Anti-MUC16 antibody, MUC16-targeted chimeric antigen receptor and application of MUC16-targeted chimeric antigen receptor

Abstract

The invention relates to the technical field of biology, and discloses an anti-MUC16 antibody, a chimeric antigen receptor targeting MUC16 and application of the chimeric antigen receptor. The anti-MUC16 antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, can be specifically combined with a human MUC16 structural domain, and can be used for treating diseases such as cancers; the MUC16-targeting chimeric antigen receptor comprises an antigen binding structural domain, a stem structural domain, a transmembrane structural domain and an intracellular structural domain, the antigen binding structural domain is specifically bound with MUC16, and an immune effector cell expressing the MUC16-targeting chimeric antigen receptor can promote secretion of IFN-gamma and effectively inhibit MUC16 positive cancer cells.

Description

[0001] Priority declaration

[0002] This application claims priority to a Chinese patent application filed on November 10, 2023 (application number: 202311502030.8, invention name: anti-MUC16 antibodies and their applications) and a Chinese patent application filed on December 26, 2023 (application number: 202311818498.8, invention name: Chimeric antigen receptor targeting MUC16 and its applications). The full texts of these two Chinese patent applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of biotechnology, and in particular to an anti-MUC16 antibody, a chimeric antigen receptor targeting MUC16 and applications thereof. Background Art

[0004] Ovarian cancer is the most deadly type of gynecological malignancy. Most cases are already in the advanced stage when they are discovered, and the prognosis of cancer is poor. Although some progress has been made in the treatment of ovarian cancer, the five-year overall survival rate is still very low for patients with advanced disease. The main treatment method is a combination of surgery and paclitaxel, but the poor prognosis makes the treatment of ovarian cancer still very difficult. In recent years, more and more studies have shown that abnormal expression of MUC16 is associated with the progression of various cancers, especially in ovarian cancer.

[0005] MUC16, a member of the mucin family (MUC), belongs to type I transmembrane mucin and is a highly glycosylated mucin composed of an extracellular domain (CA 125) that is cleaved and released and a retention domain. MUC16 is often expressed in multiple tissues and organs such as the ocular surface (including the cornea and conjunctiva), respiratory tract and female reproductive tract mucosal epithelium. MUC16 is also expressed in normal endometrial tissue, especially in glandular and epithelial cells, and in cervical mucus. MUC16 is a high-molecular-weight, highly glycosylated protein. In normal tissues, it provides sufficient hydrophilicity and lubrication for epidermal cells, and builds a protective barrier for the epidermis to resist foreign particles and infectious factors. In addition, MUC16 participates in multiple signaling pathways to regulate the occurrence of tumors. In ovarian cancer, overexpression of MUC16 can stabilize β-catenin protein and promote its entry into the cell nucleus, thereby activating the Wnt signaling pathway. In breast cancer, the interaction between MUC16 and tyrosine kinase JAK2 induces breast cancer cell proliferation. The interaction between MUC16 and mesothelin mediates tumor cell metastasis to the peritoneum. In addition, MUC16 is often used as a marker for cancer diagnosis and prognosis monitoring. For example, serum MUC16 levels are significantly elevated in ovarian cancer patients, and MUC16 levels drop rapidly in patients who respond to chemotherapy and surgery. If there is a recurrence, MUC16 elevation may precede clinical symptoms. Most of the extracellular domain of MUC16 is cleaved and secreted, which limits the practicality of this part of MUC16 as a target antigen.

[0006] Chimeric antigen receptor T cell immunotherapy, referred to as CAR-T technology, is a method of transforming the patient's T cells in vitro so that the patient's T cells have the ability to recognize tumor cells, and then infusing them back into the patient's body for treatment after expanding the culture in vitro. At present, CAR-T targeting CD19 has achieved great results in the treatment of B-cell blood tumors. If it can be done, it will help to specifically kill ovarian cancer cells and improve the treatment level of ovarian cancer patients.

[0007] Therefore, for diagnostic and therapeutic purposes, it is necessary to generate antibodies against the unshed region of MUC16 or develop CAR-T cells targeting MUC16. Summary of the invention

[0008] One of the purposes of the present invention is to provide an anti-MUC16 antibody and its application.

[0009] The present invention is achieved in that:

[0010] In the first aspect of the present invention, the present invention provides an anti-MUC16 antibody, the antibody comprising: (a) a heavy chain variable region comprising HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof of the heavy chain variable region shown in any one of SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.11; and, (b) a light chain variable region comprising LCDR1 or a variant thereof, LCDR2 or a variant thereof, and LCDR3 or a variant thereof of the light chain variable region shown in any one of SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.12;

[0011] Wherein, the variant has 1, 2 or 3 conservative amino acid substitutions, deletions or additions relative to the parent sequence, and the antibody comprising the variant specifically binds to MUC16;

[0012] In some specific embodiments, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are determined according to the IMGT definition, the Kabat definition, the Chothia definition, the AbM definition or the Contact definition.

[0013] In a second aspect of the present invention, the present invention provides an anti-MUC16 antibody, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the complementarity determining region of the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

[0014] Wherein, the above HCDR1 includes SEQ ID NO.1 (DSEVFPIX 1-8 X 1-9 ) or SEQ ID NO.2 (GYX2-3FTX 2-6 YX 2-8 ) shown in the amino acid sequence, wherein X 1-8 is V or A; X 1-9 is Y or F; X 2-3 is T or A; X 2-6 is S or N; X 2-8 is W or L;

[0015] The above HCDR2 comprises an amino acid sequence as shown in SEQ ID NO.14 (IIPSIGRT) or SEQ ID NO.3 (INPX3-4NGDT), wherein X 3-4 is S or G;

[0016] The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO.15 (ARDSYGTTYGFAY), SEQ ID NO.19 (ARPEGSSYGGFAY), SEQ ID NO.24 (TIWGNYN) or SEQ ID NO.27 (TRAGGYDAMDY);

[0017] The LCDR1 includes SEQ ID NO.4 (QSX 4-3 VHSNGNTY) or SEQ ID NO.28 (SSINY), wherein X 4-3 is L or I;

[0018] The LCDR2 comprises an amino acid sequence as shown by amino acid residues KV or DT;

[0019] The LCDR3 mentioned above includes an amino acid sequence as shown in SEQ ID NO.17 (SQSTHVPLT), SEQ ID NO.21 (FQGSHVPPT) or SEQ ID NO.29 (HQRSSSYPWT).

[0020] In the third aspect of the present invention, the present invention provides an anti-MUC16 antibody or an antigen-binding fragment thereof, which competitively binds to MUC16 with the antibody described in the first or second aspect of the present invention, or the epitope that binds to the MUC16 antigen is the same as the epitope of the antibody described in the first or second aspect of the present invention.

[0021] In a fourth aspect of the present invention, the present invention provides a chimeric antigen receptor (CAR), characterized in that the chimeric antigen receptor comprises an antigen binding domain; and the antigen binding domain contains the anti-MUC16 antibody or antigen binding fragment thereof as described in any one of the aforementioned items.

[0022] In a fifth aspect of the present invention, the present invention provides a multispecific antibody comprising the aforementioned anti-MUC16 antibody or an antigen-binding fragment thereof.

[0023] In the sixth aspect of the present invention, the present invention provides a conjugate comprising (I) and (II) connected:

[0024] (I) the anti-MUC16 antibody or antigen-binding fragment thereof according to the first, second or third aspect, or the multispecific antibody according to the fourth aspect;

[0025] (II) Functional molecules, which include drugs and / or signal substances.

[0026] In the seventh aspect of the present invention, the present invention provides a fusion protein characterized in that it contains at least two domains, one of which contains the anti-MUC16 antibody or antigen-binding fragment thereof as described in the first, second or third aspect above.

[0027] In the eighth aspect of the present invention, the present invention provides an engineered immune cell, which expresses the aforementioned chimeric antigen receptor or contains a nucleic acid encoding the aforementioned chimeric antigen receptor.

[0028] In a ninth aspect of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof, or the aforementioned multispecific antibody, or the aforementioned chimeric antigen receptor.

[0029] In the tenth aspect of the present invention, the present invention provides a vector comprising the aforementioned nucleic acid molecule.

[0030] In the eleventh aspect of the present invention, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule and / or the aforementioned vector.

[0031] In a twelfth aspect of the present invention, the present invention provides a method for stimulating the proliferation and / or survival of engineered immune effector cells, comprising:

[0032] (a) obtaining cells from a subject, wherein the cells obtained from the subject comprise immune effector cell precursor cells;

[0033] (b) transfecting the cells obtained from the subject with the aforementioned vector to provide engineered immune cells expressing the chimeric antigen receptor described in any of the preceding items;

[0034] (c) and optionally, culturing the transfected cells in vitro.

[0035] In a thirteenth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned engineered immune cell, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned recombinant cell;

[0036] In some specific embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0037] In the fourteenth aspect of the present invention, the present invention provides the use of the aforementioned anti-MUC16 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned engineered immune cell, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned recombinant cell in the preparation of a medicament for preventing, treating, or diagnosing cancer.

[0038] In the fifteenth aspect of the present invention, the present invention provides a method for treating, preventing or alleviating cancer, comprising administering to a subject a therapeutically effective amount of the aforementioned pharmaceutical composition; the present invention also provides an anti-MUC16 antibody or antigen-binding fragment as described in any of the aforementioned aspects for use as a medicament;

[0039] In some specific embodiments, the above cancer is a cancer expressing MUC16;

[0040] In some specific embodiments, the cancer is selected from ovarian cancer, breast cancer, cervical cancer, pancreatic cancer, uterine cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, lung cancer, nasopharyngeal cancer, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, liver cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T or B cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor and adenocarcinoma;

[0041] In some specific embodiments, the cancer is selected from ovarian cancer or breast cancer.

[0042] In the sixteenth aspect of the present invention, the present invention provides a method for producing the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof, comprising the following steps:

[0043] (a) culturing the aforementioned recombinant cell under expression conditions for expressing an anti-MUC16 antibody or an antigen-binding fragment thereof;

[0044] (b) isolating and purifying the anti-MUC16 antibody or antigen-binding fragment thereof obtained in step (a).

[0045] In the seventeenth aspect of the present invention, the present invention provides a kit for detecting MUC16, comprising the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof.

