Anti-MUC16 antibodies, chimeric antigen receptors targeting MUC16 and their applications

Abstract

This invention relates to the field of biotechnology, and discloses an anti-MUC16 antibody, a chimeric antigen receptor targeting MUC16, and their applications. The anti-MUC16 antibody disclosed in this invention includes a heavy chain complementarity-determining region (CDR) and a light chain CDR, which can specifically bind to the human MUC16 domain and can be used to treat diseases such as cancer. The chimeric antigen receptor targeting MUC16 includes an antigen-binding domain, a stem domain, a transmembrane domain, and an intracellular domain. The antigen-binding domain specifically binds to MUC16. Immune effector cells expressing this chimeric antigen receptor targeting MUC16 can promote the secretion of IFN-γ and effectively inhibit MUC16-positive cancer cells.

Description

[0001] Priority Statement

[0002] This application claims priority to two Chinese patent applications filed on November 10, 2023 (application number: 202311502030.8, invention title: Anti-MUC16 antibody and its application) and December 26, 2023 (application number: 202311818498.8, invention title: Chimeric antigen receptor targeting MUC16 and its application), the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to an anti-MUC16 antibody, a chimeric antigen receptor targeting MUC16, and their applications. Background Technology

[0004] Ovarian cancer is one of the most deadly gynecological malignancies, often diagnosed at an advanced stage with a poor prognosis. Despite advancements in ovarian cancer treatment, the five-year overall survival rate remains very low for patients with advanced disease. Current treatment primarily combines surgery with paclitaxel, but the poor prognosis makes ovarian cancer treatment extremely challenging. In recent years, increasing research has shown that aberrantly expressed MUC16 is associated with the progression of various cancers, particularly ovarian cancer.

[0005] MUC16 is a member of the mucin family (MUC), belonging to type I transmembrane mucins. It is a highly glycosylated mucin composed of a cleaved and released extracellular domain (CA 125) and a retained domain. MUC16 is commonly expressed in various tissues and organs, including the ocular surface (cornea and conjunctiva), respiratory tract, and female reproductive tract mucosal epithelium. It is also expressed in normal endometrial tissue, particularly in glandular and epithelial cells, as well as in cervical mucus. MUC16 is a high-molecular-weight, highly glycosylated protein that provides sufficient hydrophilicity and lubrication for epidermal cells in normal tissues, forming a protective barrier against foreign particles and infectious agents. Furthermore, MUC16 participates in multiple signaling pathways that regulate tumorigenesis. In ovarian cancer, overexpression of MUC16 can stabilize β-catenin protein and promote its entry into the nucleus, thereby activating the Wnt signaling pathway. In breast cancer, the interaction between MUC16 and the tyrosine kinase JAK2 induces breast cancer cell proliferation. The interaction between MUC16 and mesothelin mediates tumor cell metastasis to the peritoneum. Furthermore, MUC16 is frequently used as a biomarker for cancer diagnosis and prognostic monitoring. For example, serum MUC16 levels are significantly elevated in ovarian cancer patients, and MUC16 levels decline rapidly in patients who respond to chemotherapy and surgery. In cases of recurrence, MUC16 elevation may precede clinical symptoms. The fact that most of the extracellular domain of MUC16 is cleaved and secreted limits the practical applicability of this portion of MUC16 as a target antigen.

[0006] Chimeric antigen receptor T-cell immunotherapy, or CAR-T therapy for short, is a method that involves modifying a patient's T cells in vitro to enable them to recognize tumor cells. These modified T cells are then cultured and expanded in vitro before being reinfused into the patient for treatment. Currently, CAR-T therapy targeting CD19 has achieved significant success in treating B-cell hematological malignancies. If successful, it could help specifically kill ovarian cancer cells and improve the treatment outcomes for ovarian cancer patients.

[0007] Therefore, for diagnostic and therapeutic purposes, there is a need to generate antibodies against the non-shedded regions of MUC16 or to develop CAR-T cells that target MUC16. Summary of the Invention

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

[0009] This invention is implemented as follows:

[0010] In a first aspect, 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] Among them, the above variants have one, two or three conserved amino acid substitutions, deletions or additions relative to the parental sequence, and the antibodies containing the above variants specifically bind to MUC16;

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

[0013] In a second aspect, 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 regions of the heavy chain variable region include HCDR1, HCDR2 and HCDR3, and the complementarity-determining regions of the light chain variable region include LCDR1, LCDR2 and LCDR3.

[0014] Among them, the aforementioned HCDR1 includes, for example, SEQ ID NO.1 (DSEVFPIX) 1-8 X 1-9 ) or SEQ ID NO.2 (GYX2-3FTX) 2-6 YX 2-8 The amino acid sequence shown is X. 1-8 It is V or A; X 1-9 Is it Y or F; X 2-3 Is it T or A; X 2-6 It is S or N; X 2-8 It is W or L;

[0015] The aforementioned HCDR2 includes the amino acid sequence shown in SEQ ID NO.14 (IIPSIGRT) or SEQ ID NO.3 (INPX3-4NGDT), wherein X 3-4 Is it S or G;

[0016] The aforementioned HCDR3 includes the amino acid sequences 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 aforementioned LCDR1 includes, for example, SEQ ID NO.4 (QSX) 4-3 VHSNGNTY) or SEQ ID NO.28 (SSINY), the amino acid sequence shown, wherein X 4-3 It is L or I;

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

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

[0020] In a third aspect of the 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 binding 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, the present invention provides a chimeric antigen receptor (CAR), characterized in that the chimeric antigen receptor includes an antigen-binding domain; and the antigen-binding domain contains the anti-MUC16 antibody or its antigen-binding fragment as described in any one of the preceding claims.

[0022] In a fifth aspect, the present invention provides a multispecific antibody containing the aforementioned anti-MUC16 antibody or its antigen-binding fragment.

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

[0024] (I) The anti-MUC16 antibody or its antigen-binding fragment described in the first, second or third aspects above, or the multispecific antibody described in the fourth aspect;

[0025] (II) Functional molecules, including drugs and / or signaling molecules.

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

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

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

[0029] In a tenth aspect of the invention, the invention provides a carrier comprising the aforementioned nucleic acid molecules.

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

[0031] In a twelfth aspect of the invention, the 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) Transfect 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, the transfected cells are cultured 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 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 carrier or the aforementioned recombinant cell.

[0036] In some specific embodiments, the above-described pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0037] In a fourteenth aspect, 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 the prevention, treatment or diagnosis of cancer.

[0038] In a fifteenth aspect, 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 preceding aspects as a medicament.

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

[0040] In some specific embodiments, the cancers mentioned above are selected from ovarian cancer, breast cancer, cervical cancer, pancreatic cancer, uterine cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, lung cancer, nasopharyngeal carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, bronchial cancer, bone cancer, hepatobiliary duct cancer, pancreatic cancer, liver cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral 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 cancers mentioned above are selected from ovarian cancer or breast cancer.

