Antibody targeting BCMA and application thereof

Abstract

The present application provides an antibody that targets BCMA. The invention also provides different forms of recombinant receptor molecules constructed through the antigen binding domain of the antibody, and nucleic acid molecules and expression vectors encoding the antibody and the recombinant receptor molecules. The present application also relates to the use of said antibodies, including recombinant receptors of CAR molecules, in the treatment of disease, in particular in the treatment of multiple bone marrow cancer.

Description

Technical Field

[0001] The present invention relates to a fully human antibody or a single-chain antibody or fragment thereof that specifically binds to a human BCMA antigen protein and a BCMA antigen in a natural state on the surface of a cell membrane. Background Art

[0002] Multiple myeloma is a plasma cell tumor that occurs in the bone marrow. The tumor can cause hypercalcemia, anemia, renal dysfunction, osteonecrosis and bone marrow failure. Currently, multiple myeloma is the second most common blood tumor. According to the 2020 statistics on the incidence of multiple myeloma published by the World Health Organization (WHO), the incidence and mortality rates of Asians accounted for 36% and 42% respectively, ranking first among all continents. The global incidence and mortality ratio of multiple myeloma is 1.8:1.1, and that of Asia is 1.1:0.76. It can be seen that the survival rate of multiple myeloma in the onset population is low. The main reason is that the median age of the onset population is high at around 66 years old, and the incidence rate of people under 40 years old is about 2%. Second, multiple myeloma is almost impossible to cure under commonly used chemotherapy, immunomodulators and monoclonal antibody therapy, and the barely relieved is also accompanied by an extremely high relapse rate, that is, relapsed / refractory multiple myeloma (RRMM), with a five-year survival rate of only 51%.

[0003] bb2121, a CAR-T product developed by Bristol-Myers Squibb (BMS) targeting the BCMA target for multiple myeloma, was approved by the FDA in May 2021 as the first CAR-T product for multiple myeloma to be marketed. The overall response rate (ORR) of this product is 72%, and 28% of patients can achieve strict complete remission (sCR). Although this product has brought hope for the cure of multiple myeloma, according to the published data, the 22-month progression-free survival rate of patients with complete remission (CR) is less than 50%, which shows that there is also a high recurrence rate in the later stages of treatment. In addition, since this therapy removes all plasma cells, there are still side effects caused by hematopoiesis and reduced immunoglobulins.

[0004] The medical market still needs CAR-T products with wider patient coverage, better efficacy and lower recurrence rate. Summary of the invention

[0005] The present application provides an antibody that can specifically bind to BCMA, or its application on a recombinant receptor (such as a CAR molecule) and the medical uses of the above molecules.

[0006] That is, the present invention mainly relates to the following aspects:

[0007] In one aspect, the present invention provides an antibody, an antigen-binding fragment thereof, or a derivative thereof targeting BCMA, comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises:

[0008] (a) a heavy chain complementarity determining region 1 (HCDR1) comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6; and

[0009] (b) a heavy chain complementarity determining region 2 (HCDR2) comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7; and

[0010] (c) a heavy chain complementarity determining region 3 (HCDR3), which comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 8.

[0011] In some embodiments, the BCMA-targeting antibody, antigen-binding fragment thereof, or derivative thereof, wherein:

[0012] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1;

[0013] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2;

[0014] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0015] In some embodiments, the BCMA-targeting antibody, antigen-binding fragment thereof, or derivative thereof, wherein:

[0016] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6;

[0017] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0018] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8.

[0019] In some embodiments, the heavy chain variable region (VH) comprises any amino acid sequence selected from the following sequences: SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequences.

[0020] In some embodiments, the antibody is a fully human antibody or a humanized antibody.

[0021] In some embodiments, the antibody is a single domain antibody.

[0022] In some embodiments, the BCMA-targeting antibody, antigen-binding fragment thereof, or derivative thereof further comprises: an Fc fragment.

[0023] In some embodiments, the Fc fragment comprises any amino acid sequence selected from the following sequences: the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0024] In one aspect, the present invention provides a recombinant microorganism that expresses the above-mentioned antibody, antigen-binding fragment thereof or derivatives thereof.

[0025] In one aspect, the present invention provides a recombinant cell that expresses the above-mentioned antibody, antigen-binding fragment thereof or derivatives thereof.

[0026] In one aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody, antigen-binding fragment thereof or derivatives thereof.

[0027] In one aspect, the present invention provides a kit comprising the above-mentioned antibody, antigen-binding fragment thereof or derivatives thereof.

[0028] In one aspect, the present invention provides a nucleic acid molecule comprising a sequence encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0029] In one aspect, the present invention provides use of the above-mentioned antibodies, antigen-binding fragments thereof or derivatives thereof and / or the above-mentioned nucleic acid molecules for treating proliferative diseases or autoimmune diseases.

[0030] In some embodiments, the proliferative disease comprises a lymphoproliferative disease or a tumor.

[0031] In some embodiments, the tumor is a plasma cell malignancy or a B cell malignancy, preferably, the tumor is a myeloma.

[0032] In some embodiments, the autoimmune disease is an antibody-mediated autoimmune disease. Preferably, the autoimmune disease comprises systemic lupus erythematosus, myasthenia gravis, neuromyelitis optica, and lupus nephritis.

[0033] In one aspect, the present invention provides a chimeric antigen receptor (CAR) molecule, comprising an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain, wherein the antigen binding domain comprises a heavy chain variable region targeting BCMA (anti-BCMA VH), and the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises:

[0034] (a) a heavy chain complementarity determining region 1 (HCDR1), comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6;

[0035] (b) a heavy chain complementarity determining region 2 (HCDR2), comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7;

[0036] (c) a heavy chain complementarity determining region 3 (HCDR3), which comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 8.

[0037] In some embodiments, the chimeric antigen receptor (CAR) molecule, wherein the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises:

[0038] (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 1;

[0039] (b) a heavy chain complementarity determining region 2 (HCDR2), comprising the amino acid sequence shown in SEQ ID NO: 2;

[0040] (c) a heavy chain complementarity determining region 3 (HCDR3), which comprises the amino acid sequence shown in SEQ ID NO: 3.

[0041] In some embodiments, the chimeric antigen receptor (CAR) molecule, wherein the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises:

[0042] (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 6;

[0043] (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 7;

[0044] (c) a heavy chain complementarity determining region 3 (HCDR3), comprising the amino acid sequence shown in SEQ ID NO:8.

[0045] In some embodiments, the BCMA-targeting heavy chain variable region (anti-BCMA VH) comprises any amino acid sequence selected from the following sequences: SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0046] In some embodiments, the chimeric antigen receptor (CAR) molecule is arranged in the following order: antigen binding domain, hinge region, transmembrane domain, co-stimulatory domain, and signal transduction domain.

[0047] In some embodiments, the chimeric antigen receptor (CAR) molecule further comprises an extracellular signal peptide sequence.

[0048] In some embodiments, the extracellular signal peptide sequence is an extracellular signal peptide sequence derived from CD8α.

[0049] In some embodiments, the hinge region is a hinge region derived from CD8α.

[0050] In some embodiments, the transmembrane domain is a transmembrane domain derived from CD8α.

[0051] In some embodiments, the costimulatory domain comprises a costimulatory domain derived from 4-1BB or a costimulatory domain derived from CD28.

[0052] In some embodiments, the signaling domain comprises a signaling domain derived from CD3ζ.

[0053] In some embodiments, the extracellular signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:17.

[0054] In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:19.

[0055] In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:21.

[0056] In some embodiments, the costimulatory domain derived from 4-1BB comprises the amino acid sequence shown in SEQ ID NO:23.

[0057] In some embodiments, the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:25.

[0058] In some embodiments, the chimeric antigen receptor (CAR) molecule comprises any sequence selected from the following sequences: an amino acid sequence as shown in SEQ ID NO: 12, SEQ ID NO: 14, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0059] In one aspect, the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule as described in some embodiments.

[0060] In some embodiments, the nucleic acid molecule further expresses a tag molecule.

[0061] In some embodiments, in the polypeptide chain encoded by the nucleic acid molecule, the tag molecule is connected to the signal transduction region of the chimeric antigen receptor (CAR) through a self-cleaving peptide sequence.

[0062] In some embodiments, the tag molecule is tEGFR, and / or the self-cleaving peptide is P2A or T2A.

[0063] In some embodiments, the tag molecule comprises an amino acid sequence as shown in SEQ ID NO:31.

[0064] In some embodiments, the self-cleaving peptide comprises the amino acid sequence shown in SEQ ID NO:33.

[0065] In some embodiments, the nucleic acid molecule comprises any sequence selected from the following sequences: a nucleic acid sequence as shown in SEQ ID NO: 11, SEQ ID NO: 13, or a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequences.

[0066] In one aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule described in some embodiments.

