Antibodies having specificity for CD38 and uses thereof

Abstract

To provide antibodies with specificity for CD38 for treatment of multiple myeloma (MM), and to provide uses thereof.SOLUTION: The inventors have reported the development of Bi38-3 which is a new bispecific T cell engager targeting CD38 on MM cells and recruiting cytotoxic T cells through CD3ε. Bi38-3 does not have an Fc region of natural mAb, which contributes to resistance processes, but triggers T cells to proliferate, release cytokine and lyse CD38 positive MM cells in vitro. Similarly, Bi38-3 induces autologous T cells to eliminate tumor plasma cells isolated from MM patients both at diagnosis and at relapse. The cytotoxicity triggered by Bi38-3 is restricted to cells expressing high levels of CD38 and preserves the integrity of T, B and NK lymphocytes in vitro.SELECTED DRAWING: Figure 3

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. The functions of CD38 are adhesion and signaling. CD38 is normally expressed on hematopoietic cells, including hematopoietic stem cells, hematopoietic stem cells, and hematopoietic stem cells. In hematopoietic cells, the majority of thymic medullary cells express CD38 + in 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 in the germinal centers of lymph nodes. It is expressed in lymphoblasts, plasma B cells, and some intrafollicular cells. It is also expressed in CD3 8 can also be expressed on dendritic cells. A high proportion of normal bone marrow cells, especially progenitor cells, are expressed on dendritic cells. Furthermore, 50-80% of cord blood cells express CD38 + and human blood CD38 is also involved in lymphoid cell division. In addition to progenitor cells, CD38 is expressed on erythrocytes and platelets. In the intestine, lymphocytes from the epithelial cells and lamina propria mediate the development of Purkinje cells and neurofibrillary changes. in the brain by mitochondrial cleavage, in the prostate by epithelial cells, in the pancreas by beta cells, and in osteoclasts by It is expressed in bone, in the eye by retinal cells, and in the sarcolemma of smooth and striated muscles.

[0003] CD38 is also expressed in multiple myeloma, B-cell chronic lymphocytic leukemia, and B-cell acute lymphoblastic leukemia. Leukemia, Waldenström's macroglobulinemia, primary systemic amyloidosis , mantle cell lymphoma, prolymphocytic / myelocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia A variety of malignancies, including myeloid leukemia, follicular lymphoma, NK cell leukemia, and plasma cell leukemia It also occurs in other hematological disorders. For example, multiple myeloma (MM) is a monoclonal immunoglobulin It is characterized by the accumulation of tumor plasma cells secreting steroids in the patient's bone marrow and osteolytic lesions. It is a heterogeneous hematological malignancy that is characterized by 1 Current treatments result in a median overall survival of approximately 6 years. In recent years, elotuzumab (anti-SLAMF7) and daratumumab ( The development of monoclonal antibodies (mAbs) such as anti-CD38 has further improved the prognosis. R 2-4 However, proteasome inhibitors (PIs), immunomodulatory agents (IMIDs), and Overall survival for patients whose disease recurs after mAb treatment remains extremely poor, and MM is incurable. Thus, there is a need to improve patient care and ultimately develop a cure. Therefore, new treatment strategies are needed.

[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. is doing. [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 provides antibodies and and its use. [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 demonstrated CD in vitro, ex vivo, and in vivo. A novel anti-CD38 / CD3 antibody induces specific T cell-mediated lysis of CD38-positive MM cells We developed a bispecific T cell engager antibody. This novel anti-CD38 / CD3 bispecific antibody T cell-mediated activation of MM cells by T cells mediated by the T cell engager antibody Bi38-3 Killing by anti-CD38 mAb (for the treatment of MM) is associated with binding of the therapeutic antibody to FcγR. are not affected by mechanisms of resistance to approved anti-CD38 mabs (such as daratumumab) The present inventors have demonstrated that Bi38-3 has a similar effect on patients at the time of diagnosis and recurrence. Furthermore, the inventors have demonstrated that the autologous T cell-mediated killing of tumor plasma cells in patients with leukemia is mediated by the autologous T cell-mediated killing of tumor plasma cells in patients with leukemia. Bi38-3 had significant effects on T cells, B cells, and NK cells in vitro. and protects B cells from T cell cytotoxic activity while inhibiting T cell-mediated proliferation of MM cells. The inventors have shown that Bi38-3 rapidly induces killing in vivo. We demonstrated that this method can induce a six-fold reduction in tumor burden in just three days. The inventors believe that Bi38-3 is a selective and effective compound in the treatment of MM and has been shown to be effective in situ. and may be used in both frontline and relapse settings to aid further evaluation in MM patients. showed.

[0010] (Main definitions) As used herein, the term "CD38" refers to its known function in the art. ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase The amino acid sequence of an example of CD38 is shown in SEQ ID NO: 1. The domain is from the 43rd amino acid residue to the 300th amino acid residue in SEQ ID NO: 1. The range is. SEQ ID NO: 1 >sp|P28907|CD38_HUMAN ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1 O S=Homo sapiens OX=9606 GN=CD38 PE=1 SV=2 MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVL AVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRH VDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPC NKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTW CGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEA ACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLE AWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIY RPDKFLQCVKNPEDSSCTSEI

[0011] As used herein, the term "CD3" is used in the art. It has a general meaning and is used to express cytotoxic T cells (CD8+ naive T cells) and also helper T cells. This refers to the CD3 (cluster of differentiation 3) T cell co-receptor, which promotes activation of both T cells and T cells. It consists of a protein complex, composed of four specific chains. In mammals, this complex The CD3 receptor contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains are It associates with the TCR and zeta chain to generate activation signals for T lymphocytes. The TCR, ζ chain, and CD3 molecule together comprise the TCR complex. Example amino acids of CD3ε The sequence is shown in SEQ ID NO: 2. The extracellular domain of CD3ε is located at the 23rd position in SEQ ID NO: 2. The range is from the amino acid residue to the 207th amino acid residue. Sequence number 2 >sp|P07766|CD3E_HUMAN T-cell surf ace glycoprotein CD3 epsilon chain OS=Ho mo sapiens OX=9606 GN=CD3E PE=1 SV=2 MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVS ISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVC ENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKA KPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI

[0012] As used herein, the term "antibody" thus refers to a polypeptide having an antigen-binding region. The term is used to refer to any antibody-like molecule having a Fab', Fab, F(ab antibody fragments containing antigen-binding domains such as single domain antibodies (DABs); TandAb dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd , linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (bispecific or trispecific scFv-Fab fusions, respectively), c-diabody, κ(λ)body (scFv-CL fusion), BiTE (bispecific antibody) Heterologous T cell engager (scFv-scFv tandem type that attracts T cells), DVD-I g (dual variable domain antibodies, bispecific formats), SIP (small molecule immunoprotein , a type of minibody), SMIP ("small molecule modular immunopharmaceutical" scFv-Fc dimer ), DART (ds stabilized diabody "dual affinity retargeting"), Various antibody-based constructs and fragments are available. Techniques for preparing and using acetaldehyde are known in the art (see Kabat et al., 1991). (The disclosure of which is expressly incorporated herein by reference.) Diabodies are in particular those described in European Patent No. 4 and further described in WO 93 / 11161, linear antibodies. The antibody was further described by Zapata et al. (1995). For example, F(ab')2 fragments can be produced by cleaving an antibody with pepsin. The resulting F(ab')2 fragment can be prepared by Fab' fragments can be produced by treatment to reduce sulfide bridges. Papain digestion can produce Fab fragments. Fab, Fab ', and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, d s-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and and other fragments can be synthesized recombinantly or chemically. Techniques for producing antibody fragments are known and described in the art. For example, Beckman et al., 2006; Holliger and Hudson, 20 05;Le Gall et al., 2004;Reff and Heard, 2001;Reiter et al., 1996; and Young et al., 1995, each of which reported on the production of effective antibody fragments. The manufacturing process is further described to make it possible.

[0013] In natural antibodies, the two heavy chains are linked to each other by disulfide bonds, and each heavy chain The chains are linked to one light chain by a disulfide bond: lambda (1) and kappa (k). There are five major classes of heavy chains (also known as heavy chains) that determine the functional activity of antibody molecules. IgM, IgD, IgG, IgA, and IgE are the isotypes. The light chain contains a variable domain (VL) and a constant domain (CL). The heavy chain contains two domains: a variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4, collectively referred to as CH) It contains five (μ, ε) domains, one for each light chain (VL) and one for each heavy chain (VH). The variable regions determine binding recognition and specificity to the antigen. Light chain (CL) and heavy chain (CH) The constant region domains of the antibody are involved in antibody chain assembly, secretion, transplacental transport, complement fixation, and Fc receptor (F Fv fragments confer important biological properties, such as binding to immunoglobulins (e.g., Fv receptors). It is the N-terminal part of the Fab fragment of a human antibody, and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody-combining site and the antigenic determinant. The site is composed primarily of residues from hypervariable regions or complementarity-determining regions (CDRs). As such, residues in the non-hypervariable or framework regions (FR) are likely to be involved in the antibody binding site. CDRs may be involved in or affect the whole domain structure, i.e., the binding site. Amino acids that define the binding affinity and specificity of the natural Fv region of a human immunoglobulin combining site. The light and heavy chains of an immunoglobulin each refer to a sequence of L-CDR1, L-CDR2, L-CDR3 and three CDRs called H-CDR1, H-CDR2, and H-CDR3 Thus, the antigen-binding site typically comprises the CD1 and CD2 domains of the heavy and light chain V regions. The framework region (FR) is located between the CDRs. The residues of antibody variable domains are traditionally identified by Kabat et al. The numbers are numbered according to a system devised by Kabat et al. 1987, Sequences of Proteins of Immunology cal Interest, U.S. Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al.") This numbering system is used herein. Kabat residue names does not always correspond directly to the linear numbering of amino acid residues in the SEQ ID NO sequence. The actual linear amino acid sequence is not the framework region of the basic variable domain structure. Truncation of or insertion into structural elements, whether they are amino acid sequences or complementarity determining regions (CDRs), Depending on the input, the amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering. The correct Kabat numbering of groups is determined by the homologous residues in the antibody sequence for a given antibody. by aligning with the "standard" Kabat numbered sequence The CDRs of the heavy chain variable domain are located at residue 3 according to the Kabat numbering system. 1-35B (VH-CDR1), residues 50-65 (VH-CDR2), and residues 95-1 The CDRs of the light chain variable domain are located at 02 (VH-CDR3) according to the Kabat numbering system. According to the system, residues 24–34 (VL-CDR1), residues 50–56 (VL-CDR 2), and located at residues 89 to 97 (VL-CDR3).

[0014] As used herein, the term "BB51 antibody" refers to the BB51 antibody as set forth in SEQ ID NO:3. and a light chain variable domain as shown in SEQ ID NO: 4. It refers to a mouse antibody that can be used to treat rhesus mastitis. SEQ ID NO: 3 > IgH VH1.87-D1.1-J1: QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQ RPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTA YMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTVTV SS SEQ ID NO: 4 >Igk Vk12.44-Jk5: DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQK QGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQ PEDFGSYYCQHHYGIPLTFGAGTKLELK

[0015] As used herein, the term "scFv" refers to a small fragment comprising the variable region of a light chain. At least one antibody fragment and at least one antibody fragment containing the variable region of the heavy chain The term "fusion protein" refers to a fusion protein comprising a light chain and a heavy chain variable region, the light chain and the heavy chain variable region being linked together via, for example, a synthetic linker, For example, a single polypeptide chain is formed by linking the polypeptide chains in close proximity via a small flexible polypeptide linker. scFvs can be expressed as scFvs with the specificity of the intact antibody from which they are derived. Unless otherwise specified, as used herein, scFv refers to a polypeptide, e.g., The polypeptide has variable regions VL and VH in either order relative to the N-terminus and C-terminus. The scFv can comprise a VL-linker-VH, or a VH-linker As used herein, a "monoclonal antibody" may comprise a CAR-VL. "," "monoclonal Ab," "monoclonal antibody composition," "mAb," etc. The term as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to antibodies derived from human germline immunoglobulins. The term "antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from the sequence of the antibody.

[0016] As used herein, the term "chimeric antibody" refers to a chimeric antibody that is composed of the VH domain of a non-human antibody. refers to an antibody that contains the VL and VL domains of a human antibody, as well as the CH and CL domains of a human antibody. In some embodiments, a "chimeric antibody" comprises (a) a constant region (i.e., a heavy chain and / or or light chain) or parts thereof to modify, substitute, or exchange the antigen binding site (variable region) differ or change class, effector function, and / or species constants antibody molecules that bind to the domain, or e.g., enzymes, toxins, hormones, growth factors, drugs, etc. (b) a completely different molecule that confers new properties to the chimeric antibody; or (b) a variable region, or and changing, substituting, or exchanging a portion of the variable region with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized antibodies and, in particular, humanized antibodies. Additionally, chimeric antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody function. Jones et al., Nature 321:522-525 (1986); Riech mann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. Acad. Sci. USA, 81:6851-6855 (1984).

