Antibodies specifically targeting CLDN6 and their application in the preparation of CAR-T cells

Abstract

The present invention provides antibodies that specifically target CLDN6 and their use in preparing CAR-T cells. The CLDN6 antibodies provided by the present invention specifically target CLDN6, but do not recognize CLDN3, CLDN4, or CLDN9, or have significantly different recognition modes. Antibody sequences based on specific targeting of CLDN6 can be used to construct chimeric antigen receptors (CARs) and bivalent antibodies (bispecific antibodies). CARs constructed based on these antibody sequences can be transduced into T cells to create CAR-T cells that specifically target CLDN6, which can be used to treat solid tumors such as ovarian cancer.

Description

Technical Field

[0001] The present invention relates to antibodies specifically targeting CLDN6 and applications thereof in preparing CAR-T cells, and relates to the fields of genetic engineering and antibody technology. Background Art

[0002] CLDN6 (Claudin-6) is a tumor-specific protein that is expressed in a variety of solid tumors, including ovarian, endometrial, lung, gastric, and testicular cancers, but is barely expressed in healthy adult tissues. This specific expression pattern makes CLDN6 a promising target for tumor therapy.

[0003] CLDN6 belongs to the claudin protein family, which has 24 members expressed in mammals. Among them, CLDN3 is widely expressed in various epithelial tissues, including the transverse colon mucosa and gastrointestinal tract of the digestive system, the endometrial epithelium and right fallopian tube of the reproductive system, various glandular tissues, and type II alveolar epithelial cells. CLDN4 has a more widespread expression range, with peak expression in the gastrointestinal tract, but also in adipose tissue, adrenal glands, tonsils, appendix, basal ganglia, bone marrow, mammary gland, bronchi, cerebellum, cerebral cortex, cervix, choroid plexus, colon, duodenum, and endometrium. In contrast, CLDN9 has more restricted expression and primarily plays an important role in the cochlea of ​​the inner ear, where it regulates ion homeostasis and hearing function. CLDN6 shares a high degree of structural homology with CLDN3, CLDN4, and CLDN9, with the extracellular domains of CLDN6 and CLDN9 differing by only three amino acids. The widespread expression of these family proteins also affects the safety of the use of CLDN6 antibodies.

[0004] Based on the above background, there is an urgent need for an antibody that can specifically target CLDN6 without binding to CLDN3, CLDN4 and CLDN9, so as to not affect other normal physiological functions of the claudin protein family while treating tumors and improve the safety of antibody use. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an antibody that specifically targets CLDN6 without binding to CLDN3, CLDN4, and CLDN9. Specifically, the present invention includes the following contents:

[0006] A first aspect of the present invention provides an antibody specifically targeting CLDN6, comprising:

[0007] 1) HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:4; or 2) HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:8; or 3) HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:12; or 4) HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:16; or 5) HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:20.

[0008] Furthermore, the numbering schemes for the CDRs include IMGT, Chothia, Kabat, AbM, and Contact.

[0009] In certain embodiments, the sequences of HCDR1-3 of the heavy chain variable region of the antibodies described in the present invention can be obtained based on the full-length sequence of the above-mentioned heavy chain variable region according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also within the scope of protection of the present invention.

[0010] Furthermore, the numbering scheme of the CDR is IMGT.

[0011] Further, the sequence of HCDR1-HCDR3 of the heavy chain variable region includes: the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO: 1-3; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO: 5-7; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO: 9-11; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO: 13-15; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO: 17-19.

[0012] Furthermore, the antibody further comprises a heavy chain variable region in which some amino acids in the framework region of 4) or 5) are replaced with humanized amino acids.

[0013] In certain embodiments, " humanization " of the present invention generally refers to humanizing non-human antibodies to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, the CDR (or part thereof) of the humanized antibody is derived from the non-human antibody sequence, and the FR (or part thereof) is derived from the human antibody sequence. Optionally, the humanized antibody also includes at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0014] Furthermore, the heavy chain variable region in which some amino acids in the framework region of 4) are replaced with humanized amino acids is as shown in any one of the following groups: a) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21; b) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22; c) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; d) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24.

[0015] Furthermore, the heavy chain variable region in which some amino acids in the framework region of 5) are replaced with humanized amino acids is as shown in any one of the following groups: e) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25; f) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; g) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; h) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29; j) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30; k) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31.

