ID NO: 6, and (iii) an H-CDR3 as set forth in SEQ ID NO: 7, and - a monoclonal antibody having binding specificity to the extracellular domain of CD38, comprising a light chain comprising (i) L-CDR1 as set forth in SEQ ID NO: 8, (ii) L-CDR2 as set forth in SEQ ID NO: 9, and (iii) L-CDR3 as set forth in SEQ ID NO: 10. SEQ ID NO: 5 (H-CDR1): GYTFTSYW SEQ ID NO: 6 (H-CDR2): IYPGDGDT SEQ ID NO: 7 (H-CDR3): ARERTTGAPRYFDV SEQ ID NO: 8 (L-CDR1): ENIYSF SEQ ID NO: 9 (L-CDR2): NTK SEQ ID NO: 10 (L-CDR3): QHHYGIPLT

[0036] In some embodiments, the monoclonal antibodies of the invention comprise a VH domain having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:3.

[0037] In some embodiments, the monoclonal antibodies of the invention comprise a VL domain having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:4.

[0038] In the present invention, a first amino acid sequence having at least 70% identity to 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 to the second amino acid sequence. In the present invention, a first amino acid sequence having at least 90% identity to a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% identity to the second amino acid sequence. Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods for aligning sequences 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, Proc. Natl. Acad. Sci. 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, provides a detailed discussion of sequence alignment methods and homology calculations.For example, sequence comparisons can be performed using the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) (Internet Program, 1996, W.R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, published December 1996). ALIGN compares entire sequences to each other, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet, for example, at the NCSA website. Alternatively, for comparisons of amino acid sequences longer than approximately 30 amino acids, the Blast2 sequences function can be used with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, per-residue gap cost of 1). For alignments of short peptides (fewer than approximately 30 amino acids), alignments should be performed using the Blast2 sequences function with the PAM30 matrix set to default parameters (start gap penalty of 9, extension gap penalty of 1). The BLAST sequence comparison system is available, for example, from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish and 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.

[0039] Thus, the present invention provides antibodies comprising functional variants of the VL region, VH region, or one or more functional variants of the CDRs of the BB51 antibody. Functional variants of the VL, VH, or CDRs used in the context of the monoclonal antibodies of the present invention enable the antibody to still retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the affinity / avidity and / or specificity / selectivity of the parent antibody (i.e., the BB51 antibody); in some cases, such monoclonal antibodies of the present invention may be associated with higher affinity, selectivity, and / or specificity than the parent Ab. Such functional variants typically retain high sequence identity to the parent Ab. The sequence of a CDR variant may differ from the sequence of a CDR of a parent antibody sequence by mostly 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%, e.g., about 92%, 93%, or 94%) of the substitutions in the variant are conservative substitutions of amino acid residues. The sequence of a CDR variant may differ from the sequence of a CDR of a parent antibody sequence by mostly conservative substitutions, for example, at least 10, e.g., at least 9, 8, 7, 6, 5, 4, 3, 2, or 1, of the substitutions in the variant are conservative substitutions of amino acid residues. In the context of the present invention, conservative substitutions may be defined by substitutions within amino acid classes, as represented below: Aliphatic residues I, L, V, and M Residues F, H, W, and Y associated with cycloalkenyl Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in the turn: A, C, D, E, G, H, K, N, Q, R, S, P; and residue T involved in the formation of the turn. Mobile residues Q, T, K, S, G, P, D, E, and R

[0040] Further conservative substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation of the hydrophobic / hydrophilic properties and residue weight / size in the variant CDRs is also substantially maintained compared to the CDRs of the BB51 antibody. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is recognized that the relative hydropathicity characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein 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 characteristics. These are 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also or alternatively be determined by similarity scores, as determined by use of a BLAST program (e.g., BLOSUM62 with standard settings, starting gap = 11 and extension gap = 1, available at NCBI, BLAST 2.2.8). Suitable variants typically exhibit at least about 70% identity with the parent peptide.

[0041] In some embodiments, the monoclonal antibody of the invention is a chimeric antibody. In some embodiments, the monoclonal antibody of the invention is a chimeric antibody having a heavy chain set forth in SEQ ID NO: 3. In some embodiments, the monoclonal antibody is a chimeric antibody having a light chain set forth in SEQ ID NO: 4. In some embodiments, the monoclonal antibody of the invention is a chimeric antibody having a heavy chain set forth in SEQ ID NO: 3 and a light chain set forth in SEQ ID NO: 4.

[0042] In some embodiments, the monoclonal antibodies of the invention are humanized antibodies.

[0043] The monoclonal antibodies of the invention can be characterized by one or more of the functional or structural characteristics in the above-described aspects, or by any combination of selected functional and structural characteristics.

[0044] The antibodies of the present invention can be of any isotype. The choice of isotype will typically be guided by the desired effector function, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the kappa or lambda human light chain constant region can be used. If necessary, the class of the monoclonal antibodies of the present invention can be switched by known methods. Exemplary class switching techniques can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. In this way, the effector function of the human monoclonal antibodies of the present invention can be changed by isotype switching to, for example, an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In some embodiments, the antibodies of the present invention are full-length antibodies. In some embodiments, the full-length antibody is an IgG1 antibody. In some embodiments, the full-length antibody is an IgG4 antibody. In some embodiments, the IgG4 antibody is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4 antibodies include antibodies in which arginine at position 409 in the heavy chain constant region of human IgG4 (as indicated in the EU index, as in Kabat et al., supra) is substituted with lysine, threonine, methionine, or leucine, preferably lysine (as described in WO 2006 / 033386), and / or antibodies in which the hinge region comprises the Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO 2008 / 145142, which is incorporated herein by reference in its entirety. In some embodiments, the monoclonal antibodies of the invention are non-IgG4 antibodies, e.g., IgG1, IgG2, or IgG3, that have been mutated to reduce or even eliminate their ability to mediate effector functions such as ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006), and Hezareh M, J Virol. 75(24):12161-12168 (2001).

[0045] In addition to, or instead of, modifications made within the framework or CDR regions, antibodies of the present invention can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as, for example, serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, 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 their glycosylation, again to alter one or more functional properties of the antibody. For example, it will be understood that the affinity of antibodies provided by the present invention can be altered using any suitable method known in the art. Accordingly, the present invention also relates to variants of the antibody molecules of the present invention that have improved affinity for CD38. Such variants can be obtained by a number of affinity maturation protocols, including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), using mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (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). Vaughan et al. (supra) discuss these affinity maturation methods.

[0046] In some embodiments, the Fc region is modified by substituting 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 substituted with a different amino acid residue so that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The affinity-altered effector ligand can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0047] In some embodiments, one or more selected amino acid residues can be substituted with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0048] In some embodiments, one or more amino acid residues are altered to alter the antibody's ability to fix complement. This approach is further described in WO 94 / 29351 by Bodmer et al. In some embodiments, the Fc region is modified to improve the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to improve the antibody's affinity for an Fc receptor by modifying one or more amino acids. This approach is further described in WO 00 / 42072 by Presta. Additionally, the binding sites in human IgG1 for FcyRI, FcyRII, FcyRIII, and FcRn have been mapped, and mutants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chen. 276:6591-6604; WO 2010 / 106180).