[0046] In the eighteenth aspect of the present invention, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned recombinant cell or the aforementioned pharmaceutical composition in the preparation of a kit, wherein the aforementioned kit is used to detect MUC16 or diagnose cancer expressing MUC16.

[0047] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0049] Figure 1 A schematic diagram of the structure of MUC16-ectodomain114 is shown.

[0050] Figure 2 The differences in MUC16-ectodomain114 expression levels in cell lines were evaluated using a cytoflex flow cytometer. A represents K562 cells transfected and stably expressing hMUC16-ectodomain114 at three different expression levels, and B represents SKOV3 cells transfected and stably expressing hMUC16-ectodomain114 at three different expression levels.

[0051] Figure 3 The binding activity of MUC16 monoclonal antibody to K562-MUC16-ectodomain114 cells is shown.

[0052] Figure 4 The binding activity of MUC16 monoclonal antibody to SKOV3-MUC16-ectodomain114 cells is shown.

[0053] Figure 5 The binding activity of MUC16 monoclonal antibody to OVCAR3 cells is shown.

[0054] Figure 6 The results of non-specific binding of MUC16 monoclonal antibody to PBMCs are shown.

[0055] Figure 7 This is a schematic diagram of the structure of the CAR portion encoded by plasmid pCDHF-R2512 in Example 3;

[0056] Figure 8 This is a schematic diagram of the structure of the positive control CAR portion encoded by plasmid pCDHF-R2514 in Example 3;

[0057] Fig. 9The percentage result of the positive rate of 293T cells obtained after the lentivirus infected 293T cells in Example 4;

[0058] Fig.10 The positive rate test results of CAR-T cells prepared in Example 5;

[0059] Fig.11 The results of CAR-T cells killing MUC16 positive cells OVCAR-3 in vitro in Example 6;

[0060] Fig.12 The results of CAR-T cells killing MUC16-negative SKOV-3 cells in vitro in Example 6;

[0061] Fig.13 The secretion of IFN-γ in the culture supernatant after co-culture of CAR-T and MUC16-negative SKOV-3 cells in implementation 6;

[0062] Fig.14 The secretion of IFN-γ in the culture supernatant after co-culture of CAR-T and MUC16-positive OVCAR-3 cells in implementation 6;

[0063] Fig.15 This is the curve of the change of mouse tumor volume over treatment time after injection of CAR-T cells into tumor-bearing mice in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0065] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined in the present disclosure, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Unless otherwise indicated, the methods disclosed herein using commercially available kits and reagents are generally performed according to the protocols and / or parameters defined by the manufacturer.

[0066] The articles "a", "an", "said", "above", "aforementioned" and "above" herein include plural references unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

[0067] In the present invention, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0068] In the first aspect of the present invention, the present invention provides an anti-MUC16 antibody. In some specific embodiments, the above-mentioned antibody comprises: (a) a heavy chain variable region, which comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof of the heavy chain variable region shown in any one of SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.7; and (b) a light chain variable region, which comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, and LCDR3 or a variant thereof of the light chain variable region shown in any one of SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.8;

[0069] Wherein, the variant has 1, 2 or 3 conservative amino acid substitutions, deletions or additions relative to the parent sequence, and the antibody comprising the variant specifically binds to MUC16;

[0070] In some specific embodiments, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are determined according to the IMGT definition, the Kabat definition, the Chothia definition, the AbM definition or the Contact definition.

[0071] MUC16 has a large number of glycosylation sites outside the cell, and is accompanied by cleavage, thereby exposing the sites near the membrane. Current conventional antibodies usually bind to the cleaved and dissociated parts of MUC16, and cannot recognize and bind to the near-membrane domain of MUC16. It is also difficult to screen antibodies with high affinity and specific recognition of the near-membrane domain of MUC16. For cancer patients with different degrees of malignancy, the level of exposure of the near-membrane end of MUC16 varies, and existing antibodies are difficult to exert therapeutic effects.

[0072] The anti-MUC16 antibody or antigen-binding fragment thereof provided by the present invention can bind to MUC16, especially to the proximal membrane domain of MUC16, and can be widely used in treating various tumors.

[0073] In the present invention, the term "MUC16" refers to full-length MUC16, and its amino acid sequence is shown in the exemplary human MUC16 amino acid sequence of GenBankTM Accession No. NP_078966.2 (SEQ ID NO.59). Full-length MUC16 includes a large extracellular domain that is cleaved and released (i.e., CA 125, whose amino acid sequence is shown in SEQ ID NO.60) and a domain retained on the membrane (i.e., MUC CD, also known as "MUC16-ectodomain114", whose amino acid sequence is shown in SEQ ID NO.57). MUC16-ectodomain114 includes an extracellular domain near the cleavage site (i.e., MUC16-ectodomain58, whose amino acid sequence is shown in SEQ ID NO.55), a transmembrane domain (whose amino acid sequence is shown in SEQ ID NO.61) and a cytoplasmic tail (whose amino acid sequence is shown in SEQ ID NO.62). The structural schematic diagram of MUC16-ectodomain114 is shown in Figure 1 As shown;

[0074] Amino acid sequence of MUC16-ectodomain114:

[0075] NFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLP FWAVILIGLAGLLGVITCLICGVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ(SEQ ID NO.57);

[0076] Amino acid sequence of MUC16-ectodomain58:

[0077] NFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLP(SEQ IDNO.55);

[0078] Amino acid sequence of the transmembrane domain:

[0079] FWAVILIGLAGLLGVITCLICGVLV(SEQ ID NO.61);

[0080] Amino acid sequence of the cytoplasmic tail:

[0081] TTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ(SEQ ID NO.62)

[0082] In the present invention, the term "anti-MUC16 antibody", "MUC16 monoclonal antibody", "anti-human MUC16 mouse monoclonal antibody" or "anti-human MUC16 proximal membrane antibody" refers to an antibody that can specifically bind to the extracellular domain of human MUC16 (MUC16-ectodomain58). In particular, antibodies targeting full-length MUC16 (including MUC16-ectodomain58) also fall within the scope of the "anti-MUC16 antibody" of the present invention.

[0083] In the present invention, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The above term "antibody" is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies / polyclonal antibodies, monospecific antibodies / multispecific antibodies, full-length antibodies\antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.

[0084] Typically, a natural complete antibody (i.e., full-length antibody) contains two heavy (H) chains and two light (L) chains. Antibodies can be divided into five major categories or isotypes: IgA, IgD, IgE, IgG, and IgM, depending on whether they contain α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). Each heavy chain consists of a variable region (VH) and the first, second, third, and fourth (optional) constant regions (CH1, CH2, CH3, CH4, respectively). Mammalian light chains can be divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region (CL).

[0085] In some specific embodiments, the variable region is a rodent (e.g., mouse or rat) antibody variable region. In some specific embodiments, the variable region is a human variable region. In some specific embodiments, the variable region comprises a rodent (e.g., mouse or rat) CDR and a human framework region (FR). In some specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some specific embodiments, the variable region comprises a rodent or murine CDR and a primate (e.g., non-human primate) framework region (FR).

[0086] In the present invention, the terms "variable region" or "variable domain" are used interchangeably and are conventional in the art. The variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, and its sequence is different in different antibodies. The heavy chain variable region and the light chain variable region generally contain three hypervariable regions, called "complementarity determining regions (CDRs)", wherein the light chain CDR comprises LCDR1, LCDR2, LCDR3, and the heavy chain CDR comprises HCDR1, HCDR2, HCDR3. The portion of the variable region other than the CDR is called the framework region (FR), and the FR is distributed on both sides of the CDR. Generally, the structure of the CDR and FR from the N-terminus to the C-terminus is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0087] CDR is defined in many ways in the art. In the present invention, the CDR boundaries of the disclosed antibodies or antigen-binding fragments thereof are defined or identified according to the IMGT, Kabat or Chothia definitions. CDRs defined in other ways, such as AbM, Contact, etc., also fall within the scope of protection of the present invention (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); RM MacCallum et al.,. Antibody-antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, ACR Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell Receptor variable domains and Igsuperfamily V-like domains,Developmental and Comparative Immunology 27(2003)55–77).

[0088] In some specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the above anti-MUC16 antibody can be selected from Table 1.

[0089] Table 1 shows the CDR amino acid sequences defined in different ways for exemplary antibodies FC006-10 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.6), FC006-9 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10), FC006-14 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.12) and FC006-2 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8).

[0090] Table 1. CDR amino acid sequences of exemplary antibodies

[0091]

[0092]

[0093] In the present invention, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Common natural amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., α carbon bound to hydrogen, carboxyl, amino and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid analogs typically have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0094] In the present invention, the term "parent sequence" refers to the amino acid sequence contained in the exemplary antibody of the present invention, and "variant" refers to the sequence obtained after substitution, deletion or addition of at least one, two or three amino acids on the basis of the parent sequence. In some specific embodiments, the parent sequence of CDR refers to the CDR amino acid sequence contained in the exemplary antibody of the present invention. For example, in some specific embodiments, the parent sequence refers to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.5; HCDR1 variant, HCDR2 variant, and HCDR3 variant refer to the sequence obtained after substitution, deletion or addition of at least one, two or three amino acids on the basis of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.5. For example, in some specific embodiments, the parent sequence of HCDR1 refers to the amino acid sequence shown in SEQ ID NO.13, and the HCDR1 variant refers to the sequence obtained after substitution, deletion or addition of one, two or three amino acids on the basis of the amino acid sequence shown in SEQ ID NO.13.

[0095] In some specific embodiments, the above-mentioned amino acid substitution refers to conservative amino acid substitution.

[0096] In the present invention, the term "conservative amino acid substitution" refers to the substitution of an amino acid with another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the MUC16 antibody or the MUC16 antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic, such as the substitution of hydrophobic residues isoleucine, valine, leucine or methionine, or the use of polar amino acids such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, etc.