[0067] In one aspect, the present invention provides a recombinant cell that expresses a chimeric antigen receptor (CAR) molecule as described in some embodiments.

[0068] In some embodiments, the recombinant cell is a T cell or a NK cell.

[0069] In some embodiments, the recombinant cell further expresses a tag molecule.

[0070] In some embodiments, the tag molecule is tEGFR.

[0071] In one aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid molecule described in some embodiments or the recombinant cell described in some embodiments.

[0072] In one aspect, the present invention provides the use of the above-mentioned chimeric antigen receptor (CAR) molecule and / or the above-mentioned nucleic acid molecule for treating a proliferative disease or an autoimmune disease.

[0073] In some embodiments, the proliferative disease comprises a lymphoproliferative disease or a tumor.

[0074] In some embodiments, the tumor is a plasma cell malignancy or a B cell malignancy, preferably, the tumor is a myeloma.

[0075] In some embodiments, the autoimmune disease is an antibody-mediated autoimmune disease. Preferably, the autoimmune disease comprises systemic lupus erythematosus, myasthenia gravis, neuromyelitis optica, and lupus nephritis.

[0076] In one aspect, the present invention provides a bispecific antibody, an antigen-binding fragment thereof, or a derivative thereof, comprising a heavy chain variable region targeting BCMA (anti-BCMA VH); and an antigen-binding region targeting a second target, wherein the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises:

[0077] (a) a heavy chain complementarity determining region 1 (HCDR1), comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6;

[0078] (b) a heavy chain complementarity determining region 2 (HCDR2), comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7;

[0079] (c) a heavy chain complementarity determining region 3 (HCDR3), comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 8,

[0080] Among them, the second target targets the surface protein of immune cells.

[0081] In some embodiments, the second target is a T cell or B cell surface protein.

[0082] In some embodiments, the second target is CD3 or GPRC5D.

[0083] In some embodiments, the antigen binding region of the second target is a heavy chain variable region, or an antibody variable region comprising a heavy chain variable region and a light chain variable region.

[0084] In some embodiments, the bispecific antibody, antigen-binding fragment thereof or derivative thereof, wherein:

[0085] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1;

[0086] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2;

[0087] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0088] In some embodiments, the bispecific antibody, antigen-binding fragment thereof or derivative thereof, wherein:

[0089] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6;

[0090] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0091] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8.

[0092] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The general process of the present invention for screening specific antibodies targeting BCMA from a phage antibody library is shown.

[0094] Figure 2 The results of enzyme-linked immunosorbent assay (ELISA) of some selected phage monoclones with target antigen and control antigen are shown.

[0095] Figure 3 Flow cytometric analysis results of binding of some phage monoclones to 293CT-BCMA and 293CT cells are shown.

[0096] Figure 4The results of ELISA analysis of the screened phage monoclonals with BCMA antigen proteins of various species and non-related antigens are shown. Negative control is a negative control phage antibody clone, anti-M13phage mouse Ab / anti-mouse HRP Ab is a negative antibody control with only the first antibody and the second antibody, anti-mouse HRP Ab is a negative antibody control with only the second antibody, and anti-his-HRP Ab is a positive antibody control for detecting antigen tags. Positive control is a positive antibody control for detecting the target antigen BCMA, and anti-rabbit HRP Ab is a negative control for the second antibody of the positive control antibody. Figure 5 In the figure, the bar graphs corresponding to each test antibody and the control group represent the test results of the proteins Human-BCMA-his-Bio, Mouse-BCMA-his, cyno-BCMA-his, Human-BAFFR-his-Bio, Human-IL10-his-Bio, and SA from left to right.

[0097] Figure 5 The results of flow cytometric analysis of the binding of the screened phage monoclonals to various BCMA positive and negative cell lines are shown. NC1 is a negative control phage antibody clone, and NC2 is a secondary antibody staining cell group used for positive control antibody staining cells. Figure 5 A is the test result of positive control antibody against cell lines expressing BCMA antigen. Figure 5 B is the flow cytometric analysis result (peak graph) of the binding of the screened phage monoclonal clone No. 11 to BCMA positive and negative cell lines at the phage level. Figure 5 C is the flow cytometric analysis result (peak graph) of the binding of the screened phage monoclonal clone 54 to BCMA-positive and -negative cell lines at the phage level.

[0098] Figure 6 The results of the ELISA analysis of the screened phage monoclonals at the protein level with the target antigen and non-related antigens are shown. Anti-human IgG HRP Ab is a negative antibody control with only secondary antibody added, anti-his tagHRP Ab is a positive antibody control for detecting antigen tags, Positive control is a positive antibody control for detecting the target antigen BCMA, and anti-rabbit HRP Ab is a negative control for the secondary antibody of the positive control antibody. Figure 6In the figure, the bar graphs corresponding to each test antibody and the control group represent the test results of proteins Human-BCMA-his-Bio, Mouse-BCMA-his, cyno-BCMA-his, Human-BAFFR-his-Bio, Human-IL10-his-Bio, and SA from left to right.

[0099] Figure 7 A shows the flow cytometric analysis results of the binding of the screened phage monoclonal clone 54 to BCMA-positive and -negative cell lines at the protein level (peak graph).

[0100] Figure 7 B shows the flow cytometric analysis results (peak graph) of the binding of the screened phage monoclonal clone No. 11 to BCMA positive and negative cell lines at the protein level. NC is a negative control with only the secondary antibody FITC anti-human IgG Ab added.

[0101] Figure 8 The degranulation results of BCMA CAR-T cells in each group on various target cells (8226-luc, MM1.s-luc, H929-luc, CCRF-luc, Raji-luc) are shown.

[0102] Among them, effector cells B11 and B54 are BCMA CAR-T cells constructed using BCMA clones No. 11 and No. 54, respectively. 0037 is the BCMA CAR-T cell 0037 in the submitted patent (CN201980002363.X), which is used as a positive control, and MockT is a negative control cell.

[0103] Figure 9 shows the killing results of BCMA CAR-T cells in each group against various target cells (8226-luc, MM1.s-luc, H929-luc, CCRF-luc). Fig.9A The killing results of positive target cells, Fig. 9B The definition of each effector cell in Figure 9 is the same as Figure 8 same.

[0104] Figure 10 shows the anti-tumor efficacy of CAR-T cells corresponding to different BCMA antibody clones in H929-luc tumor-bearing NCG mice. Figure A shows the change of photon number over time after CAR-T cell infusion, Figure B shows the survival rate of mice infused with CAR-T cells, and Figure C shows the change of T cell to mouse lymphocyte ratio after CAR-T cell infusion. The definition of each effector cell in Figure 10 is the same as Figure 8 same. DETAILED DESCRIPTION

[0105] The following describes various aspects of the present application in detail. It should be noted that the following description is not limiting, and those skilled in the art can make any modifications and substitutions based on the following description and the technical common sense in the art without significantly hindering the technical effect of the present application.

[0106] The practice of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, which are within the skill of the art. Descriptions of these methods can be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols in Immunology (Q. E. Coligan, AM Kruisbeek, DH Margulies, E M Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and journal monographs such as Advances in Immunology.

[0107] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this application, the following terms are defined below.

[0108] In this application, the term "single domain antibody" generally refers to a type of antibody that lacks the light chain of an antibody and only has a heavy chain variable region. Studies have found that there is a heavy chain antibody (hcAb) composed only of heavy chains but with complete functions in Bactrian camels, dromedaries, alpacas and llamas. The molecular weight of its variable region (variable domains of the hcAb, referred to as VHH) is only 1 / 10 of that of conventional antibodies. It is the smallest molecular fragment with complete antibody function that can be obtained at present, called a single domain antibody (sdAb). Compared with other antibodies, single domain antibodies have the advantages of low immunogenicity, small molecules, and strong penetration. Therefore, they have broad application prospects in basic research, drug development, disease treatment and other fields. For example, single domain antibodies can come from alpacas. Single domain antibodies can be composed of heavy chain variable regions (VH). The term "heavy chain variable region" generally refers to the amino terminal domain of the heavy chain of the antigen binding fragment. The heavy chain variable region can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino-terminus to carboxyl-terminus: H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3 and H-FR4. The heavy chain variable region contains a binding domain that interacts with an antigen (e.g., HSA). The exact boundaries of the CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antigen binding protein, but also provides precise residue boundaries defining the CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 500:1-106). 342: 877-883 (1989)) found that despite the great diversity at the amino acid sequence level, certain sub-portions within Kabat CDRs adopt almost identical peptide main chain conformations. These sub-portions are named L1, L2 and L3 or H1, H2 and H3, where "L" and "H" refer to the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have borders that overlap with Kabat CDRs.Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262 (5): 732-45 (1996)). In addition, other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although they may be shortened or lengthened according to predictions or experimental findings that specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. In this application, CDRs may be defined using the Kabat numbering system. In this application, the term "single domain antibody" may be used interchangeably with "nanobody" and "VHH".