[0017] As used herein, the term "humanized antibody" refers to an antibody in which the variable regions are derived from a human antibody. It has the regional framework regions and constant regions, but retains the CDRs of the previous non-human antibody. In some embodiments, a humanized antibody refers to an antibody that is derived from minimal non-human immunoglobulins. In many cases, humanized antibodies and antibody fragments thereof contain sequences similar to those of the recipient. Mice whose complementarity determining region (CDR) residues have the desired specificity, affinity, and potency; Human immunoglobulins substituted with residues from the CDRs of a non-human species (donor antibody), such as rat or rabbit. globulin (recipient antibody or antibody fragment). Residues in the Fv framework regions (FR) of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody / antibody fragment also contains the C The antibody may contain residues that are not found in the DR or framework sequences. The binding region is derived from a non-human antibody, but is not specifically engineered to retain the binding specificity of the non-human antibody from which it was derived. These modifications are designed to prevent an immune response to the antibody. In general, humanized antibodies or their antibodies can be further refined and optimized. A human immunoglobulin fragment is one in which all or substantially all of the CDR regions are those of a non-human immunoglobulin. and wherein all or most of the FR regions are from human immunoglobulin sequences. The humanized antibody may comprise at least one, and typically two, variable domains. The antibody or antibody fragment is typically of a human immunoglobulin, For further details, see J. ones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988;Presta, Curr.Op. See Struct.Biol., 2:593-596, 1992.

[0018] As used herein, the term "antibody binds to a given antigen or epitope" refers to a specific antigen or epitope that binds to a specific antigen or epitope. The term "binding" in this context typically refers to binding that is achieved by, for example, surface plasmon resonance (SPR) techniques. The antigen was used as the ligand and the analyte was used as the analyte in a BIAcore 3000 instrument. When measured using a soluble form of the antibody, approximately 10 -6 K of M D The corresponding low affinity BIACORE® (GE Healthcare, Piscaat away, NJ) is conventionally used for epitope bins of monoclonal antibodies. One of a variety of surface plasmon resonance assay formats. Typically, the antibody is Nonspecific antibodies that are not identical to or closely related to a specific antigen (e.g., BSA, casein) K in binding to D at least 10 times lower, for example at least 100 times lower, e.g., at least 1000 times lower, e.g., at least 10,000 times lower, e.g., at least 100,000 times lower than 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 but, Typically, K against non-specific antigens D At least 10,000 times lower than The binding is not detectable (e.g., BIAco using surface plasmon resonance (SPR) technology). Using the antigen as the ligand and the soluble form of the antibody as the analyte in the re3000 instrument or the antibody and an antigen or antigens having different chemical structures or amino acid sequences. is 100-fold, 500-fold, 1000-fold, or 10-fold greater than the binding detected by the epitope. If the antibody is more than 00 times less, it can be said that it does not substantially bind to the antigen or epitope.

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

[0020] As used herein, "bispecific T cell engagers" or "BiTEs" The term "antibody" refers to a recombinant antibody consisting of two movably linked single-chain fragments (scFv). It refers to a bispecific antibody that is a protein construct, in which one of the scFv antibodies is a selected The second binds specifically to a target cell-expressed tumor antigen, and the second binds to a T cell receptor in a T cell. It specifically binds to other molecules, such as CD3, a subunit of the CD4 complex. In this state, BiTE antibodies transiently bind T cells to target cells and simultaneously bind T cells to target cells. BiTE-mediated activation of T cells can activate specific lytic activity in T cells. Neither the specific T cell receptor nor the MHC I molecule, peptide antigen, or costimulatory molecule on the target cell. Not necessary.

[0021] As used herein, the term "CAR-T cells" refers to cells that express a CAR. CAR-T cells are T lymphocytes genetically engineered to target specific CD4+ T cells. , CD8+ T cells, γδ T cells, and effector T cells, memory T cells, and regulatory T cells. Includes all classes and subclasses of T lymphocytes, including those that are genetically modified. The T lymphocytes are "derived" from a subject undergoing treatment using genetically modified T cells. can be "derived" or "obtained" from, or "derived" or "obtained" from, a different object "You can do it."

[0022] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor (CAR) that is a chimeric antigen receptor (CAR). Immune effector cells have specificity for target cells, typically cancer cells, and A set of polypeptides, typically in the simplest embodiment, that provide an intracellular signal generation to the cell. In some embodiments, a CAR comprises at least two polypeptides. an extracellular antigen-binding domain, a transmembrane domain, and a stimulatory molecule and / or Cytoplasmic signaling domains containing functional signaling domains derived from costimulatory molecules (Honmei In some embodiments, the polypeptide further comprises an "intracellular signaling domain" (also referred to herein as an "intracellular signaling domain"). The polypeptides of the set are proximal to one another. In some embodiments, the polypeptides of the set are adjacent to one another. The peptides can bind to each other in the presence of a dimerization molecule, e.g., For example, a dimerization sequence that can link an antigen-binding domain to an intracellular signaling domain. In some embodiments, the stimulatory molecule comprises a ζ-cell receptor complex associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain is a cytoplasmic signaling domain, as defined below. and further comprising one or more functional signaling domains derived from at least one costimulatory molecule such as In some embodiments, the costimulatory molecule is a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In this embodiment, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, a stimulatory molecule-derived domain, and a Chimeric fusion proteins containing intracellular signaling domains containing functional signaling domains of interest. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane protein. domains, and functional signaling domains derived from costimulatory molecules and functional signaling domains derived from stimulatory molecules. and a chimeric fusion protein comprising an intracellular signaling domain. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and two functional signaling domains derived from one or more costimulatory molecules and a functional domain derived from a stimulatory molecule. and a chimeric fusion protein comprising an intracellular signaling domain and a functional signaling domain. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and and at least two functional signaling domains from one or more costimulatory molecules. 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. In some embodiments, the polypeptide further comprises an optional leader sequence at the amino terminus (N-terminus) of the polypeptide. The CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, The driver sequence optionally binds to the antigen-binding domain during cellular processes and localization of the CAR to the cell membrane. In certain embodiments, the CAR is cleaved from a transmembrane domain (e.g., scFv). and a single-chain variable fragment derived from a monoclonal antibody fused to the CD3-ζ endodomain. In some embodiments, the CAR comprises a fusion of a CD3-ζ fragment (scFv). , FcR, CD27, CD28, CD137, DAP10, and / or OX40 In some embodiments, the costimulatory molecule comprises a domain for costimulatory signaling comprising: reporter genes for imaging (e.g., for positron emission tomography), prodrugs Gene products that conditionally eliminate T cells upon addition of homing receptors, chemokines, and chemokines Molecules including kine receptors, cytokines, and cytokine receptors are co-expressed with CAR. It is possible.

[0023] As used herein, the term "T cells" is used in conjunction with the term "T cells" as used in the art. They have general significance and represent important components of the immune system that play a central role in cell-mediated immunity. T cells express their TCRs through presentation or restriction by molecules of the major histocompatibility complex. They are known as general lymphocytes because they recognize antigens with their T cell receptors (antigen receptors). Several T cells, including D8+ T cells, CD4+ T cells, and γδ T cells, each with a specific function, are There are several subsets of T cells.

[0024] As used herein, the term "CD8+ T cells" is used in the art. It has its general meaning of "cancerous T cells" and refers to a subset of T cells that express CD8 on their surface. are MHC class I restricted and function as cytotoxic T cells. "T cells" refers to 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. It is a member of the IL-1 receptor and the associated recognition element in class I-restricted interactions of the major histocompatibility complex. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to tumor-infiltrating CD8+ T cells that are derived from the bloodstream. This refers to the pool of CD8+ T cells in a patient that have left the tumour and migrated to the tumour.

[0025] As used herein, "CD4+ T cells" (also known as T helper cells or TH cells) The term CD4 (also called CD4+) refers to the B-cell plasma cells and Immunity, including maturation into memory B cells and activation of cytotoxic T cells and macrophages CD4+ T cells are T cells that help other white blood cells in the process. When peptide antigens are presented by MHC class II molecules expressed on the surface of APCs, Once activated, they divide rapidly to regulate or support the active immune response. These cells secrete cytokines that promote various types of immune responses. TH1, TH2, TH3, TH17, TH9, TFH, or They can differentiate into one of several subtypes, including Tregs. In addition to CD4, this technology also induces T cells into specific subtypes. The known TH cell surface biomarkers in the field are CXCR3 (Th1), CCR4, and Crth 2 (Th2), CCR6 (Th17), CXCR5 (Tfh), and cytokines and Translocations including T-bet, GATA3, EOMES, RORγT, BCL6, and FoxP3 Includes subtype-specific expression of transcription factors.

[0026] As used herein, the term "γδ T cells" refers to the γδT cells are normally found in the peripheral blood of healthy individuals (humans, monkeys). They account for 1-5% of lymphocytes, which are involved in the initiation of protective immune responses and are involved in the MHC Recognizes antigenic ligands by interacting directly with the antigen without being presented by a molecule It has been shown that γ9δ2 T cells (sometimes called γ2δ2 T cells) , γδ T cells carrying TCR receptors with variable domains Vγ9 and Vδ2. They constitute the majority of γδ T cells in human blood. Upon activation, γδ T cells can produce various It exhibits potent non-MHC-restricted cytotoxic activity, effective in killing various cell types, especially pathogen cells. It is a virus (Poccia et al., J. Leukocyte Biology ology, 1997, 62:1-5) or other intracellular parasites such as mycobacteria ( Constant et al., Infection and Immunity,D October 1995, Vol. 63, No. 12: 4628-4633) or protozoa (Behr et al., Infection and Immunity, 1996 , vol.64, no.8:2892-2896). This may be cancer cells (Poccia et al., J. Immunol., 15 9:6009-6015; Fournie and Bonneville, Res. Immunol.,66th Forum in Immunology,147:33 8-347). Therefore, in vitro, ex vivo, or in vivo The possibility of modulating the activity of these cells in infectious diseases (especially viral or parasitic diseases) ), cancer, allergies, and even autoimmune and / or inflammatory disorders New and effective therapeutic approaches in treatment may be available.

[0027] As used herein, the term "treatment" or "treating" refers to the treatment of a disease. Patients at risk of or suspected of having a disease, and those who are ill or have a disease or Preventive or preventative treatment, including treatment of patients diagnosed with a medical condition, and curative treatment Treatment refers to both a disease-modifying or a prophylactic treatment, including the prevention of clinical recurrence. Prevent, cure, or delay the onset of recurrent disorders, or reduce the severity of disorders or recurrent disorders or to improve one or more symptoms of the disorder or recurring disorder, or to treat such to extend a patient's survival beyond that expected in the absence of a medical disability Therapeutic regimens can be administered to patients who have or may eventually develop such disorders. " means a pattern of treatment for a disease, for example, a pattern of medication used during treatment. The regimen can include an induction regimen and a maintenance regimen. The expression "induction period" refers to the period during which the therapeutic regimen (or regimens) used to treat the disease initially is initiated. The overall purpose of an induction regimen is to induce high levels of The induction regimen is a "loading regimen" (partial or overall), which is more than a physician may use during a maintenance regimen. administering a lower dose of the drug, or administering the drug more frequently than a physician may administer during a maintenance regimen; "Maintenance regimen" or "maintenance period" may include administering a The term "therapeutic drug" refers to a drug that is used to treat a disease, for example, to keep a patient in a state of remission for an extended period of time (months or years). The therapeutic regimen (or part of the therapeutic regimen) used for the maintenance of the patient during treatment A maintenance regimen refers to continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly). or annually), or intermittent treatment (e.g., treatment with breaks, intermittent treatment, repeated Treatment for the onset of a disease or when certain prescribed conditions are met (e.g., pain, symptoms of a disease, etc.) The above procedure can be used.

[0028] As used herein, the term "cancer" refers to its general meaning in the art. It has a specific meaning and is capable of autonomous growth, i.e., invading or spreading to other parts of the body. an abnormal condition characterized by rapidly growing cell proliferation with the potential to spread or Refers to abnormal cells that have a pathological condition. This term refers to the type or stage of histopathological invasion. Regardless of all types of cancer growth or oncogenic process, metastatic tissue or malignant cancerous cells, The term "cancer" is meant to include affected lung, breast, Malignant lesions of various organ systems, including the thyroid, lymphoid, gastrointestinal, and genitourinary tract, as well as large intestinal partial colon cancer, renal cell carcinoma, prostate and / or testicular cancer, glioblastoma, non-small cell lung cancer, This includes, but is not limited to, adenocarcinoma, including malignant lesions such as small intestine cancer and esophageal cancer. The term "cancer" includes, without limitation, solid tumors and blood-borne tumors.

[0029] The term "solid tumor" has its general meaning in the art, including head and neck tumors. 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 bone tumors, chordoma, multiple myeloma), brain and central nervous system cancers (e.g., meningioma, astrocytoma, oligodendroma, Dendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germinoma, craniopharynx cephaloma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ) cancer, 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 gastrointestinal carcinoid tumors (e.g., choriocarcinoma, destructive chorioadenocarcinoma), Kaposi's sarcoma, kidney Cancer of the liver (e.g., renal cell carcinoma), pharynx and hypopharynx, 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), cytoma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g., esthesioneuroblastoma, midline granuloma), nasopharynx Neuroblastoma, oral and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, Prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), stomach cancer testicular cancer (e.g., seminoma, non-seminomatous germ cell cancer), thymic cancer, thyroid cancer ( For example, follicular carcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer. a solid tumor selected from the group consisting of, but not limited to, uterine cancer (e.g., uterine leiomyosarcoma) Refers to a tumour.