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

[0043] (a) The aforementioned recombinant cells were cultured under conditions expressing anti-MUC16 antibody or its antigen-binding fragment;

[0044] (b) Separation and purification of the anti-MUC16 antibody or its antigen-binding fragment obtained in step (a).

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

[0046] In an eighteenth aspect, 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 for detecting MUC16 or diagnosing cancers expressing MUC16.

[0047] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0048] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[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 assessed using Cytoflex flow cytometry. A represents K562 cells transfected with three different expression levels and stably expressing hMUC16-ectodomain114, and B represents SKOV3 cells transfected with three different expression levels and stably expressing hMUC16-ectodomain114.

[0051] Figure 3 The binding activity of the MUC16 monoclonal antibody to K562-MUC16-ectodomain114 cells was demonstrated.

[0052] Figure 4 The binding activity of the MUC16 monoclonal antibody to SKOV3-MUC16-ectodomain114 cells was demonstrated.

[0053] Figure 5 The binding activity of the MUC16 monoclonal antibody to OVCAR3 cells was demonstrated.

[0054] Figure 6 The results show the detection results of nonspecific binding of MUC16 monoclonal antibody to PBMC.

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

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

[0057] Figure 9This is the percentage of 293T cells positive after lentivirus infection of 293T cells in Example 4;

[0058] Figure 10 The results show the positivity rate of CAR-T cells prepared in Example 5;

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

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

[0061] Figure 13 To determine the amount of IFN-γ secreted in the culture supernatant after co-culturing 6 types of CAR-T cells with MUC16 negative SKOV-3 cells;

[0062] Figure 14 To determine the amount of IFN-γ secreted in the culture supernatant after co-culturing 6 types of CAR-T cells with MUC16 positive cells using OVCAR-3;

[0063] Figure 15 The curves showing the change in tumor volume over time in 7 tumor-bearing mice after injection of CAR-T cells. Invention Details

[0065] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise stated, methods using commercially available kits and reagents in this disclosure are generally performed according to manufacturer-defined protocols and / or parameters.

[0066] The articles “a / an,” “an,” “the,” “the,” and “the aforementioned” used in this invention include plural references unless the context clearly indicates otherwise. For example, “an antibody” refers to one or more antibodies.

[0067] In this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0068] In a first aspect, the present invention provides an anti-MUC16 antibody. In some specific embodiments, the 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;

[0069] Among them, the above variants have one, two or three conserved amino acid substitutions, deletions or additions relative to the parental sequence, and the antibodies containing the above variants specifically bind to MUC16;

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

[0071] MUC16 has numerous extracellular glycosylation sites, accompanied by cleavage, thus exposing its juxtamembrane domain. Current conventional antibodies typically bind to the cleaved portion of MUC16 and cannot recognize or bind to the juxtamembrane domain. Furthermore, screening for antibodies with high affinity and specificity to recognize the juxtamembrane domain of MUC16 is challenging. For cancer patients with different degrees of malignancy, the level of juxtamembrane exposure of MUC16 varies, making it difficult for existing antibodies to achieve therapeutic effects.

[0072] The anti-MUC16 antibody or its antigen-binding fragment provided by this invention can bind to MUC16, especially to the juxtamembranous domain of MUC16, and can be widely used to treat various tumors.

[0073] In this invention, the term "MUC16" refers to full-length MUC16, whose amino acid sequence is shown in the exemplary human MUC16 amino acid sequence as indicated in GenBank™ accession number NP_078966.2 (SEQ ID NO. 59). Full-length MUC16 comprises 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 membrane-retained domain (i.e., MUC16 CD, also referred to as "MUC16-ectodomain114", whose amino acid sequence is shown in SEQ ID NO. 57). MUC16-ectodomain114 comprises 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). A schematic diagram of the structure of MUC16-ectodomain114 is shown below. Figure 1 As shown;

[0074] The amino acid sequence of MUC16-ectodomain114:

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

[0076] The 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] The amino acid sequence of the cytoplasmic tail:

[0081] TTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ(SEQ ID NO.62)

[0082] In this invention, the terms "anti-MUC16 antibody," "MUC16 monoclonal antibody," "anti-human MUC16 mouse monoclonal antibody," or "anti-human MUC16 juxtamembrane antibody" refer to antibodies capable of specifically binding to the extracellular domain (MUC16-ectodomain58) of human MUC16. Specifically, antibodies targeting the full-length MUC16 (including MUC16-ectodomain58) also fall within the scope of the term "anti-MUC16 antibody" in this invention.

[0083] In this invention, the term "antibody" includes any immunoglobulin capable of binding to a specific antigen. The term "antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal / polyclonal antibodies, monospecific / 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., a full-length antibody) consists of two heavy (H) chains and two light (L) chains. Based on the presence or absence of α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five main categories or isotypes: IgA, IgD, IgE, IgG, and IgM. Several major antibody categories can also be subclassed, 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), etc. Each heavy chain consists of a variable region (VH) and first, second, third, and fourth (optionally) 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 frame 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 mouse CDR and a primate (e.g., non-human primate) frame region (FR).

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

[0087] CDRs are defined in various ways in the art. In this invention, the CDR boundaries of disclosed antibodies or their antigen-binding fragments are defined or identified according to the definition of IMGT, Kabat, or Chothia. CDRs defined in other ways, such as AbM, Contact, etc., are also within the scope of protection of this invention (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily 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-mentioned anti-MUC16 antibody can be selected from Table 1.

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

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

[0091]

[0092]

[0093] In this invention, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, 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 are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0094] In this invention, the term "parental sequence" refers to the amino acid sequence contained in the exemplary antibody itself, and "variant" refers to a sequence obtained by substituting, deleting, or adding at least one, two, or three amino acids based on the parental sequence. In some specific embodiments, the parental sequence of CDR refers to the CDR amino acid sequence contained in the exemplary antibody itself. For example, in some specific embodiments, the parental 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 sequences obtained by substituting, deleting, or adding at least one, two, or three amino acids based on HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 5. For example, in some specific embodiments, the parental sequence of HCDR1 refers to the amino acid sequence shown in SEQ ID NO. 13, and the HCDR1 variant refers to a sequence obtained by substituting, deleting, or adding one, two, or three amino acids based on the amino acid sequence shown in SEQ ID NO. 13.

[0095] In some specific implementations, the substitution of the aforementioned amino acids refers to the substitution of conserved amino acids.