[0109] In this application, the term "BCMA" refers to B cell maturation antigen, also known as CD269, which is a type III transmembrane protein, does not contain a signal peptide, contains a cysteine-rich extracellular domain, is only expressed on the surface of mature B cells, and is not expressed in T cells or monocytes. It is an important B cell biomarker. The gene encoding BCMA belongs to a member of the TNF receptor superfamily. The receptor is preferentially expressed in mature B lymphocytes and plays an important role in B cell development and autoimmune response. The receptor specifically binds to tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B / TALL-1 / BAFF) and leads to NF-κB and MAPK8 / JNK activation. The receptor also binds to various TRAF family members to mediate cell survival and proliferation signals. In multiple myeloma, the expression of BCMA increases, the pro-proliferation signal is enhanced, and finally canceration occurs. Therefore, the expression level of BCMA on multiple myeloma cells is significantly higher than that of healthy plasma cells.

[0110] In the present application, the term "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains interconnected (coordinated) by disulfide bonds, and includes any molecule comprising its antigen-binding portion. The term "antibody" includes monoclonal antibodies, polyclonal antibodies, and also antibody fragments or antibody derivatives. In addition, the term "antibody" includes human antibodies, humanized antibodies, and also includes chimeric antibodies, single-domain antibodies (e.g., dAb or VHH), single-chain antibodies (e.g., scFv) and antibody fragments that bind to antigens (e.g., Fab, Fab' and F(ab')2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any of the antibody fragments and derivatives thereof that bind to antigens. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region CH. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region CL. The VH and VL regions can be further divided into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conservative regions called framework regions (FRs). It should be noted that in this specification, the complementary determining regions (CDRs) in the heavy chain variable region are called heavy chain complementary determining regions (HCDRs), and the complementary determining regions (CDRs) in the light chain variable region are called light chain complementary determining regions (LCDRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy chain constant region usually includes at least three heavy chain constant region domains, namely, CH1 domain, CH2 domain and CH3 domain. The heavy chain constant regions of IgG, IgA and IgD include three domains of CH1, CH2 and CH3, and the heavy chain constant regions of IgM and IgE include four domains of CH1, CH2, CH3 and CH4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0111] The term "antibody fragment" or "antigen binding fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and may refer to the antigen binding domain of an intact antibody, e.g., an antigenicity-determining variable region, which is sufficient to confer recognition and specific binding to a target, such as an antigen, on the antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (VH or VL), camelid VHH domains, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated herein, a scFv may have the VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, a scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0112] In the present application, the term "antigen binding moiety" or "Fab binding domain" refers to an antibody portion comprising a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains comprise a VL antibody domain, a CL antibody domain, a VH antibody domain, and a CH1 antibody domain. The VL and CL antibody domains may be located on one chain, and the VH and CH1 antibody domains may be located on another chain; or the VL and CH1 antibody domains may be located on one chain, and the VH and CL antibody domains may be located on another chain.

[0113] The term "functional variant" of a protein or polypeptide sequence refers in this application to an amino acid sequence having a sequence modification compared to a parent by 1 or more, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions, additions and / or insertions, and the functional variant substantially retains the biological properties of the protein or polypeptide sequence before modification. In one embodiment, the present application encompasses variants of any protein or polypeptide sequence described in the present application. In certain embodiments, the functional variant of a protein or polypeptide sequence retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the parent before modification. The functional variant described in the present application can be a functional variant of an antibody, an extracellular signal peptide, or a self-cleaving peptide. When referring to the functional variants of antibodies, it also includes the antibody variable region (such as VH or VL), antibody constant region (such as CH or CL), heavy chain CDR region (HCDR1, HCDR2 or HCDR3), light chain CDR region (LCDR1, LCDR2 or LCDR3), heavy chain CH1, CH2 or CH3 domain, etc. Functional variants. For antibodies, their biological activity includes, for example, antigen binding ability. In certain embodiments, the amino acid modifications contained in the functional variants of antibodies do not cause the antibody variants to lose binding to the antigen, but can optionally confer properties such as increased antigen affinity and different effector functions. It is understandable that the heavy chain variable region or light chain variable region of the antibody, or each CDR region, can be modified individually or in combination. In one embodiment, the amino acid modifications in one or more or all three HCDRs are no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications are made in one or more or all three LCDRs. In certain embodiments, the amino acid modifications may be conservative sequence modifications, such as conservative amino acid substitutions. In certain embodiments, the functional variant has at least 70%, 80%, 85%, 90% or 95% or 99% or higher sequence identity with the parent. Similarly, the "functional variant" of a nucleic acid molecule refers in this application to a nucleic acid molecule that can encode the same amino acid as the parent nucleic acid molecule or a nucleic acid molecule that encodes the functional variant of the protein.

[0114] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or modify the binding characteristics of the antibody or antibody fragment comprising the amino acid sequence. Conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the present application's antibodies or antibody fragments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions of an amino acid residue by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the present application CAR can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assay described herein.

[0115] In this application, the term "isolated" generally refers to an antibody that has been separated from components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0116] In the present application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide or ribonucleotide or its analog in an isolated form of any length separated from its natural environment or artificially synthesized. The nucleic acid molecule described in the present application can be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies or antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific operations, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0117] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, for transferring the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product. In the present application, one or more of the nucleic acid molecules may be included in the vector. In addition, other genes may also be included in the vector, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that are involved in the initiation of transcription and translation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functional connection nucleic acid. The vector described in the present application may be selected from plasmids, retroviral vectors, and lentiviral vectors. The plasmids, retroviral vectors, and lentiviral vectors described in the present application may include CAR.

[0118] In this application, the term "plasmid" generally refers to a DNA molecule other than chromosomes or nucleoids in bacteria, yeast, and other organisms, which exists in the cytoplasm and has the ability to replicate autonomously, enabling it to maintain a constant copy number in daughter cells and express the genetic information it carries. Plasmids are used as gene carriers in genetic engineering research.

[0119] In this application, the term "retroviral vector" generally refers to a type of RNA virus whose genetic information is stored in ribonucleic acid, and most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains genes for proteins that make up the center and structure of the virus; pol, which contains genes for reverse transcriptase and env, which contains genes that make up the viral shell. Through retroviral transfection, retroviral vectors can randomly and stably integrate their own genome and the exogenous genes they carry into the host cell genome, for example, CAR molecules can be integrated into host cells.

[0120] In this application, the term "lentiviral vector" generally refers to a diploid RNA viral vector belonging to a retrovirus. The lentiviral vector is based on the genome of the lentivirus, and multiple sequence structures related to viral activity are removed to make it biologically safe, and then the sequence and expression structure of the target gene required for the experiment are introduced into this genome skeleton to prepare the vector. Compared with other retroviruses, lentiviral vectors have a wider range of hosts and have the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, and undifferentiated cells, the transduction efficiency of the target gene can be greatly improved (see Chen Chen and Wan Haisu, "Lentiviral Vectors and Research Progress, Chinese Journal of Lung Cancer 17.12 (2014): 870–876. PMC). Through lentiviral vector transfection, the retroviral vector can randomly and stably integrate its own genome and the exogenous genes it carries into the host cell genome, for example, the CAR molecule can be integrated into the host cell.

[0121] In the present application, the term "immune effector cells" generally refers to immune cells that participate in the clearance of foreign antigens and perform effector functions in immune responses, such as plasma cells, cytotoxic T cells, NK cells, APSC multipotent cells, mast cells, etc.

[0122] In the present application, the term "recombinant receptor" or "recombinant receptor molecule" refers to an artificially designed receptor molecule obtained by biological methods such as genetic engineering. Specifically, chimeric antigen receptor (CAR) molecules, antibody-T cell receptor chimeric molecules (abTCR), synthetic T cell receptor antigen receptor (STAR ​​CAR) molecules, etc. can be cited. It should be noted that the term "receptor" or "receptor molecule" (such as a chimeric antigen receptor (CAR) molecule) used in this application may include all molecular forms that appear in the process of obtaining a mature protein with a spatial conformation from an unmodified polypeptide chain through modification, that is, including an intermediate between a polypeptide chain and a mature protein, rather than just referring to a mature receptor protein.

[0123] In the present application, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a signaling domain (also referred to herein as an "intracellular signaling sequence"), wherein the signaling domain comprises a functional signaling domain derived from a stimulatory molecule defined below. In one embodiment, the stimulatory molecule is a ζ chain associated with a T cell receptor complex, for example, CD3ζ can be cited. In one embodiment, CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, a co-stimulatory domain, and a signaling domain, wherein the co-stimulatory domain may have one or more. In one embodiment, the co-stimulatory domain is selected from 4-1BB (ie, CD137), CD27, and / or CD28. In one embodiment, CAR comprises an optional leader sequence at the amino terminus (N terminus) of the CAR fusion protein, and the leader sequence may be, for example, an extracellular signal peptide. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.