[0016] Furthermore, the antibody further comprises an Fc sequence.

[0017] Furthermore, the Fc sequence is shown in SEQ ID NO: 32.

[0018] Furthermore, the antibody also includes an export signal peptide.

[0019] Furthermore, the sequence of the membrane export signal peptide is shown in SEQ ID NO: 33.

[0020] Furthermore, the antibody includes a single domain antibody, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody or a bivalent antibody.

[0021] Furthermore, the antibody is a single domain antibody.

[0022] The second aspect of the present invention provides an antibody derivative, wherein the antibody derivative comprises any one of the following:

[0023] a. an antibody-marker conjugate, the antibody-marker conjugate comprising the antibody according to the first aspect of the present invention and a detectable marker conjugated thereto;

[0024] b. a pharmaceutical composition comprising the antibody according to the first aspect of the present invention and a pharmaceutically acceptable carrier;

[0025] c. A chimeric antigen receptor comprising the heavy chain variable region of the antibody according to the first aspect of the present invention.

[0026] Furthermore, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme.

[0027] Furthermore, the chimeric antigen receptor further includes one or more of an extracellular hinge region, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory domain.

[0028] Furthermore, the extracellular hinge region is selected from the extracellular hinge regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0029] Furthermore, the extracellular hinge region is CD8 Hinge.

[0030] Furthermore, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO: 34.

[0031] Furthermore, the transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0032] Furthermore, the transmembrane domain is CD8 TM.

[0033] Furthermore, the amino acid sequence of the CD8 TM is shown in SEQ ID NO: 35.

[0034] Furthermore, the intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and variants thereof.

[0035] Furthermore, the intracellular signaling domain is the CD3ζ intracellular signaling domain.

[0036] Furthermore, the amino acid sequence of CD3ζ is shown in SEQ ID NO:36.

[0037] Furthermore, the chimeric antigen receptor also includes a co-stimulatory signaling domain.

[0038] Furthermore, the costimulatory domain is selected from the costimulatory domains of the following molecules: 4 1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof.

[0039] Furthermore, the co-stimulatory signaling domain is 4-1BB.

[0040] Furthermore, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 37.

[0041] Furthermore, the chimeric antigen receptor further comprises a suicide gene or a detectable tag.

[0042] Furthermore, the suicide gene or detectable tag includes EGFRt.

[0043] Furthermore, the amino acid sequence of the EGFRt is shown in SEQ ID NO: 38.

[0044] Furthermore, the suicide gene or detectable tag also includes an EGFRt membrane exit signal.

[0045] Furthermore, the amino acid sequence of the EGFRt export signal is shown in SEQ ID NO: 39.

[0046] Furthermore, the chimeric antigen receptor further comprises a linker.

[0047] Furthermore, the linker includes 2A peptide and IRES.

[0048] Furthermore, the 2A peptide includes P2A, T2A, E2A and F2A.

[0049] Furthermore, the linker is a 2A peptide.

[0050] Furthermore, the 2A peptide is T2A.

[0051] Furthermore, the amino acid sequence of T2A is shown in SEQ ID NO:40.

[0052] Furthermore, the chimeric antigen receptor also includes a signal peptide.

[0053] Furthermore, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 41.

[0054] In certain embodiments, those skilled in the art can change the combination category and sequence of the signal peptide, the extracellular hinge region and the transmembrane domain, the costimulatory domain and the intracellular signaling domain according to actual conditions or needs. Regardless of the form of change, as long as the chimeric antigen receptor has the CDR sequence or heavy chain variable region sequence of the heavy chain variable region of the above-mentioned humanized antibody of the present invention, it falls within the scope of protection of the present invention. Most preferably, the chimeric antigen receptor is selected from any one of the following groups: 1. A chimeric antigen receptor obtained by sequentially connecting the signal peptide, the antibody described in the first aspect of the present invention, CD8 Hinge, CD8 TM, 4 1BB costimulatory domain, CD3ζ intracellular signaling domain, T2A, EGFRt signal peptide, and EGFRt; 2. A derivative chimeric antigen receptor formed by replacing, deleting or adding one or more amino acids based on the amino acid sequence of the chimeric antigen receptor described in 1.