[0097] In the second aspect of the present invention, the present invention provides an anti-MUC16 antibody. In some specific embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the complementarity determining region of the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

[0098] Wherein, the above HCDR1 includes SEQ ID NO.1 (DSEVFPIX 1-8 X 1-9 ) or SEQ ID NO.2(GYX2- 3 FTX 2-6 YX 2-8 ) shown in the amino acid sequence, wherein X 1-8 is V or A; X 1-9 is Y or F; X 2-3 is T or A; X 2-6 is S or N; X 2-8 is W or L;

[0099] The above HCDR2 includes SEQ ID NO.14 (IIPSIGRT) or SEQ ID NO.3 (INPX 3-4 NGDT), the amino acid sequence shown, wherein X 3-4 is S or G;

[0100] The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO.15 (ARDSYGTTYGFAY), SEQ ID NO.19 (ARPEGSSYGGFAY), SEQ ID NO.24 (TIWGNYN) or SEQ ID NO.27 (TRAGGYDAMDY);

[0101] The LCDR1 includes SEQ ID NO.4 (QSX 4-3 VHSNGNTY) or SEQ ID NO.28 (SSINY), wherein X 4-3 is L or I;

[0102] The LCDR2 comprises an amino acid sequence as shown by amino acid residues KV or DT;

[0103] The LCDR3 mentioned above includes an amino acid sequence as shown in SEQ ID NO.17 (SQSTHVPLT), SEQ ID NO.21 (FQGSHVPPT) or SEQ ID NO.29 (HQRSSSYPWT).

[0104] In some specific embodiments, X 1-8 It's V.

[0105] In some specific embodiments, X 1-8 It is A.

[0106] In some specific embodiments, X 1-9 It is Y.

[0107] In some specific embodiments, X 1-9 It's F.

[0108] In some specific embodiments, X 2-3It's T.

[0109] In some specific embodiments, X 2-3 It is A.

[0110] In some specific embodiments, X 2-6 It’s S.

[0111] In some specific embodiments, X 2-6 It is N.

[0112] In some specific embodiments, X 2-8 It's W.

[0113] In some specific embodiments, X 2-8 It's L.

[0114] In some specific embodiments, X 3-4 It’s S.

[0115] In some specific embodiments, X 3-4 It's G.

[0116] In some specific embodiments, X 4-3 It's L.

[0117] In some specific embodiments, X 4-3 It's I.

[0118] In some specific embodiments, the anti-MUC16 antibody is characterized in that the HCDR1 comprises an amino acid sequence as shown in SEQ ID NO.13 or a variant thereof, an amino acid sequence as shown in SEQ ID NO.22 or a variant thereof, an amino acid sequence as shown in SEQ ID NO.25 or a variant thereof, or an amino acid sequence as shown in SEQ ID NO.18 or a variant thereof; and

[0119] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof, the amino acid sequence shown in SEQ ID NO.23 or a variant thereof, or the amino acid sequence shown in SEQ ID NO.26 or a variant thereof; and

[0120] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO.15 or a variant thereof, the amino acid sequence shown in SEQ ID NO.24 or a variant thereof, the amino acid sequence shown in SEQ ID NO.27 or a variant thereof, or the amino acid sequence shown in SEQ ID NO.19 or a variant thereof; and

[0121] The LCDR1 comprises the amino acid sequence shown in SEQ ID NO.16 or a variant thereof, the amino acid sequence shown in SEQ ID NO.20 or a variant thereof, or the amino acid sequence shown in SEQ ID NO.28 or a variant thereof; and

[0122] The LCDR2 comprises an amino acid sequence as shown by amino acid residues KV or DT; and

[0123] The LCDR3 includes the amino acid sequence shown in SEQ ID NO.17 or a variant thereof, the amino acid sequence shown in SEQ ID NO.21 or a variant thereof, or the amino acid sequence shown in SEQ ID NO.29 or a variant thereof.

[0124] In some specific embodiments, the complementarity determining region of the above antibody is selected from any one of the following (a)-(d):

[0125] (a):

[0126] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.13 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.15, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.16 or a variant thereof, the LCDR2 comprises the amino acid sequence shown in amino acid residue KV or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.17 or a variant thereof; or

[0127] (b)

[0128] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.22 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.23 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.24 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.20 or a variant thereof, the LCDR2 comprises the amino acid sequence shown in amino acid residue KV or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.21 or a variant thereof; or

[0129] (c):

[0130] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.25 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.26 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.27 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.28 or a variant thereof, the LCDR2 comprises the amino acid sequence shown in amino acid residue DT or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.29 or a variant thereof; or

[0131] (d):

[0132] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.18 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.19 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.20 or a variant thereof, the LCDR2 comprises the amino acid sequence shown in amino acid residue KV or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.21 or a variant thereof;

[0133] Wherein, the above variants are substituted, deleted or added with 1, 2 or 3 amino acids based on the original CDR sequence, and the antibody comprising the above variants specifically binds to MUC16;

[0134] In some specific embodiments, the above-mentioned amino acid substitution refers to conservative amino acid substitution.

[0135] In some specific embodiments, the above-mentioned antibody contains a heavy chain framework region and / or a light chain framework region, and the above-mentioned heavy chain framework region and / or light chain framework region are derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0136] In some specific embodiments, the heavy chain variable region of the above-mentioned antibody has an amino acid sequence as shown in any one of SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.7, or a sequence having at least 75% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.7; and the light chain variable region of the above-mentioned antibody has an amino acid sequence as shown in any one of SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.8, or a sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.8;

[0137] In some specific embodiments, the above-mentioned antibody comprises a heavy chain variable region and a light chain variable region as shown in any one of the following (1)-(4):

[0138] (1): a heavy chain variable region sequence as shown in SEQ ID NO.5 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.5, and a light chain variable region sequence as shown in SEQ ID NO.6 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.6; or

[0139] (2) a heavy chain variable region sequence as shown in SEQ ID NO.9 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.9, and a light chain variable region sequence as shown in SEQ ID NO.10 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.10; or

[0140] (3) a heavy chain variable region sequence as shown in SEQ ID NO.11 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.11, and a light chain variable region sequence as shown in SEQ ID NO.12 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.12; or

[0141] (4): A heavy chain variable region sequence as shown in SEQ ID NO.7 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.7, and a light chain variable region sequence as shown in SEQ ID NO.8 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.8.

[0142] In the present invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. The comparison of amino acid sequence identity percentages can be performed in various ways in the art, such as software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA, which are well known in the art. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the comparison sequence.

[0143] In some specific embodiments, the heavy chain constant region of the above antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region of the above antibody is κ or λ chain.

[0144] In the present invention, the anti-MUC16 antibody can be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some specific embodiments, the antibodies described herein are IgG antibodies or one of their classes or subclasses. In some specific embodiments, the antibodies described herein are IgG2 antibodies. In some specific embodiments, the antibodies described herein are a mixture of IgG2a and IgG2b antibodies. In some specific embodiments, the antibodies described herein are IgG2a antibodies. In some specific embodiments, the antibodies described herein are IgG2b antibodies. In some specific embodiments, the antibodies described herein are IgG1 antibodies.

[0145] In some specific embodiments, the above-mentioned antibody is a full-length antibody or an antigen-binding fragment thereof, and the above-mentioned antigen-binding fragment is any one selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv, scFv and single-domain antibody.

[0146] The term "antigen-binding fragment" refers to a portion that contains an intact antibody, but unlike an intact antibody, the portion can specifically bind to the antigen bound by the intact antibody. Some examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dsFv, (dsFv)2, single-chain Fab (scFab), single-chain antibodies (e.g., scFv), diabodies, linear antibodies, and single-domain antibodies, as well as multispecific antibodies formed from antigen-binding fragments. Among them, "Fab" is a monovalent fragment composed of VL, VH, CL and CH1 domains; "Fv" is composed of VH and VL; "Fab'" is a Fab fragment containing part of the hinge region; "F(ab')2" is a bivalent fragment containing two Fab' fragments connected by a disulfide bond at the hinge region; "scFab" is a polypeptide composed of VH, CH1, VL, CL and a linker, wherein the antibody domain and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; "scFv" is a polypeptide containing the light chain variable region and A fusion protein of a heavy chain variable region, in which the light chain variable region and the heavy chain variable region are connected by a peptide linker, can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv herein can have VL and VH variable regions in any order, for example, from the N-terminus to the C-terminus of the scFv polypeptide, and can include: a) VL-linker-VH or b) VH-linker-VL; "dsFv" is a disulfide bond-stabilized Fv fragment; (dsFv)2 is a dimerized dsFv; "Fab'-SH" is a cysteine ​​residue in the hinge region of the Fab' fragment carrying a free thiol group; a "single domain antibody" comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0147] In the third aspect of the present invention, the present invention provides an anti-MUC16 antibody or an antigen-binding fragment thereof, which competitively binds to MUC16 with the anti-MUC16 antibody mentioned above in the first or second aspect, or the epitope thereof that binds to the MUC16 antigen is the same as the epitope that the anti-MUC16 antibody mentioned above in the first or second aspect binds to the MUC16 antigen.

[0148] In the present invention, the term "epitope" refers to any antigenic determinant on an antigen that is bound by the paratope of an antibody. Antigenic determinants are typically special chemical groups with a certain composition and structure. An epitope can be linear (i.e., continuous) or conformational (i.e., comprising spaced amino acid residues, non-continuous). An epitope defines the minimum binding site of an antibody and is therefore a specific target of an antibody or its antigen-binding fragment. An epitope can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy).

[0149] In some specific embodiments, the above antibodies have one or more of the following properties (a)-(d):

[0150] (a) The membrane-proximal domain that specifically binds to MUC16;

[0151] (b) binds to K562-MUC16-ectodomain114 cells with an EC50 of no more than 0.6 nM (e.g., no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM, no more than 0.09 nM, no more than 0.08 nM, or no more than 0.05 nM), as measured by a Cytoflex flow cytometer;

[0152] (c) binds to SKOV3-MUC16-ectodomain114 cells with an EC50 of no more than 11 nM (e.g., no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM, no more than 1 nM, no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM or no more than 0.4 nM) as measured by a Cytoflex flow cytometer;

[0153] (d) No nonspecific binding to PBMCs.

[0154] In the present invention, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, for example, a reaction between an antibody and an antigen. In some specific embodiments, it can be determined according to flow cytometry fluorescence sorting technology.

[0155] In the present invention, the term "affinity" or "avidity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen. The strength or affinity of the immune binding interaction can be expressed as "half-maximal effective concentration (EC50)", where EC50 refers to the concentration of a drug or antibody that can achieve 50% of the maximum biological effect after a specific exposure time. Generally, the smaller the EC50, the better the affinity, indicating that it can bind to the target protein at a lower concentration. The EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technology, and other binding assays).

[0156] In the present invention, the term "K562-MUC16-ectodomain114 cells" refers to a human immortalized myeloid leukemia cell line that stably expresses a human MUC16 extracellular 114 amino acid protein (MUC16-ectodomain114, SEQ ID NO. 57) on the cell membrane.