[0049] In some embodiments, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody has no glycosylation). Glycosylation can be altered, for example, to improve the affinity of the antibody for antigen. Such carbohydrate modifications can be obtained, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more variable region framework glycosylation sites can be removed by making one or more amino acid substitutions that eliminate glycosylation at that site. Such glycosylation can improve the affinity of the antibody for antigen. Such approaches are described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al. Additionally or alternatively, antibodies can be generated with altered glycosylation types, for example, hypofucosylated or nonfucosylated antibodies with reduced or no fucosyl residues, or antibodies with increased biantennary GlcNac structures. Such altered glycosylation patterns have been shown to improve the ADCC ability of antibodies. Such carbohydrate modifications can be obtained, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies of the invention to produce glycosylated antibodies. For example, European Patent No. 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 no fucosyl residues. Thus, in some embodiments, human monoclonal antibodies of the invention can be produced by recombinant expression in a cell line that exhibits a hypofucosylated or non-fucosylated pattern, e.g., a mammalian cell line deficient in expression of the FUT8 gene, which encodes a fucosyltransferase.WO 03 / 035835 by Presta describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in improved ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with an altered mammalian glycosylation pattern that lacks fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human monoclonal antibodies of the invention can be produced in yeast or filamentous fungi that have been engineered with a mammalian-like glycosylation pattern and are capable of producing antibodies that lack fucose as a glycosylation pattern (see, e.g., EP 1297172).

[0050] In some embodiments, the antibody is an antigen-binding fragment. Antibody fragments can be obtained by conventional techniques, for example, by fragmenting full-length antibodies or by expressing nucleic acids encoding the antibody fragment in recombinant cells (see, e.g., Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). The fragments can then be tested or screened for their properties in the same manner as described herein for full-length antibodies.

[0051] In some embodiments, the monoclonal antibody of the present invention is an scFv fragment comprising the VH domain and VL domain of an antibody of the present invention. In some embodiments, the scFv fragment of the present invention consists of the amino acid sequence set forth in SEQ ID NO:11. SEQ ID NO: 11; > scFv antibody DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGGGG SQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTVTVSS

[0052] Nucleic Acid Molecules for Producing Antibodies of the Invention and Their Use Monoclonal antibodies of the present invention can be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. For example, knowing the amino acid sequence of the desired sequence, one of skill in the art can readily produce the antibody by standard methods for producing polypeptides. For example, it can be synthesized using the known solid-phase method, preferably using a commercially available peptide synthesizer (such as those manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques known in the art. For example, antibodies can be obtained as DNA expression products by incorporating a DNA sequence encoding the antibody into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host capable of expressing the desired antibody, from which the antibody can then be isolated using known methods.

[0053] Therefore, a further object of the present invention relates to nucleic acid sequences encoding the monoclonal antibodies of the present invention. In some embodiments, the nucleic acid sequences encode the heavy and / or light chains of the monoclonal antibodies of the present invention.

[0054] Typically, the nucleic acid is a DNA or RNA molecule, which can be contained in any suitable vector. The term "vector," as used herein, is used to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicate along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors for use in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector, although the invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0055] Therefore, a further object of the present invention relates to a vector comprising a nucleic acid according to the invention.

[0056] Such vectors may contain regulatory elements such as promoters, enhancers, and terminators to express or induce expression of the antibody upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin heavy chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983). Any expression vector for animal cells can be used as long as it allows insertion and expression of a gene encoding a human antibody C region. 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), and pSG1βd2-4- (Miyaji H et al. 1990). Other examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenovirus, retrovirus, herpesvirus, and AAV vectors. Such recombinant viruses can be produced by methods known in the art, for example, by transfecting packaging cells or by transiently transfecting helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO 94 / 19478.

[0057] A further object of the present invention relates to host cells transfected, infected or transformed with the nucleic acids and / or vectors according to the invention.

[0058] The term "transformation" means the introduction of a "foreign" (i.e., exogenous 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 introduced DNA or RNA has been "transformed."

[0059] The nucleic acids of the invention can be used to produce the monoclonal antibodies of the invention in a suitable expression system. The term "expression system" refers to a host cell and corresponding vector under appropriate conditions for expression of a protein encoded by foreign DNA, for example, carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, and plant cells). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., made from lymphoblast cells, fibroblast cells, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Other examples include mouse SP2 / 0-Agl4 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al; 1980), and rat YB2 / 3HL.P2.G1 1.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells").

[0060] The present invention also relates to a method for producing a recombinant host cell expressing an antibody of the invention, the method comprising the steps of (i) introducing, in vitro or ex vivo, a recombinant nucleic acid or vector as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, the resulting recombinant host cell, and (iii) optionally selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used to produce the antibody of the invention.

[0061] (Multispecific antibodies of the present invention) A further object of the present invention relates to a multispecific antibody comprising a first antigen-binding site from a monoclonal antibody of the invention and at least one second antigen-binding site.

[0062] In the present invention, the multispecific antibody of the present invention binds to the extracellular domain of CD38 and to the extracellular domain of another antigen of interest.

[0063] In some embodiments, the second antigen-binding site is used to recruit killing mechanisms, for example, by binding an antigen on a human effector or by binding a cytotoxic agent or second therapeutic agent.

[0064] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (B cells and T cells, including cytolytic T cells (CTLs)), 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 are capable of inducing ADCC, such as natural killer cells. For example, monocytes and macrophages express FcRs and are involved in the specific killing of target cells and presenting antigens to other components of the immune system. In some embodiments, effector cells are capable of phagocytosing target antigens or cells. The expression of specific FcRs on effector cells can be regulated by humoral factors, such as cytokines. Effector cells are capable of phagocytosing a target antigen or phagocytosing or lysing a target cell. Suitable cytotoxic agents and second therapeutic agents are exemplified below and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs.

[0065] In some embodiments, a second antigen-binding site is used to recruit T cells. In some embodiments, the second antigen-binding site has specificity for the extracellular domain of CD3ε.

[0066] 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 an antibody of the invention.

[0067] In some embodiments, the antigen-binding domain comprises a linker peptide, which may be disposed between the light chain variable region and the heavy chain variable region.

[0068] Exemplary formats for the multispecific antibody molecules of the invention include: (i) two antibodies cross-linked by chemical heteroconjugation, one with specificity for CD38 and the other with specificity for another antigen, such as CD3ε; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, e.g., two scFvs linked in tandem by an additional peptide linker; and (iv) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig) Molecule, In: Antibody Engineering, Springer Berlin). (v) chemically linked bispecific (Fab') fragments; (vi) Tandabs, which are fusions of two single-chain diabodies resulting in tetravalent bispecific antibodies with two binding sites for each of the target antigens; (vii) Flexibodies, which are combinations of scFvs and diabodies resulting in multivalent molecules; (viii) so-called "dock-and-lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fabs, can generate trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (ix) so-called Scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab-arm; and (x) diabodies.

[0069] Another exemplary format for bispecific antibodies is an IgG-like molecule with complementary CH3 domains for heterodimerization. Such molecules can be prepared using known technologies, such as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche), and electrostatically matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A / S) technologies.