[0124] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information into cells to regulate cellular activity through a defined signaling pathway by producing second messengers, or a protein that functions as an effector by responding to such messengers.

[0125] The term "stimulation" refers to an initial response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β, and / or reorganization of cytoskeletal structures, etc.

[0126] The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a first cytoplasmic signaling sequence, wherein for at least some aspects of the T cell signaling pathway, the sequence regulates the initial activation of the TCR complex in a stimulating manner. On the one hand, the first signal is triggered by the binding of, for example, a TCR / CD3 complex to an MHC molecule loaded with a peptide, thereby mediating a T cell response (including but not limited to, proliferation, activation, differentiation, etc.). A signaling domain that acts in a stimulating manner (also referred to as an "intracellular signaling sequence") may include a signaling motif called an immunoreceptor tyrosine activation motif or ITAM. Examples of signaling domain sequences that are particularly useful in the present application include, but are not limited to, sequences derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), and CD66d. In a specific CAR of the present application, the signaling domain derived from CD3ζ can be a CD3ζ signaling domain from a human, or an equivalent residue from a non-human species such as a mouse, rodent, monkey, ape, etc.

[0127] The term "costimulatory molecule" refers to an associated binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules or their ligands that are not antigen receptors and are required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) and 4-1BB (CD137).

[0128] The part containing an antibody or its antibody fragment in the CAR composition of the present application can exist in various forms, including, for example, single domain antibodies, single domain antibody fragments (sdAb), single chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426), wherein the antigen binding domain is expressed as part of a continuous polypeptide chain. In one embodiment, the antigen binding domain of the CAR composition of the present application includes an antibody fragment. In another embodiment, CAR includes a fragment of a single domain antibody.

[0129] In this application, the term "CAR-T cell" refers to a T cell with a CAR molecule, which is usually obtained by transducing a T cell with an expression vector encoding CAR. Commonly used expression vectors are viral vectors, such as lentiviral expression vectors. T cells (CAR-T) modified with chimeric antigen receptors are not restricted by the major histocompatibility complex and have specific targeted killing activity and the ability to proliferate persistently. In addition to T cells, other lymphocytes such as NK cells can also be transformed with an expression vector encoding CAR to obtain targeted killer cells expressing the CAR.

[0130] In the present application, the term "extracellular signal peptide" generally refers to a short (5-30 amino acids in length) peptide chain that guides newly synthesized proteins to transfer to the secretory pathway.

[0131] In the present application, the term "self-cleaving peptide" refers to a self-cleaving 2A peptide, which can achieve the function of cleaving protein through ribosome jumping rather than protease hydrolysis, and may include but is not limited to T2A, F2A and P2A.

[0132] In the present application, the term "label molecule" generally refers to a gene, protein or other molecule of known function or sequence that can play a specific marking role and emit a detectable signal. The label molecule can be a fluorescent protein, such as GFP, RFP and YFP, etc. The label molecule can be tEGFR. The term "tEGFR" refers to a gene encoding a truncated human epidermal growth factor receptor polypeptide, which lacks the distal membrane EGF binding domain and the cytoplasmic signaling tail, but retains the extracellular epitope recognized by the anti-EGFR antibody. tEGFR can be used as a non-immunogenic selection tool and a tracking marker with genetically modified cell functions. In the present application, it can be used as a marker molecule for CAR-T cells, which is used to clear the ADCC pathway (cetuximab mediated ADCC pathway) mediated by EGFR antibodies (e.g., cetuximab) in the body when necessary (see US8802374B2).

[0133] In the present application, "sequence identity" generally refers to the degree of sequence identity based on nucleotide or amino acid by nucleotide in a comparison window. "Sequence identity percentage ratio" can be calculated in the following manner: the sequences of two optimal comparisons are compared in a comparison window, it is determined that there are identical nucleic acid bases (for example, A, T, C, G, I) or identical amino acid residues (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in two sequences The number of positions is to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (that is, window size), and the result is multiplied by 100, to produce the sequence identity percentage ratio. In order to determine the optimal comparison carried out for the sequence identity percentage ratio, it can be realized in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared, or over a region of interest.

[0134] The term "endogenous" refers to a gene or protein sequence or portion thereof that originates from an organism (eg, the human body) itself.

[0135] In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.

[0136] In this application, the term "cancer" generally refers to or describes the physiological condition of mammals, which is typically characterized by cell proliferation or survival disorders. In this application, the hyperproliferative diseases referred to as cancer include but are not limited to solid tumors, such as cancers occurring in the breast, respiratory tract, brain, reproductive organs, digestive tract, urethra, eyes, liver, skin, head and neck, thyroid, parathyroid glands, and their distal metastases. Such diseases also include lymphomas, sarcomas, and leukemias. Examples of breast cancer include but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, breast ductal carcinoma in situ, and breast lobular carcinoma in situ. Examples of respiratory tract cancer include but are not limited to small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancer include but are not limited to brain stem and hypothalamic keratinoma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Male genital tumors include but are not limited to prostate and testicular cancer. Female genital tumors include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer and vulvar cancer, and uterine tumors. Digestive tract tumors include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach, pancreas, rectum, small intestine and salivary gland cancer. Urinary tract tumors include, but are not limited to, bladder, penis, kidney, renal pelvis, ureter and urethral cancer. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatoma with or without fibrolamellar variation), cholangiocarcinoma (intrahepatic bile duct carcinoma) and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancers, and lip and oral cancers. Lymphomas include, but are not limited to, AIDS-related lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Hodgkin's disease, and central nervous system lymphomas. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcoma, and rhabdomyosarcomas. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0137] The term "therapeutically effective amount" refers to the amount of active compound sufficient to cause the biological or medical response desired by the clinician in the subject. The "therapeutically effective amount" of the antibody of the present application can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight. The administration of the antibody of the present application includes, but is not limited to, injection, such as intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, etc.

[0138] The term "subject" encompasses mammals, including human subjects in need of treatment, and also includes non-human mammals, such as primates, cows, sheep, cats, dogs, and the like.

[0139] The term "and / or" should be understood to mean either or both of the alternatives.

[0140] As used in this application, the term "comprising" or "including" means including the described elements, integers or steps, but does not exclude any other elements, integers or steps. In this application, when the term "comprising" or "including" is used, unless otherwise indicated, the situation consisting of the described elements, integers or steps is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0141] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0142] Single domain antibodies targeting BCMA

[0143] In the present invention, a single-domain antibody targeting BCMA was obtained through screening, which can effectively bind to BCMA only through the heavy chain variable region (VH). In one aspect, the present invention provides a single-domain antibody targeting BCMA, an antigen-binding fragment thereof, or a derivative thereof, comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises: (a) a heavy chain complementary determining region 1 (HCDR1), which comprises an amino acid sequence selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 6; and (b) a heavy chain complementary determining region 2 (HCDR2), which comprises an amino acid sequence selected from the following sequences: SEQ ID NO: 2, SEQ ID NO: 7; and (c) a heavy chain complementary determining region 3 (HCDR3), which comprises an amino acid sequence selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 8. In one embodiment, the single-domain antibody is clone 11 described below, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3. In one embodiment, the single-domain antibody is clone 54 described below, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; in one embodiment, the single-domain antibody is clone 11 described below, wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequences. In one embodiment, the single domain antibody is clone 54 described below, wherein the heavy chain variable region (VH) comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the above sequence. In one embodiment, the single domain antibody or its antigen binding fragment is a fully human antibody, a humanized antibody, or an antigen binding fragment thereof. In one embodiment, the single domain antibody, its antigen binding fragment, or its derivative targeting BCMA further comprises an Fc fragment.In one embodiment, the Fc fragment comprises any amino acid sequence selected from the following sequences: the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0144] Chimeric Antigen Receptor (CAR)

[0145] In one aspect, the present invention provides a chimeric antigen receptor (CAR) molecule, comprising a hinge region, a transmembrane domain, a co-stimulatory domain and a signal transduction domain, and an antigen binding domain, wherein the antigen binding domain comprises a heavy chain variable region targeting BCMA (anti-BCMA VH), wherein the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises: (a) a heavy chain complementary determining region 1 (HCDR1), which comprises any amino acid sequence selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 6; (b) a heavy chain complementary determining region 2 (HCDR2), which comprises any amino acid sequence selected from the following sequences: SEQ ID NO: 2, SEQ ID NO: 7; (c) a heavy chain complementary determining region 3 (HCDR3), which comprises any amino acid sequence selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 8. In one embodiment, the heavy chain variable region targeting BCMA (anti-BCMA VH) is based on clone 11 described later, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3. In one embodiment, the heavy chain variable region targeting BCMA (anti-BCMA VH) is based on clone 54 described later, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; in one embodiment, the heavy chain variable region targeting BCMA (anti-BCMA VH) is based on clone 11 described later, wherein the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence. In one embodiment, the heavy chain variable region targeting BCMA (anti-BCMA VH) is based on clone 54 described later, wherein the heavy chain variable region targeting BCMA (anti-BCMA VH) comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0146] In the chimeric antigen receptor (CAR) involved in one embodiment of the present application, it further comprises a leader sequence. In one embodiment of the present application, the leader sequence is an extracellular signal peptide. In one embodiment of the present application, the extracellular signal peptide sequence is an extracellular signal peptide sequence derived from CD8α. The extracellular signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO: 17