[0157] In the present invention, the term "SKOV3-MUC16-ectodomain114 cells" refers to human ovarian cancer cells that stably express the human MUC16 extracellular 114 amino acid protein (MUC16-ectodomain114, SEQ ID NO: 57) on the cell membrane.

[0158] In a fourth aspect of the present invention, the present invention provides a chimeric antigen receptor (CAR), characterized in that the chimeric antigen receptor comprises an antigen binding domain; and the antigen binding domain contains the anti-MUC16 antibody or antigen binding fragment thereof as described in any one of the aforementioned items.

[0159] In the present invention, the term "chimeric antigen receptor (CAR)" is a recombinant transmembrane molecule mainly composed of an extracellular domain, a transmembrane domain and an intracellular domain. It is an artificially constructed receptor that can recognize specific antigens. After the tumor cell antigen binds to the antigen binding domain in the extracellular domain, the signal is transmitted to the cell through the transmembrane domain and the intracellular domain and converted into an activation signal and activates effector cells, causing immune cells to produce cytokines or perforins that kill tumor cells, and at the same time proliferate to further expand the killing effect.

[0160] [Extracellular domain]

[0161] The extracellular domain of the chimeric antigen receptor provided by the present invention includes an antigen binding domain and a stem domain.

[0162] [Antigen binding domain]

[0163] The antigen binding domain can specifically bind to MUC16, and the antigen binding domain contains at least one set of complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or, at least one set of complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region.

[0164] The antigen binding domain can specifically bind to MUC16, and the antigen binding domain contains the complementary determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or the complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region; the HCDR1, HCDR2 and HCDR3 include amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.5, and the LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.6.

[0165] In an optional embodiment, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable region is defined by any one of the systems of Kabat, Chothia, IMGT, ABM or Contact or a combination of multiple definition systems, as shown in FC006-10 of Table 1.

[0166] In an optional embodiment, the antigen binding domain can specifically bind to MUC16, and the antigen binding domain contains the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region.

[0167] The HCDR1 includes the amino acid sequence shown as SEQ ID NO.13, the HCDR2 includes the amino acid sequence shown as SEQ ID NO.14, the HCDR3 includes the amino acid sequence shown as SEQ ID NO.15, the LCDR1 includes the amino acid sequence shown as SEQ ID NO.16, the LCDR2 includes the amino acid residue KV, and the LCDR3 includes the amino acid sequence shown as SEQ ID NO.17.

[0168] In an optional embodiment, the above-mentioned antigen binding domain contains a heavy chain framework region and / or a light chain framework region, and the above-mentioned heavy chain framework region and / or light chain framework region are derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0169] In an alternative embodiment, the three CDRs of the above antigen binding domain are separated by flanking portions called framework regions (FR, light chain FR comprises LFR1, LFR2, LFR3 and LFR4, heavy chain FR comprises HFR1, HFR2, HFR3 and HFR4).

[0170] In an optional embodiment, 1, 2, 3 or 4 of HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO. 5. The CDR region is determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition or IMGT definition in Table 1, and the FR region of the corresponding definition is obtained according to the structure of the heavy chain variable region.

[0171] In an optional embodiment, 1, 2, 3 or 4 of LFR1, LFR2, LFR3 and LFR4 are selected from the framework region of the light chain variable region whose amino acid sequence is shown in SEQ ID NO. 6. The CDR region is determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition or IMGT definition in Table 1, and the FR region of the corresponding definition is obtained according to the structure of the light chain variable region.

[0172] In an optional embodiment, the antigen binding domain has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.5.

[0173] In an optional embodiment, the antigen binding domain has a light chain variable region having an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.6.

[0174] In an alternative embodiment, the heavy chain variable region and the light chain variable region are connected via a peptide linker.

[0175] In an optional embodiment, the antigen binding domain has the following structure from N-terminus to C-terminus: VH-linker-VL or VL-Linekr-VH; the VH is the heavy chain variable region, the VL is the light chain variable region, and the linker is a peptide linker;

[0176] In an optional embodiment, the peptide linker is a flexible connecting peptide;

[0177] In an optional embodiment, the amino acid sequence of the flexible connecting peptide is (GGGGS)n (SEQ ID NO.79), n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0178] In an optional embodiment, the peptide linker has an amino acid sequence as shown in SEQ ID NO.67 or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.4.

[0179] In an alternative embodiment, the antigen binding domain comprises a single-chain antibody scFV.

[0180] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO.5, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.5.

[0181] In an optional embodiment, the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO.6, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.6.

[0182] In an optional embodiment, the single-chain antibody has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence that has at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.5; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence that has at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.6; and the heavy chain variable region and the light chain variable region are connected by a peptide linker. In an optional embodiment, the peptide linker has an amino acid sequence as shown in SEQ ID NO.67 or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.67.

[0183] In an optional embodiment, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.68 or an amino acid sequence having at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.68.

[0184] [Stem domain]

[0185] The stem domain is the part connecting the antigen binding domain and the transmembrane domain, which usually maintains the stability required for robust chimeric antigen receptor expression and activity in immune effector cells. In an alternative embodiment, the stem domain is derived from the extracellular region of CD8 or CD28 or the hinge of IgG.

[0186] In an alternative embodiment, the stalk domain is derived from the extracellular region of CD8.

[0187] In an optional embodiment, the stem domain comprises an amino acid sequence as shown in SEQ ID NO.69 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.69.

[0188] [Transmembrane domain]

[0189] The transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain, and the transmembrane domain can be any sequence or artificial sequence derived from a natural molecule or a combination thereof that helps insert CAR into the cell membrane. Examples of transmembrane domains include, but are not limited to, α, β or ζ chains, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9 transmembrane domains or any derivatives, variants or fragments thereof, any synthetic sequences with the same function, and any combination thereof.

[0190] Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In an exemplary embodiment, one or both termini of the synthetic transmembrane domain are triplets of phenylalanine, tryptophan and valine.

[0191] In an optional embodiment, the transmembrane domain is derived from CD8, comprising an amino acid sequence as shown in SEQ ID NO.70 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.70.

[0192] [Intracellular domain]

[0193] The intracellular domain includes a signaling domain and, in an optional embodiment, also includes a co-stimulatory signaling domain.

[0194] [Co-stimulatory signaling domain]

[0195] The costimulatory signaling domain contributes to CAR-T cell expansion, function, persistence and anti-tumor activity. The example of the costimulatory signaling domain includes but is not limited to CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand specifically bound to CD83 or a costimulatory molecule of any fragment thereof.

[0196] In an alternative embodiment, the co-stimulatory signaling domain comprises 4-1BB (CD137) and / or CD28.

[0197] In an optional embodiment, the co-stimulatory signaling domain is derived from 4-1BB (CD137), including an amino acid sequence as shown in SEQ ID NO.71 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.7.

[0198] [Signaling domain]

[0199] The signaling domain is responsible for activating at least one effector function of cells expressing CAR. The intracellular signaling domain transduces effector function signals and instructs cells (such as immune effector cells) to perform their specific functions, such as damaging and / or destroying target cells. The example of the intracellular signaling domain includes but is not limited to fragments or domains from one or more molecules or receptors, including but not limited to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP 10, DAP 12. T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1Id, ITGAE, CD103, ITGAL, CD11 a, LFA-l, ITGAM, CD lib, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD 96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and any derivatives, variants or fragments thereof, any synthetic sequence of a signal transduction domain having the same functional capability, and any combination thereof.

[0200] In an optional embodiment, the signaling domain includes CD3ζ, including the amino acid sequence shown in SEQ ID NO.72 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.72.

[0201] In an alternative embodiment, the co-stimulatory signaling domain of the chimeric antigen receptor is derived from 4-1BB (CD137) and the signaling domain is derived from CD3ζ.

[0202] In an optional embodiment, the chimeric antigen receptor comprises:

[0203] (a) An antigen-binding domain comprising an amino acid sequence as shown in SEQ ID NO.68 or a single-chain antibody having an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.68.

[0204] (b) a stem domain comprising the amino acid sequence shown in SEQ ID NO. 69 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 69.

[0205] (c) Transmembrane domain: comprising the amino acid sequence shown in SEQ ID NO.70 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.70.

[0206] and (d) an intracellular domain: comprising a co-stimulatory signaling domain 4-1BB and a signaling domain CD3ζ, 4-1BB comprising an amino acid sequence as shown in SEQ ID NO.71 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.71; CD3ζ comprising an amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.8.

[0207] In an optional embodiment, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO.65 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.65.

[0208] The chimeric antigen receptor of the fourth aspect includes the following beneficial effects:

[0209] In some embodiments, immune effector cells modified with the chimeric antigen receptor of the present invention can effectively target and kill MUC16-positive tumor cells, and compared with T cells modified with chimeric antigen receptors containing existing antibody fragments that can target MUC16, T cells modified with the chimeric antigen receptor provided by the present invention can secrete more IFN-γ when co-cultured with tumor cells. Animal experiments also show that immune effector cells modified with the chimeric antigen receptor provided by the present invention can significantly inhibit tumor growth.

[0210] In a fifth aspect of the present invention, the present invention provides a multispecific antibody comprising the aforementioned anti-MUC16 antibody or an antigen-binding fragment thereof.

[0211] In the sixth aspect of the present invention, the present invention provides a conjugate comprising (I) and (II) connected:

[0212] (I) the anti-MUC16 antibody or antigen-binding fragment thereof according to the first, second or third aspect above,

[0213] or, the multispecific antibody according to the fourth aspect;

[0214] (II) Functional molecules, which include drugs and / or signal substances.

[0215] In the present invention, the term "conjugate" refers to a compound formed by at least two molecules linked to each other.

[0216] In the present invention, the term "signal substance" refers to a substance that can provide a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art; the signal substance can directly provide a signal, such as color (such as colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation or luminescence; it can also indirectly provide a signal through a subsequent reaction in which the signal substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0217] In the seventh aspect of the present invention, the present invention provides a fusion protein characterized in that it contains at least two domains, one of which contains the anti-MUC16 antibody or antigen-binding fragment thereof as described in the first, second or third aspect above.

[0218] The "fusion protein" in the present invention is a recombinant protein with multiple functions expressed by connecting two or more protein gene fragments from different sources through genetic engineering technology; or a recombinant protein with improved function of one of the proteins, such as a fusion protein fused with a tag or a fusion protein that can form a multimeric domain. In the present invention, the term "multispecific antibody" refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.