[0070] In some embodiments, bispecific antibodies are obtained or obtainable by controlled Fab-arm exchange, typically using DuoBody technology. In vitro methods for generating bispecific antibodies by controlled Fab-arm exchange are described in WO 2008 / 119353 and WO 2011 / 131746 (both by Genmab A / S). In one example method described in WO 2008119353, bispecific antibodies are formed by "Fab-arm" or "half-molecule" exchange (swapping of heavy chains and attached light chains) between two monospecific antibodies, both of which contain IgG4-like CH3 regions, upon incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences. In another example method described in WO 2011 / 131746, a bispecific antibody of the invention is prepared by a method wherein at least one of the first and second antibodies is an antibody of the invention, the method comprising the steps of: (a) providing a first antibody comprising an immunoglobulin Fc region, wherein the Fc region comprises a first CH3 region; (b) providing a second antibody comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region, wherein the first and second CH3 regions have different sequences and wherein the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between the first and second CH3 regions, respectively; (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. Reducing conditions can be provided by adding a reducing agent, such as one selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step (d) can further comprise restoring non-reducing or less reducing conditions, for example, by removing the reducing agent, e.g., by desalting.Preferably, the sequences of the first and second CH3 regions are different and contain only a few, somewhat conservative, asymmetric mutations so that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between the first and second CH3 regions, respectively. Further details about this interaction and how it is obtained are described in WO 2011 / 131746, which is incorporated herein by reference in its entirety. In some other embodiments, the bispecific antibody of the invention is a symmetric class IgG4 bispecific antibody, which comprises two heavy chains, each comprising a variable domain, a CH1 domain, and a hinge region, in which in each heavy chain, the cysteine ​​in the CH1 domain that forms an interchain disulfide bond with a cysteine ​​in the light chain is substituted with another amino acid, and optionally one or more amino acids located in the upper hinge region are substituted with cysteine, the constant region sequences of each heavy chain are similar or identical, and the variable regions of each heavy chain are different. Antibodies in this bispecific format are described in International Publication No. WO 2013 / 124450. In some embodiments, the bispecific antibodies of the present invention are asymmetric antibodies comprising two heavy chains or heavy chain fragments, each comprising at least a variable region, a hinge region, and a CH1 domain, wherein the first heavy chain or fragment thereof is of class IgG4 and characterized in that (a) in the CH1 domain, the interchain cysteine ​​at position 127, as 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 cysteines, and the second heavy chain or fragment thereof has an amino acid sequence in part or all of its chains that differs from that of the first heavy chain in at least a region outside the variable region (e.g., the constant region). Antibodies in this bispecific format are described in International Publication No. WO 2013 / 124451.

[0071] In some embodiments, the multispecific antibodies of the invention are bispecific T cell engager (BiTE) antibodies.

[0072] In some embodiments, the multispecific antibody of the invention is a BiTE® antibody.

[0073] In some embodiments, the multispecific antibody of the invention comprises the sequence shown in SEQ ID NO:12. Sequence number 12 > Sequence of Bi38-3 DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGGGSQVQ LQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTVTVSSGGGGSGGG GSGGGGSDIKLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS GGGGSGGGGSGGGGSVDDIQLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLELKAAA

[0074] The present invention also provides nucleic acids encoding the multispecific antibodies of the present invention. In some embodiments, the nucleic acids are incorporated into vectors as described above.

[0075] Chimeric Antigen Receptors (CARs) and Uses Thereof for Generating Host Cells Expressing the CARs The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of the antibody of the present invention.

[0076] 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 comprising the antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. Characteristics of CARs include their ability to exploit the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and responsiveness to a selected target in a non-MHC-restricted manner. Furthermore, 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 comprises at least one VH and / or VL sequence of an antibody of the present invention. The chimeric antigen receptor of the present invention also comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0077] In some embodiments, the antigen-binding domain comprises a linker peptide, which may be disposed between the light chain variable region and the heavy chain variable region.

[0078] In some embodiments, the invention provides a CAR comprising an antigen-binding domain comprising, consisting of, or consisting essentially of a single-chain variable fragment (scFv) of an antibody of the invention.

[0079] 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. SEQ ID NO: 13 > CAR CD38 1G DIQMTQSPASLSASVGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKTLTGEVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSNLTSEDASAVYCARE RTTGAPRYFDVWGAGTTTVVSSLEHFVPVFLAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:14 > CAR CD38 3G DIQMTQSPASLSASVGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKTLTGEVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSNLTSEDASAVYCARERTTGAPRYFDVWGAGTTVTVSSLEIEVMYPPPY LDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0080] In some embodiments, the CAR comprises 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 important for T cell costimulation. 4-1BB delivers a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. CD3ζ associates with TCR to generate signals and contains an immunoreceptor tyrosine-based activation motif (ITAM).

[0081] In some embodiments, the chimeric antigen receptors of the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or polymerized.

[0082] The present invention also provides nucleic acids encoding the chimeric antigen receptor antibodies of the present invention. In some embodiments, the nucleic acids are incorporated into vectors as described above.

[0083] Therefore, a further object of the present invention relates to a host cell engineered to express a chimeric antigen receptor (CAR) as described above.

[0084] In some embodiments, the host cell is a cytotoxic lymphocyte.

[0085] As used herein, the term "cytotoxic lymphocyte" has its general meaning in the art and refers to lymphocytes that target the destruction of intracellular pathogens, such as viral pathogens, where a lethal attack on infected target cells is required to limit the spread of infection. In the present invention, "cytotoxic lymphocytes" include cytotoxic T cells and natural killer cells.

[0086] In some embodiments, the host cell is a natural killer cell.

[0087] As used herein, the term "natural killer cell" has its general meaning in the art and refers to a type of cytotoxic lymphocyte that is important in the innate immune system. The role of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells respond quickly to virus-infected cells and respond to tumor formation.

[0088] In some embodiments, the host cells are T cells isolated, for example, from peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. If obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cells can be any type of T cell and can be at any developmental stage, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. The T cells can be CD8+ T cells or CD4+ T cells.

[0089] Therefore, a further object of the present invention is a CAR-T cell comprising a chimeric antigen receptor (CAR) of the present invention.

[0090] In some embodiments, the host cell is a pluripotent stem cell (PSC). PSCs can actually be modified with a CAR and then used to induce T cells (see, e.g., International Application No. 2017100403). PSCs include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). iPSCs can be generated directly from adult cells (e.g., somatic cells). iPSCs can typically be induced or generated by introducing a set of specific pluripotency-associated 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, 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.

[0091] In some embodiments, the host cells are hematopoietic stem cells. As used herein, the term "hematopoietic stem cell" or "HSC" refers to blood cells that can self-renew and differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem and progenitor cells exhibit numerous phenotypes, including 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. III: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). Within the bone marrow microenvironment, stem cells self-renew, maintaining a continuous generation of hematopoietic stem cells that 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.

[0092] In some embodiments, CAR activity can be controlled as needed to optimize the safety and efficacy of CAR therapy. There are many ways in which CAR activity can be regulated. For example, inducible apoptosis using, for example, a caspase fused with a dimerization domain (see, e.g., 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.

[0093] Treatment Methods and Pharmaceutical Compositions The antibodies, multispecific antibodies and CAR-T cells of the invention are particularly suitable for use in therapy.