[0147] In one embodiment of the present application, the chimeric antigen receptor (CAR) involved in the present application is arranged in the following order: extracellular signal peptide sequence, antigen binding domain, hinge region, transmembrane domain, co-stimulatory domain, signal transduction domain. In one embodiment of the present application, the hinge region is a hinge region derived from CD8α. In one embodiment of the present application, the transmembrane domain is a transmembrane domain derived from CD8α. In one embodiment of the present application, the co-stimulatory domain comprises a co-stimulatory domain derived from 4-1BB or a co-stimulatory domain derived from CD28. In one embodiment of the present application, the signal transduction domain comprises a signal transduction domain derived from CD3ζ. In one embodiment of the present application, the antigen binding domain comprises an amino acid sequence as shown in SEQ ID NO: 4 or 9. In one embodiment of the present application, the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 19. In one embodiment of the present application, the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 21. In one embodiment of the present application, the costimulatory domain derived from 4-1BB comprises the amino acid sequence shown in SEQ ID NO: 23. In one embodiment of the present application, the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 25.

[0148] In one embodiment of the present application, the CAR can also be formed by cleavage of a complete polypeptide, and the polypeptide comprises the polypeptide chain of the CAR and the truncated EGFR. In one embodiment of the present application, the polypeptide may include an extracellular signal peptide sequence, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain from the amino terminus to the carboxyl terminus. In one embodiment of the present application, the polypeptide may also include an extracellular signal peptide sequence, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, a signal transduction domain, a self-cleavage peptide, and a truncated EGFR (tEGFR) from the amino terminus to the carboxyl terminus. In one embodiment, the tEGFR has an amino acid sequence as shown in SEQ ID NO: 31 or a functional variant thereof. In certain embodiments, the self-cleavage peptide is selected from T2A, F2A or P2A. In certain embodiments, the self-cleavage peptide is selected from P2A and has an amino acid sequence as shown in SEQ ID NO: 33 or a functional variant thereof.

[0149] Nucleic acid, vector, cell, preparation method and composition

[0150] In one embodiment, the present application provides an isolated nucleic acid molecule that can encode an antibody or chimeric antigen receptor (CAR) molecule described in the present application. For example, the isolated nucleic acid molecule encoding the antibody may include a nucleic acid sequence shown in SEQ ID No: 5, 10 or a functional variant thereof; the isolated nucleic acid molecule encoding the chimeric antigen receptor may include a nucleic acid sequence shown in SEQ ID No: 11, 13 or a functional variant thereof.

[0151] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0152] In one embodiment, the present application provides a vector, which may include the nucleic acid molecule. In one embodiment of the present application, the vector may be selected from one or more of a plasmid, a retroviral vector, and a lentiviral vector. The lentiviral vector described in the present application may include a nucleic acid sequence encoding a recombinant receptor of the present application. In addition, the vector may also include other genes, such as marker genes that allow the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that are involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, a 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functionally connected to a nucleic acid. One or more nucleic acid molecules described herein may be operably linked to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector, including a CAR plasmid.

[0153] In one embodiment, the present application provides a kind of immune effector cell, which may include the recombinant receptor described in the present application, the nucleic acid molecule or the vector. In one embodiment of the present application, the immune effector cell may be a mammalian cell. In one embodiment of the present application, the immune effector cell may be selected from T cells and natural killer (NK) cells.

[0154] In one embodiment, the present application provides a method for preparing an immune effector cell, comprising introducing a vector described in the present application into an immune effector cell. For example, the vector described in the present application can be introduced into the immune effector cell, such as a T cell or a natural killer (NK) cell. In certain embodiments, each or each cell may include one or more vectors described in the present application. In certain embodiments, each or each cell may include multiple (e.g., 2 or more) or multiple (e.g., 2 or more) vectors described in the present application. In one embodiment of the present application, the vector can be introduced into an immune effector cell and the vector described in the present application can be introduced into the cell by methods known in the art. For example, immune effector cells can be transfected by a retroviral vector, and the viral genome with a CAR molecule can be integrated into the host genome to ensure long-term and stable expression of the target gene. For another example, a transposon is used to introduce a plasmid carrying a CAR (transposon) and a plasmid carrying a transposase into a target cell. For another example, a CAR molecule can be added to the genome by gene editing (e.g., CRISPR, Cas9). In one embodiment of the present application, the vector carrying the CAR molecule described in the present application can be introduced into the cell by methods known in the art, such as electroporation, liposome transfection (lipofectamine2000, Invitrogen), etc.

[0155] In one embodiment, the application provides a composition, which may include the immune effector cells and a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex and or a nonionic surfactant, etc. In one embodiment of the application, the pharmaceutical composition may be formulated for oral administration, intravenous administration (e.g., intravenous injection, IV), intramuscular administration (e.g., intramuscular injection, IM), in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration by subcutaneous storage depot.

[0156] Pharmaceutical use

[0157] In one embodiment, the present application provides the use of the different forms of antibody molecules, the nucleic acid molecules, the vectors or the recombinant cells for preparing a drug, wherein the drug is used to treat a proliferative disease. In one embodiment of the present application, the proliferative disease may be cancer or a malignant tumor. In one embodiment of the present application, the proliferative disease may be multiple myeloma.

[0158] In one embodiment, the present application provides the antibody molecule, the nucleic acid molecule, the vector or the recombinant cell for use in treating a proliferative disease.

[0159] In one embodiment, the present application provides a method for treating a proliferative disease, comprising administering the antibody molecule, the nucleic acid molecule, the vector or the recombinant cell to a subject.

[0160] In one embodiment, the present application provides a method for treating a proliferative disease, comprising administering the antibody molecule, the nucleic acid molecule, the vector or the recombinant cell to a subject.

[0161] Without intending to be bound by any theory, the following examples are merely intended to illustrate the workings of the chimeric antigen receptors, vectors, cells, and compositions of the present application, and are not intended to limit the scope of the invention of the present application.

[0162] Example

[0163] The present application is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not used to limit the scope of the present application. After reading the present application, various equivalent forms of modifications to the present application by those skilled in the art fall within the scope defined by the claims attached to the present application.

[0164] The present invention uses fully human phages for antibody screening to directly obtain fully human BCMA monoclonal antibodies. Compared with traditional hybridoma technology, the difficult step of humanizing mouse antibodies is omitted, and fully human antibodies have lower immunogenicity than humanized mouse antibodies, and have better potential in the application of antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), detection reagents, etc.

[0165] The present invention is described in detail below with reference to specific embodiments.

[0166] Example 1. Enrichment of specific antibody clones targeting BCMA protein from phage antibody library by affinity panning

[0167] During the antibody screening process, we found that the antibody clones directly screened using recombinantly expressed BCMA protein could not bind to the cell line 293CT-BCMA that highly expresses BCMA. This may be due to the fact that the recombinantly expressed BCMA protein antigen and the natural BCMA on the cell membrane surface have great differences in conformation and accessible antigen epitopes. To overcome this problem, we used the protein / cell line alternating panning method to enrich phage antibodies that can bind to both recombinantly expressed BCMA protein and 293CT-BCMA cells, and screened out monoclonal antibodies that can specifically bind to the BCMA antigen on the cell membrane surface.

[0168] BCMA protein panning

[0169] Multiple rounds of panning were performed using BCMA-His as the positive panning protein to obtain a phage pool enriched with the target antibody clone. The experimental steps are briefly described as follows:

[0170] 1) The SA magnetic beads were first blocked with blocking solution for 2 hours, and then the target antigen (BCMA-His) was combined with the blocked SA magnetic beads;

[0171] 2) Add phage library (containing 5x10 12 phage particles) and a clean SA magnetic bead were incubated together to remove the phage antibody clones that non-specifically bound to the SA magnetic beads;

[0172] 3) After incubation, the supernatant is transferred to SA magnetic beads bound to the target antigen and incubated further to allow the phage to bind to the target antigen;

[0173] 4) Wash the magnetic beads with washing solution to remove unbound phages;

[0174] 5) Elute the positive phage from the target antigen with an elution solution and add a neutralization solution to neutralize it;

[0175] 6) Re-infect the host bacteria XL1-blue with the eluted phages to amplify the recovered phages. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phages;

[0176] 7) Repeat steps 1) to 6), usually 2 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0177] 293CT-BCMA / 293CT cell selection

[0178] Multiple rounds of panning were performed using 293CT cells with negative BCMA expression as negative panning cells and 293CT-BCMA cells with positive BCMA expression as positive panning cells to obtain a phage pool enriched with the target antibody clone.