[0219] In the eighth aspect of the present invention, the present invention provides an engineered immune cell, which expresses the aforementioned chimeric antigen receptor or contains a nucleic acid encoding the aforementioned chimeric antigen receptor.

[0220] In the present invention, the term "engineered immune cells" refers to immune cells expressing CAR or immune cells modified by CAR, wherein the immune cells include but are not limited to T cells, natural killer cells (NK cells), macrophages (M cells), and Treg cells.

[0221] In the ninth aspect of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof or the aforementioned chimeric antigen receptor or the aforementioned fusion protein.

[0222] In the present invention, the term "nucleic acid molecule" is used interchangeably with the term "polynucleotide" in this disclosure to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or connections, and nucleic acids can be synthetic, naturally occurring, and non-naturally occurring, such as non-natural nucleic acids that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to reference nucleotides. Including but not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA) modified nucleic acids.

[0223] In the tenth aspect of the present invention, the present invention provides a vector comprising the aforementioned nucleic acid molecule.

[0224] In the present invention, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the above-mentioned genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells. The vector may be an expression vector or a cloning vector. In some embodiments, the vector provided by the present invention (e.g., expression vector) contains a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof as described in the present invention, at least one promoter (e.g., SV40, CMV, EF 1α) operably linked to the above nucleic acid sequence, and at least one selection marker.

[0225] In the eleventh aspect of the present invention, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule and / or the aforementioned vector.

[0226] In the present invention, the term "recombinant cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced. The recombinant cell contains the vector, which can be introduced into a mammalian cell to construct a recombinant cell, and then the recombinant cell is used to express the antibody or antigen-binding fragment provided by the present invention. The recombinant cell is cultured to obtain the corresponding antibody. The mammalian cell that can be used can be a CHO cell, etc.

[0227] In the twelfth aspect of the present invention, the present invention provides a pharmaceutical composition containing the aforementioned anti-MUC16 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned engineered immune cell, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned recombinant cell.

[0228] In the present invention, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the above composition will be administered.

[0229] In some embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0230] In the present invention, the term "pharmaceutically acceptable carrier" may include any physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are used to prolong the shelf life or efficacy of the antibody.

[0231] In the thirteenth aspect of the present invention, the present invention provides the use of the aforementioned anti-MUC16 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned engineered immune cell, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned recombinant cell in the preparation of a drug for preventing, treating, or diagnosing and treating tumor diseases related to MUC16.

[0232] In the fourteenth aspect of the present invention, the present invention provides a method for treating, preventing or alleviating cancer, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof and / or the aforementioned pharmaceutical composition. The present invention also provides the aforementioned anti-MUC16 antibody or antigen-binding fragment of any of the aforementioned aspects for use as a medicament.

[0233] In the present invention, the term "subject" or "patient" refers to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0234] In the present invention, the term "effective amount" refers to a therapeutic amount, which is sufficient to reduce or improve the severity and / or duration of a disease or one or more symptoms thereof; prevent the progression of the disease; cause the disease to regress; prevent the recurrence, development or progression of one or more symptoms associated with the disease; detect the disease; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a preventive or therapeutic agent). The therapeutically effective dose of the above-mentioned antibody or antigen-binding fragment thereof of the present invention depends on a variety of factors known in the art, such as body weight, age, past medical history, current treatment, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the degree of tumor development. A person skilled in the art (e.g., a doctor or veterinarian) may reduce or increase the dose in proportion to these or other conditions or requirements.

[0235] In some embodiments, the dosage may vary over the course of treatment. For example, in some embodiments, the initial dosage may be higher than the subsequent dosage. In some embodiments, the dosage is adjusted during the course of treatment based on the response of the subject.

[0236] In some embodiments, the immune effector cells modified by CAR can be administered alone or co-administered with a second therapeutic agent of a therapeutically effective amount. For example, co-administered with a second therapeutic agent (e.g., chemotherapeutic agents, anticancer agents, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators or cytokines).

[0237] In some embodiments, when CAR-modified immune effector cells are used in combination with one or more additional therapeutic agents, they can be administered simultaneously with the one or more additional therapeutic agents. In some such embodiments, CAR-modified immune effector cells and the additional therapeutic agents can be administered simultaneously as part of the same pharmaceutical composition. However, CAR-modified immune effector cells "used in combination" with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "combination" in the present invention also includes that CAR-modified immune effector cells administered before or after another therapeutic agent are also considered to be "combined" with the therapeutic agent, that is, CAR-modified immune effector cells and a second substance are administered by different administration methods.

[0238] In some specific embodiments, the above-mentioned cancer is a cancer expressing MUC16.

[0239] In the present invention, the term "MUC16-expressing" cancer refers to a cancer in which MUC16 is expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells, and the level of MUC16 expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells is significantly higher than that in normal cells.

[0240] In some specific embodiments, the above-mentioned cancer is selected from the following group: ovarian cancer, breast cancer, cervical cancer, pancreatic cancer, uterine cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, lung cancer, nasopharyngeal cancer, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, bronchial cancer, bone cancer, hepatobiliary cancer, liver cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T or B cell lymphoma, gastrointestinal stromal tumor and soft tissue tumor.

[0241] In some specific embodiments, the cancer is ovarian cancer or breast cancer.

[0242] In some specific embodiments, the above-mentioned antibodies or antigen-binding fragments of the present invention can be administered at a therapeutically effective dose of about 0.001 mg / kg to about 1000 mg / kg. In some embodiments, the dosage can vary with the course of treatment. For example, in some embodiments, the initial dosage can be higher than the subsequent dosage. In some embodiments, the dosage is adjusted during the course of treatment according to the response of the subject being administered.

[0243] In some specific embodiments, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer agent, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator or a cytokine).

[0244] In some specific embodiments, when the antibodies or antigen-binding fragments thereof disclosed in the present invention are used in combination with one or more additional therapeutic agents, they can be administered simultaneously with the above-mentioned one or more additional therapeutic agents. In some such embodiments, the above-mentioned antibodies or antigen-binding fragments thereof and the above-mentioned additional therapeutic agents can be administered simultaneously as part of the same pharmaceutical composition. However, the antibodies or antigen-binding fragments thereof "used in combination" with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "used in combination" in the present invention also includes that antibodies or antigen-binding fragments thereof administered before or after another therapeutic agent are also considered to be "used in combination" with the therapeutic agent, that is, the above-mentioned antibodies or antigen-binding fragments thereof and the second substance are administered by different administration methods.

[0245] In a fifteenth aspect of the present invention, the present invention provides a method for producing the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof, comprising the following steps:

[0246] (a) culturing the aforementioned recombinant cell under conditions for expressing an anti-MUC16 antibody or an antigen-binding fragment thereof;

[0247] (b) isolating and purifying the anti-MUC16 antibody or antigen-binding fragment thereof obtained in step (a).

[0248] In the sixteenth aspect of the present invention, the present invention provides a kit for detecting MUC16, comprising the aforementioned anti-MUC16 antibody or antigen-binding fragment thereof.

[0249] In the seventeenth aspect of the present invention, the present invention provides the use of the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned recombinant cell in the preparation of a kit for detecting MUC16 or diagnosing cancer expressing MUC16.

[0250] In some specific embodiments, the aforementioned anti-MUC16 antibody or its antigen-binding fragment can specifically target and bind to MUC16. The kit according to the embodiment of the present invention can achieve specific detection of MUC16. For example, when the antibody is bound to a fluorescent group, a fluorescent detection device can be used to achieve the positioning or real-time detection of MUC16. For example, it can be used for immunoblotting, immunoprecipitation, and other kits that involve the use of the specific binding properties of MUC16 antigens and antibodies for detection. These kits may contain any one or more of the following: antagonists, anti-MUC16 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. The above kits can also be used to detect cancers that express MUC16. DETAILED DESCRIPTION

[0251] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0252] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0253] The relevant reagent information in the following examples is shown in Table 2 below:

[0254] Table 2

[0255]

[0256]

[0257] Example 1. Generation of mouse monoclonal antibodies against MUC16

[0258] 1. Immunogen

[0259] The 58 amino acids of the extracellular membrane-proximal domain of human MUC16 (MUC16-ectodomain58, SEQ ID NO.55) were used as an immunogen to produce antibodies against the membrane-proximal end of human MUC16. In the immunization process, the human MUC16-ectodomain58 extracellular domain fusion protein MUC16-ectodomain58-mFc (SEQ ID NO.56) carrying a mouse Fc tag was used as an immunogen.

[0260] Amino acid sequence of MUC16-ectodomain58:

[0261] NFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLP(SEQ IDNO.55)

[0262] MUC16-ectodomain58-mFc amino acid sequence:

[0263] MGWSCIILFLVATATGVHSNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFN STFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO.56)

[0264] 2. Immunization process

[0265] The commonly used mouse hybridoma system was used to produce anti-human MUC16 mouse monoclonal antibody. The process is as follows:

[0266] The human MUC16-ectodomain58 extracellular domain fusion protein MUC16-ectodomain58-mFc carrying a mouse Fc tag was prepared into a solution with a concentration of 1 mg / mL, and then mixed with titermax adjuvant (sigma, Cat.T2684) in equal volumes to obtain an oily emulsion. A dose of 0.1 mL was subcutaneously administered to the back site of 6-week-old female BALB / c mice (Guangdong Medical Experimental Animal Center). Seven days after the first immunization, MUC16-ectodomain58-mFc was mixed with alum adjuvant (Thermo, Cat.77161) in equal volumes for intraperitoneal immunization. After 4-5 injections of immunization, tail blood was collected for titer detection. After the titer reached the established titer, 0.1 mL of the immunogen diluted with physiological saline was used for immunization enhancement, and spleen cell fusion was performed 3 days later. The immunization scheme is shown in Table 3:

[0267] Table 3. Immunization scheme for generating monoclonal antibodies

[0268]

[0269] 3. Preparation of hybridoma cells

[0270] The spleen cells of immunized BALb / c mice were fused with mouse myeloma cells, and then the obtained hybridoma cells were screened for antigen-specific antibodies. The spleen cells of mice were isolated based on the standard protocol, and the immune spleen cells were mixed with mouse myeloma SP2 / 0 cells at a cell number ratio of 1:1, transferred to a 50mL centrifuge tube, and washed once with DMEM basal medium. The supernatant was discarded, and the cells were mixed with 40mL of electrofusion solution (BTX, Cat.47-0001), added to the electrofusion tank, and electrofused according to the established parameters. After fusion, 200mL of DMEM complete medium containing HAT was added and gently suspended, and divided equally into 96-well plates, a total of 40 pieces, 50μL / well, 37°C, 5% CO 2 The cells were cultured in a static incubator. On the sixth day, the DMEM complete medium containing HAT was replaced once.