[0094] Therefore, a further object of the present invention relates to a method of treatment in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of an antibody of the invention and / or a multispecific antibody of the invention and / or a population of CAR-T cells of the invention.

[0095] In particular, the multispecific antibodies and CAR-T cells of the invention are particularly suitable for the treatment of cancer, more particularly for the treatment of CD38-positive hematological malignancies.

[0096] Accordingly, the present invention relates to an antibody of the invention and / or a multispecific antibody of the invention and / or a population of CAR-T cells of the invention for use in the treatment of cancer in a subject in need thereof.

[0097] In some embodiments, the cancer is a CD38-positive cancer.

[0098] In some embodiments, the CD38-positive cancer is a CD38-positive hematological malignancy.

[0099] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma.

[0100] In some embodiments, the cancer is a cancer that exhibits no or very low levels of B cell maturation antigen (BCMA).

[0101] In some embodiments, the cancer is a CD38-positive cancer that exhibits no or very low levels of B-cell maturation antigen (BCMA).

[0102] In some embodiments, the cancer is a CD38-positive hematological malignancy that exhibits no or very low levels of B-cell maturation antigen (BCMA).

[0103] In some embodiments, the multispecific antibodies of the invention and / or the population of CAR-T cells of the invention induce specific T cell-mediated lysis of CD38-positive cancers.

[0104] In some embodiments, the multispecific antibodies of the invention and / or the population of CAR-T cells of the invention induce specific T cell-mediated lysis of CD38-positive cancers while protecting B cells and NK cells from the cytotoxic activity of T cells.

[0105] As used herein, the term "subject" refers to any mammal, such as a rodent, cat, dog, or primate. 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.

[0106] In some embodiments, the subject has a recurrence of cancer. In some embodiments, the subject has a recurrence of a CD38-positive cancer. In some embodiments, the subject has a recurrence of a CD38-positive hematological malignancy.

[0107] In some embodiments, the subject has resistance to a monoclonal antibody that targets CD38 (such as daratumumab).

[0108] In some embodiments, the subject has undergone treatment with a monoclonal antibody that targets CD38 and has developed resistance to the anti-CD38 monoclonal antibody.

[0109] In certain embodiments, the antibodies of the invention and / or the multispecific antibodies of the invention and / or the population of CAR-T cells of the invention can be used in combination with an anti-cancer treatment.

[0110] Accordingly, the present invention relates to a method of treatment in a subject in need thereof, the method comprising administering to the subject (ii) a therapeutically effective amount of an antibody of the invention and / or a multispecific antibody of the invention and / or a population of CAR-T cells of the invention as a combined formulation to treat cancer, and (ii) a conventional treatment.

[0111] As used herein, the term "anti-cancer therapeutic" has its general meaning in the art and refers to any compound, natural or synthetic, used in the treatment of cancer.

[0112] In certain embodiments, anti-cancer therapy refers to radiation therapy, antibody therapy, or chemotherapy.

[0113] As used herein, the term "chemotherapeutic agent" refers to a chemical compound effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multi-kinase inhibitors, such as sorafenib and sunitinib, alkylating agents, such as thiotepa and cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelanamine); acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; cristatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estranustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin 11 and calicheamicin 211, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemycins (including dynemycin A); esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin, puromycin, chiramycin , lodorubicin, streptomugin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid acid); aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;Fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogenanium; tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, rhodilin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, 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; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antihormonal agents that act to regulate or inhibit hormone action in tumors, such as antiestrogens (including, for example, tamoxifen, raloxifene, 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 above;

[0114] As used herein, the term "radiotherapy" has its common meaning in the art and refers to the treatment of cancer with ionizing radiation. Ionizing radiation provides energy that damages or destroys cells in the treated area ("target tissue") by damaging genetic material, preventing these cells from continuing to grow. One type of radiation therapy commonly used involves photons, such as X-rays. Depending on the amount of energy they contain, the rays can be used to destroy cancer cells on the surface or deeper within the body. The greater the energy of the X-ray beam, the deeper the X-rays can penetrate into the target tissue. Linear accelerators and betatrons produce X-rays with increasingly higher energies. The use of machines to focus radiation (such as X-rays) at the cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are naturally produced when certain elements (such as radium, uranium, and cobalt-60) emit radiation during decomposition or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers beams shaped to closely match the shape of the tumor from different directions; intensity-modulated radiation therapy (IMRT), such as helical tomotherapy, which shapes the radiation beam to closely match the shape of the tumor and varies the dose of radiation according to the shape of the tumor; conformal proton radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation techniques to provide a real-time image of the tumor to guide radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to the tumor during surgery; stereotactic radiosurgery, which delivers many precise radiation doses to a small tumor area in a single session; hyperfractionated radiation therapy, such as sequential hyperfractionated accelerated radiation therapy (CHART), which targets more than one radiation therapy treatment (fraction) per day; and hypofractionated radiation therapy, which delivers radiation therapy in fewer fractions but with a higher dose per fraction.

[0115] 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.

[0116] As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule expressed by T cells to either signal up (stimulatory checkpoint molecules) or signal down (inhibitory checkpoint molecules).

[0117] Examples of stimulatory checkpoints 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.

[0118] As used herein, the terms "combination treatment," "combination therapy," or "combination therapy" refer to treatment using more than one agent. Combination treatment can be dual therapy or bi-therapy.

[0119] The agents used in the combination treatment of the present invention may be administered to a subject together, separately or sequentially.

[0120] As used herein, the term "co-administration" refers to the 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 the administration of two active ingredients by different routes at the same time or substantially the same time. The term "sequential administration" refers to the administration of two active ingredients at different times by the same or different routes of administration.

[0121] In particular, the population of CAR-T cells prepared as described above can be used in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those skilled in the art based on this disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al., and U.S. Patent No. 4,690,915 to Rosenberg. Currently, most adoptive immunotherapies are autologous lymphocyte therapies (ALT), which involve treatment using the patient's own immune cells. This treatment involves processing the patient's own lymphocytes. Typically, treatment is achieved by removing the patient's lymphocytes and converting them into a population of CAR-T cells as described above. Once CAR-T cells have been prepared with the CAR of the present invention, the ex vivo cells are reinfused into the patient to enhance the immune system and kill tumor cells. In some embodiments, the cells are first harvested from their culture medium and then formulated by washing and concentrating the cells in an appropriate medium and container system (a "pharmaceutically acceptable" carrier) for administration in a therapeutically effective amount. Suitable infusion media can be any isotonic media formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but also 5% dextrose in water or lactated Ringer's solution. The infusion media can be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition depends on the relative amount of T cells with the desired specificity, the age and weight of the recipient, the severity of the condition being targeted, and the immunogenicity of the target Ag. This amount of cells is approximately 10 3 / kg, preferably 5 × 10 3 / kg, and can be as low as 10 7 / kg, preferably 10 8The number of cells can be as high as 1 / kg. The number of cells, as well as the type of cells contained therein, will depend on the intended end use of the composition. For example, if cells specific for a particular Ag are desired, the population may contain more than 70%, typically more than 80%, 85%, and even 90-95% of such cells. For uses provided herein, the cells will generally be in a volume of 1 liter or less, and can be 500 ml or less, even 250 ml or 100 ml or less. A clinically relevant number of immune cells can be distributed over multiple infusions that cumulatively equal or exceed the desired total cell volume.