[0096] In this invention, the term "substitution of a conserved amino acid" refers to the substitution of an amino acid by another amino acid with biologically, chemically, or structurally similar residues. Biological similarity means that the substitution does not impair the biological activity of the MUC16 antibody or the MUC16 antigen. Structural similarity means that the amino acid has a side chain of similar length, such as alanine, glycine, or serine, or a side chain of similar size. Chemical similarity means that the amino acids have the same charge or are all hydrophilic or hydrophobic, for example, the hydrophobic residues isoleucine, valine, leucine, or methionine are substituted for each other, or polar amino acids are used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

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

[0098] Among them, the aforementioned HCDR1 includes, for example, SEQ ID NO.1 (DSEVFPIX) 1-8 X 1-9 ) or SEQ ID NO.2 (GYX2- 3FTX 2-6 YX 2-8 The amino acid sequence shown is X. 1-8 It is V or A; X 1-9 Is it Y or F; X 2-3 Is it T or A; X 2-6 It is S or N; X 2-8 It is W or L;

[0099] The aforementioned HCDR2 includes, for example, SEQ ID NO.14 (IIPSIGRT) or SEQ ID NO.3 (INPX). 3-4 The amino acid sequence shown in NGDT is X. 3-4 Is it S or G;

[0100] The aforementioned HCDR3 includes the amino acid sequences 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 aforementioned LCDR1 includes, for example, SEQ ID NO.4 (QSX) 4-3 VHSNGNTY) or SEQ ID NO.28 (SSINY), the amino acid sequence shown, wherein X 4-3 It is L or I;

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

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

[0104] In some specific implementations, X 1-8 It is V.

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

[0106] In some specific implementations, X 1-9 It's Y.

[0107] In some specific implementations, X 1-9 It is F.

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

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

[0110] In some specific implementations, X 2-6 It is S.

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

[0112] In some specific implementations, X 2-8 It is W.

[0113] In some specific implementations, X 2-8 It is L.

[0114] In some specific implementations, X 3-4 It is S.

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

[0116] In some specific implementations, X 4-3 It is L.

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

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

[0119] The aforementioned HCDR2 includes 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 aforementioned HCDR3 includes 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 aforementioned LCDR1 includes 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 aforementioned LCDR2 includes an amino acid sequence as indicated by amino acid residues KV or DT; and

[0123] The aforementioned 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 antibody is selected from any one of the following (a)-(d):

[0125] (a):

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

[0127] (b):

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

[0129] (c):

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

[0131] (d):

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

[0133] Among them, the above variants are based on the original CDR sequence by substitution, deletion or addition of 1, 2 or 3 amino acids, and the antibodies containing the above variants specifically bind to MUC16;

[0134] In some specific implementations, the substitution of the aforementioned amino acids refers to the substitution of conserved amino acids.

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

[0136] In some specific embodiments, 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 an 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 an 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 antibody includes a heavy chain variable region and a light chain variable region as shown in any one of (1)-(4) below:

[0138] (1): The 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 the 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): Heavy chain variable region sequences as shown in SEQ ID NO.9 or sequences having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.9, and light chain variable region sequences as shown in SEQ ID NO.10 or sequences having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.10; or

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

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

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

[0143] In some specific embodiments, 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.

[0144] In this invention, the anti-MUC16 antibody can be 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) of immunoglobulin molecule. In some specific embodiments, the antibody described herein is an IgG antibody or one of its classes or subclasses. In some specific embodiments, the antibody described herein is an IgG2 antibody. In some specific embodiments, the antibody described herein is a mixture of IgG2a and IgG2b antibodies. In some specific embodiments, the antibody described herein is an IgG2a antibody. In some specific embodiments, the antibody described herein is an IgG2b antibody. In some specific embodiments, the antibody described herein is an IgG1 antibody.

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

[0146] The term "antigen-binding fragment" refers to a portion of a complete antibody that, unlike the complete antibody, specifically binds to the antigen bound by the complete antibody. 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), bispecific antibodies, linear antibodies, and single-domain antibodies, as well as multispecific antibodies formed from antigen-binding fragments. Wherein, “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 a portion of the hinge region; “F(ab’)2” is a divalent fragment containing two Fab’ fragments connected by disulfide bonds in the hinge region; “scFab” is a polypeptide composed of VH, CH1, VL, CL, and a linker, wherein the antibody domains 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 a light chain variable region and Fusion proteins with heavy chain variable regions, where the light chain variable regions and heavy chain variable regions are linked by peptide linkers, can be expressed as single-chain polypeptides, and scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv may have VL and VH variable regions in any order, for example, the N-terminus to C-terminus of the scFv polypeptide may contain: a) VL-linker-VH or b) VH-linker-VL; “dsFv” is a disulfide-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; “single-domain antibody” contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

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

[0148] In this invention, the term "epitaph" refers to any antigenic determinant on an antigen that is bound to the complementary site of an antibody. An antigenic determinant is typically a specific chemical group with a defined composition and structure. Epitopes can be linear (i.e., continuous) or conformational (i.e., consisting of spaced-apart amino acid residues, discontinuous). Epitopes define the minimum binding site of an antibody and are therefore specific targets for antibodies or their antigen-binding fragments. Epitopes 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 assays (e.g., nuclear magnetic resonance spectroscopy).

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

[0150] (a) The lipomembrane domain that specifically binds to MUC16;

[0151] (b) K562-MUC16-ectodomain114 cells were bound with EC50 at a concentration not exceeding 0.6 nM (e.g., not exceeding 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, or 0.05 nM), the EC50 being measured by Cytoflex flow cytometry.

[0152] (c) SKOV3-MUC16-ectodomain114 cells were bound 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), the EC50 being measured by Cytoflex flow cytometry.

[0153] (d) It does not bind nonspecifically to PBMCs.

[0154] In this invention, the term "specific binding" or "specifically bound" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen. In some specific embodiments, it can be determined using flow cytometry fluorescence sorting technology.

[0155] In this invention, the term "affinity" or "compatibility" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen. The strength or affinity of the immunobinding interaction can be expressed as the "half-maximum effective concentration (EC50)," which refers to the concentration of a drug or antibody that, after a specific exposure time, achieves 50% of the maximum biological effect. Generally, a lower EC50 indicates better affinity, meaning that binding to the target protein can occur at a lower concentration. EC50 values ​​can be determined using binding detection methods known in the art, such as direct or indirect binding detection methods (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometry, and other binding detection methods).

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

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

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

[0159] In this invention, the term "chimeric antigen receptor (CAR)" refers to a recombinant transmembrane molecule mainly composed of an extracellular domain, a transmembrane domain, and an intracellular domain. It is an artificially constructed receptor capable of recognizing specific antigens. After tumor cell antigens bind to the antigen-binding domain in the extracellular domain, the signal is transmitted through the transmembrane and intracellular domains to the intracellular space, where it is converted into an activation signal and activates effector cells. This causes immune cells to produce cytokines or perforin that kill tumor cells, and simultaneously proliferate to further amplify the killing effect.

[0160] [Extracellular domain]

[0161] The extracellular domains of the chimeric antigen receptor provided by this invention include 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 complementary determinant regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or at least one set of complementary determinant regions LCDR1, LCDR2 and LCDR3 of the light chain variable region.

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

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

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

[0167] 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, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.17.

[0168] In an optional embodiment, the antigen-binding domain contains a heavy chain framework region and / or a light chain framework region, wherein the 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 optional embodiment, the three CDRs of the antigen-binding domain are separated by flanking portions called frame regions (FR, where the light chain FR includes LFR1, LFR2, LFR3 and LFR4, and the heavy chain FR includes HFR1, HFR2, HFR3 and HFR4).