[0055] The third aspect of the present invention provides a biomaterial comprising:

[0056] (I) a nucleic acid molecule encoding the antibody according to the first aspect of the present invention or the antibody derivative according to the second aspect of the present invention; or

[0057] (II) a vector comprising the nucleic acid molecule described in (I); or

[0058] (III) a recombinant host cell comprising the nucleic acid molecule of (I) and / or the vector of (II); or

[0059] (IV) A CAR-T cell comprising the chimeric antigen receptor according to the second aspect of the present invention.

[0060] The recombinant host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. Host cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. Preferably, the recombinant host cell includes a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell includes bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, or rickettsia; more preferably, the eukaryotic cell includes mammalian cells, insect cells, plant cells, or yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes a T cell, a NK cell, an iNKT cell, a B cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, a macrophage, or any combination thereof; most preferably, the immune cell is a T cell.

[0061] Furthermore, the nucleic acid molecule comprises a base sequence encoding HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NOs: 43-58.

[0062] Furthermore, the base sequences encoding HCDR1-HCDR3 are shown in SEQ ID NOs: 59-73.

[0063] Furthermore, the nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 74.

[0064] Furthermore, the nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO: 75.

[0065] Furthermore, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO: 76.

[0066] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 77.

[0067] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:78.

[0068] Furthermore, the nucleic acid molecule also comprises a base sequence encoding 4-1BB as shown in SEQ ID NO: 79.

[0069] Furthermore, the nucleic acid molecule also comprises a base sequence encoding EGFRt as shown in SEQ ID NO:80.

[0070] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an EGFRt export signal as shown in SEQ ID NO: 81.

[0071] Furthermore, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 82.

[0072] Furthermore, the nucleic acid molecule further comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO: 83.

[0073] The fourth aspect of the present invention provides a use of the antibody according to the first aspect of the present invention, the chimeric antigen receptor in the antibody derivative according to the second aspect of the present invention, and / or the nucleic acid molecule in the biomaterial according to the third aspect of the present invention, wherein the use comprises any one of the following:

[0074] Application in the preparation of products for detecting CLDN6 protein.

[0075] Application in constructing bivalent antibodies targeting CLDN6.

[0076] Application in constructing immune cells that specifically target CLDN6.

[0077] Application in the treatment of CLDN6-positive related diseases.

[0078] Application in the preparation of products for treating CLDN6-positive related diseases.

[0079] Furthermore, the products for treating CLDN6-positive related diseases include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips.

[0080] Furthermore, the CLDN6-positive related diseases include solid tumors.

[0081] Furthermore, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer.

[0082] Furthermore, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors.

[0083] Furthermore, the ovarian epithelial tumor includes ovarian adenocarcinoma tumor.

[0084] Furthermore, the solid tumor is an ovarian adenocarcinoma tumor.

[0085] A fifth aspect of the present invention provides a method, comprising any one of the following:

[0086] (1) An in vitro method for detecting CLDN6 in a sample for non-therapeutic purposes, the method comprising: contacting the sample with the antibody described in the first aspect of the present invention or the antibody-marker conjugate in the composition described in the second aspect of the present invention, and detecting the formation of a complex between the antibody and CLDN6.

[0087] (2) A method for producing the antibody of the first aspect of the present invention, comprising: culturing the recombinant host cells in the biological material of the third aspect of the present invention, and isolating the antibody of the first aspect of the present invention from the culture.

[0088] (3) A method for promoting apoptosis of ovarian adenocarcinoma cells in vitro for non-therapeutic purposes, the method comprising co-culturing the CAR-T cells in the biomaterial described in the third aspect of the present invention with ovarian adenocarcinoma cells.

[0089] Furthermore, the ovarian adenocarcinoma cell is SKOV3.

[0090] Another aspect of the present invention provides a method or pharmaceutical composition for treating a CLDN6-positive disease in a subject, the method comprising administering to the subject the antibody of the first aspect of the present invention, the antibody derivative of the second aspect of the present invention, and / or the CAR-T cells in the biomaterial of the third aspect of the present invention. In some embodiments, the subject comprises a mammal, and in specific embodiments of the present invention, the subject is preferably a human.

[0091] Furthermore, the CLDN6-positive related diseases include solid tumors.

[0092] Furthermore, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer.

[0093] Furthermore, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors.

[0094] Furthermore, the ovarian epithelial tumor includes ovarian adenocarcinoma tumor.