[0122] For administration, the antibodies of the present invention are formulated as pharmaceutical compositions. Pharmaceutical compositions containing the antibodies of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, thereby combining therapeutic molecules in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by the recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc. The dosage form, route of administration, dosage, and regimen of the pharmaceutical composition will necessarily depend on the condition being treated, the severity of the disease, the age, weight, and sex of the patient, etc. The pharmaceutical compositions of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and the like.

[0123] Typically, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for injectable formulations, which may in particular be an isotonic solution, a sterile solution, a saline solution (such as monosodium or di-sodium phosphate, sodium, potassium, calcium, or magnesium chloride, or a mixture of such salts), or a dried, in particular a lyophilized composition, which, when added with sterile water or saline, can constitute an injectable solution.

[0124] The dosage used for administration can be adapted depending on various parameters, in particular depending on the mode of administration used, the pathology involved or the duration of treatment desired.

[0125] To prepare pharmaceutical compositions, an effective amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0126] Pharmaceutical dosage forms suitable for use in injections include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid to the extent that it can be easily syringable. It must be stable under the conditions of manufacture and storage and must be protected against the contamination of microorganisms such as bacteria and fungi.

[0127] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0128] The antibodies of the present invention can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, and mandelic acids. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0129] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

[0130] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent containing various other ingredients as listed above, and then, if necessary, filter sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients as listed above that are required.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any other desired ingredients from the solution that has been previously sterile-filtered.

[0131] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as a solvent is expected to result in extremely rapid penetration, delivering high concentrations of the active agent to small tumor areas.

[0132] Once formulated, solutions may be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug-release capsules and the like are also available.

[0133] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, available sterile aqueous vehicles will be known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion fluid, or injected at the intended site of infusion (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dosage for the individual subject.

[0134] The antibodies of the invention can be formulated into therapeutic mixtures containing about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or even about 10 milligrams per dose, and multiple doses can also be administered.

[0135] In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable dosage forms include, for example, tablets or other solids for oral administration, extended release capsules, and any other dosage form currently in use.

[0136] In some embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies into host cells. The formation and use of liposomes and / or nanoparticles is known to those of skill in the art.

[0137] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, these ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily produced.

[0138] Liposomes are formed from phospholipids that disperse in aqueous media and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs), which contain aqueous solution in their cores and have diameters in the range of 200 to 500 Å. The physical characteristics of liposomes are determined by pH, ionic strength, and the presence of divalent cations.

[0139] The present invention is further described by the following figures and examples, which, however, should not be construed in any way as limiting the scope of the present invention. [Example]

[0140] Example 1: Novel CD38 / CD3 bispecific T cell engagers for the treatment of multiple myeloma method Construction and purification of Bi38-3 Bi38-3 was generated by fusing two scFvs derived from mouse hybridomas producing anti-human CD38 and CD3ε (B51 and OKT3, respectively), linked 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 and His tag sequences were introduced at the C-terminus. The sequence encoding Bi38-3 was cloned into the pCDNA3 expression vector (ThermoFisher) and confirmed by sequencing. This vector was transiently transfected into HEK-293T cells, and a 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 analysis with an anti-Myc tag antibody.

[0141] cell line The 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 (KMS11luc and MM1.Sluc) were generated by lentiviral transduction with a luciferase expression vector (Addgene, pLenti CMV Puro 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. 6MM1.Sluc cells were nucleofected with 2 μg of each Cas9 vector using Nucleofector-II (Lonza) and incubated in culture medium for 24 hours. GFP-positive cells were sorted by FACS and cloned in 96-well plates. Subclones were analyzed for CD38 expression by flow cytometry, and CD38-negative clones were selected for further analysis.

[0142] Blood and bone marrow samples Peripheral blood samples from healthy donors were obtained from the French Hematology Agency (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 the approval of the Saint-Louis Hospital Internal Research Committee.

[0143] Flow cytometry and cytotoxicity To assess lysis of the KMS-11luc or MM1Sluc MM cell lines, purified peripheral T cells (effectors) were incubated with various concentrations of Bi38-3 and luciferase-expressing MM1.S or KMS-11 cells (targets) at an effector-to-target ratio of 1:5 in 96-well flat-bottom plates in medium (RPMI, 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin, 10 μg / mL streptomycin, and 2 mM L-glutamine). Luciferase signals generated by viable MM cells were determined 24 h later using a CLARIOstar Plus luminometer plate reader (BMG LABTECH GmbH, Ortenberg, Germany) within 20 min after addition of firefly luciferase substrate (Bright-Glo Luciferase Assay System, Promega) according to the manufacturer's instructions. T cell cytotoxicity in primary cells was assayed by flow cytometry using similar coculture conditions. Effector T cells were incubated 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 / cyanin 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 viable cell counts were determined by flow cytometry using Brightcount beads (ThermoFisher). All FACS analyses were performed on a Canto II (Beckon Dickinson) column. Proliferation index analysis and calculations were performed on Flowjo.

[0144] T cell activation and proliferation assays For activity detection, effector (T) cells and target (MM1.S) cells were cocultured at a 5:1 ratio for 24 h and stained with anti-human CD4-APC-cyanine 7 (clone RPA-T4), anti-CD8-BV421 (clone RPA-T8), anti-CD25-phycoerythrin (Pe) / cyanine 5 (clone BC96), and anti-CD69-BV711 (clone FN 50) antibodies (all Biolegend) and analyzed by flow cytometry. For proliferation analysis, T cells were labeled with CellTrace Violet dye (ThermoFisher) and stimulated with MM1.S or MM1.S-CD38KO cells with or without Bi38-3 (10 ng / mL) for 96 h. Cells were stained with anti-CD4-APC / Cy7 (clone RPA-T4) and anti-CD8-BV421 (clone RPA-T8) antibodies and analyzed by flow cytometry.

[0145] Quantification of cytokines in cell culture supernatants Cytokine concentrations in the supernatants of the cytotoxicity assays were analyzed using the BD CBA Human Th1 / Th2 Cytokine Kit II (Beckon Dickinson). Data were acquired on a Canto II and analyzed with FCAP Array software (Beckon Dickinson).

[0146] Systemic tumor mouse model Under a protocol approved by the Animal Care and Use Committee (Comite d'ethique Paris-Nord), 6- to 12-week-old NOD / SCID / IL-2Rγ null mice (The Jackson Laboratory) were used. Mice were injected with 5 × 10 IL-2Rγ-null IgG via tail vein. 6 of MM1.Sluc cells were inoculated, followed 14 days later by 5×10 6Purified human T cells (purified using Miltenyi Biotec's pan T cell isolation kit) were injected into the mice. Tail vein injections of Bi38-3 (or control PBS) were repeated daily for 9 days. No randomization or blinding was used. Bioluminescence imaging was performed every 3 or 4 days. Mice were intraperitoneally injected with 240 μL of D-luciferin (15 mg / mL) (XenoLight D-luciferin potassium salt, Perkin Elmer), and images were acquired 15 minutes later using an IVIS imaging system (Perkin Elmer) with Living Image software (Perkin Elmer) at a medium binning level and various exposure times, in a 25 cm field of view. 22 days after MM cell inoculation, all mice were sacrificed.