[0179] The brief experimental steps are as follows:

[0180] 1) The phage pool (containing 5x10 11 phage particles) and 1x10 7 Mix the negative selection cells 293CT and incubate them at room temperature for 2 hours on a rotating mixer to allow the antibody clones that bind to the negative selection cell line to fully bind to these cells;

[0181] 2) Centrifuge at 1500 rpm for 5 minutes to pellet the cells, transfer the supernatant to a new tube, and mix with 1x10 7 293CT-BCMA cells (BCMA-positive cells) were mixed and allowed to bind on a rotating mixer at room temperature for 2 hours;

[0182] 3) Wash the cells 6 times with PBS, discard the supernatant each time, resuspend and centrifuge at 1500 rpm for 5 minutes to remove unbound phages;

[0183] 4) Elute the positive phage from the target antigen with an elution solution and add a neutralization solution to neutralize it;

[0184] 5) Use the eluted phage to re-infect the host bacteria and amplify the recovered phage. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phage;

[0185] 6) Repeat steps 1) to 5), usually 2 to 3 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0186] The enriched phage pool can be used for the next step of monoclonal selection and ELISA / FACS screening.

[0187] Main materials and reagents:

[0188] Fully human phage antibody library;

[0189] Helper phage KO7, Thermo / Invitrogen, 18311019;

[0190] Biotinylated Human BCMA / TNFRSF17 Protein(hBCMA-bio-his),

[0191] ACRObiosystem, BCA-H82E4

[0192] 293CT-BCMA cell line (BCMA-positive cell line);

[0193] 293CT cell line (BCMA-negative cell line);

[0194] BeaverBeads TM Streptavidin, Beaver Bio, 22307-10;

[0195] High binding ELSIA plate,Costar,#3590

[0196] Blocking solution: PBS + 3% BSA

[0197] Rinse solution: PBS + 0.1% Tween20

[0198] Eluent: 0.2M Glycine, pH 2.2

[0199] Neutralization solution: 1M Tris, pH 9.1

[0200] Experimental results:

[0201] Using different antibody libraries, three rounds of protein panning and two rounds of cell panning were performed, and a significant increase in the recovery rate was observed in each panning (Table 1), demonstrating that the antibody clones were effectively enriched.

[0202] In our experiment, we used the method of protein and cell alternating panning to enrich specific antibody clones that can bind to both BCMA protein and BCMA in its natural state on the cell surface from the phage antibody library. Table 1 shows the results of co-panning using recombinant BCMA protein and 293CT-BCMA / 293CT cell line. From the recovery rate, all two pannings were enriched and can be used for the next step of selecting single clones.

[0203] Table 1 Protein / cell selection experiment results

[0204]

[0205] Example 2. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) from enriched phage Pool screening for specific clones

[0206] Purpose and principle: The phage pool enriched by affinity panning contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. In order to obtain specific clones, we need to isolate monoclonal clones, package them into monoclonal phages, and screen a large number of monoclonal clones by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), and select monoclonal clones that specifically bind to BCMA protein and BCMA-positive cell line 293CT-BCMA at the same time. The specific monoclonal clones are further sequenced by DNA to determine the unique antibody sequence contained therein.

[0207] In the ELISA primary screening, the biotinylated target protein (BCMA-his-Bio) is closer to the antigen conformation of the natural state in the reaction solution through the binding of streptavidin and biotin. Those that only bind to BCMA-his-Bio but not to the control antigen SA are identified as specific clones. The FACS primary screening is carried out using the BCMA-positive cell line 293CT-BCMA with high expression of BCMA and the BCMA-negative cell line 293CT. Those that only bind to the cell 293CT-BCMA and not to the 293CT cells are identified as specific clones. Through the two primary screenings of ELISA and FACS, we can obtain candidate antibodies that can bind to the recombinantly expressed BCMA protein and recognize the natural state BCMA molecules on the cell surface for further screening.

[0208] Brief steps of ELISA screening experiment:

[0209] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0210] 2) Dilute Strepavidin to 2 μg / mL with PBS, add 100 μL / well to the high binding ELISA plate, and bind for 2 h at room temperature;

[0211] 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C;

[0212] 4) Wash the plate twice with 250 μL of rinse solution;

[0213] 5) Dilute the biotin-tagged target protein and control protein to 2 μg / mL with PBS, add 100 μg / well to the ELISA plate pre-coated with Strepavidin, and bind at room temperature for 1 hour;

[0214] 6) Wash the plate twice with 250 μL of rinse solution;

[0215] 7) Add 100 μL of the cultured phage supernatant in step 1) to the wells coated with the target antigen and allow to bind for 2 h at room temperature;

[0216] 8) Wash the plate 4 times with 250 μL of rinse solution;

[0217] 9) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, and incubate at room temperature for 45 min;

[0218] 10) Wash the plate 4 times with 250 μL of rinse solution;

[0219] 11) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL / well, and incubate at room temperature for 45 min;

[0220] 12) Wash the plate 6 times with 250 μL of rinse solution;

[0221] 13) Add 100 μL TMB colorimetric substrate and allow color to develop for 5 to 10 min;

[0222] 14) Add 100 μL 2M H2SO4 to terminate the reaction and read the results on a microplate reader.

[0223] Brief steps of FACS screening experiment:

[0224] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0225] 2) Wash 293CT-BCMA and 293CT cells twice with PBS and resuspend them in PBS to 1x10 7 / mL concentration, and dispensed into 96-well deep-well plates at 50 μL;

[0226] 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and allow to bind at 4°C for 2 h;

[0227] 4) Wash twice with 200 μL PBS;

[0228] 5) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0229] 6) Wash twice with 200 μL PBS;

[0230] 7) Add 1:300 diluted FITC horse anti mouse-IgG (H+L), 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0231] 8) Wash twice with 200 μL PBS; finally resuspend the cells with 200 μL PBS;

[0232] 9) Detect the fluorescence intensity of the sample FITC channel on a flow cytometer and analyze the results.

[0233] Main materials and reagents:

[0234] Helper phage KO7, Thermo / Invitrogen, 18311019

[0235] Streptavidin,Pierce,21125

[0236] Biotinylated Human BCMA / TNFRSF17 Protein,His,Avitag,ACRObiosystem,BCA-H82E4

[0237] High binding ELSIA plate,Costar,#3590

[0238] Corning 96Well Clear Round Bottom TC-Treated Microplate,Costar,#3799

[0239] Blocking solution: PBS + 3% BSA

[0240] Rinse solution: PBS + 0.1% Tween20

[0241] Soluble one-component TMB substrate solution, Tiangen, PA-107-02

[0242] Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225

[0243] HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306

[0244] Fluorescein(FITC)AffiniPure Goat Anti-Mouse IgG(H+L),JacksonImmunoReseach,115-095-003

[0245] Experimental results:

[0246] Monoclonal clones were randomly selected from the enriched phage antibody pool, packaged into phage, and then the binding of monoclonal phage to BCMA-His-Bio protein and SA protein was detected by phage ELISA to find BCMA-specific phage antibody clones. The ELISA results of some clones are shown in Figure 2. Figure 2As shown in the figure. As can be seen from the figure, clones G1 to G3 and G5 to G8 bind strongly to the target antigen BCMA (BCMA-His-Bio) and do not bind to the control antigen streptavidin, with good specificity. Clones G4, G9 and G10 do not bind to the target antigen BCMA (BCMA-His-Bio) and streptavidin, and are negative clones. Negative phage control is a negative control phage antibody clone that does not bind to either the target antigen or the control antigen. Anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative antibody control with only the first antibody and the second antibody added. Anti-mouse HRP Ab is a negative antibody control with only the second antibody added. They do not bind to either the target antigen or the control antigen. Positive control is a positive antibody control for the target antigen (BCMA-His-Bio), which binds to the target antigen but not to the control antigen.

[0247] The results of the initial FACS screening of antibody clones corresponding to ELISA are as follows: Figure 3 As shown in the figure, G1-G3 and G5-G8 clones bind to 293CT-BCMA cells but not to 293CT cells, and are specific clones; the other clones are negative clones (do not bind to either cell type).

[0248] Through ELISA detection and FACS initial screening, we obtained a total of 25 ELISA and FACS double-positive clones with good specificity. We then sequenced the 25 double-positive clones with good specificity and obtained 5 different monoclonal sequences. These 4 monoclonal clones with different sequences were then further identified by FACS of multiple cell lines and ELISA of multiple antigens to detect the binding specificity of the candidate clones.