[0095] Furthermore, the solid tumor is an ovarian adenocarcinoma tumor.

[0096] Furthermore, the pharmaceutical composition comprises an effective amount of the antibody described in the first aspect of the present invention, the antibody derivative described in the second aspect of the present invention and / or the CAR-T cells in the biomaterial described in the third aspect of the present invention and a pharmaceutically acceptable carrier.

[0097] The term "effective amount" herein relates to that amount of an active compound or a material, composition or dosage form comprising an active compound which, when administered according to the desired treatment regimen, is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0098] Advantages and beneficial effects of the present invention: The CLDN6 antibodies provided herein specifically target CLDN6, but do not recognize CLDN3, CLDN4, or CLDN9, or their recognition patterns differ significantly. Antibody sequences specifically targeting CLDN6 can be used to construct chimeric antigen receptors (CARs) and bivalent antibodies (bispecific antibodies). CARs constructed based on these antibody sequences can be transduced into T cells to create CAR-T cells that specifically target CLDN6, which can be used to treat solid tumors such as ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 Diagram of the construction of a phage display library for alpaca immunization;

[0100] Figure 2 This is the result of FACS binding verification of CLDN6 transfection supernatant;

[0101] Figure 3 This is a diagram showing the validation results of the candidate antibodies binding to cells expressing CLDN6-related homologous proteins;

[0102] Figure 4 This is a diagram showing the results of binding validation of purified candidate antibodies to CLDN6 homologous proteins;

[0103] Figure 5 This is the result of affinity analysis of CLDN6 antibodies;

[0104] Figure 6 This is the FACS result of 2D11 humanized sequence;

[0105] Figure 7 This is the result of affinity testing of 2D11 humanized antibody;

[0106] Figure 8 This is the FACS result of 1H07 humanized sequence;

[0107] Figure 9 This is the affinity result diagram of the humanized sequence of 1H07;

[0108] Figure 10 Schematic diagram for the construction of CAR-T vector;

[0109] Figure 11 This is a graph showing the killing rate of CLDN6-2D11 and humanized antibody 2D11-HM7 CAR-T;

[0110] Figure 12 Figure 2 shows the killing rate of CLDN6-2D11, humanized 2D11-HM4, 1H07, and humanized 1H07-HM2 CAR-T cells.

[0111] Figure 13This is a graph showing the killing rate of candidate antibodies against SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9. DETAILED DESCRIPTION

[0112] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and do not constitute a limitation of the present invention in any way.

[0113] In the context of the present invention, the term "antibody" is used in the broadest sense and specifically covers single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies and multispecific antibodies (e.g., diabodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity.

[0114] In the present invention, the term "single-domain antibodies" (sdAbs), also known as VHH antibodies or camelid antibodies, are artificially designed antibody molecules. They are heavy-chain antibodies (HCAbs) that are naturally devoid of light chains and found in camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. They include two constant regions (CH2 and CH3), a hinge region, and a variable heavy chain domain (VHH). Single-domain antibodies containing only one heavy chain variable domain, namely VHH antibodies, are then cloned. The crystal structure of VHH antibodies is an oval shape of 4nm×2.5nm×3nm. The molecular weight is only 1 / 10 of that of ordinary antibodies, approximately 12-14kDa. It is the smallest complete antigen-binding fragment and is therefore also called a nanobody. The term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen.

[0115] In the present invention, the terms "specific," "binding," and "targeting" refer to binding that is selective for the antigen and can be distinguished from undesired or nonspecific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art. In specific embodiments of the present invention, specificity and targeting refer to the ability of the antibodies described in the first aspect of the present invention to bind to CLDN6 but not to CLDN3 / 4 / 9, or to bind in a manner that is significantly different from CLDN6.

[0116] In the present invention, antibody sequences that have been modified to have an amino acid sequence identity of 80% or greater to the antibody described in the first aspect of the present invention also fall within the scope of protection of the present invention. The term "modification" refers to any form of modification to an amino acid sequence, such as amino acid substitution, deletion, insertion, and / or addition. The term "substitution" refers to the replacement of one or more amino acids in the original amino acid sequence with a different amino acid. The term "deletion" refers to the removal of one or more amino acids from the original amino acid sequence. The term "insertion" or "addition" refers to a change in an amino acid sequence that results in the addition of one or more amino acids compared to the original amino acid sequence. The term "identity," also known as "homology," refers to an amino acid sequence that is at least 80% identical to a sequence provided herein. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment across the full-length sequences being compared.