[0147] result Construction, production, and binding properties of Bi38-3 Bi38-3 is derived from mouse hybridomas that produce anti-human CD38 and CD3ε (B51 and OKT3, respectively), and consists of two scFvs linked by a 15-amino acid glycine-serine (G4S1x3) spacer (not shown). The amino acid sequences corresponding to the variable domains of the anti-CD38 heavy and light chains, as well as the amino acid sequences of the anti-CD38 scFv and Bi38-3, are shown in Table 1. Table 1 shows the amino acid sequences of the immunoglobulins (IgH and IgK) of the BB515 hybridoma (anti-CD38), the corresponding scFv, and Bi38-3. [Table 1]

[0148] The anti-CD38 scFv is located at the N-terminus, and the anti-CD38 ε 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 revealed a unique protein with the expected size of 55.6 kDa recognized by anti-Myc tag antibodies (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 demonstrated by gel electrophoresis followed by Coomassie blue staining (data not shown). Binding of Bi38-3 to CD38-expressing MM1.S, KMS11, and NCI-H939 MM cells was analyzed by flow cytometry using an anti-Fab antibody recognizing the scFv domain. We observed that Bi38-3 was detected on the surface of MM cell lines, with less intense staining in KMS11 cells, which express lower levels of CD38, and stronger signals in MM1.S and NCI-H929 cells, which express 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 revealed that Bi38-3 was not detectable on the surface of CD38-negative MM1.S-KO cells (data not shown). Thus, purified Bi38-3 effectively and specifically recognizes CD38 on MM cells.

[0149] Bi38-3 induces T cell activation and proliferation in response to MM cells in vitro Next, we examined the T cell response to MM cells induced by Bi38-3. First, we performed FACS analysis to measure the proliferation index of violet-stained T cells. Stimulation of donor effector T cells (E) with Bi38-3 in the presence of MM1.S target cells (T) resulted in robust proliferation, with an average of five cell divisions (proliferation index) after 4 days, a level slightly higher than that induced by treatment with anti-CD38 / CD28 beads (data not shown). T lymphocytes cultured with CD38-deficient MM1.S cells and Bi38-3 did not proliferate, indicating that proliferation required CD38 expression on target cells. Furthermore, culture with Bi38-3 or MM1.S cells alone did not induce significant T cell proliferation.

[0150] Next, we analyzed the expression of early activation markers, CD69 and CD25, on donor T cells. After overnight coculture with MM1.S cells, CD4 and CD8 T cells rapidly upregulated both markers in a Bi38-3 dose-dependent manner, with up to 80% of CD69-positive T cells detected at the highest concentration (data not shown). On the other hand, upon stimulation with 100 ng / mL Bi38-3 alone, lower percentages of CD25- and CD69-expressing T cells (15% and 30%, respectively) were observed. Furthermore, coculture with MM1.S target cells alone did not induce the expression of activation markers (data not shown). Consistent with this, coculture of MM1.SKO cells with Bi38-3 induced less CD69 and CD25 than coculture with wild-type MM1.S cells (data not shown), indicating that the upregulation of activation markers was enhanced by CD38 expression on target cells.

[0151] Finally, we monitored the induction of cytokine production by Bi38-3. Coculture of MM1.S with donor T cells induced the production of interferon-γ (IFNg), tumor necrosis factor-α (TNFa), interleukin-2 (IL-2), IL-4, and IL-10 in a dose-dependent manner (data not shown). In contrast, stimulation with Bi38-3 alone or coculture with MM1.S alone failed to induce T cell secretion of any of these cytokines (data not shown). These results together demonstrate that Bi38-3 induces T cell proliferation, activation, and cytokine release in vitro in response to CD38-expressing MM cells.

[0152] Bi38-3 induces CD38-dependent T cell-mediated killing of MM cells in vitro To assess the function of Bi38-3, a co-culture assay was performed to measure the cytotoxic activity of effector T cells isolated from PBMCs of healthy donors on firefly luciferase-expressing target MM cell lines KMS11 and MM1.S. To measure the percentage killing in the presence of various concentrations of Bi38-3, the level of luciferase, which indicates the number of viable MM target cells remaining, was compared to the luciferase observed in untreated controls. T cells rapidly killed KMS11 target cells in a Bi38-3 dose-dependent manner, reaching a 50% effective concentration (EC 50 ) was approximately 5 ng / mL, which corresponds to 0.09 nM for this 55.6 Kd protein (data not shown). Bi38-3-mediated T cell cytotoxicity was also observed in coculture with MM1.S cells. However, in this cell line, which expresses higher levels of CD38, EC 50The concentration of Bi38-3 was 10-fold lower (0.5 ng / mL), indicating a stronger effect of Bi38-3. Meanwhile, the viability of MM1.S or KMS11 MM cells was not affected by coculture with T cells or Bi38-3 alone (data not shown). Furthermore, Bi38-3 resulted in impaired T cell-mediated killing of MM1.S-KO cells, and even at the highest dose of Bi38-3 (1 ng / mL), approximately half of the CD38-deficient MM1.S cells survived coculture (data not shown). Thus, Bi38-3 induced effective T cell cytotoxicity in CD38-expressing MM cells.

[0153] Bi38-3 induces autologous T cell-mediated killing of tumor plasma cells in vitro Next, we analyzed the potential of Bi38-3 to induce lysis of MM cells 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 various concentrations of Bi38-3. FACS analysis of overnight cocultures revealed a dose-dependent reduction in the number of viable CD138-positive MM cells, resulting in an increase in EC 50 We found that the cytotoxicity of Bi38-3 ranged from 0.5 to 1 ng / mL depending on the patient (Figure 1). Importantly, in the absence of T cells, Bi38-3 showed no toxicity to fresh primary MM cells. The cytotoxicity of Bi38-3-induced autologous T cells was further investigated in tumor plasma cells from MM patients at relapse and showed similar efficacy, compared with EC 50 ranged from 0.2 to 1 ng / mL (Figure 1). Thus, in this in vitro experiment, Bi38-3 induced autologous T cell-mediated killing of patients' tumor plasma cells both at diagnosis and at relapse.