[0271] 4. Detection of anti-human MUC16 hybridoma supernatant

[0272] Dilute the his-tagged MUC16-ectodomain58-his fusion protein with 0.05M carbonate buffer to a final concentration of 1μg / mL, add 100μL / well to a 96-well ELISA test plate, and coat overnight at 2℃-8℃. Discard the supernatant, add blocking solution (1×PBS+1%BSA) at 200μL / well, and block at 37℃ for 0.5h. On the 7th day after fusion, add cell supernatant at 100μL / well and incubate at 37℃ for 30min. Wash 3 times with 1×PBS. Add HRP-labeled goat anti-mouse IgG (sigma, Cat.A0168-1ML) at 100μL / well, incubate at 37℃ for 30min, wash 3 times with 1×PBS, and then perform color reaction.

[0273] Human MUC16 binding screening was performed using K562-muc114 (overexpressing the proximal membrane domain of human MUC16) or K562 cells (human immortalized myeloid leukemia cell line). 2E5 cells / well were added to a 96-well flow cytometry plate at 100 μL / well, and cell supernatant was added at 100 μL / well and incubated at 4°C for 1 hour. After washing 3 times with 200 μL / well 2% BSA, fluorescently labeled goat anti-mouse IgG antibody was added at 100 μL / well, incubated at 4°C for 1 hour, washed 3 times with 2% BSA, and resuspended with 100 μL / well PBS before detection.

[0274] 5. Screening of subclones

[0275] The fusions with positive binding to human MUC16 were selected and cloned by limiting dilution. The hybridoma clones were detected by the experimental process of step 4 above. The obtained positive clones were cultured in vitro for seed preservation and expression.

[0276] 6. Produce monoclonal antibodies

[0277] 2×10 6 Hybridoma cells were inoculated into a dialysis-based bioreactor at 10 cells / ml, and the antibody-containing supernatant was harvested once a week. Mouse monoclonal antibodies were purified using Protein A (GE-MabSelectSuRe LX, CatFC006-17-5438-03) by FPLC. Antibody concentration was determined by BCA kit or A280 absorbance, antibody purity was determined by SEC (size exclusion chromatography), and purity was checked by SDS (sodium dodecyl sulfate) gel electrophoresis and Coomassie brilliant blue staining.

[0278] 7. Antibody subtype identification

[0279] A total of 15 anti-MUC16 monoclonal antibodies were generated during the entire immunization of mice. After screening, 14 monoclonal antibodies were finally selected and subjected to isotype ELISA tests to determine the isotype of the antibodies. The mouse monoclonal antibody Ig class / subclass identification ELISA kit (IgG1\IgG2a\IgG2b\IgG3\IgM\IgA) (Biolon, Cat#BF06001) was used to determine the Ig subclass of the identified MUC16 reactive monoclonal antibodies. The antibody isotype results are as follows:

[0280] Table 4. Mouse monoclonal antibody subtypes

[0281] Serial number name Subtype 1 FC006-1 mIgG1 2 FC006-2 mIgG1 3 FC006-3 mIgG1 4 FC006-4 mIgG1 5 FC006-5 mIgG1 6 FC006-6 mIgG1 7 FC006-7 mIgG1 8 FC006-8 mIgG2b 9 FC006-9 mIgG1 10 FC006-10 mIgG1 11 FC006-11 mIgG1 12 FC006-12 mIgG1 13 FC006-13 mIgG1 14 FC006-14 mIgG1

[0282] 8. MUC16 monoclonal antibody sequencing

[0283] After screening clones and DNA sequencing analysis, the heavy chain variable region sequences and light chain variable region sequences of the four MUC16 monoclonal antibodies FC006-2, FC006-9, FC006-10 and FC006-14 were obtained, as shown in Table 5:

[0284] Table 5. Mouse monoclonal antibody variable region sequences

[0285]

[0286]

[0287] Example 2. Binding characteristics of monoclonal antibodies against MUC16

[0288] 1. Quality Control of Transfected Cells

[0289] In order to generate cells stably expressing the human MUC16 extracellular 114 amino acid protein (MUC16-ectodomain114, SEQ ID NO.57), K562 cells / SKOV3 cells were transfected with a vector encoding MUC16-ectodomain114, and then stable cell lines were screened by flow cytometry using K562 cells (human immortalized myeloid leukemia cell line) / SKOV3 cells (human ovarian cancer cell line) as negative controls using a control antibody 4H11 targeting human MUC16 protein (anti-MUC16 monoclonal antibody 4H11 in International Patent Application Publication No. WO2011 / 119979, the amino acid sequence of its heavy chain variable region VH is shown in SEQ ID NO.85, and the amino acid sequence of its light chain variable region VL is shown in SEQ ID NO:59) and an antibody targeting mouse IgG (PE-labeled goat anti-mouse IgG).

[0290] Amino acid sequence of MUC16-ectodomain114:

[0291] NFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLP FWAVILIGLAGLLGVITCLICGVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ(SEQ ID NO.57)

[0292] The amino acid sequence of the heavy chain variable region of the control antibody 4H11:

[0293] VKLQESGGGSVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVATIISSAGGYI FYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQ GTTVTVSS(SEQ ID NO.58)

[0294] The amino acid sequence of the light chain variable region of the control antibody 4H11:

[0295] DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSPELLIYWA STRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEVKR(SEQ ID NO.31)

[0296] K562 cells / SKOV3 cells and cells stably expressing human MUC16-ectodomain114 (hMUC16-ectodomain114) were collected separately, centrifuged at 350g for 5 minutes, resuspended in PBS, plated into V-shaped 96-well plates at 2E5 / well, added with control antibody 4H11, and incubated at 4°C for 50 minutes. After centrifugation, the cells were washed once with PBS, and then PE goat anti-mouse IgG was added and incubated at 4°C for 30 minutes. After centrifugation, the cells were washed once with PBS, and then 150μL PBS was added to each well to resuspend the cells. The differences in the expression of MUC16-ectodomain114 in the cell lines were evaluated using a cytoflex flow cytometer (Beckman Countler). Figure 2 -A shows that K562 cells transfected and stably expressing hMUC16-ectodomain114 showed three different rightward shift trends, representing three cell lines with different expression levels. Figure 2-B shows that SKOV3 cells transfected and stably expressing hMUC16-ectodomain114 showed two different rightward shift trends, representing two cell lines with different expression levels. Based on the flow cytometry results, the high-expressing K562-MUC16-ectodomain114 and SKOV3-MUC16-ectodomain114 cell lines were selected for subsequent expansion.

[0297] 2. The binding activity of MUC16 monoclonal antibody to K562-MUC16-ectodomain114 cells.

[0298] K562-MUC16-ectodomain114 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, plated on a V-shaped 96-well plate at 2E5 / well, and MUC16 monoclonal antibody and control antibody 4H11 (R1336) were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL / well of PE-labeled goat anti-mouse IgG Fc (1:500 dilution) was added. After resuspending the cells, the cells were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL of PBS was added to each well. After resuspending the cells, the binding differences of MUC16 antibody to K562-MUC16-ectodomain114 cells were evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 3 As shown, the EC50 of antibodies FC006-5 and FC006-14 binding to K562-MUC16-ectodomain114 positive cells were 0.1975 nM and 0.4067 nM, respectively, which had better affinity than the positive control antibody R1336.

[0299] 3. The binding activity of MUC16 monoclonal antibody to SKOV3-MUC16-ectodomain114 cells.

[0300] SKOV3-MUC16-ectodomain114 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated into V-shaped 96-well plates at 2E5 / well. MUC16 monoclonal antibody and control antibody 4H11 (R1336) were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL / well of PE-labeled goat anti-mouse IgG Fc (1:500 dilution) was added. After resuspending the cells, the cells were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL of PBS was added to each well to resuspend the cells, and the binding differences of MUC16 antibody to K562-MUC16-ectodomain114 cells were evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 4 As shown, antibody FC006-9 has a similar affinity to the positive antibody 4H11 for SKOV3-MUC16-ectodomain114 cells, while antibodies FC006-2, FC006-3, FC006-9, FC006-10, and FC006-14 bind to SKOV3-MUC16-ectodomain114 cells with EC50 of 0.1928 nM, 0.1824 nM, 0.2162 nM, and 0.1242 nM, respectively, having better affinity than the positive antibody.

[0301] 4. Binding activity of MUC16 monoclonal antibody to OVCAR3 (human ovarian cancer cell line) cells.

[0302] OVCAR3 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated in a V-shaped 96-well plate at 2E5 / well. MUC16 monoclonal antibody, proximal epitope control antibody 4H11 (R1336) and distal epitope positive control antibody CA125-25 (Guangdong Feipeng Biological Co., Ltd., catalog number CA125 McAb1#) were added respectively. All of the above antibodies were labeled with FITC and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL of PBS was added to each well to resuspend the cells and the binding difference of MUC16 antibody to OVCAR3 cells was evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 5 As shown, antibody FC006-10 has a better affinity with OVCAR3 cells.

[0303] 5. Detection of non-specific binding of MUC16 monoclonal antibody to PBMC.

[0304] After PBMC (peripheral blood mononuclear cells) were revived, counted, and centrifuged at 300g for 5 minutes. The cell density was adjusted to 5E6 based on the count results, and the cells were plated in a V-shaped 96-well plate at 5E5 / well. MUC16 monoclonal antibody, isotype control, and control antibody 4H11 were added respectively, and one well was reserved as a positive control. The cells were incubated at 4°C for 30 minutes. After centrifugation, 200uL PBS + 3% BSA was added to each well to wash the cells, and then 100μL / well of PE-labeled goat anti-mouse IgG Fc (1:500 dilution) was added. APC goat anti-mouse CD3 was added to the positive control. The cells were resuspended and incubated at 4°C for 30 minutes. After centrifugation, 200μL PBS was added to each well to wash the cells, and then 100μL PBS was added to each well to resuspend the cells and the binding differences of MUC16 antibody to PBMC cells were evaluated using a Cytoflex flow cytometer (BeckmanCountler). Figure 6 As shown, all antibodies showed no non-specific binding to PBMCs.