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

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

[0032] As used herein, the term "CD38-positive hematological malignancies" refers to leukemia, These tumors are characterized by the presence of tumor cells that express CD38, including leukemia, lymphoma, and myeloma. Examples of such CD38-positive hematologic malignancies include precursor B-cell lymphomas (PCL) and B-cell lymphomas (BLM). acute myelocytic 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), follicular lymphoma (FL) (including low-grade, intermediate-grade, and high-grade FL), cutaneous follicle center lymphoma, peripheral Marginal zone B-cell lymphoma (MALT, nodal and splenic types), hairy cell leukemia, diffuse large cell type DLBCL, Burkitt lymphoma (BL), plasmacytoma, multiple myeloma leukemia, plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom macroglobulinemia This includes leukemia, 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 a dose that achieves a desired therapeutic result. A therapeutically effective amount of an active agent is an amount effective for the treatment of an individual's disease, at the dosage and for the period of time necessary. The individual's condition, age, sex, and weight, and the ability of the active agent to induce the desired response in the individual. A therapeutically effective amount may vary depending on factors such as the severity of the disease, the severity of the illness, and the severity of the underlying condition. A therapeutically effective amount may also vary depending on factors such as the severity of the disease, the severity of the underlying condition, and the severity of the underlying condition. A therapeutically effective amount may also vary depending on factors such as the severity of the illness, severity ... The therapeutically beneficial effects outweigh the adverse effects. The amount of hydroxybenzoates used will depend on the disease or condition being treated and can be determined by one skilled in the art. A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician may prescribe a lower level than is required to achieve the desired therapeutic effect. and increasing the dosage until the desired effect is achieved. Generally, the appropriate dose of the composition of the present invention will depend on the particular administration. The regimen may be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. For example, for therapeutic use, A therapeutically effective amount can be determined by its ability to stabilize the progression of the disease. In addition, the tumor suppressive potential of compounds is evaluated in animal model systems that are predictive of efficacy in human tumors, for example. A therapeutically effective amount of a therapeutic compound can reduce tumor size in a patient. Those skilled in the art will be able to determine such amounts based on the size of the patient. The dosage will be determined based on factors such as 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 0.1 to 100 mg / kg, for example about 0.1 to 50 mg / kg, for example about 0.1 to 20 mg / kg, for example about 0.1 to 10 mg / kg, for example about 0.5, for example about 0.3, about 1, about The therapeutic efficacy of the inhibitors of the present invention is about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. An exemplary, non-limiting range of amounts is 0.02 to 100 mg / kg, for example, about 0.02 to 30 mg / kg, for example, about 0.05 to 10 mg / kg, or 0.1 to 3 mg / kg, for example, about The dose is 0.5 to 2 mg / kg. The administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous. The above-described methods and uses of treatment can be used in a variety of applications, for example, by administering the agent proximal to the target site. Dosage regimens may be adjusted throughout the administration of the compound to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or The dosage may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, treatment efficacy is monitored during therapy, e.g., at predetermined time points. In some embodiments, efficacy can be assessed by visualization of diseased areas or by other methods as further described herein. By other diagnostic methods described, for example, the labeled inhibitors of the present invention, the inhibitors of the present invention fragments or miniantibodies derived from the agent can be used to perform, for example, one or more PET-CT scans. If necessary, the efficacy of the pharmaceutical composition can be monitored by carrying out a test. The effective daily doses are administered separately at appropriate intervals throughout the day, optionally in single dosage forms. The compound may be administered in 3, 4, 5, 6, or more divided doses. In this embodiment, the human monoclonal antibodies of the present invention are effective in minimizing any undesirable side effects. To achieve this, the drug is administered by slow continuous infusion over an extended period of time, for example, more than 24 hours. Effective doses of the inhibitors of the present invention may also be administered using weekly, biweekly, or every three week dosing periods. The administration period can be extended, for example, to 8 weeks, 12 weeks, or until clinical progression is observed. As a non-limiting example, the treatment in the present invention can be limited to the treatment start time. After the start 1, 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, At least on the 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th or 40th day Also on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th At least one week of the 14th, 15th, 16th, 17th, 18th, 19th, or 20th week, or as otherwise determined In any combination, about 0.1 to 100 mg / kg per day, for example, 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 The daily dose of the inhibitor of the present invention may be administered in a single dose or in an amount of 24, 12, 8, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 3 , 4, or 2 hourly divided doses, or any combination thereof can be provided.

[0035] (Antibody of the present invention) The first object of the present invention is to - (i) H-CDR1 as shown in SEQ ID NO: 5, (ii) H-CDR2 as shown in SEQ ID NO: 6, and (iii) a heavy chain comprising the H-CDR3 set forth in SEQ ID NO: 7, and - (i) L-CDR1 as shown in SEQ ID NO: 8, (ii) L-CDR2 as shown in SEQ ID NO: 9, and (iii) a light chain comprising the L-CDR3 set forth in SEQ ID NO: 10. It relates to monoclonal antibodies with predominant binding specificity. 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 the amino acid sequence set forth in SEQ ID NO:3. It contains a VH domain that has at least 70% identity to the sequence.

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

[0038] In the present invention, a first amino acid sequence having at least 70% identity with a second amino acid sequence is The amino acid sequence is 70;71;72;73;74;75, where the first sequence is identical to the second amino acid sequence. ;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% In the present invention, the term "having at least one amino acid sequence identical to a second amino acid sequence" means that the second amino acid sequence has at least one amino acid sequence identical to the first amino acid sequence. A first amino acid sequence that has at least 90% identity with a second amino acid sequence is 90;91;92;93;94;95;96;97;98;99; or 100% identity Sequence identity is often understood as identity (or similarity or The higher the ratio, the more similar the two sequences are. Methods for sequence alignment for this purpose are well known in the art and include various programs and The alignment algorithm is based on Smith and Waterman, Adv. Appl. M ath., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. atl.Acad.Sci.USA, 85:2444, 1988;Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sh arp, CABIOS, 5:151-153, 1989;Corpet et al., Nuc.Ac ids 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, A detailed discussion of sequence alignment methods and homology calculations is provided in The alignment tool ALIGN (Myers and Miller, CABIO S 4:11-17, 1989) or LFASTA (Pearson and Lipm Sequence comparisons can be performed using the Internet Program am,1996,WRPearson and the University o f Virginia, fasta20u63 version 2.0u63,199 (Released in December 2016). ALIGN compares entire sequences with each other, while LFASTA compares local sequences. Compare areas of similarity. These alignment tools and their respective tutorials are available at Available on the internet, for example, at the NCSA website. For comparison of amino acid sequences exceeding 100, the default parameters (gap existence cost) are used. 11, the default BLOSUM62 matrix was set to a per-residue gap cost of 1). You can use the Blast2 sequences function with the When aligning peptides (less than about 30 amino acids), the default PAM set to parameters (start gap penalty 9, extension gap penalty 1) Align the sequences using the Blast2 function with the 30 matrix. The BLAST sequence comparison system is available from, for example, the NCBI website. Altschul et al., J. Mol. Biol., 215:403-4 10, 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 a functional variant of the VL region, VH region, or CD of the BB51 antibody. The present invention provides an antibody comprising one or more functional variants of R. Context of the monoclonal antibodies of the present invention The functional variants of VL, VH or CDRs used in at least a significant proportion (at least) of the affinity / avidity and / or specificity / selectivity of the antibody (even about 50%, 60%, 70%, 80%, 90%, 95% or more) if the antibody is still In some cases, such a monoclonal antibody of the present invention can be maintained as Such functional variants may have higher affinity, selectivity and / or specificity than the parent Ab. The CDR variants typically retain high sequence identity to the parent Ab. Conservative substitutions of portions may differ from the sequences of the CDRs of the parent antibody sequence, e.g., At least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more of substitutions Above, about 80% or more, about 85% or more, about 90% or more (e.g., about 65-95%, e.g., about 92%) 93%, or 94%) are conservative substitutions of amino acid residues. Conservative substitutions of portions may differ from the sequences of the CDRs of the parent antibody sequence, e.g., At least 10, for example at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions In the context of the present invention, conservative substitutions are defined as follows: It can be defined by substitutions within the amino acid classes represented. 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 turn formation are A, C, D, E, G, H, K, N, Q, R, S, P, and Residue T involved Mobile residues Q, T, K, S, G, P, D, E, and R

[0040] Further conservative substitution classifications include valine-leucine-isoleucine, phenylalanine-thymine, These include lysine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation of hydrophobic / hydrophilic character and weight / size of residues in variant CDRs is also important. Substantially retained compared to the CDRs of the BB51 antibody. Protein-interactive biology The importance of the hydropathic amino acid index in conferring functional function has been well recognized in the art. It is generally understood that the relative hydrophilicity of amino acids determines the structure of the resulting protein. contributes to secondary structure, which in turn interacts with proteins and other molecules, e.g., enzymes, substrates, receptors, It is recognized that each amino acid determines the interaction with DNA, antibodies, antigens, etc. Each molecule is assigned a hydrophilicity index based on its hydrophobicity and charge characteristics. They found that isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenyl Calcium (+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); As Partate (-3.5); Asparagine (-3.5); Lysine (-3.9); and Arg The retention of similar residues can also be achieved by using the BLAST program. (e.g., BLOSUM62 with standard settings, starting gap = 11 and extension gap = 1 The results were determined using BLAST (available at NCBI using BLAST 2.2.8). Suitable variants can be determined by a similarity score, such as: It exhibits at least about 70% identity with the parent peptide.

[0041] In some embodiments, the monoclonal antibodies of the invention are chimeric antibodies. In an embodiment, the monoclonal antibody of the invention is a chimeric antibody having a heavy chain shown in SEQ ID NO:3. In some embodiments, the monoclonal antibody comprises a light chain set forth in SEQ ID NO:4. In some embodiments, the monoclonal antibodies of the present invention are chimeric antibodies having the following structure: It is a chimeric antibody having a heavy chain shown in sequence number 3 and a light chain shown in sequence number 4.

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

[0043] The monoclonal antibodies of the present invention may have one or more of the functional or structural characteristics in the above-mentioned aspects. Characterized by a feature or by any combination of selected functional and structural features It is possible.

[0044] The antibodies of the present invention can be of any isotype. Typically, the desired effector function, such as ADCC induction, can be guided by the desired effector function. The isotypes are IgG1, IgG2, IgG3, and IgG4. Either the kappa or lambda region can be used. The class of an antibody can be switched by known methods. Exemplary class switching The conversion of one IgG subclass to another, e.g., IgG1 to IgG2, is a common technique used in immunofluorescence. Thus, the human monoclonal antibody of the present invention can be used for Vector function can be enhanced by isotype switching to allow for various therapeutic uses, e.g. For IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies In some embodiments, the antibodies of the invention are full-length antibodies. In some embodiments, the full-length antibody is an IgG1 antibody. The antibody is an IgG4 antibody. In some embodiments, the IgG4 antibody is a stabilized IgG An example of a suitable stabilized IgG4 antibody is a human IgG4 heavy chain constant region. Arginine 409 in the ATPase (as indicated in the EU index by Kabat et al. is substituted with lysine, threonine, methionine, or leucine, preferably lysine. and / or an antibody having a hinge region (described in WO 2006 / 033386). Other suitable stabilized IgG4 antibodies include those containing the Cys-Pro-Pro-Cys sequence. The antibodies are disclosed in WO 2008 / 145142, which is incorporated herein by reference. and is hereby incorporated by reference in its entirety. Null antibodies have reduced or even eliminated ability to mediate effector functions such as ADCC. non-IgG4 types, such as IgG1, IgG2, or IgG 3 antibody. Such mutations are described, for example, in Dall'Acqua WF et al., J Imm unol.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, the present invention The antibody of interest typically has characteristics, e.g., serum half-life, complement fixation, Fc receptor binding, and / or in the Fc region to modify one or more functional properties of the antibody, such as antigen-dependent cellular cytotoxicity. Furthermore, the monoclonal antibodies of the present invention can be engineered to contain modifications as described herein. However, antibodies can be chemically modified (e.g., , which can attach one or more chemical moieties to the antibody), or its glycosylation. For example, the affinity of the antibodies provided by the present invention can be modified to alter The modifications can be made using any suitable method known in the art. Therefore, the present invention also provides a method for producing a medicament for the treatment of psoriasis, which has improved affinity for CD38. The present invention also relates to variants of the antibody molecules of the invention. Such variants can be obtained by mutating the CDRs (Ya ng et al., J. Mol. Biol., 254, 392-403, 1995), Chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), the use of mutagenized 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) These can be obtained by a number of affinity maturation protocols, including those described by Vaughan et al. (see above) explores these affinity maturation methods.

[0046] In some embodiments, the Fc region may contain at least one amino acid sequence that modifies the effector functions of the antibody. It is modified by replacing at least one amino acid residue with a different amino acid residue. For example, one or more amino acids may be added to the antibody to provide altered affinity for an effector ligand. However, different amino acid residues may be substituted so as to retain the antigen-binding ability of the parent antibody. The affinity-modified effector ligand can be, for example, an Fc receptor or a complement C1 receptor. This approach is based on the US study by Winter et al. As described in more detail in Patent Nos. 5,624,821 and 5,648,260 It is listed.

[0047] In some embodiments, the antibody comprises one or more amino acids selected from the amino acid residues Altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (C1q) The amino acid sequence can be substituted with different amino acid residues to give a nucleotide sequence (DC). This approach , as further described in U.S. Patent No. 6,194,551 to Idusogie et al. It has been done.

[0048] In some embodiments, one or more amino acid residues are modified to fix complement. This approach is described in WO 99 / 043000 by Bodmer et al. In some embodiments, the Fc region of an antibody is further described in US Pat. improve the ability of the antibody to mediate ADCC and / or enhance one or more Amino acid modifications to improve antibody affinity for Fc receptors This approach is further described in WO 00 / 42072 by Presta. Furthermore, FcyRI, FcyRII, FcyRIII, and FcRn The binding site in human IgG1 has been mapped, and mutants with improved binding activity have been identified. has been described (Shields, R.L. et al., 2001 J.Biol.Chen. 276:6591-6604, see WO 2010 / 106180).