[0154] Specific activity of Bi38-3 against CD38-highly expressing MM cells in vitro While CD38 is highly expressed on plasma cells, it is also expressed on various cell types, including hematopoietic cell subsets. To examine the effects of Bi38-3 on blood cells, donor PBMCs were treated with various concentrations of Bi38-3 for 24 hours, and various cell populations were analyzed by FACS (data not shown). We observed a significant dose-dependent decrease in the percentage of CD14-expressing monocytes falling within the viability gate (data not shown). Meanwhile, while the percentage of CD14-positive cells decreased, the percentages of CD4 and CD8 T lymphocytes, which together accounted for approximately 60% of the PBMC population, increased slightly in response to Bi38-3. Similarly, B (CD19+) cell and NK (CD56+) cell populations increased slightly or remained at similar levels (approximately 10% and 5%, respectively) even at high concentrations of Bi38-3 (100 ng / mL) (data not shown). We next investigated whether Bi38-3 impaired CD38 expression on the surface of blood cells. FACS analysis showed that the mean fluorescence intensity (MIF) of CD38 on T cells, B cells, and NK cells remained similar in cultures containing increasing Bi38-3 doses (data not shown). Consistent with this, CD38 expression was not significantly reduced on CD14+ myeloid cells, but analysis could not be performed with higher doses of Bi38-3 (1 and 100 ng / mL) because no cells or very few cells were detectable. To compare the activity of Bi38-3 on CD38-high (CD38hi) versus CD38-intermediate (CD38int) cells, we performed coculture assays with MM1.S cells expressing high levels of CD38 (data not shown), freshly isolated B cells expressing intermediate amounts of CD38 (data not shown), and autologous T cells. After overnight culture, the percentages of viable CD20+ B cells and CD138+ MM1.S cells were analyzed by flow cytometry. We observed that the percentage of MM1.S cells decreased at a Bi38-3 concentration of 0.1 ng / mL, and this decrease was even more pronounced at higher doses (data not shown). Meanwhile, compared with the untreated condition, the percentage of viable CD20+ B cells remained unchanged even at high Bi38-3 concentrations (data not shown).

[0155] 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, we developed three similar autologous coculture assays. We found that while Bi38-3 rapidly induced MM cell killing at low concentrations (10 to 2 ng / mL or higher), it did not induce significant T cell-mediated cytotoxicity in Foxp3+ Tregs (Figure 2A). Similarly, there was no significant cytotoxicity in CD34+ hematopoietic progenitor cells at concentrations below 10 ng / mL, and moderate cytotoxicity (>40% viability) at the highest concentration (Figure 2B). These results together indicate that Bi38-3 does not impair CD38 surface expression and induces only T cell-mediated killing of cells expressing high levels of CD38, with no or limited cytotoxicity against cells expressing moderate levels of CD38, such as hematopoietic progenitor cells, B cells, T cells, or NK cells.

[0156] Together, these results demonstrate that Bi38-3 does not impair surface expression of CD38 and induces T cell-mediated killing of CD38hi cells without significant activity against CD38int cells.

[0157] Bi38-3 regulates MM cell proliferation in vivo The in vivo antitumor 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 intravenously transplanted with or without Bi38-3 (0.08 mg / kg). Treatment with Bi38-3 or vehicle was repeated daily for 7 days (Figure 2A). 11 days after tumor cell injection, all mice showed similar levels of luciferase, indicating that MM cells had effectively engrafted in the host animals prior to Bi38-3 treatment (Figure 2B). While control mice showed rapid tumor progression, all animals treated with Bi38-3 showed a 5-fold reduction in tumor growth within the first 4 days of Bi38-3 treatment (Figure 2C). After 7 days, the level of luciferase-expressing MM cells in Bi38-3-treated mice was only 10-fold lower than the initial level and 50-fold lower than untreated controls. These results demonstrate that Bi38-3 is effective in controlling MM tumor progression in vivo.

[0158] Consider Herein, we report the development of Bi38-3, a novel anti-CD38 / CD3 bispecific T cell engager antibody, which induces specific T cell-mediated lysis of CD38-positive MM cells in vitro, ex vivo, and in vivo.

[0159] Monoclonal antibodies (Mabs) targeting CD38 have shown therapeutic efficacy in the treatment of MM 13 Daratumumab, an anti-CD38 Mab approved for MM, is a monotherapy 14 or in combination with standard of care regimens 2、15, and has shown good therapeutic efficacy. These clinical data indicate that CD38, which is abundantly expressed on tumor plasma cells, is a target of choice for immunotherapy of MM. However, despite significant survival improvements, many patients treated with daratumumab ultimately 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 does not have the Fc region found in natural immunoglobulins and recruits cytotoxic T cells via its anti-CD3 scFv without downregulating CD38 expression on target cells (data not shown). Therefore, Bi38-3-mediated T cell killing of MM cells appears not to be affected by mechanisms of resistance to anti-CD38 mAbs such as daratumumab, which are related to binding of therapeutic antibodies to FcγR. Similarly, the mechanism of resistance observed during relapse and 17 Bi38-3 treatment does not appear to upregulate the complement inhibitors CD55 and CD59 on cytotoxic cells, which is thought to contribute to resistance. Furthermore, MM is characterized by immune system abnormalities, and standard treatment regimens involving IMID and dexamethasone may limit the effectiveness of cytotoxic cells. However, these data indicate that Bi38-3 mediates autologous T cell-mediated killing of patients' tumor plasma cells at diagnosis and relapse with similar efficacy (Figure 1). Together, these data suggest that Bi38-3 can effectively eliminate MM cells in patients resistant to standard treatments, including those involving daratumumab.

[0160] CD38 is expressed on the surface of blood cells, including T lymphocytes, B lymphocytes, and NK lymphocytes. 18 Anti-CD38 mAb may target these cells and impair their function. Indeed, daratumumab has been shown to eliminate regulatory T cells. 19 , a process that may be associated with an increase in T cell number and activity during the early stages of treatment. 16Furthermore, daratumumab treatment resulted in the depletion of NK cells. 20 , which may increase a patient's susceptibility to infections 21 These data indicate that Bi38-3 has no significant effects on T cells, B cells, and NK cells in vitro (data not shown). We also report that it rapidly induced T cell-mediated killing of MM cells, even at high doses (10 ng / mL), 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 induces T cell cytotoxicity in B cells, T cells, and NK cells in vitro. 22 Although further experiments are needed to evaluate the toxicity of Bi38-3, especially in in vivo models against bone marrow cells, these results suggest that Bi38-3 can effectively induce the elimination of MM cells without affecting cells expressing low levels of CD38.

[0161] Recently, bispecific antibodies directed against Fc receptor-like 5 (Fcrl5 or FcHR5) or B cell maturation antigen (BCMA) have been reported. 10、12、23 BI836909, 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 mutated Fc region, effectively eliminated NCI-H929 cells in immunocompromised mice at the same dose (0.5 mg / kg). 10 BCMA expression is restricted to post-germinal center B cells, including memory B cells and both conventional 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 in tumor plasma cells are heterogeneous among patients. 25、26 Furthermore, in MM patients treated with T cells expressing anti-BCMA chimeric antigen receptors, BCMA was downregulated in tumor plasma cells. 27, a process that may contribute to tumor escape and recurrence. Together, these data reinforce the need to identify and evaluate additional targets in MM. Indeed, the development of effective and safe bispecific antibodies may contribute to improved treatment of MM.

[0162] These data demonstrate that targeting CD38 with Bi38-3 is effective in xenograft models at a dose of 0.1 mg / Kg (Figure 2), a dose significantly lower than that reported for BCMA bispecific antibodies in similar mouse models. 10、23 Therefore, Bi38-3 may represent an attractive treatment option in MM cases that do not express BCMA or express very low levels of BCMA.