[0249] Example 3. Identification of monoclonal specificity by ELISA using multiple unrelated antigens

[0250] Experimental purpose and principle: Antibodies used for treatment must have very good target specificity, only binding to target antigens, and not binding to any unrelated antigens; on the other hand, whether our antibodies can bind to target antigens of different species is related to the design of animal experiments. In order to further analyze the specificity and species cross-reaction of these monoclonal clones and find the best candidate clones, we further evaluated the specificity of the initial screening clones by enzyme-linked immunosorbent assay (ELISA). In this experiment, we used human, mouse and monkey BCMA antigens and a variety of BCMA-unrelated antigens to react with these monoclonal phage antibodies to analyze whether these clones can bind to BCMA antigens of different species and whether there is any non-specific binding with other BCMA-unrelated antigens. Through this experiment, we obtained several clones with excellent specificity.

[0251] Experimental method: Same as ELISA initial screening;

[0252] Main samples and reagents:

[0253]

[0254] The remaining reagents are the same as those for the ELISA primary screening.

[0255] Experimental results:

[0256] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we used enzyme-linked immunosorbent assay (ELISA) to identify multiple clones obtained in Example 2 on multiple antigens. The results are shown in Figure 4In the figure, Negative control is a negative control phage antibody clone, which does not bind to either the target antigen or the control antigen. Positive control (sequence comes from patent ZL201980002363.X) is a positive antibody control for detecting the target antigen BCMA, which binds to the target antigen and has monkey-mouse cross-activity, and does not bind to non-related antigens. Anti-M13phage mouse Ab / anti-mouse HRP Ab are negative antibody controls with only the first antibody and the second antibody added. Anti-mouse HRP Ab is a negative antibody control with only the second antibody added. They do not bind to either the target antigen or the control antigen. Anti-rabbit HRP Ab is a negative control for the second antibody of the positive control antibody, indicating that the binding of the positive control antibody to the target antigen is specific. Anti-his HRP Ab is a positive control antibody for detecting the antigen label, indicating that the coated antigen has been bound to the ELISA plate. Clone11 and 54 only bind to human-BCMA-his-Bio, but not to Cyno-BCMA-his and Mouse-BCMA-his antigens, indicating that they can only bind to human BCMA, have no monkey-mouse species crossover, and have good specificity.

[0257] Example 4. Identification of monoclonal specificity by FACS using multiple cell lines

[0258] Purpose and principle of the experiment: Antibodies used for treatment must have very good target specificity, only bind to the target antigen, and not bind to any unrelated antigens; on the other hand, the amino acid sequence of the same antigen on different cell lines will be different (isomers or mutants) or the bound ligands will be different, and it is also necessary to examine whether our antibodies can bind to cells that are positive for various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, we further evaluated the specificity of the initial screening clones by flow cytometry. In this experiment, we used a variety of BCMA-positive cell lines and a variety of BCMA-negative cell lines to react with these monoclonal phage antibodies to analyze whether these clones can bind to BCMA antigens on different cell lines, and whether there is any non-specific binding with other cell lines that do not express BCMA. Through this experiment, we obtained several clones with excellent specificity.

[0259] Experimental method: Same as FACS initial screening;

[0260] Main samples and reagents:

[0261] 293CT-BCMA cell line, BCMA-positive cell line;

[0262] 293CT cell line, BCMA-negative cell line;

[0263] MM1S cell line, BCMA-positive cell line;

[0264] K562 cell line, BCMA-negative cell line;

[0265] The remaining reagents were the same as those for the FACS primary screening.

[0266] Experimental results:

[0267] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we used enzyme-linked immunosorbent assay and flow cytometry to identify the unique specific clone obtained in Example 2 on more antigens and cell lines. The results are shown in Figure 5 In the figure, NC1 is the negative control phage antibody clone stained cell. NC2 is the second antibody stained cell group used for the positive control antibody stained cell. PC (sequence from ZL201980002363.X patent) is the positive antibody stained cell group. Figure 5 A is the result of staining cells with positive antibodies, indicating that both BCMA-positive cells and negative cells used in this experiment express BCMA target antigen normally. Figure 5 B is the binding result of Clone11 to cells. Clone 11 binds to 293CT-BCMA, a cell line with high BCMA expression, and MM1S, a BCMA-positive cell line, but does not bind to 2 BCMA-negative cell lines, 293CT and K562, showing good specificity. Figure 5 C is the binding result of Clone54 with cells. Clone54 binds to the BCMA high-expressing cell line 293CT-BCMA, binds weakly to MM1S cells, and does not bind to the two BCMA-negative cell lines 293CT and K562, indicating good specificity.

[0268] Example 5. Further determination of the binding specificity of monoclonal clones at the protein level by ELISA and FACS

[0269] Experimental purpose and principle

[0270] Through affinity panning, we enriched specific antibody clones targeting BCMA antigen from the phage antibody library, screened and identified clones that bind to BCMA antigen. However, after the antibody molecules expressed in the prokaryotic system were converted into IgG antibody molecules expressed in the eukaryotic system, their binding ability and specificity needed to be further confirmed. To this end, we prepared IgG expression plasmids of these clones, expressed them by transient transfection of CHOS cells, and purified the antibodies by Protein A. Then, the binding specificity of the monoclonal was further determined by ELISA and FACS.

[0271] The binding specificity of the monoclonal antibody was further confirmed by ELISA at the protein level.

[0272] Main samples and reagents:

[0273] Same as ELISA reagents for phage levels;

[0274] HRP Donkey anti-Human IgG (minimal x-reactivity) Antibody, biolegend, 410902 Experimental results:

[0275] In order to further analyze whether these monoclonal antibodies can still maintain the binding ability and specificity of the original antibodies after being expressed as IgG in the eukaryotic system, we expressed the two clones obtained by CHOS, purified the antibodies into IgG form by Protein A, and identified them by enzyme-linked immunosorbent assay (ELISA) on various antigens. The results are shown in Figure 6 In the figure, Positive control (sequence comes from patent ZL201980002363.X) is the positive antibody control of the target antigen (BCMA), which binds to the target antigen but not to the control antigen. Anti-human IgG HRP Ab is the negative antibody control with only secondary antibody added, anti-rabbit HRP Ab is the secondary antibody negative control of the positive control antibody, indicating that the binding of the positive control antibody to the target antigen is specific, and the secondary antibody used does not bind to the target antigen. Anti-his HRP Ab is the positive antibody control for detecting antigen tags, which binds to the antigen with his tag, indicating that the coated antigen has been bound to the plate. Clone11 and 54 clones only bind to human-BCMA-his-Bio, not Cyno-BCMA-his and Mouse-BCMA-his antigens, indicating that they can only bind to human BCMA, without monkey-mouse species crossover, and have good specificity.

[0276] The binding specificity of the monoclonal antibody was further confirmed by FACS at the protein level.

[0277] Main samples and reagents:

[0278] Fluorescein(FITC)AffiniPure Goat Anti-Human IgG,Fcγfragmentspecific,Jackson ImmunoReseach,109-095-190

[0279] Experimental results:

[0280] In order to further analyze whether these monoclonal antibodies can still maintain the binding ability and specificity of the original antibodies after being expressed as IgG in the eukaryotic system, we expressed the two clones obtained in Example 3 through CHOS, purified the antibodies into IgG form by Protein A, and identified them by enzyme-linked immunosorbent assay and flow cytometry on more antigens and cell lines. The results showed Figure 7 middle, Figure 7 A is the result of Clone54 binding to cells. Figure 7 B is the result of Clone11 binding to cells, indicating that Clone11 and 54, a total of 2 clones, bound to BCMA-positive cell lines 293CT-BCMA and MM1S, but did not bind to BCMA-negative cell line 293CT, with good specificity. NC is the negative control with only the secondary antibody FITC anti-human IgG Ab added.

[0281] Example 6. BCMA single CAR-T CD107a degranulation experiment

[0282] The specific antibody clones 11 and 54 screened from the phage library were constructed on lentiviral vectors with CAR structures (the CAR molecular sequences are shown in SEQ ID NOs: 12 and 14), respectively. The in vitro CD107a degranulation and killing function activity of the corresponding CAR-T cells were studied in vitro by lentiviral packaging and transduction of human T cells.

[0283] Experimental principle and purpose:

[0284] CD107a is a marker of intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, CD107a on the cell membrane will increase. When monesin (purchased from BioLegend) is used to block its recovery, the intensity of microvesicle release can be quantitatively reflected. When CAR-T is stimulated by target antigens on target cells, granzymes will be released, and the activation of T cells can be determined by flow cytometry to detect the increase in CD107a.

[0285] Brief experimental steps of CD107a degranulation:

[0286] 1) The CAR-T cells and target cells to be tested were centrifuged at 300g for 5 min at room temperature, and the supernatant was discarded and resuspended in 1640 medium + 10% FBS to 2x10 6 cells / mL;

[0287] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate and mix well;

[0288] 3) Add 1.5 μL PE / Cy7 anti-human CD107a antibody and 0.2 μL monensin to each well of cells, and then place in a cell culture incubator at 37°C, 5% CO2 for 4 h;

[0289] 4) After incubation, centrifuge at 300g for 5 min at 4°C, discard the supernatant, and wash the cells twice with 200ul PBS;

[0290] 5) Resuspend the cells with 100 μL PBS, add 1.5 μL BV421 anti-human CD8 and 1.5 μL APC anti-human EGFR antibodies respectively, mix well and incubate on ice in the dark for 20 min;

[0291] 6) After incubation, wash the cells three times with 200ul PBS; resuspend in 100μL PBS and detect using flow cytometry.