[0049] In some embodiments, the glycosylation of the antibody is modified. For example, aglycosylated Antibodies can be made that are glycosylated (i.e., the antibodies have no glycosylation). Sylation can be modified, for example, to improve the affinity of the antibody for antigen. Such carbohydrate modifications can be made, for example, by altering one or more glycosylation sites within the antibody sequence. For example, glycosylation of one or more variable region frameworks can be achieved by one or more enzymes that remove glycosylation at the site, thereby eliminating glycosylation at that site Such glycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in U.S. Patent No. 5,714, by Co et al. Further details are described in US Pat. Nos. 3,350 and 6,350,861. Alternatively, antibodies with altered glycosylation types, e.g., antibodies with reduced amounts of fucosyl residues. a hypofucosylated antibody or a non-fucosylated antibody having no fucosyl residues, or a bisecting antibody Antibodies can be produced that contain increased amounts of bisecting GlcNac structures. The silylating pattern has been shown to improve the ADCC ability of antibodies. Modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with modified glycosylation machinery can be obtained by the methods described in the art. By expressing the recombinant antibody of the present invention, it is possible to obtain an antibody with modified glycosylation. For example, the host cells described in European Patent No. 1,247,299 by Hang et al. No. 176,195 describes a cell line with a functionally disrupted FUT8 gene. This gene is expressed in such cell lines to show hypofucosylation or fucosylation. Therefore, some fucosyltransferases are encoded so that the fucosyltransferase does not have any fucosyl residues. In one embodiment, the human monoclonal antibodies of the invention are hypofucosylated or nonfucosylated. cell lines that exhibit a specific fusion pattern, e.g., the FUT8 gene encoding fucosyltransferase It can be produced by recombinant expression in a mammalian cell line that has been deficient in expression of the gene. Presta, International Publication No. 03 / 035835, discloses a method for linking fucose to Asn(297). The ability of antibodies to attach to synthetic carbohydrates is reduced, and the low fucosylation of antibodies expressed in the host cells have described a mutant CHO cell line, Lec13 cells, that also results in Shield s, RL et al., 2002 J.Biol.Chem.277:26733-26740 Umana et al., in WO 99 / 54342, report on glycoprotein-modifying glycoproteins. Cosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferases) We have described cell lines engineered to express GnTIII (GnTIII). Thus, antibodies expressed in engineered cell lines exhibit a bipartite structure that results in improved ADCC activity of the antibody. It shows an increase in branched GlcNac structures (Umana et al., 1999 Nat. Biol. tech.17:176-180). Eureka Therapeutics , generating antibodies with modified mammalian glycosylation patterns that lack fucosyl residues Further genetically engineered CHO mammalian cells are described (http: / / www.eurekainc.com / a&boutus / companyover Alternatively, the human monoclonal antibodies of the present invention may be derived from mammalian-like glycoproteins. The glycosylation pattern of the antibody was engineered to lack fucose. The enzymes can be produced in yeast or filamentous fungi, which are capable of producing the enzymes (see, for example, European Patent No. 129 See No. 7172).

[0050] In some embodiments, the antibody is an antigen-binding fragment. Antibodies can be produced by conventional techniques, for example, by fragmenting full-length antibodies or in recombinant cells. The fragment can be obtained by expressing a nucleic acid encoding the fragment (e.g., Eva ns et al., J. Immunol. Meth. 184, 123-38 (1995)). The fragments are then purified in the same manner as described herein for full-length antibodies. It can be tested or screened for that property.

[0051] In some embodiments, the monoclonal antibodies of the invention comprise the VH domain of the antibodies of the invention. In some embodiments, the fragment is an scFv fragment comprising a VL domain and a VL domain. The scFv fragment consists of the amino acid sequence shown in SEQ ID NO:11. SEQ ID NO: 11; > scFv antibody DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQK QGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQ PEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGG GGSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWV KQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSS TAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTV TVSS

[0052] Nucleic Acid Molecules for Producing Antibodies of the Invention and Their Use The monoclonal antibodies of the present invention include, but are not limited to, the following, alone or in combination: Any chemical, biological, genetic, or enzymatic technique known in the art. For example, the amino acid sequence of the desired sequence can be known. Therefore, those skilled in the art can easily prepare such antibodies by standard methods for producing polypeptides. For example, this can be done using known solid phase methods, preferably on a commercially available peptide synthesizer (A pplied Biosystems, Foster City, California can be synthesized using amines (e.g., manufactured by Sigma-Aldrich) according to the manufacturer's instructions. Alternatively, the antibodies of the present invention can be produced by recombinant DNA techniques known in the art. For example, antibodies can be synthesized by inserting a DNA sequence encoding the antibody into an expression vector. and expressing the desired antibody, which can then be isolated using known methods. After introducing such vectors into a suitable eukaryotic or prokaryotic host capable of expressing DNA It can be obtained as an object.

[0053] Therefore, a further object of the present invention is a nucleic acid encoding a monoclonal antibody of the invention. In some embodiments, the nucleic acid sequence is related to the sequence of the monoclonal antibody of the present invention. The nucleotides encode the ribonucleotide and / or light chain.

[0054] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in any suitable vector. The term "vector" as used herein means a vector that can be linked to It is used to refer to a nucleic acid molecule capable of transporting other nucleic acids. One type of vector is a "pla a "mid," which is a circular bilayer to which additional DNA segments can be ligated. The term refers to a single-stranded DNA loop. Another type of vector is a viral vector, which can ligate additional DNA segments into the viral genome. The vector is capable of autonomous replication in a host cell into which it is introduced (e.g., a bacterial replication origin). bacterial vectors and episomal mammalian vectors). Other vectors (non-episomal When introduced into a host cell, the vector (e.g., a mammalian vector) is integrated into the genome of the host cell. In addition, certain vectors can be operably Such vectors are capable of directing the expression of genes to which they are linked. These vectors are called "recombinant expression vectors" (or simply "expression vectors"). Expression vectors of use in DNA technology are often in the form of plasmids. As used herein, "plasmid" and "vector" refer to a vector, with the plasmid being the most commonly used However, the present invention also provides a vector in which Equivalent functional viral vectors (replication-defective retroviruses, adenoviruses, and It is intended to include such other forms of expression vectors, such as adeno-associated viruses. .

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

[0056] Such vectors, when administered to a subject, can be used to express or induce expression of the antibody. Therefore, it may contain regulatory elements such as promoters, enhancers, and terminators. Examples of promoters and enhancers used in expression vectors for human cells include the SV40 promoter and the SV40 promoter. Early promoter and enhancer (Mizukami T. et al. 1987) , Moloney murine leukemia virus long terminal repeat promoter and enhancer (Kuwana Y et al.1987), immunoglobulin heavy chain promoter (Mason JO et al.1985) and enhancers (Gillies SD et al.19 83)) and the like. Any expression vector for animal cells may contain a gene encoding a human antibody C region. Any suitable vector can be used as long as it can insert and express the gene. Examples are pAGE107 (Miyaji H et al. 1990), pAGE103 ( Mizukami T et al.1987), pHSG274(Brady G e t al. 1984), pKCR (O'Hare K et al. 1981), pSG Including lβd2-4- (Miyaji H et al. 1990). Examples of such plasmids include replicative plasmids containing an origin of replication, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenovirus, retrovirus, and integrating plasmids. Such recombinant viruses include viruses, herpes viruses, and AAV vectors. By methods known in the art, for example, by transfecting packaging cells. or by transient transfection with a helper plasmid or virus. Typical examples of virus packaging cells are PA317 cells, Psi These include CRIP cells, GPenv+ cells, 293 cells, etc. Such replication-deficient recombinant viruses Detailed protocols for producing the fusion protein are described, for example, in WO 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6, Nos. 013,516, 4,861,719, and 5,27 No. 8,056, and WO 94 / 19478.

[0057] A further object of the present invention is to provide a method for gene transfer using the nucleic acid and / or vector of the present invention. It relates to a host cell that has been infected, infected or transformed.

[0058] The term "transformation" refers to the introduction of "foreign" (i.e., exogenous or extracellular) genes, DNA A, or RNA sequence into a host cell, the host cell can then The sequence is expressed to produce a desired substance, typically encoded by the introduced gene or sequence. It means that the cells produce proteins or enzymes by accepting introduced DNA or RNA. A host cell expressing the vector has been "transformed."

[0059] The nucleic acids of the invention can be used in a suitable expression system to produce the monoclonal antibodies of the invention. The term "expression system" refers to a vector-carried, host-hosted vector. Suitable vectors for the expression of proteins encoded by exogenous DNA introduced into host cells. A common expression system is E. coli. li host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells Other examples of host cells include prokaryotic cells (such as bacteria) and mammalian host cells and vectors. Eukaryotic cells include, but are not limited to, yeast cells, mammalian cells, insect cells, plant cells, etc. Specific examples include E. coli, Kluyveromyces, or Saccharomyces. myces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., lymphoblastoid cells, These include cells made from cells such as blasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, and adipocytes. Examples include mouse SP2 / 0-Agl4 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), dihydrofolate reductase CHO cells (Ur Laub G et al;1980), rat YB2 / 3HL.P2.G1 1.16 Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. Also includes.

[0060] The present invention also relates to a method for producing a recombinant host cell that expresses an antibody of the present invention. The method comprises: (i) in vitro or ex vivo recombination of the above-described (ii) introducing the nucleic acid or vector into a competent host cell; Culturing the resulting recombinant host cells in vitro or ex vivo; iii) optionally selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used to produce the antibodies of the invention.

[0061] (Multispecific antibodies of the present invention) A further object of the present invention is to provide a method for producing a first antigen-binding site from a monoclonal antibody of the present invention, and at least one second antigen-binding site.

[0062] In the present invention, the multispecific antibody of the present invention comprises an extracellular domain of CD38 and a target It binds to the extracellular domain of other antigens.

[0063] In some embodiments, the second antigen-binding site binds to an antigen on, for example, a human effector. Killing by binding or by binding a cytotoxic or second therapeutic agent Used to mobilize the wound mechanism.

[0064] As used herein, the term "effector cell" refers to a cell that is involved in the recognition of an immune response. Refers to immune cells that are involved in the effector phase of the immune response, as opposed to the inflammatory and activation phases. The immune cells of the present invention include cells of myeloid or lymphoid origin, such as lymphocytes (B cells and cytolytic T cells). T cells, including CTLs), killer cells, natural killer cells, macrophages, These include monocytes, mast cells, and granulocytes, such as neutrophils, eosinophils, and basophils. Effector cells express specific Fc receptors (FcRs) and carry out specific immune functions In some embodiments, the effector cells are ADCC cells, such as natural killer cells. For example, monocytes and macrophages express FcR and can induce specific target cell responses. They are involved in the efficient killing of antigens and in presenting antigens to other components of the immune system. In embodiments, effector cells are capable of phagocytosing a target antigen or target cell. The expression of specific FcRs on effector cells is regulated by humoral factors such as cytokines. Effector cells can phagocytose target antigens, or Suitable cytotoxic and second therapeutic agents include those capable of phagocytosing or lysing target cells. Examples are listed below and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs. .

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

[0066] In some embodiments, the multispecific antibodies of the invention comprise a single chain variable flag chain of an antibody of the invention. an antigen-binding domain comprising, consisting of, or consisting essentially of a scFv Includes.

[0067] In some embodiments, the antigen binding domain comprises a linker peptide. The tide can be positioned between the light chain variable region and the heavy chain variable region.

[0068] Exemplary formats for multispecific antibody molecules of the invention include: (i) one antibody directed against CD38; one with specificity for the CD3α antigen and the other with specificity for other antigens such as CD3ε. (ii) two antibodies cross-linked by teloconjugation; and (ii) two distinct antigen-binding regions. (iii) a single antibody comprising two different antigen-binding regions, e.g., an additional peptide linker; (iv) a single-chain antibody comprising two scFvs linked in series by a linker; (iv) a light chain and a heavy chain Dual variable domains, where the chain contains two variable domains in tandem via a short peptide bond. Main antibody (DVD-Ig) (Wu et al., Generation and C haracterization of a Dual Variable Domai n Immunoglobulin (DVD-Ig) Molecule, In : Antibody Engineering, Springer Berlin H Eidelberg (2010); (v) Chemically linked bispecific (Fab')2 (vi) a tetravalent bivalent fragment having two binding sites for each of the target antigens; Tandabodies, a fusion of two single-chain diabodies resulting in specific antibodies; i) flexibodies, which are combinations of scFvs and diabodies resulting in multivalent molecules; (viii) When applied to Fab, two identical Fs linked to different Fab fragments A protease inhibitor capable of generating trivalent bispecific binding proteins consisting of ab fragments. Based on the "dimerization and docking domain" in protein kinase A, (ix) "dock-and-lock" molecules; for example, fused to both ends of a human Fab-arm (x) a so-called Scorpion molecule containing two scFvs, and (x) a diabody. Examples include, but are not limited to:

[0069] Another exemplary format for bispecific antibodies is a method for heterodimerization. Such molecules are IgG-like molecules having complementary CH3 domains. For example, Triomab / Quadroma(Trion Pharma / Freseniu s Biotech), Knob-into-Hole (Genentech), Cro ssMAb (Roche) and electrostatically matched attached) (Amgen), LUZ-Y (Genentech), strand exchange engineered domain Strand Exchange Engineered Domain b ody)(SEEDbody)(EMD Serono), Biclonic(Meru) s), and what is known as DuoBody (Genmab A / S) technology. It can be prepared as follows.