[0163] Although BiTEs have demonstrated efficacy in several malignancies, their clinical development has been hampered by their short half-life in patients requiring continuous infusion via pump. 9 The CD19 / CD3 BiTE blinatumomab was recently approved for the treatment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Interestingly, an early phase 2 clinical trial showed that MRD negativity, associated with improved survival, could be achieved at the end of the first cycle of treatment. 28、29 Although the optimal number of cycles of blinatumomab in cases of MRD remains to be further investigated in ALL, clinical data indicate that limited BiTE treatment over time can still improve the prognosis of MRD+ patients. In this study, we show that Bi38-3 was able to induce a 6-fold reduction in tumor burden in just 3 days in vivo, despite the use of a highly proliferative MM cell line (MM1.S) (Figure 2C). Therefore, this rapid and clear activity in tumor plasma cells suggests that, similar to blinatumomab in ALL, Bi38-3 may eliminate MRD in MM patients after a limited number of cycles and improve prognosis after standard treatment.

[0164] Taken together, the data presented here identify Bi38-3 as a selective and effective compound in the treatment of MM that may be used in both frontline and relapse settings, and support its further evaluation in MM patients.

[0165] Example 2: Generation of CAR-T cells method Generation of transduced CAR-T cells HEK293 cells were transfected with 10 μg of the CAR construct and helper plasmids (psPAX2 and pMD2.G) using calcium phosphate. 12 hours after transfection, the complete medium (DMEM, 10% FVS) was refreshed. Two days after transfection, the cell-free supernatant containing retroviral particles was collected, concentrated by centrifugation, and used for transduction. T cells (purified using Miltenyi Biotec's pan T cell isolation kit) were stimulated with CD3 / CD28 beads (ThermoFisher) in medium (RPMI 1640, 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin). After 16 hours, cells were transferred to retronectin-coated (15 mg / mL) (Takara) 6-well plates (Falcon) and transduced overnight with the above lentiviral particles. Seventy-two hours after transduction, GFP and CAR expression were measured by flow cytometry to determine transduction efficiency. Transduced CAR-T cells represented more than 80% of the total cells and were used for in vitro experiments.

[0166] result Anti-CD38CAT T cells induce MM cell lysis in vitro Because Bi38-3 induced T cell-mediated lysis of MM cells (see Example 1), we investigated whether its anti-CD38 scFv could induce direct cytotoxicity of 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 the BB51-derived anti-CD38 scFv, the hinge and transmembrane regions of human CD8, and the CD3ζ signaling domain (Figure 3A). Because association of the CD3ζ active region with the costimulatory signaling domain was shown to enhance CAR activity, we also constructed a third-generation anti-CD38 CAR (CAR CD38 3G) composed of the anti-CD38 scFv, the transmembrane region of CD28, and the signaling domains of CD28, CD137 (4-1BB), and CD3ζ, in that order (Figure 3A). As controls, we also constructed a CAR lacking the scFv domain (CAR Mock) and a costimulatory CAR (CCR CD38) similar to CD38 3G but lacking the CD3ζ signaling domain. The protein sequences corresponding to these constructs are shown in Table 2. The DNA sequences encoding each CAR were cloned into lentiviral vectors capable of co-expressing GFP, viral particles were generated, and transduced into donor T cells. All CAR constructs were expressed in transduced human T lymphocytes (CAR-T), but CAR CD38 3G was expressed at lower levels than the other constructs (data not shown). Despite this difference, all transduced T cells stably expressed the CAR and were able to expand in vitro for 2 weeks, indicating the potential for cytotoxicity in CD38-expressing T cells, despite the potential for CAR-T culture (data not shown). To examine the cytotoxic function of effector CAR-T cells (E), we performed coculture experiments with various ratios of luciferase-expressing target cells (T). CD38-expressing MM1.S and RPMI8288 MM cells were rapidly lysed by both CAR CD38 1G and 3G compared with Mock and CCR CD38 negative controls (Figure 3B). Even at low E / T ratios (<2.5), anti-CD38 CAR-T killed MM cells, whereas CCR CD38 and Mock-transduced T cells exhibited poor cytotoxicity.On the other hand, CAR CD38 1G and 3G induced no or little lysis in CD38-negative HEK293 cells (Figure 3B). Thus, the anti-CD38 scFv derived from the BB51 hybridoma is effective in promoting T cell cytotoxicity in MM cells with various CAR constructs. Table 2 shows the amino acid sequences of the first and third generation anti-CD38 CARs (CAR CD38 1G and 3G, respectively) and the costimulatory CAR (CCR CD38). [Table 2] JPEG0007753184000003.jpg38165

[0167] References Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into the present disclosure. JPEG0007753184000004.jpg127165JPEG0007753184000005.jpg246169JPEG0007753184000006.jpg250165

Claims

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

10.

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

3.

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

4.

4. The monoclonal antibody of claim 1, which is a chimeric antibody.

5. The monoclonal antibody of claim 4, wherein the chimeric antibody has a heavy chain variable domain shown in SEQ ID NO: 3 and / or a light chain variable domain shown in SEQ ID NO:

4.

6. The monoclonal antibody of claim 1, which is a humanized antibody.

7. A single-chain variable fragment (scFv) comprising the VH and VL domains of the antibody of claim 1.

8. The scFv of claim 7, consisting of the amino acid sequence shown in SEQ ID NO:

11.

9. A multispecific antibody comprising a first antigen-binding site from the monoclonal antibody of claim 1 and at least one second antigen-binding site.

10. The multispecific antibody of claim 9 , wherein the second antigen-binding site is used to recruit T cells.

11. The multispecific antibody of claim 10 , wherein the second antigen-binding site has specificity for the extracellular domain of CD3ε.

12. 10. The multispecific antibody of claim 9, comprising an antigen-binding domain comprising or consisting of a single-chain variable fragment (scFv) of claim 7.

13. The multispecific antibody of claim 9 which is a bispecific T cell engager antibody.

14. 14. The multispecific antibody of claim 13, comprising the sequence shown in SEQ ID NO:

12.

15. A chimeric antigen receptor (CAR) comprising the antigen-binding domain of the antibody of claim 1.

16. The chimeric antigen receptor (CAR) of claim 15, comprising an antigen-binding domain comprising or consisting of the single-chain variable fragment (scFv) of claim 7.

17. The chimeric antigen receptor (CAR) of claim 15, 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.

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

19. A nucleic acid encoding the monoclonal antibody of claim 1, the multispecific antibody of claim 9, or the chimeric antigen receptor (CAR) of claim 15.

20. A nucleic acid encoding the heavy and light chains of the monoclonal antibody of claim 1.

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

22. 16. A host cell engineered to express the monoclonal antibody of claim 1, the multispecific antibody of claim 9, or the chimeric antigen receptor (CAR) of claim 15.

23. The host cell of claim 22, which is a CAR-T cell.

24. A pharmaceutical composition for treatment of a subject in need thereof, comprising the antibody of claim 1 and / or the multispecific antibody of claim 9 and / or a population of CAR-T cells of claim 23.

25. A pharmaceutical composition for treating cancer, comprising the antibody of claim 1 and / or the multispecific antibody of claim 9 and / or a population of CAR-T cells of claim 23.

26. 26. The pharmaceutical composition of claim 25, wherein the cancer is a CD38-positive hematological malignancy.

27. A pharmaceutical composition comprising a predetermined amount of the antibody of claim 1 and / or the multispecific antibody of claim 9 and / or the population of CAR-T cells of claim 23.

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