[0170] In an optional embodiment, one, two, three, or four of HFR1, HFR2, HFR3, and HFR4 are selected from the framework regions of the heavy chain variable region as shown in SEQ ID NO.5. These are CDR regions determined according to the Kabat, Chothia, AbM, Contact, or IMGT definitions in Table 1, and the FR regions of the corresponding definitions are obtained based on the structure of the heavy chain variable region.

[0171] In an optional embodiment, one, two, three, or four of LFR1, LFR2, LFR3, and LFR4 are selected from the framework regions of the light chain variable region as shown in SEQ ID NO.6. These are CDR regions determined according to the Kabat, Chothia, AbM, Contact, or IMGT definitions in Table 1, and the FR regions of the corresponding definitions are obtained based on 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 optional embodiment, the heavy chain variable region and the light chain variable region are connected by a peptide linker.

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

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

[0177] In an optional embodiment, the amino acid sequence of the flexible linker peptide is (GGGGS)n (SEQ ID NO.79), where 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 that has 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 optional implementation, 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 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. 5; and 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., 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. 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 with 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 portion connecting the antigen-binding domain and the transmembrane domain, and it typically maintains the stability required for robust chimeric antigen receptor expression and activity in immune effector cells. Alternatively, the stem domain may be derived from the extracellular region of CD8 or CD28, or the hinge of IgG.

[0186] In an optional implementation, the stem 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 with the amino acid sequence shown in SEQ ID NO. 69.

[0188] [Transmembrane domain]

[0189] The transmembrane domain connects the extracellular domain of the CAR to the intracellular signal transduction domain. The transmembrane domain can be any sequence derived from a natural molecule, an artificial sequence, or a combination thereof that facilitates the insertion of the CAR into the cell membrane. Examples of transmembrane domains include, but are not limited to, transmembrane domains derived from the α, β, or ζ chains of T cell receptors, 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, or any derivatives, variants, or fragments thereof, any synthetic sequence having the same function, and any combination thereof.

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

[0191] In an optional embodiment, the transmembrane domain is derived from CD8 and includes 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] Intracellular domains include signal transduction domains, and in optional embodiments, co-stimulatory signal transduction domains may also be included.

[0194] [Co-stimulatory signal transduction domain]

[0195] Co-stimulatory signaling domains contribute to CAR-T cell expansion, function, persistence, and antitumor activity. Examples of co-stimulatory signaling domains include, but are not limited to, co-stimulatory molecules such as 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, ligands that specifically bind to CD83, or any fragment thereof.

[0196] In an optional implementation, the co-stimulatory signal transduction domain includes 4-1BB (CD137) and / or CD28.

[0197] In an optional embodiment, the co-stimulatory signal transduction domain is derived from 4-1BB (CD137) and includes 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] [Signal Transduction Domain]

[0199] Signal transduction domains are responsible for activating at least one effector function in cells expressing CARs. Intracellular signal transduction domains transduce effector function signals and direct cells (e.g., immune effector cells) to perform their specific functions, such as damaging and / or destroying target cells. Examples of intracellular signal transduction domains include, but are 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, FcγR11a, DAP 10, and 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 signal transduction domains having the same functional capability, and any combination thereof.

[0200] In an optional embodiment, the signal transduction domain includes CD3ζ, comprising an amino acid sequence as 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 optional embodiment, the co-stimulatory signal transduction domain of the chimeric antigen receptor is derived from 4-1BB (CD137), and the signal transduction domain is derived from CD3ζ.

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

[0203] (a) An antigen-binding domain comprising 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.

[0204] (b) A stem domain comprising an amino acid sequence as 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: including 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 shown in SEQ ID NO.70.

[0206] (d) Intracellular domains: including the co-stimulatory signal transduction domain 4-1BB and the signal transduction domain CD3ζ, wherein 4-1BB comprises 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. 71; and CD3ζ comprises 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 with the amino acid sequence 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 fourth aspect of chimeric antigen receptors 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. Compared with T cells modified with chimeric antigen receptors containing existing antibody fragments that target MUC16, T cells modified with the chimeric antigen receptor provided by the present invention, when co-cultured with tumor cells, can secrete a greater amount of IFN-γ. Animal experiments have also shown 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, the present invention provides a multispecific antibody containing the aforementioned anti-MUC16 antibody or its antigen-binding fragment.

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

[0212] (I) The anti-MUC16 antibody or its antigen-binding fragment described in the first, second or third aspects above.

[0213] Or, the multispecific antibody described in the fourth aspect;

[0214] (II) Functional molecules, including drugs and / or signaling molecules.

[0215] In this invention, the term "conjugate" refers to a compound formed by the linkage of at least two molecules together.

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

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

[0218] In this invention, "fusion protein" refers to a recombinant protein with multiple functions, expressed by linking two or more protein gene fragments from different sources together using genetic engineering techniques; or a recombinant protein whose function has been improved, such as by fusing a tag or a fusion protein that can form a multimeric domain. In this invention, the term "multispecific antibody" refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes or multiple (two or more) different antigens of the same antigen.

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

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

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

[0222] In this invention, the term "nucleic acid molecule" is used interchangeably with the term "polynucleotide" in this disclosure, referring to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. Nucleic acids include those containing known nucleotide analogs or modified backbone residues or links, and can be synthetic, naturally occurring, or non-natural, such as non-natural nucleic acids having similar binding properties to a reference nucleic acid and metabolized in a manner similar to the reference nucleotide. This includes, but is not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleotide (PNA) modified nucleic acids.

[0223] In a tenth aspect of the invention, the invention provides a carrier comprising the aforementioned nucleic acid molecules.

[0224] In this invention, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the aforementioned genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. 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), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vector (e.g., expression vector) provided by the present invention contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in the present invention, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF1α), and at least one selection marker.

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

[0226] In this invention, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be introduced or have been introduced. The recombinant cell contains the vector, which can be introduced into mammalian cells to construct recombinant cells. These recombinant cells are then used to express the antibodies or antigen-binding fragments provided by this invention. The corresponding antibodies can be obtained by culturing the recombinant cells. Suitable mammalian cells include CHO cells, etc.

[0227] In a twelfth aspect of the present invention, the present invention provides a pharmaceutical composition comprising 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 carrier, or the aforementioned recombinant cell.

[0228] In this invention, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition is applied.

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

[0230] In this invention, the term "pharmaceutically acceptable carrier" can include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, and delayed absorption agent, etc., used to extend the shelf life or efficacy of antibodies.

[0231] In a thirteenth aspect of the invention, the 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 the prevention, treatment, or diagnosis of MUC16-related tumor diseases.

[0232] In a fourteenth aspect, 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 its antigen-binding fragment and / or the aforementioned pharmaceutical composition. The present invention also provides the aforementioned anti-MUC16 antibody or antigen-binding fragment for use as a medicament in any of the foregoing aspects.