[0070] In some embodiments, bispecific antibodies are typically synthesized using DuoBody technology. Controlled F In vitro methods for generating bispecific antibodies by ab-arm exchange have been published internationally. Publication Nos. 2008 / 119353 and 2011 / 131746 (both Genmab A / S). One example is described in WO 2008119353. In the method of claim 1, the bispecific antibody is capable of reacting with both "Fab-arms" or "half molecules" between two monospecific antibodies containing IgG4-like CH3 regions The resulting product is formed by exchange (swapping of heavy chains with attached light chains). It is a bispecific antibody having two Fab arms that may comprise a sequence of: In another exemplary method described in US Pat. No. 11 / 131746, the bispecific antibody of the invention comprises: wherein at least one of the first and second antibodies is an antibody of the present invention, the method comprising the steps of: The method comprises the steps of: (a) providing a first antibody comprising an Fc region of an immunoglobulin; (b) providing an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region; a second antibody comprising a second CH3 region, the Fc region comprising a second CH3 region, and The sequences of the CH3 regions of the first and second CH3 regions are different, and the heterodimeric interaction between the first and second CH3 regions is The homodimer interactions of the first and second CH3 regions are stronger than those of the first and second CH3 regions. (c) incubating the first antibody with the second antibody under reducing conditions. (d) detecting a first antibody that is an antibody of the present invention and a second antibody that is different from the first antibody; obtaining the bispecific antibody with a binding specificity, or vice versa. The conditions are, for example, 2-mercaptoethylamine, dithiothreitol, and Tris(2- The reaction is carried out by adding a reducing agent, such as one selected from the group consisting of methyl carboxyethyl phosphines. Step (d) can be carried out, for example, by removing the reducing agent, for example by desalting. and restoring the condition to a non-reducing or nearly non-reducing state. Preferably, the sequences of the first and second CH3 regions are different, and the first and second CH3 The heterodimeric interaction between the first and second CH3 domains is similar to the homodimeric interaction between the first and second CH3 domains. It contains only a small number of relatively conservative, asymmetric mutations, making each stronger than the others. Further details regarding the interaction and how it is obtained are provided in International Publication No. WO 2005 / 024990. No. 011 / 131746, which is incorporated herein by reference in its entirety. In some other embodiments, the bispecific antibodies of the invention are symmetric clusters. It is a bispecific IgG4 antibody, which consists of a variable domain, a CH1 domain, and and two heavy chains including a hinge region, and in each heavy chain, a cysteine ​​of a light chain and an interchain disulfide The cysteine ​​in the CH1 domain that forms the sulfide bond is replaced with another amino acid, Optionally, one or more of the amino acids located in the upper hinge region are substituted with cysteine, The constant region sequences are similar or identical, and the variable regions of each heavy chain are different. Antibodies to the antibody are described in International Publication No. WO2013 / 124450. In an embodiment, the bispecific antibodies of the invention are asymmetric antibodies, which are Two heavy chains or heavy chain flags containing at least a variable region, a hinge region, and a CH1 domain the first heavy chain or a fragment thereof is of class IgG4; and (a) CH1 The 127th number in the domain, as numbered by the Kabat numbering system, (b) the interchain cysteines in the and one or more of the amino acids at the positions corresponding to the first heavy chain or fragment thereof are substituted with cysteine. In some cases, some or all of the chains may have a structure similar to that of the first chain in at least a region outside the variable region (e.g., the constant region). The bispecific format is characterized by having an amino acid sequence different from that of the heavy chain of the Antibodies to this antibody are described in International Publication No. WO2013 / 124451.

[0071] In some embodiments, the multispecific antibodies of the invention comprise bispecific T cell engagers ( BiTE) antibody.

[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 DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQK QGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQ PEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGG GGSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWV KQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSS TAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTV TVSSGGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMS CKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYD DHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSVDDIQLT QSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKR WIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATY YCQQWSSNPFTFGSGTKLELKAAA

[0074] The present invention also provides nucleic acids encoding the multispecific antibodies of the present invention. In the method, the nucleic acid is incorporated into a vector as described above.

[0075] (Chimeric antigen receptor (CAR) for generating host cells expressing a chimeric antigen receptor (CAR) (CAR) and its use) The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of the antibody of the present invention. to provide.

[0076] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor (CAR) that is a chimeric antigen receptor (CAR). has its general meaning in the art and is linked to a T cell signaling domain. An artificially constructed hybrid antibody containing the antigen-binding domain of an antibody (e.g., scFv) CARs are characterized by the antigen-binding properties of monoclonal antibodies. Utilizing these techniques, T cell specificity and reactivity can be directed to selected targets in a non-MHC restricted manner. Furthermore, when expressed in T cells, the CAR advantageously inhibits endogenous It does not dimerize with the T cell receptor (TCR) α and β chains. Typically, the chimeric antigen receptor The antibody of the present invention comprises at least one VH and / or VL sequence of the antibody of the present invention. The La antigen receptor consists of an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain. Includes the Good domain.

[0077] In some embodiments, the antigen binding domain comprises a linker peptide. The tide can be positioned between the light chain variable region and the heavy chain variable region.

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

[0079] In some embodiments, the CAR of the present invention comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14. It consists of an amino acid sequence. SEQ ID NO: 13 > CAR CD38 1G DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQK QGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQ PEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGG GGSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWV KQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSS TAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTV TVSSLEHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLR PEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 14 > CAR CD38 3G DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQK QGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQ PEDFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGGGSGG GGSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWV KQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSS TAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTTV TVSSLEIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP GPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR

[0080] In some embodiments, the CAR comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell selected from the group consisting of CD28, 4-1BB, and CD3ζ intracellular domains; Contains a cell signaling domain. CD28 is a T cell marker that is important for T cell costimulation. 4-1BB delivers a potent costimulatory signal to T cells, promoting their differentiation and proliferation. CD3ζ enhances the long-term survival of lymphocytes. CD3ζ engages with TCR to generate signals and promote immune response. It contains an ITAM receptor tyrosine-based activation motif (ITAM).

[0081] In some embodiments, the chimeric antigen receptors of the present invention are glycosylated, amidated, carboxylated, or carboxylated. carboxylation, phosphorylation, esterification, N-acylation, e.g., via disulfide bridges It can be cyclized or converted to an acid addition salt 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 an embodiment, the nucleic acid is incorporated into a vector as described above.

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

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

[0085] As used herein, the term "cytotoxic lymphocyte" refers to a lymphocyte that is a type of lymphocyte that is expressed in the art. Its general meaning in the context of the lethal attack on infected target cells is the spread of infection. Targeting the destruction of intracellular pathogens, such as viral pathogens, required for limiting proliferation In the present invention, the term "cytotoxic lymphocyte" refers to a lymphocyte that is a cytotoxic T cell. and natural killer cells.

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

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

[0088] In some embodiments, the host cells are, for example, peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells. In some embodiments, the T cells are isolated from primary PBMCs. Cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat cells. The T cells can be any T cells, such as T cells from rats, SupT1, etc., or mammals. When obtained from a mammal, T cells may be isolated from blood, bone marrow, lymph nodes, thymus, or other tissues. It can be obtained from a number of sources, including but not limited to, bodily fluids. The cells may also be enriched or purified. The T cells may be any type of T cell. , CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g. Th2 cells, C D8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naïve The cells may be at any stage of development, including but not limited to T cells. The cells can be CD8+ T cells or CD4+ T cells.

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

[0090] In some embodiments, the host cells are pluripotent stem cells (PSCs). can be modified with a CAR and then used to induce T cells (see, for example, International Patent Application Publication No. PSCs are a type of stem cell derived from embryonic stem cells (ESCs) and induced pluripotent stem cells ( iPSCs can be generated directly from adult cells (e.g., somatic cells). iPSCs typically contain a set of pluripotency-associated genes, or "liposomes," specific to a given cell type. Reprogramming factors can be introduced into the cells to induce or generate reprogramming. The transcription factor is OCT4 (also known as "POU5FL"), which is also known as the Yamanaka factor. These include, but are not limited to, SOX2, cMYC, and KLF4. akahashi, K; Yamanaka, S (2006) “Induction of pluripotent stem cells from mouse embryo onic and adult fibroblast cultures by de "Finested factors" Cell 126(4):663-76.

[0091] In some embodiments, the host cells are hematopoietic stem cells. The term "hematopoietic stem cell" or "HSC" refers to a type of hematopoietic stem cell that is capable of self-renewal and differentiation into the precursors of blood cells. These progenitor cells are blood cells that cannot self-replicate and differentiate into mature blood cells. Hematopoietic stem and progenitor cells are immature blood cells that require specific functions. 8-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA -, Lin-CD34+CD38+IL-3aloCD45RA-, and Lin-CD3 They exhibit numerous phenotypes, including CD4+CD38+CD10+ (Daley et al., ocus 18:62-67,1996;Pimentel,E.,Ed.,Handb ook of Growth Factors Vol.III:Hematopoie tic Growth Factors and Cytokines,pp.1-2, CRC Press, Boca Raton, Fla., 1994). In this process, stem cells self-renew and give rise to all mature blood cells over the course of a person's lifetime. In some embodiments, the method comprises the steps of: maintaining continuous production of hematopoietic progenitor cells or hematopoietic stem cells; The cells are isolated from peripheral blood cells.

[0092] In some embodiments, CAR activity is measured to optimize the safety and efficacy of CAR therapy. There are many ways in which CAR activity can be regulated. For example, inducible enzymes using caspases fused to dimerization domains are available. apoptosis (e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3;365 (18):1673-1683) as a safety switch in the CAR therapy of the present invention. It can be used as such.

[0093] Treatment Methods and Pharmaceutical Compositions The antibodies, multispecific antibodies, and CAR-T cells of the present 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. The method comprises administering a therapeutically effective amount of an antibody of the invention and / or a multispecific antibody of the invention and / or a multispecific antibody of the invention. and administering to the subject a population of CAR-T cells of the invention.

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

[0096] Thus, the present invention provides a method for treating cancer in a subject in need thereof, comprising: Individuals with the antibodies of the present invention and / or the multispecific antibodies of the present invention and / or the CAR-T cells of the present invention Pertaining to a group.

[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 exhibits no or low B-cell maturation antigen (BCMA) levels. Cancer with very low levels of B-cell maturation antigens.

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

[0102] In some embodiments, the cancer exhibits no or low B-cell maturation antigen (BCMA) levels. It is a CD38-positive hematologic malignancy with extremely low levels of B-cell maturation antigens.

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

[0104] In some embodiments, the multispecific antibodies of the invention and / or the CAR-T cells of the invention The population is CD38+, protecting B cells and NK cells from the cytotoxic activity of T cells. induces specific T cell-mediated lysis of cancer.

[0105] As used herein, the term "subject" refers to any mammal, e.g., a rodent. In particular, in the present invention, the subject is a patient suffering from cancer, preferably a or CD38-positive cancer, more preferably CD38-positive hematologic malignancies. or a human susceptible to such an illness.

[0106] In some embodiments, the subject has a recurrence of the cancer. In some embodiments, the subject has recurrent CD38-positive cancer. Recurrent hematologic malignancies.

[0107] In some embodiments, the subject receives a monoclonal antibody (Darat) targeting CD38. Resistance to antivirals such as tetanus (e.g., tetanus).

[0108] In some embodiments, the subject is a patient receiving treatment with a monoclonal antibody that targets CD38. He has received chemotherapy and has developed resistance to anti-CD38 monoclonal antibodies.

[0109] In a particular embodiment, the antibodies of the invention and / or the multispecific antibodies of the invention and / or the The population of CAR-T cells of the invention can be used in combination with anti-cancer treatments.

[0110] Accordingly, the present invention relates to a method of treatment in a subject in need thereof, the method comprising: As a combined preparation for treating cancer, a therapeutically effective amount of the antibody of the present invention and / or the (ii) a population of the multispecific antibodies of the invention and / or the CAR-T cells of the invention, and (iii) a population of conventional Treatment includes administering to a subject.