[0305] Example 3. Construction of MUC16CAR lentiviral vector and its control vector

[0306] The following nucleotide sequence was synthesized by gene, and the nucleotide fragment was constructed into the lentiviral vector according to the restriction site of the lentiviral vector, primers were designed, and the correctness of the vector construction was verified by sequencing results.

[0307] The nucleotide sequence of MUC16 CAR is shown in SEQ ID NO.63 (CAR plasmid code: PCDHF-R2512), encoding the following structure (the amino acid sequence of the polypeptide encoded by SEQ ID NO.63 is shown in SEQ ID NO.64):

[0308] CD8SP-VH-linker-VL-CD8 hinge-CD8 TM-CD137-CD3ζ-SR-P2A-eGFP (such as Figure 7 The underlined portion is the chimeric antigen receptor portion targeting MUC16, and the amino acid sequence of the chimeric antigen receptor portion targeting MUC16 is shown in SEQ ID NO.65:

[0309] QVHLQQSGSELRSPGSAVKLSCKDFDSEVFPIVYMSWVRQKPGHGFEWIGDIIPSIGRTIYGDKFEDKATLDADTVSSTAYLELNSLTSEDSAIYYCARDSYGTTYGFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.65)

[0310] Signal peptide: CD8SP, with the amino acid sequence as follows:

[0311] MALPVTALLLPLALLLHAARP(SEQ ID NO.66)

[0312] Single-chain antibody scFv that specifically binds to MUC16: VH-linker-VL, with the amino acid sequences of VH, linker, and VL as follows:

[0313] VH:

[0314] QVHLQQSGSELRSPGSAVKLSCKDFDSEVFPIVYMSWVRQKPGHGFEWIGDIIPSIGR TIYGDKFEDKATLDADTVSSTAYLELNSLTSEDSAIYYCARDSYGTTYGFAYWGQGTLVT VSA(SEQ ID NO.5)

[0315] linker:

[0316] GGGGSGGGGSGGGGS(SEQ ID NO.67)

[0317] VL:

[0318] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKR(SEQ ID NO.6)

[0319] The amino acid sequence of scFV is shown in SEQ ID NO.68:

[0320] QVHLQQSGSELRSPGSAVKLSCKDFDSEVFPIVYMSWVRQKPGHGFEWIGDIIPSIGRTIYGDKFEDKATLDADTVSSTAYLELNSLTSEDSAIYYCARDSYGTTYGFAYWGQGTLVTVSAGGGGS GGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKR(SEQ ID NO.68)

[0321] Stem domain: CD8 hinge, amino acid sequence is as follows:

[0322] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.69)

[0323] Transmembrane domain: CD8 TM, amino acid sequence is as follows:

[0324] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO.70)

[0325] Costimulatory signal transduction domain: 4-1BB (CD137), the amino acid sequence is as follows:

[0326] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.71)

[0327] Signal transduction domain: CD3ζ, the amino acid sequence is as follows:

[0328] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.72)

[0329] Cleavable linker: P2A, amino acid sequence is as follows:

[0330] ATNFSLLKQAGDVEENPGP(SEQ ID NO.73)

[0331] Marker protein: eGFP, the amino acid sequence is as follows:

[0332] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.74)

[0333] The control MUC16 CAR nucleotide sequence is shown in SEQ ID NO.75 (CAR plasmid code: PCDHF-R2514, positive control antibody 4H11), encoding the following structure (the amino acid sequence of the polypeptide encoded by SEQ ID NO.75 is shown in SEQ ID NO.76):

[0334] CD8SP-VH-linker-VL-CD8 hinge-CD8 TM-CD137-CD3zeta-SR-P2A-eGFP (such as Figure 8 ), the underlined portion is the chimeric antigen receptor portion targeting MUC16, which differs from sequence 1 only in the amino acid sequences of VH and VL.

[0335] The amino acid sequences of VH and VL of the positive control antibody 4H11 are as follows:

[0336] VH of positive control antibody 4H11:

[0337] VKLQESGGGSVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVATIISSAGGYI FYSDSVQGRFTISSRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQ GTTVTVSS(SEQ ID NO.77)

[0338] Positive control antibody 4H11 VL:

[0339] DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSPELLIYWA STRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEVKR(SEQ ID NO.78)

[0340] Example 4. Lentivirus packaging and titer detection

[0341] The pCDHF-R2512 and pCDHF-R2514 plasmids constructed above were packaged into lentivirus according to the lentivirus packaging system shown in Table 6, and the steps are as follows:

[0342] (1) Inoculate 293T cells (cell cryopreservation density is 5×106 cells / mL) in a 10 cm cell culture dish, add 10 mL of DMEM medium containing 10% FBS, and culture in a CO2 incubator at 5% CO2 and 37°C for 24 h.

[0343] (2) Lentivirus packaging was performed according to Table 6; the medium was changed 16 hours after transfection, and DMEM medium containing 10% fetal bovine serum was added according to 10 ml / 100 mm2 culture dish. The virus supernatant was collected 48 hours and 72 hours after transfection, and centrifuged at 3000 rpm and 4°C for 10 minutes to remove cell debris. Then, the virus was concentrated by ultrafiltration using an Amicon Ultra-15 centrifugal filter device at 3000 rpm for 30 minutes. The concentrated virus was packaged in appropriate amounts and stored at -80°C; and the titers of the two concentrated viruses were detected by flow cytometry using human MUC16 C114-His (the amino acid sequence of human MUC16 C114-His is shown in SEQ ID NO.49, and the nucleotide sequence is shown in SEQ ID NO.50) and Alexa Fluor647 anti-His tag. The detection method is as follows:

[0344] The collected gradient volumes of lentiviral stock solution were used to infect 293T cells under the same conditions, and the percentage of 293T cell positivity was detected by flow cytometry 48 hours later (detected by human MUC16 C114-His and Alexa Fluor 647 anti-His tags).

[0345] Calculate the titer of the lentiviral stock solution according to the following formula:

[0346] The titer of lentivirus stock solution (TU / mL) = 1.5*(10×105)*293T cell positive rate percentage / lentivirus stock solution volume μL*1000.

[0347] Table 6 Lentivirus packaging system

[0348]

[0349]

[0350] Table 7 Lentivirus titer

[0351] Plasmids Lentiviral titer PCDHF-R2512 <![CDATA[1.30×10 8 TU / mL]]> PCDHF-R-2514 <![CDATA[1.35×10 8 TU / mL]]>

[0352] The results of 293T cell positive rate percentage are as follows Fig. 9 The results of the lentivirus titer detection are shown in Table 7. The results show that the titers of PCDHF-R2512 and PCDHF-R2514 lentiviruses are 1.30×108TU / mL and 1.35×108TU / mL, respectively.

[0353] Example 5. CAR-T cell preparation

[0354] T cells were isolated from PBMC (ORiCELLs, FPB-007-1, IDY1484) by positive selection using magnetic beads coupled to CD3 / CD28 antibodies (Dynabeads, CD3 / CD28 CTS, catalog number 40203D, batch number A2-011710E). The lentivirus prepared in Example 2 was used to infect T cells at an MOI of 5:1 to prepare CAR-T cells. Control T cells were also set up. After 7 days of CAR-T cell culture, the positive rate of CAR-T cells was detected by using human MUC16114aa-His and Alexa Fluor 647anti-His tag. The positive rates of 2512CAR-T and 2514CAR-T were as follows: Fig.10 As shown, they are 91.90% and 31.48% respectively.

[0355] Example 6. Evaluation of CAR-T cell activity in vitro

[0356] First, 50 μl of RPMI1640 complete medium (RPMI1640 + 20% FBS) was added to each well of the E-Plate 16PET (ACEA, batch number, 20190125) plate to measure the baseline, and then 20,000 ovarian cancer cells (SKOV-3, MUC16 negative ovarian cancer cells; OVCAR-3 cells, MUC16 positive ovarian cancer cells) were added, 100 μL / well, as shown in Table 8 below, and placed on the xCELLigence RTCAS16 instrument, 5% CO2, 37°C for about 25 hours, and mock T cells, 2512CAR-T cells, and 2514CAR-T cells were taken respectively. After counting, 2512CAR-T cells were diluted with T cells to a positive rate of 31.48%, and added to each well at a ratio of effector cells (CAR positive cells): target cells (ie, E / T) = 1:2 and 2:1, respectively. The culture medium for CAR-T cells was X-VIVO 15, 100 μL / well, CAR-T cells and tumor cells were mixed and cultured for about 42 hours, and the ability of 2512CAR-T cells to kill tumor cells in vitro was measured.

[0357] Table 8

[0358] Cell loading 1 Cell loading 2 OVCAR-3 SKOV-3 OVCAR-3+Mock T 0.5 SKOV-3+Mock T 0.5 OVCAR-3+Mock T 2 SKOV-3+Mock T 2 OVCAR-3+2514CAR-T 0.5 SKOV-3+2514CAR-T 0.5 OVCAR-3+2514CAR-T 2 SKOV-3+2514CAR-T 2 OVCAR-3+2512CAR-T 0.5 SKOV-3+2512CAR-T 0.5 OVCAR-3+2512CAR-T 2 SKOV-3+2512CAR-T 2

[0359] The results of the ability of 2512CAR-T cells prepared in Example 5 to kill tumor cells in vitro are as follows Fig.11 and Fig.12 As shown, the results showed that compared with Mock T cells (negative control, ordinary T cells) and 2514CAR-T cells, the ability of 2512CAR-T cells to specifically kill OVCAR-3 tumor cells was significantly higher than that of 2514CAR-T and MockT; 2512CAR-T and 2514CAR-T had no significant killing effect on SKOV-3 cells.

[0360] In addition, the supernatants of Mock T cells, 2514CAR-T cells, and 2512CAR-T cells co-cultured with tumor cells for about 42 hours were tested by ELISA to detect the concentration of secreted IFN-γ. Fig.13 and Fig.14 As shown, the 2512CAR-T cells prepared in Example 3 can normally secrete IFN-γ when co-cultured with tumor cells.