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

[0234] In this invention, the term "effective dose" refers to a therapeutic dose sufficient to reduce or improve the severity and / or duration of a condition or one or more of its symptoms; prevent disease progression; cause disease remission; prevent recurrence, development, or progression of one or more disease-related symptoms; detect disease; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). The therapeutically effective dose of the antibody or its antigen-binding fragment described above depends on a variety of factors known in the art, such as weight, age, medical history, current treatment, the subject's health status and potential for cross-infection, allergies, hypersensitivity, and side effects, as well as the route of administration and the extent of tumor development. Those skilled in the art (e.g., physicians or veterinarians) may proportionally reduce or increase the dose according to these or other conditions or requirements.

[0235] In some embodiments, the dosage may vary as treatment progresses. For example, in some embodiments, the initial dosage may be higher than subsequent dosages. In some embodiments, the dosage is adjusted during treatment based on the subject's response.

[0236] In some implementations, CAR-modified immune effector cells can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, anticancer drug, radiotherapy agent, immunotherapy agent, antiangiogenic agent, targeted therapy agent, cell therapy agent, gene therapy agent, hormone therapy agent, antiviral agent, antibiotic, analgesic, antioxidant, metal chelator, or cytokine).

[0237] In some embodiments, when CAR-modified immune effector cells are used in combination with one or more other therapeutic agents, they may be administered simultaneously with the other therapeutic agents. In some such embodiments, the CAR-modified immune effector cells and the other therapeutic agents may 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 "combined use" in this invention also includes CAR-modified immune effector cells administered before or after another therapeutic agent, which is also considered "combined use" with that therapeutic agent; that is, CAR-modified immune effector cells and the second substance are administered via different routes of administration.

[0238] In some specific implementations, the aforementioned cancer is a cancer expressing MUC16.

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

[0240] In some specific implementations, the aforementioned cancers are selected from the group consisting of: ovarian cancer, breast cancer, cervical cancer, pancreatic cancer, uterine cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, lung cancer, nasopharyngeal carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, bronchial cancer, bone cancer, hepatobiliary duct cancer, liver cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral 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 tumors, and soft tissue tumors.

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

[0242] In some specific embodiments, the antibody or antigen-binding fragment described above in this invention can be administered at a therapeutically effective dose between about 0.001 mg / kg and about 1000 mg / kg. In some embodiments, the dosage may vary with the course of treatment. For example, in some embodiments, the initial dosage may be higher than subsequent dosages. In some embodiments, the dosage is adjusted during the course of treatment based on the subject's response.

[0243] In some specific embodiments, the antibodies or antigen-binding fragments disclosed in this invention can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments disclosed in this invention can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer drug, a radiotherapy agent, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy 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 antibody or antigen-binding fragment disclosed in this invention is used in combination with one or more other therapeutic agents, it may be administered simultaneously with the aforementioned one or more other therapeutic agents. In some such embodiments, the antibody or antigen-binding fragment and the aforementioned other therapeutic agents may be administered simultaneously as part of the same pharmaceutical composition. However, the antibody or antigen-binding fragment "used in combination" with other therapeutic agents does not need to be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "used in combination" in this invention also includes that an antibody or antigen-binding fragment administered before or after another therapeutic agent is also considered to be "used in combination" with that therapeutic agent, i.e., the antibody or antigen-binding fragment and the second substance are administered via different routes of administration.

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

[0246] (a) The aforementioned recombinant cells were cultured under conditions expressing anti-MUC16 antibody or its antigen-binding fragment;

[0247] (b) Separation and purification of the anti-MUC16 antibody or its antigen-binding fragment obtained in step (a).

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

[0249] In a seventeenth aspect, 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 cancers 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 embodiments of the present invention can achieve specific detection of MUC16. For example, when the antibody is bound to a fluorescent group, a fluorescence detection device can be used to locate or detect MUC16 in real time. For instance, it can be used in kits involving the specific binding properties of MUC16 antigen and antibody for detection, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonist, anti-MUC16 antibody or drug reference material; protein purification column; immunoglobulin affinity purification buffer; cell assay diluent; instructions or literature, etc. The above kits can also be used to detect cancers expressing MUC16. Detailed Implementation

[0251] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0252] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0253] The relevant reagent information for 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] A 58-amino acid domain of the human MUC16 extracellular juxtamembrane domain (MUC16-ectodomain58, SEQ ID NO. 55) was used as an immunogen to produce antibodies against the human MUC16 juxtamembrane domain. During immunization, the human MUC16-ectodomain58 extracellular domain fusion protein MUC16-ectodomain58-mFc (SEQ ID NO. 56) carrying a mouse Fc tag was used as the immunogen.

[0260] The 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] Anti-human MUC16 mouse monoclonal antibody was generated using a commonly used mouse hybridoma system. The procedure is as follows:

[0266] The human MUC16-ectodomain58-mFc fusion protein carrying the mouse Fc tag was prepared into a solution with a concentration of 1 mg / mL, and then mixed with an equal volume of titermax adjuvant (Sigma, Cat. T2684) to obtain an oily emulsion. 0.1 mL of this emulsion was subcutaneously administered to the dorsal site of 6-week-old female BALB / c mice (Guangdong Provincial Medical Laboratory Animal Center). Seven days after the first immunization, intraperitoneal immunization was performed using an equal volume mixture of MUC16-ectodomain58-mFc and alum adjuvant (Thermo, Cat. 77161). After 4-5 injections, tail blood was collected for titer testing. Once the titer reached the predetermined level, a booster immunization was performed using 0.1 mL of immunogen diluted with physiological saline. Splenic cell fusion was performed 3 days later. The immunization protocol is shown in Table 3.

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

[0268]

[0269] 3. Preparation of hybridoma cells

[0270] Spleen cells from immunized BALb / c mice were fused with mouse myeloma cells, and the resulting hybridoma cells were then screened for antigen-specific antibodies. Mouse spleen cells were isolated according to a standard protocol. Immunized spleen cells were mixed with mouse myeloma SP2 / 0 cells at a 1:1 cell ratio and transferred to 50 mL centrifuge tubes. The cells were washed once with DMEM basal medium. The supernatant was discarded, and the cells were mixed with 40 mL of electrofusion buffer (BTX, Cat. 47-0001) and added to the electrofusion bath. Electrofusion was performed according to the predetermined parameters. After fusion, the cells were gently resuspended in 200 mL of DMEM complete medium containing HAT and evenly distributed into 40 wells of a 96-well plate (50 μL / well). The plates were incubated statically at 37°C in a 5% CO2 cell culture incubator. On day 6, the DMEM complete medium containing HAT was replaced once.

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

[0272] Dilute the MUC16-ectodomain58-his fusion protein carrying the his tag to a final concentration of 1 μg / mL using 0.05 M carbonate buffer. Add 100 μL / well to a 96-well ELISA plate and incubate overnight at 2℃–8℃. Discard the supernatant and add 200 μL / well of blocking buffer (1×PBS + 1% BSA). Block at 37℃ for 0.5 h. On day 7 post-fusion, add 100 μL / well of cell supernatant and incubate at 37℃ for 30 min. Wash three times with 1×PBS. Add 100 μL / well of HRP-labeled goat anti-mouse IgG (Sigma, Cat.A0168-1 mL), incubate at 37℃ for 30 min, wash three times with 1×PBS, and then perform the colorimetric reaction.