[0111] As used herein, the term "anti-cancer therapy" refers to any of the therapeutic agents known in the art. It has its general meaning 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 compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multi-kinase inhibitors, such as sorafenib and sucralose. Nitinib, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates aziridines, such as busulfan, improsulfan, and piposulfan; benzodopa, carboquone, metholedopa, and uredopa; ethyleneimine and methylamer Methylamelamine (altretamine, triethylenemelamine, Triethylene phosphoramide, triethylene thiophosphoramide, and trimethylol omer acetogenins (especially bullatacin and bullatacinone); camptothecins (c arnptothecin) (including the synthetic analog topotecan); bryostatin; chrysostatin tatin; CC-1065 (and its synthetic analogs adozelesin, carzelesin, and bizelesin) including); cryptophycins (especially cryptophycin 1 and cryptophycin 8); Statins; Duocarmycins (including synthetic analogs KW-2189 and CBI-TMI) ;Elytherobin;Pancratistatin;Sarcodictin;Spongistatin;Nitro Gen mustards, such as chlorambucil, chlornaphazine, and colofosfamide (ch olophosphamide), estranustine, ifosfamide, mechlorethamine , mechlorethamine oxide hydrochloride, melphalan, novembicine, phenesterine, prednisolone Nimustine, trofosfamide, uracil mustard; nitrosoureas, eg carmustine chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics enediyne antibiotics (e.g., calicheamicin, especially calicheamicin 11 and Calicheamicin 211, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemycins (including dynemycin A) ); esperamicin; and neocarzinostatin chromophore and related chromoprotein enzymes dyne antibiotic chromophore), aclacinomycin, actinomycin, anthramycin (authramycin), azaserine, bleomycin, cactinomycin, carabinycin Cin, caninomycin, carzinophilin, chromomycin, dactinomycin, daunomycin Rubicin, Detrubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodeoxycholic acid doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, ibuprofen Darbicin, Marcelomycin, Mitomycin, Mycophenone benzoic acid, nogalarnycin, olivomycin, peplomycin thromycin, thromycin, puromycin, chiramycin, rhodrubicin, streptomycin Muglin, 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, thioguam; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, karya Mofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, flocc sulidine, 5-FU; androgens, e.g., calcitonin, dromostanolone propionate antiadrenal substances, e.g., acrolein, epithiostanol, mepitiostane, testolactone; Minoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid (frol inic acid; aceglatone; aldophosphamide nide) glycoside; aminolevulinic acid; amsacrine; Bestrabsil; Bisantre edatraxate; defofamine; demecolcine; diaziquone; eflornithine ( elfornithine); elliptinium acetate; epothilone; etoglucide; nitrate hydroxyurea; lentinan; lonidamine; maytansinoids, e.g., maytansin ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrylamide Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide Zide; Procarbazine; PSK (registered trademark); Razoxane; Rhizoxin; Sizofiran; Spirogenanium; Tenuazonic acid, Triaziquone, 2,2',2''-trichlorotrimethylolpropane Ethylamine (2,2',2''-trichlorotriethylarnine) Trichothecenes (especially T-2 toxin, verrucarin A, and rojiru) amine A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitomycin Mitobromtol; Mitolactol; Pipobroman; Gashitoshi arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. For example, paclitaxel (TAXOL®, Bristol-Myers Squibb) bb Oncology, Princeton, NJ) and docetaxel (TAXOTE RE®, Rhone-Poulenc Rorer, Antony, Fr ance);Chlorambucil;Gemcitabine;6-Thioguanine;Mercaptopurine;Me Thotrexate; platinum analogues such as cisplatin and carboplatin; Vinbras Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitomycin Toxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposine Daunomycin; Aminopterin; Xeroda; Ibandronate; CPT-11; Polyisomeric enzyme inhibitor RFS2000; difluoromethylornithine (DMFO); capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes molecules that act to regulate or inhibit hormone action in tumors. antihormonal agents used, such as antiestrogens (e.g., tamoxifen, raloxifene) , aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trio Xifene, keoxifene, LY117018, onapristone, and toremifene ( Fareston), and antiandrogens, such as flutamide, nilutamide , bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts of any of the above. Also included are salts, acids, or derivatives which may be used.

[0114] As used herein, the term "radiation therapy" refers to a radiotherapy that is used in the art. It has its general meaning and refers to the treatment of cancer with ionizing radiation. Ionizing radiation is a chemical that destroys genetic material. The enzyme damages or destroys cells in the area to be treated (the "target tissue") by damaging the This provides energy to these cells, preventing them from continuing to grow. One type of radiation therapy involves photons, e.g., X-rays. Depending on the amount of energy they have, Light 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 of increasing energy. The use of a machine to focus radiation (such as x-rays) at a specific location is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. , when certain elements (such as radium, uranium, and cobalt-60) decompose or decay In some embodiments, radiation therapy is performed using external radiation. An example of external radiation therapy is conventional external beam radiation therapy; it is a method of treating a tumor from different directions. Three-dimensional conformal radiation therapy (3D-C) delivers a beam shaped to closely match the shape of the tumor. RT); Radiation beams are shaped to closely match and follow the shape of the tumor. Intensity-modulated radiation therapy (IMRT), which varies the radiation dose by varying the Conformal proton radiation therapy; real-time imaging of tumors to guide radiation treatment Image-guided radiation therapy (IGRT), which combines scanning and radiation techniques to deliver Intraoperative radiation therapy (IORT), which delivers radiation directly to the tumor during surgery; Stereotactic radiosurgery delivers multiple radiation doses precisely to the tumor area; more than one dose per day Radiation therapy treatment (fractionation) is targeted to hyperfractionated radiation therapy, e.g., sequential hyperfractionated accelerated radiation Treatment (CHART); and radiation with high doses per fraction but few fractions Therapies include, but are not limited to, hypofractionated radiation therapy.

[0115] As used herein, the term "immune checkpoint inhibitor" refers to one These immune checkpoint proteins are either completely or partially reduced, inhibited, interfered with, or modulated. Refers to a molecule that joins two molecules.

[0116] As used herein, the term "immune checkpoint protein" refers to a protein that is It has its general meaning in the art and raises a signal (a stimulatory checkpoint) These molecules are expressed by T cells to either downregulate the signal (inhibitory checkpoint molecules) or suppress the signal (inhibitory checkpoint molecules). It refers to the molecule that is expressed.

[0117] Examples of stimulatory checkpoints are CD27, CD28, CD40, CD122, and CD1 37, OX40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include: A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, K These include IR, PD-1, PD-L1, LAG-3, TIM-3, and VISTA.

[0118] As used herein, "combination treatment," "combination therapy," or "treatment" refers to The term "combination therapy" refers to treatment using more than one agent. is called dual therapy or bi-therapy. It is possible.

[0119] The drugs used in the combination treatment of the present invention may be administered to the subject together, individually or sequentially. It is administered.

[0120] As used herein, the term "co-administered" means by the same route and at the same time. or the administration of two active ingredients at substantially the same time. "Sequential administration" refers to the simultaneous or substantially simultaneous administration of two active ingredients by different routes. The term "subsequent administration" refers to the administration of two active ingredients at different times by the same or different routes of administration. It refers to administering.

[0121] In particular, the population of CAR-T cells prepared as described above can be used by those skilled in the art based on the present disclosure. The methods and compositions for adoptive immunotherapy are used in accordance with known techniques or modifications thereof that will be apparent. For example, Gruenberg et al., U.S. Patent Application Publication No. 2003 / 0004444, No. 170238, and Rosenberg, U.S. Pat. No. 4,690,915. Currently, most adoptive immunotherapies use the patient's own immune cells. The treatment used is autologous lymphocyte therapy (ALT). Typically, the treatment involves removing the patient's lymphocytes and This is done by converting the cells into a population of CAR-T cells, such as When RT cells are prepared with the CAR of the present invention, they enhance the immune system to kill tumor cells, The ex vivo cells are then reinfused into the patient. In some embodiments, the cells are first and then placed in a suitable medium and container system ("drug" or "drug therapy") for administration in a therapeutically effective amount. The cells are formulated by washing and concentrating them in a "pharmaceutical acceptable" carrier. The medium can be any isotonic medium formulation, typically normal saline, Normosol® ( Abbott) or Plasma-Lyte A (Baxter), Alternatively, 5% dextrose in water or lactated Ringer's solution can be used. The composition may be supplemented with human serum albumin. The specificity of the T cells varies depending on the relative abundance of T cells with the desired specificity, the age and weight of the recipient, the target The amount of cells is determined by the severity of the condition and the immunogenicity of the target Ag. 3 / kg, preferably 5 × 10 3 / kg, and can be as low as 10 7 / kg, preferably 1 0 8 The number of cells, as well as the types of cells they contain, can vary depending on the composition. This depends on the intended end use of the product. For example, cells specific for a particular Ag are required. When a population is considered to be a resident species, it is more than 70%, typically 80%, 85% and 90-95%. For the uses provided herein, cells generally comprise 1 liter of such cells. The volume is less than 100 tons, and is 500 ml or less, and furthermore 250 ml or less. A clinically relevant number of immune cells can be obtained that cumulatively equals or exceeds the desired total cell dose. can be distributed over multiple injections.

[0122] For administration, the antibody of the present invention is formulated as a pharmaceutical composition. The compositions are useful for preparing pharmaceutically useful compositions, whereby therapeutic molecules are pharmaceutically acceptable. The composition can be formulated according to known methods to be combined with a carrier. A composition is considered a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate buffered saline is an example of a pharmaceutically acceptable carrier. Suitable carriers for the above are well known to those skilled in the art. (See, for example, Gennaro (ed.), Remin gton's Pharmaceutical Sciences(Mack Publ (See Fishing Company, 19th ed. 1995) A formulation may contain one or Further excipient, preservative, solubilizer, buffer, and vial surface protein loss The pharmaceutical composition may further contain albumin to prevent the formation of granules. The dosage and regimen will necessarily depend on the condition to be treated, the severity of the disease, the age, weight, and other factors of the patient. The pharmaceutical composition of the present invention can be administered topically, orally, parenterally, intranasally, intravenously, or intravenously. The composition can be formulated for intravenous, intramuscular, subcutaneous, or intraocular administration, and the like.

[0123] Typically, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for an injectable formulation. In particular, isotonic solutions, sterile solutions, saline solutions (monosodium or disodium phosphate, sodium Thorium, potassium, calcium, or magnesium chloride, etc., or their salts or, if necessary, with the addition of sterile water or saline, to form an injectable solution. It may be a dry, particularly a freeze-dried composition, which can be constituted as follows.

[0124] The dose used for administration will depend on various parameters, in particular the mode of administration used, the associated The duration of treatment can be adapted depending on the condition being treated and the duration of treatment required.

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

[0126] Suitable pharmaceutical forms for use in injection include sterile aqueous solutions or dispersions, sesame oil, peanut oil or formulations containing aqueous propylene glycol and for the extemporaneous preparation of sterile injectable solutions or dispersions In all cases, the dosage form must be sterile and easily pourable. It must be fluid enough to be sprayable. It must be stable under the conditions of manufacture and storage. and need to be protected against microbial contamination such as bacteria and fungi. .

[0127] A solution of the active compound as a free base or a pharmacologically acceptable salt may be prepared by adding hydroxypropyl It can be prepared in water appropriately mixed with a surfactant such as cellulose. Liquids include glycerol, liquid polyethylene glycol, and mixtures thereof, and oils. Under normal conditions of storage and use, these preparations are microbially Contains a preservative to prevent growth of bacteria.

[0128] The antibodies of the present invention can be formulated into the compositions in neutral or salt forms. The salts that can be prepared are those prepared from inorganic acids such as hydrochloric acid or phosphoric acid, or from acetic acid, oxalic acid, tartaric acid, and the like. Formed with organic acids such as mandelic acid (formed with free amino groups of proteins) Also included are acid addition salts formed with free carboxyl groups, e.g., sodium Inorganic bases such as potassium, ammonium, calcium, or ferric hydroxide, as well as isopropyl alcohols. Derived from organic bases such as propylamine, trimethylamine, histidine, and procaine It is also possible.

[0129] The carrier may also be, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol), or the like. polyethylene glycol, and liquid polyethylene glycol, etc.), their appropriate mixtures, and The solvent or dispersion medium may also be an oil or vegetable oil. Suitable fluidity may be, for example, a liquid such as lecithin. By using any coating, by maintaining the required particle size in the case of dispersions, and Microbial activity can be maintained by the use of various antibacterial and antifungal agents. Antibacterial agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is necessary to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use of agents that delay absorption, e.g., For example, aluminum monostearate and gelatin may be used in the composition. can be done.

[0130] Sterile injectable solutions may be prepared by combining the active compound in the required amount with various of the other ingredients as enumerated above. The compound can be prepared by dissolving the compound in a suitable solvent, such as a cereal or vegetable oil, followed by sterilization by filtration, if necessary. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into the basic dispersion medium from those enumerated above. It is prepared by incorporating it into a sterile vehicle with the required other ingredients. In the case of a sterile powder for preparing a solution, the preferred method of preparation is to sterile-filter the powder prior to preparation. Vacuum drying and freeze drying to produce a powder of the active ingredient and any additional desired ingredients from the solution. It is a drying technique.

[0131] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, in which case the solution The use of DMSO as a solvent results in extremely rapid penetration, delivering high concentrations of the active agent to small tumors. It is envisioned that the drug will be delivered to the tumor area.

[0132] Once formulated, solutions will 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 injectable solutions of the type described above, but Drug-release capsules and the like may also be available.

[0133] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary. Therefore, the liquid diluent must first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, available sterile aqueous media will be known to those of skill in the art in light of the present disclosure. For example, one dose is dissolved in 1 mL of isotonic NaCl solution and administered subcutaneously in 1000 mL of It can either be added to an infusion solution or injected at the target site of infusion (e.g. For example, "Remington's Pharmaceutical Sciences" (See 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 practitioner will, in any event, determine the appropriate dose for the individual subject.

[0134] The antibody of the present invention is administered in an amount of about 0.0001 to 1.0 milligrams per dose, or about 0.001 About 0.1 milligrams, or about 0.1 to 1.0, or even about 10 milligrams. It can also be administered in multiple doses. do.

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

[0136] In some embodiments, the use of liposomes and / or nanoparticles may be used to induce the delivery of antibodies to host cells. The formation and use of liposomes and / or nanoparticles is known to those skilled in the art. be.

[0137] Nanocapsules generally have the ability to encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, these ultrafine particles (approximately 0. 1 μm size) generally use polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylates that meet these requirements are designed to Nanoparticles are contemplated for use in the present invention, and such particles can be easily made.

[0138] Liposomes are dispersed in an aqueous medium and are multilamellar concentric bilayer vesicles (multilamellar vesicles). They are formed from phospholipids that spontaneously form multivessels (MLVs). Generally, the diameter is 25 nm to 4 μm. By sonicating the MLV, the core contains an aqueous solution. This results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å. The physical characteristics of liposomes are determined by pH, ionic strength, and the presence of divalent cations.