[0361] Example 7. Evaluation of CAR-T cell activity in vivo

[0362] Human ovarian cancer cell line OVCAR-3-C114 (OVCAR-3 cells overexpressing the 58 amino acid sequence of the proximal end of human MUC16, the amino acid sequence and nucleotide sequence of human MUC16 C114 are shown in SEQ ID NO.51 and SEQ ID NO.52) were subcutaneously inoculated in the abdomen of 6-week-old female severely immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Vst / Vst), 3.5×106 cells / (100μL DPBS+100μL DPBS) / mouse. The tumor volume of each mouse was measured on the 30th day after the mice were inoculated with the tumor. Ten mice were evenly divided into two groups according to the tumor volume, with 5 mice in each group. Each group of mice was injected with 1×107 CAR-positive 2512CAR-T and Mock T through the tail vein. The mice after treatment were observed for 22 consecutive days, and the tumor volume of the mice was measured every 3-4 days. The results are shown in Fig.15 As shown, it is shown that 2512CAR-T cells have a significant tumor inhibition effect in the OVCAR-3-C114 tumor-bearing mouse model.

[0363] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-MUC16 antibody, characterized in that The anti-MUC16 antibody comprises: (a) a heavy chain variable region comprising HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof of the heavy chain variable region shown in any one of SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.7; and, (b) a light chain variable region comprising LCDR1 or a variant thereof, LCDR2 or a variant thereof, and LCDR3 or a variant thereof of the light chain variable region shown in any one of SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.8; wherein the variant has 1, 2 or 3 conservative amino acid substitutions, deletions or additions relative to the parent sequence, and an antibody comprising the variant specifically binds to human MUC16; Preferably, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are determined according to the IMGT definition, the Kabat definition, the Chothia definition, the AbM definition or the Contact definition.

2. An anti-MUC16 antibody, characterized in that The antibody comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the complementarity determining region of the light chain variable region comprises LCDR1, LCDR2 and LCDR3; Wherein, the HCDR1 comprises SEQ ID NO.1 (DSEVFPIX 1-8 X 1-9 ) or SEQ ID NO.2(GYX 2-3 FTX 2- 6YX 2-8 ) shown in the amino acid sequence, wherein X 1-8 is V or A; X 1-9 is Y or F; X 2-3 is T or A; X 2-6 is S or N; X 2-8 is W or L; The HCDR2 comprises SEQ ID NO.14 (IIPSIGRT) or SEQ ID NO.3 (INPX 3-4 NGDT) shown in the amino acid sequence, wherein X 3-4 is S or G; The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO.15 (ARDSYGTTYGFAY), SEQ ID NO.19 (ARPEGSSYGGFAY), SEQ ID NO.24 (TIWGNYN) or SEQ ID NO.27 (TRAGGYDAMDY); The LCDR1 comprises SEQ ID NO.4 (QSX 4-3 VHSNGNTY) or the amino acid sequence shown in SEQ ID NO.28 (SSINY), wherein X 4-3 is L or I; The LCDR2 comprises an amino acid sequence as shown by amino acid residues KV or DT; and The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO.17 (SQSTHVPLT), SEQ ID NO.21 (FQGSHVPPT) or SEQ ID NO.29 (HQRSSSYPWT).

3. The anti-MUC16 antibody according to any one of claims 1 to 2, characterized in that The complementary determining region of the antibody is selected from any one of the following (a)-(d): (a)： The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.13 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.15 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.16 or a variant thereof, the LCDR2 comprises the amino acid sequence shown as amino acid residue KV, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.17 or a variant thereof; or (b)： The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.22 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.23 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.24 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.20 or a variant thereof, the LCDR2 comprises the amino acid sequence shown as amino acid residue KV or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.21 or a variant thereof; or (c)： The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.25 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.26 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.27 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.28 or a variant thereof, the LCDR2 comprises the amino acid sequence shown in amino acid residue DT or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.29 or a variant thereof; or (d)： The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.18 or a variant thereof, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.19 or a variant thereof, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.20 or a variant thereof, the LCDR2 comprises the amino acid sequence shown in amino acid residue KV or a variant thereof, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.21 or a variant thereof; The variant is a substitution, deletion or addition of 1, 2 or 3 conservative amino acids based on the parent CDR sequence, and the antibody containing the above variant specifically binds to human MUC16.

4. The anti-MUC16 antibody according to any one of claims 1 to 3, characterized in that The antibody contains a heavy chain framework region and / or a light chain framework region, wherein the heavy chain framework region and / or the light chain framework region are derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof; Preferably, the heavy chain variable region of the antibody has an amino acid sequence as shown in any one of SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.7, or a sequence having at least 75% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.7; and the light chain variable region of the antibody has an amino acid sequence as shown in any one of SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.8, or a sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.8; Preferably, the antibody comprises a heavy chain variable region and a light chain variable region as shown in any one of (1)-(4) below: (1): a heavy chain variable region sequence as shown in SEQ ID NO.5 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.5, and a light chain variable region sequence as shown in SEQ ID NO.6 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.6; or (2) a heavy chain variable region sequence as shown in SEQ ID NO.9 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.9, and a light chain variable region sequence as shown in SEQ ID NO.10 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.10; or (3) a heavy chain variable region sequence as shown in SEQ ID NO.11 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.11, and a light chain variable region sequence as shown in SEQ ID NO.12 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.12; or (4): A heavy chain variable region sequence as shown in SEQ ID NO.7 or a sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.7, and a light chain variable region sequence as shown in SEQ ID NO.8 or a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.

8.

5. The anti-MUC16 antibody according to any one of claims 1 to 4, characterized in that The antibody comprises a constant region, and the constant region is derived from at least one of a mouse antibody, a human antibody, a primate antibody or a mutant thereof; Preferably, the heavy chain constant region of the antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region of the antibody is a κ or λ chain; Preferably, the antibody is a full-length antibody or any antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv, scFv and single-domain antibody; Preferably, the anti-MUC16 antibody competes with the antibody of any one of claims 1 to 4 for binding to MUC16, or has the same epitope as the antibody of any one of claims 1 to 4 for binding to MUC16; Preferably, the antibody has one or more of the following properties (a)-(d): (a) Specifically binds to the membrane-proximal domain of MUC16; (b) binds to K562-MUC16-ectodomain114 cells with an EC50 of no more than 0.6 nM as measured by Cytoflex flow cytometer; (c) binds to SKOV3-MUC16-ectodomain114 cells with an EC50 of no more than 11 nM as determined by Cytoflex flow cytometer; (d) No nonspecific binding to PBMCs.

6. A chimeric antigen receptor (CAR), characterized in that The chimeric antigen receptor comprises an antigen binding domain; and the antigen binding domain comprises the anti-MUC16 antibody or antigen binding fragment thereof according to any one of claims 1 to 5; Preferably, the chimeric antigen receptor comprises: (a) antigen binding domain; (b) stem domain; (c) transmembrane domain; and (d) intracellular domain; The antigen binding domain can specifically bind to MUC16, and the antigen binding domain contains complementary determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region; the HCDR1, HCDR2 and HCDR3 include amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.5, and the LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.6; Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable region are defined by any one of the systems of Kabat, Chothia, IMGT, ABM or Contact or a combination of multiple definition systems. Preferably, the antigen binding domain can specifically bind to MUC16, and the antigen binding domain contains the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region; The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.13; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.14; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.15; The LCDR1 comprises the amino acid sequence shown in SEQ ID NO.16; the LCDR2 comprises the amino acid residue KV; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.17; Preferably, the antigen binding domain has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.5; And / or, the antigen binding domain has a light chain variable region having an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.

6. Preferably, the heavy chain variable region and the light chain variable region are connected via a peptide linker; Preferably, the antigen binding domain has the following structure from N-terminus to C-terminus: VH-linker-VL or VL-Linker-VH; the VH is the heavy chain variable region, the VL is the light chain variable region, and the linker is a peptide linker; Preferably, the peptide linker is a flexible connecting peptide; Preferably, the amino acid sequence of the flexible connecting peptide is (GGGGS)n (SEQ ID NO.79), n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Preferably, the peptide linker has an amino acid sequence as shown in SEQ ID NO.67 or a sequence having at least 75% sequence identity with the amino acid sequence as shown in SEQ ID NO.67; Preferably, the antigen-binding domain comprises a single-chain antibody, the heavy chain variable region of the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence that has at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.5, and the light chain variable region of the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence that has at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.

6.

7. The chimeric antigen receptor according to claim 6, characterized in that The antigen binding domain comprises a single-chain antibody having an amino acid sequence as shown in SEQ ID NO.68 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.68; Preferably, the stem domain is derived from the extracellular region of CD8 or CD28 or the hinge of IgG; Preferably, the stalk domain is derived from the extracellular region of CD8; Preferably, the stem domain comprises the amino acid sequence shown in SEQ ID NO.69 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.69; Preferably, the amino acid sequence of the transmembrane domain is derived from CD8, α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX-40, 4-1BB, CD154, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9; Preferably, the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO.70 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.70; Preferably, the intracellular domain comprises a signaling domain; or the intracellular domain comprises a co-stimulatory signaling domain and a signaling domain; Preferably, the co-stimulatory signaling domain comprises: CD3, CD4, CD8, TCR, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a co-stimulatory molecule of a ligand that specifically binds to CD83 or any fragment thereof; Preferably, the co-stimulatory signaling domain includes 4-1BB and / or CD28; Preferably, the signaling domain comprises CD3ζ; Preferably, 4-1BB comprises the amino acid sequence shown in SEQ ID NO.71 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.71; Preferably, CD3ζ comprises the amino acid sequence shown in SEQ ID NO.72 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.72; Preferably, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.65 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.

65.

8. An engineered immune cell, characterized in that: The immune cell expresses the chimeric antigen receptor according to claim 6 or 7 or contains a nucleic acid encoding the chimeric antigen receptor according to claim 6 or 7.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-MUC16 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the chimeric antigen receptor according to claim 6 or 7; Preferably, the nucleic acid molecule further comprises a signal peptide coding sequence; and / or, further comprises a coding sequence encoding a marker protein; Preferably, the signal peptide is selected from a heavy chain signal peptide, a granulocyte-macrophage colony stimulating factor receptor 2 signal peptide, an IL2 signal peptide, or a CD8 signal peptide; preferably, the signal peptide is CD8SP; Preferably, the marker protein is selected from EGFP, mCherry, EYFP, Puro, Hygro or Neo; Preferably, the marker protein is EGFP.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-MUC16 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the chimeric antigen receptor according to claim 6 or 7, the engineered immune cell according to claim 8, and the nucleic acid molecule according to claim 9; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Images