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

[0274] 5. Subcloning screening

[0275] Human MUC16-positive fusion molecules were selected and cloned using a limiting dilution method. Hybridoma cloning was then performed using the experimental procedure described in step 4 above. The resulting positive clones were cultured in vitro for preservation and expression.

[0276] 6. Production of monoclonal antibodies

[0277] With 2×10 6 Hybridoma cells were seeded at a density of 1 cell / ml into a dialysis-based bioreactor, and the supernatant containing antibodies was harvested weekly. Mouse monoclonal antibodies were purified by FPLC using Protein A (GE-MabSelectSuRe LX, CatFC006-17-5438-03). Antibody concentrations were determined by a BCA kit or A280 absorbance, and antibody purity was determined by SEC (size exclusion chromatography). Purity was also checked by SDS (sodium dodecyl sulfate) gel electrophoresis and Coomassie brilliant blue staining.

[0278] 7. Antibody subtype identification

[0279] Fifteen monoclonal antibodies against MUC16 were generated during the entire immunization process in mice. After screening, 14 monoclonal antibodies were selected and subjected to an isotype ELISA assay to determine their isotypes. The Ig subclasses of the identified MUC16-reactive monoclonal antibodies were determined using a mouse monoclonal antibody Ig class / subclass identification ELISA kit (IgG1\IgG2a\IgG2b\IgG3\IgM\IgA) (Bio-Lon, Cat#BF06001). The antibody isotype results are as follows:

[0280] Table 4. Mouse monoclonal antibody subtypes

[0281] 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 cloning and DNA sequencing analysis, the heavy chain variable region sequences and light chain variable region sequences of four MUC16 monoclonal antibodies, FC006-2, FC006-9, FC006-10 and FC006-14, were obtained, as shown in Table 5.

[0284] Table 5. Variable region sequences of mouse monoclonal antibodies

[0285]

[0286]

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

[0288] 1. Quality control of transfected cells

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

[0290] The 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 control antibody 4H11:

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

[0296] K562 / SKOV3 cells and cells stably expressing human MUC16-ectodomain114 (hMUC16-ectodomain114) were collected separately, centrifuged at 350g for 5 minutes, resuspended in PBS, and seeded into 96-well V plates at a ratio of 2E5 cells / well. Control antibody 4H11 was added, and the plates were 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, followed by incubation at 4°C for 30 minutes. After centrifugation, the cells were washed once with PBS, and then 150 μL of PBS was added to each well to resuspend the cells. The differences in MUC16-ectodomain114 expression levels in the cell lines were assessed using a Cytoflex flow cytometry system (Beckman Countler). Figure 2 As can be seen in Figure -A, K562 cells transfected with and stably expressing hMUC16-ectodomain114 exhibited three different right-shift trends, representing three cell lines with different expression levels. Figure 2As shown in Figure -B, SKOV3 cells transfected with and stably expressing hMUC16-ectodomain114 exhibited two different rightward shifts, representing two cell lines with different expression levels. Based on the flow cytometry results, cell lines highly expressing K562-MUC16-ectodomain114 and SKOV3-MUC16-ectodomain114 were selected for subsequent expansion culture.

[0297] 2. 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, and seeded into 96-well V plates at a ratio of 2E5 cells / well. MUC16 monoclonal antibody and control antibody 4H11 (R1336) were added to each well, and 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, followed by 100 μL of PE-labeled goat anti-mouse IgG Fc (1:500 dilution) per well. 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, followed by 100 μL of PBS per well. The cells were resuspended and the binding difference of the MUC16 antibody to K562-MUC16-ectodomain114 cells was assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figure 3 As shown, the EC50 values ​​of antibodies FC006-5 and FC006-14 binding to K562-MUC16-ectodomain114 positive cells were 0.1975 nM and 0.4067 nM, respectively, which showed better affinity than the positive control antibody R1336.

[0299] 3. 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 seeded into 96-well V plates at a ratio of 2E5 cells / well. MUC16 monoclonal antibody and control antibody 4H11 (R1336) were added to each well, and 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, followed by 100 μL of PE-labeled goat anti-mouse IgG Fc (1:500 dilution) per well. 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, followed by 100 μL of PBS per well to resuspend the cells. The binding difference of MUC16 antibody to K562-MUC16-ectodomain114 cells was assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figure 4 As shown, antibody FC006-9 has a similar affinity for SKOV3-MUC16-ectodomain114 cells to the positive antibody 4H11, while antibodies FC006-2, FC006-3, FC006-9, FC006-10, and FC006-14 bind to SKOV3-MUC16-ectodomain114 cells with EC50 values ​​of 0.1928 nM, 0.1824 nM, 0.2162 nM, and 0.1242 nM, respectively, exhibiting superior affinity to 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 seeded into 96-well V plates at a ratio of 2E5 cells / well. MUC16 monoclonal antibody, juxta-membrane epitope control antibody 4H11 (R1336), and distal-membrane epitope positive control antibody CA125-25 (Guangdong Feipeng Biotechnology Co., Ltd., catalog number CA125 McAb1#) were added to each well. All antibodies were FITC-labeled, and 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. The binding difference of MUC16 antibody to OVCAR3 cells was assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figure 5 As shown, antibody FC006-10 exhibits superior affinity for OVCAR3 cells.

[0303] 5. Detection of nonspecific binding of MUC16 monoclonal antibody to PBMC.

[0304] After resuscitating PBMCs (peripheral blood mononuclear cells), they were counted and centrifuged at 300g for 5 min. The cell density was adjusted to 5E6 cells per well, and the cells were seeded into 96-well V plates at a density of 5E5 cells per well. MUC16 monoclonal antibody, isotype control, and control antibody 4H11 were added to each well, with one well reserved as a positive control. Cells were incubated at 4°C for 30 min. After centrifugation, each well was washed with 200 μL PBS + 3% BSA. Then, 100 μL of PE-labeled goat anti-mouse IgG Fc (1:500 dilution) was added to each well. For the positive control, APC goat anti-mouse CD3 was added to the empty well. After resuspending the cells, they were incubated at 4°C for 30 min. After centrifugation, each well was washed with 200 μL PBS. Then, 100 μL PBS was added to each well to resuspend the cells. The binding difference of the MUC16 antibody to PBMCs was assessed using a Cytoflex flow cytometer (Beckman Countler). Figure 6 As shown, none of the antibodies exhibited nonspecific binding to PBMCs.