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

[0140] Example 1: Novel CD38 / CD3 bispecific T cell enzyme for the treatment of multiple myeloma Engager method Construction and purification of Bi38-3 Bi38-3 was generated as anti-human CD38 and CD3ε (B51 and OKT3, respectively). It is derived from a mouse hybridoma and consists of 15 amino acids, glycine-serine (G4S1x3). It was created by fusion of two scFvs linked by a spacer. The primer peptide was genetically linked to the N-terminus of the fusion fragment, and a Myc tag and Hi The sequence encoding Bi38-3 was inserted into the pCDNA3 expression vector. The fragments were cloned into a vector (ThermoFisher) and confirmed by sequencing. This vector was transiently transfected into HEK-293T cells, and the cells were then transfected with the vector against Bi38-3. The corresponding 55.6 Kd protein was purified by HisTrap HP column (GE). The integrity of Bi38-3 was confirmed by Coomassie blue staining and anti-Myc tag antibody. Analyzed by Eastern blot.

[0141] cell line MM1.S, NCI-H929, and KMS-11 MM cell lines were cultured in 10% heat-inactivated water. Fetal bovine serum, 100 units / mL penicillin, 10 μg / mL streptomycin All cells were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine. All cell lines were monitored for mycoplasma contamination. Enzyme-expressing MM1.S and KMS-11 cells (KMS11luc and MM1.Sluc) were generated by lentiviral transduction with a luciferase expression vector (A ddgene, pLenti CMV Puro LUC (w168-1), Eric (A gift from Campeau and Paul Kaufman). To generate the cells, two pairs of RNA guides encode exons 2 and 3 of the CD38 gene. The annealed oligonucleotides were then inserted into the pX458 vector. (Addgene Plasmid ID 48138, provided by Dr. Feng Zhang) The clones were cloned into the 2× (gift) and verified by sequencing. 10 6 MM1.Sluc cells were transfected using Nucleofector-II (Lonza). 2 μg of each Cas9 vector was nucleofected into the cells and incubated in the medium for 24 hours. GFP-positive cells were sorted by FACS and cloned into 96-well plates. Subclones were analyzed for CD38 expression by flow cytometry. 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). Plasma cells were obtained from the buffy coat of bone marrow aspirates of myeloma patients and further analyzed by anti-CD13 Purification was performed using 8-coated beads (Miltenyi). In accordance with the Declaration of Helsinki and the approval of the Saint-Louis Hospital Internal Research Committee, patients and volunteers Informed consent has been obtained.

[0143] Flow cytometry and cytotoxicity To assess the lysis of MM cell lines KMS-11luc or MM1Sluc, purified Peripheral T cells (effector) were cultured in a flat-bottom 96-well plate in medium (RPMI, 10% Heat-inactivated fetal bovine serum, 100 units / mL penicillin, 10 μg / mL streptomycin Various concentrations of Bi38-3 and Luciferase were tested in a 1000-well plate containing 1000 µg of ATP (1000 µg / ml of ATP, 1000 µg / ml of ATP, and 2 mM L-glutamine). The effector was administered in a 1:5 ratio to the target along with the target-expressing MM1.S or KMS-11 cells. The luciferase signal produced by viable MM cells was measured using a 2000-well platelet count (2000-20 ... The cells were incubated within 20 minutes after addition of the firefly luciferase substrate (Bright t-Glo Luciferase Assay System (Promega), CLARIOsta r Plus luminometer plate reader (BMG LABTECH GmbH, O The results were evaluated after 24 hours using a chromatographic method (Germany). The cytotoxicity of T cells was measured by flow cytometry using similar co-culture conditions. Effector T cells were cultured in serial dilutions of purified target MM cells and Bi38-3. After incubation, the cells were incubated with anti-human CD138-allophycocyanin. (APC)(clone 44F9-Miltenyi Biotec), anti-CD20- Brilliant Violet(BV)605(Clone 2H7-BioLeg end), anti-CD4-APC / cyanine 7 (Clone RPA-T4-BioLege nd), and anti-CD8-BV421 (Clone RPA-T8-BioLegend) Antibodies are added to the cells to distinguish target cells from effector cells, and the number of live cells is counted using Briggs Flow cytometry was performed using htcount beads (ThermoFisher). All FACS were performed using a Canto II (Beckon Dickinson) Analysis and calculation of proliferation indices were performed using Flowjo.

[0144] T cell activation and proliferation assays For activity detection, effector cells (T cells) and target cells (MM1.S) were mixed at a ratio of 5:1. The mice were co-cultured for 24 hours with anti-human CD4-APC-cyanine 7 (Clone RPA-T) 4), anti-CD8-BV421 (Clone RPA-T8), anti-CD25-Phycoerythrosine Phosphorus (Pe) / Cyanine 5 (clone BC96), and anti-CD69-BV711 (c Isolate FN 50) antibody (all Biolegend) and analyzed by flow cytometry. For proliferation analysis, T cells were stained with CellTrace Violet dye. Labeling with ATP (ThermoFisher) was performed with Bi38-3 (10 ng / mL) or (none) and stimulated with MM1.S cells or MM1.S-CD38KO for 96 hours. The cells were incubated with anti-CD4-APC / Cy7 (clone RPA-T4) and anti-CD8-BV. Stained with 421 (clone RPA-T8) antibody and analyzed by flow cytometry did.

[0145] Quantification of cytokines in cell culture supernatants Cytokine concentrations in the supernatants of the cytotoxicity assay were measured using BD CBA human Th1 / T Analysis was performed using the h2 cytokine kit II (Beckon Dickinson). Data were acquired on a Canto II and analyzed using FCAP Array software (Be The analysis was performed using the CKON Dickinson method.

[0146] Systemic tumor mouse model Animal Experimentation Committee (Comite d'ethique Paris-Nord) Under a protocol approved by the National Institute of Infectious Diseases, 6-12 week-old NOD / SCID / IL-2Rγ null mice were Mice (The Jackson Laboratory) were used. 5 × 10 by injection 6MM1.Sluc cells were inoculated, followed by 0.08% MM1.Sluc cells after 14 days. mg / Kg with or without Bi38-3, 5 x 10 6 Purified human T cells (Mi (purified using a pan-T cell isolation kit from Iltenyi Biotec) were injected. Tail vein injections of -3 (or control PBS) were repeated daily for 9 days. No blinding or blinding method was used. Bioluminescence imaging was performed every 3 or 4 days. , 240 μL of D-luciferin (15 mg / mL) (XenoLight D-Lucif The animals were given an intraperitoneal injection of 100 mg of riboflavin (potassium salt, Perkin Elmer) and the animals were given a Living Immunoglobulin (LII) injection. age software (PerkinElmer) with an IVIS imaging system ( PerkinElmer) at medium binning level and various exposure times. Image acquisition was performed 15 minutes after inoculation of MM cells. The mice died.

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

[0148] Anti-CD38 scFv is located at the N-terminus, anti-CD38 ε scFv is located at the C-terminus, and My The Bi38-3 expression vector was transiently transfected with PBS containing the c-tag and Hisx6-tag sequences (not shown). Western blot analysis of HEK-293 cells transfected with 55.6 Kd Identify a unique protein with the predicted size and recognized by the anti-Myc tag antibody (Data not shown). Bi38-3 was added to transiently transfected HEK-293 cells. Monomeric Bi38-3 was purified from the culture supernatant using a HisTrap HP column (GE). The purity of the protein was demonstrated by gel electrophoresis followed by Coomassie blue staining (data not shown). (Data not shown). CD38-expressing MM1.S, KMS11, and NCI-H939 MM cells The binding of Bi38-3 to the scFv domain was confirmed by flow cytometry using an anti-Fab antibody that recognizes the scFv domain. Bi38-3 was detected on the surface of MM cell lines. and less intense staining in KMS11 cells, which express lower levels of CD38. The signal was stronger in MM1.S and NCI-H929 cells, which exhibit higher levels of CD38. To verify the specificity of Bi38-3 for CD38, , to inactivate the CD38 gene in MM1.S cells (MM1.S-KO), The CRISPR / Cas9 method was used (data not shown). 8-3 was not detectable on the surface of CD38-negative MM1.S-KO cells. Therefore, purified Bi38-3 inhibited CD38 on MM cells. Recognize effectively and specifically.

[0149] Bi38-3 inhibits T cell activation and proliferation in response to MM cells in vitro to induce Next, we examined the T cell response to MM cells induced by Bi38-3. First, FACS analysis was performed to measure the proliferation index of violet fluorescent stained T cells. Bi38-3 induces the proliferation of donor effector T cells in the presence of M1.S target cells (T). Stimulation of the cells (E) resulted in robust proliferation, with an average of 5 cell divisions (proliferation index) after 4 days. which is slightly lower than that induced by treatment with anti-CD38 / CD28 beads. The levels were high (data not shown). T lymphocytes cultured with IgG did not proliferate, suggesting that proliferation requires CD38 expression on target cells. Furthermore, culture of Bi38-3 cells alone or MM1.S cells alone was also effective. did not induce significant T cell proliferation.

[0150] Next, we analyzed the expression of CD69 and CD25 early activation markers on donor T cells. After overnight co-culture with MM1.S cells, CD4 and CD8 T cells immediately expressed both MM1.S cells and CD8 T cells. Bi38-3 dose-dependently upregulated CD69 receptors, resulting in up to 80% CD69 positivity. T cells were detected at the highest concentration (data not shown). Lower percentage of CD25 and CD69 expressing T cells when stimulated with 38-3 alone Furthermore, co-culture with MM1.S target cells alone resulted in a significant reduction in the number of MM1.S cells compared with control cells (15% and 30%, respectively). did not induce the expression of activation markers (data not shown). 1. Co-culture with SKO cells and Bi38-3 cells compared with co-culture with wild-type MM1.S cells As a result, CD69 and CD25 were induced to a lesser extent (data not shown), and activation markers The upregulation was enhanced by CD38 expression on target cells.

[0151] Finally, we monitored the cytokine production induced by Bi38-3. Co-culture of MM1.S with donor T cells resulted in a dose-dependent increase in interleukin-1 (IL-1) expression in Bi38-3 cells. Interferon-γ (IFNg), tumor necrosis factor-α (TNFa), interleukin-2 ( induced the production of IL-2, IL-4, and IL-10 (data not shown). Stimulation with Bi38-3 alone or co-culture with MM1.S alone did not result in the production of any of these cytokines. Both of these results suggest that Bi38- 3 inhibited T cell proliferation, activation, and proliferation in vitro in response to CD38-expressing MM cells. These results demonstrate that IL-1 induces IL-1 activation and cytokine release.

[0152] Bi38-3 inhibits CD38-dependent T cell-mediated killing of MM cells in vitro. induce wounds To evaluate the function of Bi38-3, we used the firefly luciferase target KMS11 and M Effector T cells isolated from PBMCs of healthy donors in M1.S MM cell lines Co-culture assays were performed to measure the cytotoxic activity of various concentrations of Bi38-3. To measure the percentage of killing in the presence of MM, the number of remaining viable MM target cells was determined. The luciferase levels shown are the luciferase levels observed in untreated controls. T cells rapidly killed KMS11 target cells in a Bi38-3 dose-dependent manner. , 50% effective concentration (EC 50 ) is approximately 5 ng / mL, and this 55.6 Kd protein corresponds to 0.09 nM (data not shown). Cytotoxic activity was observed in co-culture with MM1.S cells. However, at higher levels In this cell line, which expresses CD38, EC 50 is 10 times lower (0.5 ng / mL ), indicating that the effect of Bi38-3 is stronger. On the other hand, the M M cell viability was not affected by co-culture with T cells or Bi38-3 alone. (Data not shown). Furthermore, Bi38-3 inhibited T cell-mediated mitochondrial proliferation in MM1.S-KO cells. CD38 deficiency resulted in poor killing of the leukocytes, even at the highest dose of Bi38-3 (1 ng / mL). Approximately half of the MM1.S cells survived the coculture (data not shown). 8-3 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. Lead Next, we investigated the potential of Bi38-3 to induce lysis of MM cells by autologous T cells. Target tumor plasma cells isolated from patients at the time of diagnosis were analyzed in the presence of various concentrations of Bi38-3. In the presence of IL-1, the cells were incubated with purified autologous effector T cells at an E:T ratio of 1:5. FACS analysis of overnight co-cultures revealed that the number of viable CD138-positive MM cells was B i38-3 decreased in a dose-dependent manner, and EC 50 ranges from 0.5 to 1 ng / mL depending on the patient Importantly, in the absence of T cells, Bi3 Bi38-3 showed no toxicity to fresh primary MM cells. The cytotoxicity of the cells was further investigated in tumor plasma cells of MM patients at relapse, and similar efficacy was observed. Show your strength, EC 50 The values ​​ranged from 0.2 to 1 ng / mL (Fig. 1). In vitro studies have shown that Bi38-3 significantly improves the survival of patients both at diagnosis and at relapse. It induced autologous T cell-mediated killing of tumor plasma cells in patients.