[0292] Main samples and reagents:

[0293] Target cells: human multiple myeloma cells U266 (BCMA+), RPMI 8226 (BCMA+), MM.1S (BCMA+), H929 (BCMA+), human Burkitt's lymphoma cells Raji-luc (BCMA+), human acute lymphoblastic leukemia cells CCRF (BCMA-), human acute T-lymphoblastic leukemia cells molt4 (BCMA-);

[0294] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0295] Monensin,BioLegend,Cat.No.420701;

[0296] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0297] BV421 mouse anti-human CD8,BD,Cat.No.301036;

[0298] APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No. 35290.

[0299] Experimental results:

[0300] CAR-T cells (B11, B54) were obtained by lentiviral transduction. The CAR-T cells were cultured in vitro for 9-12 days before CD107a degranulation experiment. BCMA CAR-T cell 0037 in the patent submitted by the applicant (CN201980002363.X) was selected as the positive control. The CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibody for 4 hours. The cell density of CAR-T cells and target cells was 2×10 5 cells / mL. The samples were then labeled with CD8 antibodies and EGFR antibodies and flow cytometry was performed. In the Flowjo software, the live cell gate (P1) was selected in the scatter plot to remove cell debris; the cells in the P1 gate were analyzed and the single scattered cell gate (P2) was selected; then, CD8-positive cells (P3) were further selected in the P2 gate; finally, in the P3 gate, the proportion of CD107a-positive cells in cells stained positive for EGFR antibodies (i.e., CAR-positive cells) was analyzed. The analysis results are as follows: Figure 8 As shown in the figure, the degranulation level of BCMA CAR-T increased significantly only after co-incubation with BCMA-positive cells, while there was no significant increase in degranulation level after co-incubation with negative cell lines. Among them, CAR-T cells B11 constructed using clone 11 and CAR-T cells B54 constructed using clone 54 were higher after co-incubation with BCMA-positive cells, and the degranulation level of B54 cells was similar to that of the positive control 0037 cells.

[0301] Example 7. BCMA single CAR-T cell killing experiment in vitro

[0302] Experimental purpose and principle:

[0303] In vitro cell killing experiments used MM1.s, 8226, and H929 as BCMA-positive target cells, and CCRF cells as BCMA-negative target cells to evaluate the antigen-specific killing ability of BCMA CAR-T cells. Among them, the above target cells were transduced by lentivirus to obtain target cells that stably expressed firefly luciferase, so the activity of luciferase in the sample can reflect the number of target cells. CAR-T cells and target cells were co-incubated and cultured. When target cells are killed by CAR-T cells, luciferase will be released and quickly inactivated (the half-life of firefly luciferase is about 0.5h). If the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells proliferate and the luciferase continues to be expressed. Therefore, the activity of luciferase can be used to detect the killing of target cells by CAR-T.

[0304] Brief experimental steps of in vitro cell killing:

[0305] 1) The above target cells were centrifuged at 500g for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1640+10% FBS medium to a concentration of 1x10 5 cells / mL; 100 μL of target cells were added to each well of a 96-well plate;

[0306] 2) According to the CAR positivity rate and effector-target ratio of the CAR-T sample to be tested, 100uL CAR-T cells were added to each well of the 96-well plate and mixed with the target cells; then placed in a carbon dioxide incubator for incubation for 24h;

[0307] 3) Use a luciferase detection kit to detect the luciferase activity in each well of the sample.

[0308] Main samples and reagents:

[0309] BCMA positive target cells MM1.s-luc 8226-luc, H929-luc

[0310] BCMA-negative target cells CCRF-luc;

[0311] Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

[0312] Experimental results:

[0313] CAR-T cell samples and a fixed number of target cells (1x10 4 After mixing at different effector-target ratios (E:T), the samples were incubated for 24 hours, and then the luciferase activity (RLU) in the samples was detected. Since the luciferase activity can reflect the number of target cells in the sample, the killing / inhibition ability of CAR-T cells on target cells can be obtained by the change in luciferase activity in the sample. The lower the luciferase activity reading (RLU), the more target cells are killed.

[0314] As shown in Figure 9, all CAR-T cell samples can effectively kill BCMA-positive target cells. Among them, the killing of B54 cells corresponding to clone 54 was higher than that of positive control 0037 in 8226-luc, H929-luc, and MM1.s-luc. B54 cells, B11 cells, and positive control 0037 had no non-specific killing of negative cells.

[0315] Example 8. Anti-tumor efficacy experiment of BCMA single CAR-T in NCG tumor-bearing mice

[0316] In order to further verify the anti-tumor efficacy of the two candidate clones of BCMA CAR-T in mice, the tumor cell line H929-Luc expressing firefly luciferase (ffLuc) was identified to be able to normally express firefly luciferase, and H929 itself is a natural cell line with high expression of BCMA. H929-luc cells were inoculated into immunodeficient NCG mice through the tail vein. After the mice formed tumors, the candidate clone CAR-T cells were inoculated to verify their anti-tumor effects.

[0317] brief test process :

[0318] Establish a cell line H929-luc that overexpresses firefly luciferase (ffLuc) and identify that it can normally express firefly luciferase;

[0319] After the above cells were cultured to the logarithmic growth phase, the cells were collected;

[0320] 48 NCG mice were purchased and fed for 1 week. H929-luc cells were injected into the tail vein at a rate of 5×10 6 cells / 200 μL PBS, and the general condition of the mice was observed every day after injection;

[0321] On the 10th day, the mice were divided into groups and injected with CAR-T cells (3E+06CAR+ cells / mouse), control non-transfected Mock-T cells (2E+07 cells / mouse) and PBS buffer via the tail vein.

[0322] Regular imaging observations, body weight measurement, observation and record of the general condition of the mice were performed, and the proliferation of T cells in the mouse blood was measured regularly.

[0323] Experimental results:

[0324] The drug efficacy of the mouse experiment is shown in Figure 10. Fig. 10A After the PBS and mockT CAR cells were reinfused, the tumor burden continued to increase. The tumor burden of clones 11 and 54 was significantly lower than that of PBS and mockT, and they could significantly inhibit the tumor. Their tumor inhibition effect was similar to that of the control 0037. The 7-week survival rate of clones 11, 54 and control 0037 mice was 100%, and the clinical safety was expected to be good ( Fig. 10B ), Fig. 10C The changes in the content of CD3 in the blood of the experimental group showed that clones 11, 54 and the control 0037 can ensure effective expansion in vivo, and clone 11 is the best. In summary, the CAR-T cells constructed using clones 11 and 54 have the best in vivo anti-tumor efficacy and good mouse safety, and are better candidate clones for later clinical development.

Claims

1. An antibody targeting BCMA, an antigen-binding fragment thereof, or a derivative thereof, comprising a heavy chain variable region (VH), wherein: The heavy chain variable region (VH) comprises: (a) a heavy chain complementarity determining region 1 (HCDR1), comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6; and (b) a heavy chain complementarity determining region 2 (HCDR2), which comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7; and (c) a heavy chain complementarity determining region 3 (HCDR3), comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:

8.

2. The BCMA-targeting antibody, antigen-binding fragment thereof or derivative thereof according to claim 1, wherein: The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

3. The BCMA-targeting antibody, antigen-binding fragment thereof or derivative thereof according to claim 1, wherein: The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

8.

4. The BCMA-targeting antibody, antigen-binding fragment thereof, or derivative thereof according to any one of claims 1 to 3, wherein: The heavy chain variable region (VH) comprises any amino acid sequence selected from the following sequences: the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 9, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequences.

5. The BCMA-targeting antibody, antigen-binding fragment thereof, or derivative thereof according to any one of claims 1 to 4, wherein: The antibody is a fully human antibody or a humanized antibody.

6. The BCMA-targeting antibody, antigen-binding fragment thereof, or derivative thereof according to any one of claims 1 to 5, wherein: The antibody is a single domain antibody.

7. The BCMA-targeting antibody, antigen-binding fragment thereof or derivative thereof according to any one of claims 1 to 6, further comprising: an Fc fragment.

8. The BCMA-targeting antibody, antigen-binding fragment thereof or derivative thereof according to claim 7, in, The Fc fragment comprises any amino acid sequence selected from the following sequences: the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

9. A recombinant microorganism expressing the antibody, antigen-binding fragment thereof or derivatives thereof according to any one of claims 1 to 8.

10. A recombinant cell expressing the antibody, antigen-binding fragment thereof or derivative thereof according to any one of claims 1 to 8.

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