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

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

[0307] The MUC16 CAR nucleotide sequence 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 As shown in the image, the underlined portion represents the chimeric antigen receptor portion targeting MUC16. 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, the amino acid sequence is as follows:

[0311] MALPVTALLLPLALLLHAARP(SEQ ID NO. 66)

[0312] Single-chain antibody scFv that specifically binds to MUC1: VH-linker-VL, the amino acid sequences of VH, linker and VL are 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 as follows:

[0322] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.69)

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

[0324] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO.70)

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

[0326] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.71)

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

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

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

[0330] ATNFSLLKQAGDVEENPGP(SEQ ID NO.73)

[0331] The marker protein is eGFP, and its 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 As shown in the image, the underlined portion represents the chimeric antigen receptor portion targeting MUC16. Its difference from sequence 1 lies solely 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 FYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQ GTTVTVSS(SEQ ID NO.77)

[0338] VL of positive control antibody 4H11:

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

[0340] Example 4. Lentiviral Packaging and Titer Detection

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

[0342] (1) 293T cells (cell cryopreservation density of 5×106 cells / mL) were seeded in a 10cm cell culture dish, and 10mL of DMEM medium containing 10% FBS was added. The cells were then cultured in a CO2 incubator at 37℃ for 24h with 5% CO2.

[0343] (2) Lentiviral 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 to 10 ml / 100 mm² culture dish. Viral supernatant was collected at 48 and 72 hours after transfection, centrifuged at 3000 rpm and 4°C for 10 minutes to remove cell debris, and then concentrated using an Amicon Ultra-15 centrifuge filter at 3000 rpm for 30 minutes. The concentrated virus was aliquoted and stored at -80°C. 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 methods are as follows:

[0344] The collected lentivirus stock solution was used to infect 293T cells under the same conditions with gradient volumes. After 48 hours, the percentage of 293T cells with positive positivity was detected by flow cytometry (using human MUC16 C114-His and Alexa Fluor 647 anti-His tag).

[0345] Calculate the lentivirus stock solution titer using the following formula:

[0346] Lentiviral stock solution titer (TU / mL) = 1.5 * (10 × 10⁵) * 293 / (Lentivirus stock solution volume μL) * 1000.

[0347] Table 6 Lentiviral Packaging Systems

[0348]

[0349]

[0350] Table 7 Lentiviral titers

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

[0352] The percentage of 293T cell positivity results are as follows: Figure 9 As shown in Table 7, the titers of each lentivirus were 1.30×10⁸ TU / mL and 1.35×10⁸ TU / mL, respectively.

[0353] Example 5. Preparation of CAR-T cells

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

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

[0356] First, 50 μl of RPMI 1640 complete medium (RPMI 1640 + 20% FBS) was added to each well of an E-Plate 16 PET (ACEA, batch number, 20190125) plate to determine the baseline. Then, 20,000 ovarian cancer cells (SKOV-3, MUC16-negative ovarian cancer cells; OVCAR-3 cells, MUC16-positive ovarian cancer cells) were added at 100 μl / well, as shown in Table 8 below. The plate was placed on an xCELLigence RTCAS16 instrument and cultured at 37°C with 5% CO2 for approximately 25 hours. Mock T cells, 2512 CAR-T cells, and 2514 CAR-T cells were collected and counted. After counting, the 2512 CAR-T cells were diluted with T cells to a positivity rate of 31.48%. The effector cells (CAR-positive cells): target cells (E / T) ratios were added to each well at 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 co-cultured for about 42 h, and the ability of 2512 CAR-T cells to kill tumor cells in vitro was measured.

[0357] Table 8

[0358] 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 the 2512 CAR-T cells prepared in Example 5 to kill tumor cells in vitro are as follows: Figure 11 and Figure 12 As shown, the results indicated that, compared with Mock T cells (negative control, ordinary T cells) and 2514CAR-T cells, 2512CAR-T cells had a significantly higher ability to specifically kill OVCAR-3 tumor cells than 2514CAR-T and MockT; 2512CAR-T and 2514CAR-T cells had no significant killing effect on SKOV-3 cells.

[0360] In addition, the supernatant of Mock T cells, 2514 CAR-T cells, and 2512 CAR-T cells co-cultured with tumor cells for approximately 42 hours was analyzed, and the concentration of secreted IFN-γ was detected by ELISA. Results are as follows: Figure 13 and Figure 14 As shown, the 2512CAR-T cells prepared in Example 3 can secrete IFN-γ normally when co-cultured with tumor cells.

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

[0362] Six-week-old female severely immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Vst / Vst) were subcutaneously inoculated with the human ovarian cancer cell line OVCAR-3-C114 (OVCAR-3 cells overexpressing the 58 amino acid sequences of the juxtamembranous end of human MUC16; the amino acid and nucleotide sequences of human MUC16 C114 are shown in SEQ ID NO.51 and SEQ ID NO.52), at a dose of 3.5 × 10⁶ cells / (100 μL DPBS + 100 μL DPBS) / mouse. Tumor volume was measured in each mouse on day 30 post-inoculation. Ten mice were randomly divided into two groups of five each, based on tumor volume. Each group received a tail vein injection of 1 × 10⁷ CAR-positive 2512CAR-T and a Mock T, respectively. Mice were observed for 22 consecutive days after treatment, with tumor volume measured every 3-4 days. Results are as follows: Figure 15 As shown, this demonstrates that 2512 CAR-T cells significantly inhibit tumor growth in the OVCAR-3-C114 tumor-bearing mouse model.

[0363] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-MUC16 antibody or its antigen-binding fragment, characterized in that, The anti-MUC16 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region: The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.5, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.6; or, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.9, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.10; or, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 11, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO. 12; or, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.7, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.8; or, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are determined according to the IMGT definition method, Kabat definition method, Chothia definition method, AbM definition method, or Contact definition method.

2. An anti-MUC16 antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, wherein the complementarity-determining regions of the heavy chain variable region are HCDR1, HCDR2, and HCDR3, and the complementarity-determining regions of the light chain variable region are LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are any one of (A) to (D); (A) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.13 (DSEVFPIVY), SEQ ID NO.14 (IIPSIGRT), SEQ ID NO.15 (ARDSYGTTYGFAY), SEQ ID NO.16 (QSLVHSNGNTY), KV and SEQ ID NO.17 (SQSTHVPLT); (B) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.22 (GYTFTSYW), SEQ ID NO.23 (INPSNGDT), SEQ ID NO.24 (TIWGNYN), SEQ ID NO.20 (QSIVHSNGNTY), KV and SEQ ID NO.21 (FQGSHVPPT); (C) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.25 (GYAFTNYL), SEQ ID NO.26 (INPGNGDT), SEQ ID NO.27 (TRAGGYDAMDY), SEQ ID NO.28 (SSINY), DT and SEQ ID NO.29 (HQRSSSYPWT); (D) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.18 (DSEVFPIAF), SEQ ID NO.14 (IIPSIGRT), SEQ ID NO.19 (ARPEGSSYGGFAY), SEQ ID NO.20 (QSIVHSNGNTY), KV and SEQ ID NO.21 (FQGSHVPPT).

3. The anti-MUC16 antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain framework region and a light chain framework region, wherein the heavy chain framework region and the light chain framework region are derived from at least one of a murine antibody, a primate antibody, or a mutant thereof.

4. The anti-MUC16 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain framework region and a light chain framework region, wherein the heavy chain framework region and the light chain framework region are derived from a human antibody.