[0154] Specific activity of Bi38-3 against CD38-highly expressing MM cells in vitro CD38 is highly expressed on plasma cells, but is also expressed on various cell types, including a subset of hematopoietic cells. To examine the effect of Bi38-3 on blood cells, PBMCs from donors were were treated with various concentrations of Bi38-3 for 24 hours, and various cell populations were identified by FACS. The percentage of CD14-expressing monocytes that fell into the viable gate was analyzed (data not shown). A significant dose-dependent decrease in i38-3 was observed (data not shown). On the other hand, when the percentage of CD14 positive cells declines, approximately 10% of the PBMC population is The percentage of CD4 and CD8 T lymphocytes corresponding to 60% corresponds to Bi38-3 Similarly, B (CD19+) cell and NK (CD56+) cell populations were significantly increased. The groups showed a slight increase or similar increase even at high concentrations of Bi38-3 (100 ng / mL). The levels remained at approximately 10% and 5%, respectively (data not shown). We investigated whether surface CD38 expression was impaired by Bi38-3. ACS analysis was performed to measure the mean fluorescence intensity (MIF) of CD38 in T cells, B cells, and NK cells. ) were similarly maintained in cultures containing increasing doses of Bi38-3. (Data not shown). Consistent with this, CD38 expression was predominant in CD14+ myeloid cells. Does this mean that the number of cells is not significantly reduced, but that no cells or only a very small number of cells can be detected? However, analysis could not be performed at higher doses of Bi38-3 (1 and 100 ng / mL). CD38-high (CD38hi) versus CD38-intermediate (CD38int) MM To compare the activity of Bi38-3 in cells expressing high levels of CD38, we used MM1 .S (data not shown), freshly isolated B cells expressing moderate amounts of CD38 (data not shown). After overnight culture, viable CD20 T cells were detected. Percentage of CD138-positive B cells and CD138-positive MM1.S cells by flow cytometry The percentage of MM1.S cells was analyzed by 0.1 ng / mL Bi38-3 It was observed that the decrease in α-glucan was more pronounced at higher doses (data not shown). On the other hand, compared to untreated conditions, the percentage of viable CD20-positive B cells The page remained unchanged even at high concentrations of Bi38-3 (data not shown).

[0155] CD34+ bone marrow hematopoietic progenitor cells and regulatory T cells (T To investigate the potential damaging effects of Bi38-3 on the reg We developed a co-culture assay. Bi38-3 inhibited MM cells at low concentrations (10-2 ng / mL or higher). While this rapidly induces cell killing, it also results in significant T cell-mediated inhibition in Foxp3+ Tregs. We found that the cytotoxicity of IL-1 was not induced by IL-1 at concentrations lower than 10 ng / mL (Fig. 2A). There was no significant toxicity to CD34+ hematopoietic progenitor cells at the highest concentration, and Both of these results suggest that Bi38-3 inhibits C The surface expression of CD38 is intact, and T cell-mediated killing of cells expressing high levels of CD38 is suppressed. It induces hematopoietic progenitor cells, B cells, T cells, or NK cells at moderate levels of CD4+. 38 has been shown to have no or limited toxicity to cells expressing the compound. vinegar.

[0156] Both of these results suggest that Bi38-3 does not impair surface expression of CD38 and inhibits CD38int cells. It induces T cell-mediated killing of CD38hi cells without significant activity against This indicates that

[0157] Bi38-3 regulates MM cell proliferation in vivo In vivo antitumor activity of Bi38-3 in a human MM xenograft mouse model MM1.Sluc cells were injected into the tail vein of NSG mice and luciferase-induced leukemia was evaluated. The serum levels were measured every 4 days using an IVIS imaging system. After 14 days, purified human T cells were cultured in the presence or absence of Bi38-3 (0.08 mg / kg ) were transplanted intravenously. Treatment with Bi38-3 or vehicle was repeated daily for 7 days (Figure 2 A). Eleven days after tumor cell injection, all mice showed similar levels of radiation (luciferin). This indicates that MM cells were effectively engrafted in the host animals before Bi38-3 treatment. Control mice showed rapid tumor progression, whereas Bi38-3-treated mice showed rapid tumor progression (Figure 2B). All animals treated with Bi38-3 showed a 5-fold reduction in tumor growth within the first 4 days of treatment. After 7 days, luciferase expression in mice treated with Bi38-3 was significantly increased (Fig. 2C). Current MM cell levels were only 1 / 10 of the initial levels and significantly lower than untreated controls. These results suggest that Bi38-3 exerts a potent anti-inflammatory effect in vivo. It has been shown to be effective in controlling MM tumor progression.

[0158] Consider As used herein, in vitro, ex vivo, and in vivo A novel anti-CD38 / CD38 antibody induces specific T cell-mediated lysis of CD38-positive MM cells The development of Bi38-3, a bispecific T cell engager antibody, is reported.

[0159] Monoclonal antibodies (Mabs) targeting CD38 have therapeutic efficacy in the treatment of MM. It shows 13 Daratumumab, an anti-CD38 Mab approved for MM, , alone 14 or in combination with standard of care regimens 2、15 , good therapeutic efficacy These clinical data suggest that CD38, which is highly expressed on tumor plasma cells, is involved in M However, the significant survival rate has been shown to be Despite the improvement, many patients treated with daratumumab still had CD38 expression on tumor cells. FcγR-dependent downregulation of complement-dependent cytotoxicity and antibody-dependent cellular mediation Resistance mechanisms, including inhibition of endothelial cytotoxicity and antibody-dependent cellular phagocytosis, ultimately lead to relapse. R 16 Bi38-3 lacks the Fc region found in natural immunoglobulins and is a target The anti-CD3 scFv was administered without downregulating CD38 expression in cells. Thus, the Bi3 expression of MM cells recruits cytotoxic T cells (data not shown). 8-3-mediated T cell killing in daratumma is associated with therapeutic antibody binding to FcγR. This suggests that the mechanism of resistance to anti-CD38 mAbs, such as IgG, is not affected. It is observed at the time of occurrence and 17 complement in cytotoxic cells, which may contribute to resistance Upregulation of the cytokine inhibitors CD55 and CD59 occurred upon Bi38-3 treatment. Furthermore, MM is characterized by immune system abnormalities, and IMID and Standard treatment regimens involving tetracycline may limit the effectiveness of cytotoxic cells. However, these data suggest that Bi38-3 has a similar effect on both diagnostic and recurrent disease. We demonstrate that the tumor mediates autologous T cell-mediated killing of the patient's tumor plasma cells at the time of onset (Figure 1). These data together demonstrate that Bi38-3 is superior to standard treatments, including those containing daratumumab. This suggests that MM cells can be effectively eliminated in patients resistant to MM. There are.

[0160] CD38 is expressed on the surface of blood cells, including T lymphocytes, B lymphocytes, and NK lymphocytes. Because 18 Anti-CD38 mAb may target these and impair their function. It is possible. In fact, daratumumab has been shown to eliminate regulatory T cells. 19 , This is a process that may be associated with an increase in T cell numbers and activity during the early stages of treatment. 1 6 Furthermore, daratumumab treatment resulted in the depletion of NK cells. 20 , patients with infectious diseases may increase the sensitivity of 21 These data suggest that Bi38-3 is It has been shown that it has no significant effect on T cells, B cells, and NK cells (data not shown). Furthermore, even at high doses (10 ng / mL), this inhibited B cell cytotoxicity and T cell cytotoxicity. reported that it rapidly induced T cell-mediated killing of MM cells while protecting them from activation. Interestingly, these results were obtained in vitro using B cells and T cells. Recently, anti-CD38 BiTEs have been used to induce T cell cytotoxicity and NK cell cytotoxicity. In contrast to the described activity of AMG424 22 In vivo activity, especially against bone marrow cells Although further experiments are needed to evaluate the toxicity of Bi38-3 in this model, These results suggest that Bi38-3 does not affect cells expressing low levels of CD38. This suggests that it can effectively induce the elimination of MM cells.

[0161] Recently, Fc receptor-like 5 (Fcrl5 or FcHR5) or B cell maturation antigen (BCM) A) Bispecific antibodies targeting 10、12、23 BCMA and CD BI836909, a BiTE targeting 3ε, inhibits NCI-H929 mouse xenografts A dose of 0.5mg / Kg was shown to eliminate MM cells in a model 23 .Similar In contrast, EM801, an asymmetric bispecific antibody containing a mutated Fc region, was shown to be effective against HIV-1 induced leukemia at the same dose (0. 5mg / Kg) effectively eliminated NCI-H929 cells in immune-compromised mice 10 BCMA expression is found in embryonic cells, including memory B cells and both normal and malignant plasma cells. Limited to postcardiac B cells 24 However, the majority of MM patients express BCMA. However, 6-9% of cases are negative for this marker, and expression in tumor plasma cells is unclear. Levels are heterogeneous among patients 25、26 Furthermore, anti-BCMA chimeric antigen receptor In MM patients treated with T cells expressing BCMA, BCMA was downregulated in tumor plasma cells. Regulated 27 This is 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 suggest that targeting CD38 with Bi38-3 enhances cell proliferation in a similar mouse model. The dose was 0.1 mg, a significantly lower dose than that reported for BCMA bispecific antibodies. g / Kg (Figure 2) and demonstrated efficacy in xenograft models. 10、23 .did Therefore, Bi38-3 does not express BCMA or expresses very low levels of BCMA. This may represent an attractive treatment option in MM cases.

[0163] Although BiTE has demonstrated efficacy in several malignant lesions, its clinical development remains uncertain. Development is hampered by a short half-life in patients requiring continuous infusion by pump. R 9 In recent years, the CD19 / CD3 BiTE blinatumomab has been shown to be effective in the treatment of acute lymphoblastic leukemia ( It was approved for the treatment of minimal residual disease (MRD) in patients with ALL. A phase 2 clinical trial of BRAF-1 demonstrated that MRD negativity at the end of the first cycle of treatment was associated with improved survival. It has been shown that it can be obtained 28、29 Optimal use of blinatumomab in cases of MRD Although cycle length remains an area for further study in ALL, clinical data are available. This indicates that limited BiTE treatment at the time of initial diagnosis can still improve the prognosis of MRD+ patients. In this study, Bi38-3 was used in a highly proliferative MM cell line (MM1.S). showed that, despite the lack of a specific inhibitor, it was able to induce a six-fold reduction in tumor burden in vivo in just three days. (Figure 2C). Therefore, this rapid and pronounced activity in tumor plasma cells is consistent with the ALL Similar to blinatumomab in MM, Bi38-3 was shown to be effective in patients with MM after a limited number of cycles. These findings suggest that IFN-γ may eliminate MRD in patients with IFN-γ-γ deficiency and improve outcomes after standard treatment.

[0164] In summary, the data presented in this paper can be used both in the frontline and in relapse settings. Identifying Bi38-3 as a selective and effective compound for the treatment of MM and providing it to MM patients Support further evaluation in

[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 plasmid (ps PAX2 and pMD2.G) were transfected using calcium phosphate. After 2 days, the complete medium (DMEM, 10% FVS) was replaced with a new one. Two days after gene transfer, retrotransplantation was performed. The cell-free supernatant containing the virus particles was collected, concentrated by centrifugation, and used for transduction. T cells (purified using Miltenyi Biotec's pan T cell isolation kit) were cultured in Medium (RPMI 1640, 10% FBS, 100 U / mL penicillin, 100 U / mL streptomycin) 100mg / mL) with CD3 / CD28 beads (ThermoFisher). After 16 hours, the cells were incubated with 15 mg of retronectin-coated 1 mL) (Takara) and transferred to a 6-well plate (Falcon). Cells were transduced with CAR particles overnight. 72 hours after transduction, GFP and CAR expression were measured by flow cytometry. Transduction efficiency was determined by cytometry. The cells represent 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 Since Bi38-3 induced T cell-mediated lysis of MM cells (see Example 1), its anti- CD38scFv is a promising tool for direct transfection of transgenic T cells in the case of chimeric antigen receptors (CARs). We investigated whether BB51-derived anti-CD38 scFv, human First generation antibody containing the hinge and transmembrane regions of CD8 and the CD3ζ signaling domain An anti-CD38 CAR construct (CAR CD38 1G) was developed (Figure 3A). It has been shown that the association of the D3ζ active region with the costimulatory signaling domain enhances the activity of CAR. Therefore, anti-CD38scFv, the transmembrane domain of CD28, and CD28, CD137 (4-1BB) and the signaling domain of CD3ζ, in that order. A third-generation anti-CD38 CAR (CAR CD38 3G) was also constructed (Figure 3A). As roles, CAR without scFv domain (CAR Mock) and CD38 3 A costimulatory CAR (CCR CD4) similar to G but lacking the CD3ζ signaling domain 38) were prepared. The protein sequences corresponding to these constructs are shown in Table 2. The DNA sequence encoding CAR was cloned into a lentiviral vector capable of co-expressing GFP. The CAR constructs were then transduced into donor T cells using the CAR constructs. The construct was expressed in transduced human T lymphocytes (CAR-T), R 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. While the CD38-expressing T cells were able to expand in vitro for 2 weeks, Although there is a possibility of damaging effects on the CAR-T cells, CAR-T cells were still cultured (data not shown). To investigate the cytotoxicity of effector CAR-T cells (E), various ratios of Co-culture experiments were performed with luciferase-expressing target cells (T). RPMI8288 MM cells were used as a negative control for Mock and CCR CD38. Compared to the CAR CD38 1G and 3G, the CAR CD38 1G and 3G lysed the cells immediately (Figure 3 B). Anti-CD38 CAR-T kills MM cells even at low E / T ratios (<2.5). However, CCR CD38 and Mock transduced T cells showed poor cytotoxicity. CAR CD38 1G and 3G induced lysis in CD38-negative HEK293 cells. The BB51 hybridization induced no or little lysis (Fig. 3B). CD38 scFv derived from CD38 inhibits MM cells in various CAR constructs It is effective in promoting T cell cytotoxicity in Table 2 shows the results of first and third generation anti-CD38 CARs (CAR CD38 1 and 1 , respectively). The amino acid sequences of the CARs (G and 3G) and costimulatory CAR (CCR CD38) are shown. [Table 2] JPEG2025148447000004.jpg38165

[0167] References Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated herein by reference into the present disclosure. JPEG2025148447000005.jpg127165JPEG2025148447000006.jpg246169JPEG2025148447000007.jpg250165

Claims

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

10. 。

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