5. The anti-MUC16 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region as shown in any one of (1)-(4) below: (1): The 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 the 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): The 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 the 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): The 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 the 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): Heavy chain variable region sequences as shown in SEQ ID NO.7 or sequences having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.7, and light chain variable region sequences as shown in SEQ ID NO.8 or sequences having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.

8.

6. The anti-MUC16 antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The antibody contains a constant region derived from at least one of a murine antibody, a primate antibody, or a mutant thereof.

7. The anti-MUC16 antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The antibody contains a constant region derived from a human antibody.

8. The anti-MUC16 antibody or its antigen-binding fragment according to claim 6, characterized in that, 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.

9. The anti-MUC16 antibody or its antigen-binding fragment according to claim 6, characterized in that, The antigen-binding fragment is selected from any one of F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

10. The anti-MUC16 antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody or its antigen-binding fragment has one or more of the following properties (a)-(d): (a) Specific binding to the proximal membrane domain of MUC16; (b) K562-MUC16-ectodomain114 cells were bound with EC50 at a concentration not exceeding 0.6 nM, the EC50 being measured by Cytoflex flow cytometry; (c) SKOV3-MUC16-ectodomain114 cells were bound with EC50 of no more than 11 nM, the EC50 being measured by Cytoflex flow cytometry. (d) It does not bind nonspecifically to PBMCs.

11. A chimeric antigen receptor (CAR), characterized in that, The chimeric antigen receptor includes an antigen-binding domain; and the antigen-binding domain contains the anti-MUC16 antibody or its antigen-binding fragment as described in any one of claims 1-7.

12. The chimeric antigen receptor (CAR) according to claim 11, characterized in that, The chimeric antigen receptor comprises: (a) Antigen-binding domain; (b) Stem domain; (c) Transmembrane domain; and (d) intracellular domains; The antigen-binding domain specifically binds to MUC16. The antigen-binding domain contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region. HCDR1, HCDR2, and HCDR3 have amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 5, and LCDR1, LCDR2, and LCDR3 have amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.

6. The variable regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, ABM, or Contact.

13. The chimeric antigen receptor according to claim 11, characterized in that, The antigen-binding domain can specifically bind to MUC16, and the antigen-binding domain contains complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and complementarity-determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region. The HCDR1 is the amino acid sequence shown in SEQ ID NO.13; the HCDR2 is the amino acid sequence shown in SEQ ID NO.14; and the HCDR3 is the amino acid sequence shown in SEQ ID NO.

15. The LCDR1 has the amino acid sequence shown in SEQ ID NO.16; the amino acid sequence of the LCDR2 is KV; and the LCDR3 has the amino acid sequence shown in SEQ ID NO.

17.

14. The chimeric antigen receptor according to claim 13, characterized in that, 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 shown in SEQ ID NO.5; And the antigen-binding domain 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 shown in SEQ ID NO. 6, and a light chain variable region.

15. The chimeric antigen receptor according to claim 14, characterized in that, The heavy chain variable region and the light chain variable region are connected by peptide linkers.

16. The chimeric antigen receptor according to claim 15, characterized in that, The antigen-binding domain has the following structure from the N-terminus to the C-terminus: VH-linker-VL or VL-Linker-VH; where VH is the heavy chain variable region, VL is the light chain variable region, and the linker is a peptide linker.

17. The chimeric antigen receptor according to claim 15, characterized in that, The peptide linker is a flexible linker peptide.

18. The chimeric antigen receptor according to claim 17, characterized in that, The amino acid sequence of the flexible linker peptide is (GGGGS)n, where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

19. The chimeric antigen receptor according to claim 15, characterized in that, 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 shown in SEQ ID NO.

67.

20. The chimeric antigen receptor according to claim 11, characterized in that, The antigen-binding domain comprises a single-chain antibody, wherein 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 having at least 70% sequence identity with the amino acid sequence 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 having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.

6.

21. The chimeric antigen receptor according to claim 11, characterized in that, The antigen-binding domain comprises a single-chain antibody with 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.

22. The chimeric antigen receptor according to claim 12, characterized in that, The stem domain is derived from the extracellular region of CD8 or CD28 or the hinge of IgG.

23. The chimeric antigen receptor according to claim 12, characterized in that, The stem domain is derived from the extracellular region of CD8.

24. The chimeric antigen receptor according to claim 12, characterized in that, The stem domain comprises an amino acid sequence as 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.

25. The chimeric antigen receptor according to claim 12, characterized in that, The amino acid sequence of the transmembrane domain is derived from CD8, 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.

26. The chimeric antigen receptor according to claim 12, characterized in that, The transmembrane domain includes 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 shown in SEQ ID NO.

70.

27. The chimeric antigen receptor according to claim 12, characterized in that, The intracellular domain includes a signal transduction domain; or the intracellular domain includes a co-stimulatory signal transduction domain and a signal transduction domain.

28. The chimeric antigen receptor according to claim 27, characterized in that, The co-stimulatory signal transduction domain includes: co-stimulatory molecules of CD3, CD4, CD8, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, or ligands that specifically bind to CD83.

29. The chimeric antigen receptor according to claim 27, characterized in that, The co-stimulatory signal transduction domains include 4-1BB and / or CD28.

30. The chimeric antigen receptor according to claim 27, characterized in that, The signal conduction structural domain includes CD3ζ.

31. The chimeric antigen receptor according to claim 29, characterized in that, 4-1BB includes an amino acid sequence as 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.

32. The chimeric antigen receptor according to claim 30, characterized in that, CD3ζ includes an amino acid sequence as 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.

33. The chimeric antigen receptor according to claim 11, characterized in that, The antigen-binding domain includes an amino acid sequence as 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.

34. An engineered immune cell, characterized in that, The immune cells express the chimeric antigen receptor as described in any one of claims 11 to 33 or contain nucleic acid encoding the chimeric antigen receptor as described in any one of claims 11 to 33.

35. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-MUC16 antibody or its antigen-binding fragment as described in any one of claims 1-10, or the chimeric antigen receptor as described in any one of claims 11-33.

36. The nucleic acid molecule according to claim 35, characterized in that, The nucleic acid molecule also includes a signal peptide coding sequence; and / or, it also contains a coding sequence encoding a marker protein.

37. The nucleic acid molecule according to claim 36, characterized in that, The signal peptide is selected from granulocyte-macrophage colony-stimulating factor receptor 2 signal peptide, IL2 signal peptide, or CD8 signal peptide.

38. The nucleic acid molecule according to claim 36, characterized in that, The signal peptide is the CD8 signal peptide.

39. The nucleic acid molecule according to claim 36, characterized in that, The marker protein is selected from EGFP, mCherry, or EYFP.

40. The nucleic acid molecule according to claim 36, characterized in that, The marker protein is EGFP.

41. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-MUC16 antibody or its antigen-binding fragment as described in any one of claims 1-10, the chimeric antigen receptor as described in any one of claims 11-33, the engineered immune cell as described in claim 34, and the nucleic acid molecule as described in any one of claims 35-40.

42. The pharmaceutical composition according to claim 41, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

Images