[0117] Unless otherwise indicated, the antibodies described herein are isolated antibodies. The term "isolated" as used herein refers to a nucleic acid or antibody or fragment thereof that has been extracted from its natural environment. Nucleic acids or antibodies or fragments thereof that have been "isolated" thus include nucleic acids or antibodies or fragments thereof purified by standard purification methods. The term also includes nucleic acids or antibodies or fragments thereof prepared by recombinant expression in a host cell and chemically synthesized nucleic acids and / or antibodies.

[0118] In the present invention, the term "nucleic acid" or "nucleic acid molecule" is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Therefore, the nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the antibody of the present invention is isolated, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then isolate it from the host cells after proliferation by conventional methods.

[0119] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention or a precursor thereof, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0120] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, antibodies of the present invention are obtained by direct amino acid synthesis. In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0121] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, pharmaceutically acceptable acids, bases, or buffers may be used to adjust the pH of the formulation to enhance the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may be administered to a recipient by any route that reaches the target tissue.

[0122] The pharmaceutical compositions of the present invention may also be used in combination with other drugs for treating solid tumors. These other compounds for treating solid tumors may be administered simultaneously with the main active ingredient (e.g., the antibody described in the first aspect of the present invention), or even administered simultaneously in the same composition. The other therapeutic compounds may also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient.

[0123] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids such as water, saline, glycerol and ethanol. Auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in such vehicles.

[0124] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials and reagents used in the following examples, unless otherwise specified, are all commercially available. Simple modifications to the present invention made according to the essence of the present invention fall within the scope of protection claimed in the present invention.

[0125] Example 1 Construction of phage display library, antibody selection and humanization

[0126] 1. Construction of phage display library by alpaca immunization

[0127] Two alpacas were immunized with 293F-CLDN6, with an interval of 14 days between immunizations. Seven days after the last immunization, peripheral blood was collected and serum was separated for immune titer testing using ELISA. PBMCs were isolated using lymphocyte separation buffer after passing the test. RNA was extracted using PrimeScript TM II 1st Strand cDNA Synthesis Kit was used for reverse transcription to prepare cDNA. The VHH sequence was amplified from the cDNA sample using the single-domain antibody cloning primer combination and subcloned into the phage display vector pDisplay. SS320 competent cells were electroporated to construct a single-domain antibody phage display library. A library was constructed by mixing two alpacas and sequencing 23 clones. Two antibody sequences with a base deletion and one overlapping peak were removed. The remaining sequences were all antibody sequences with good diversity. The phage display library was as follows: Figure 1 shown.

[0128] 2. Select candidate antibody clones for specific recognition verification

[0129] Panning was performed using CLDN6-VLP and CHO-S-Lenti-CMV-hCLDN6 cells, while negative selection was performed using CHO-S-Lenti-CMV-hCLDN6 cells. Based on phage ELISA, eight different antibody sequences were screened through solid-phase and cell-based panning. After PCR, they were digested with SfiI and subcloned into the pcDNA3.4-IgG1Fc expression vector. Binding to CLDN6 transfection supernatants was verified by FACS. The experiment involved 3×10 5 Each well corresponds to overexpression or blank control cells. The primary antibody is the CLDN6 target transfection supernatant (100 μl / well), and the secondary antibody is PE-Goat anti-Human IgG Fc (Invitrogen, Cat#: 12-4998-82) (1:500 dilution). Among them, PC is a positive control (clone number: AB89A, reference patent: CN111875703A). The experimental results are shown in Figure 2. Figure 2 As shown, candidate clones 1-D05, 1-G01, 1-H06, 1-H07, 2-D11 and 3-D12 bound more strongly to CHO-S-CLDN6 cells.

[0130] The expression, purification and detection of 5 candidate antibodies were used to verify the binding of antibodies to cells overexpressing CLDN3 / 4 / 6 / 9. Control CHO-S cells and 3×10 CHO-S cells overexpressing CLDN3 / 4 / 6 / 9 were used. 5 Cells were plated per well and incubated with a candidate CLDN6 target antibody (10 μg / ml, 100 μl / well) as the primary antibody for 1 hour at room temperature. After washing the cells three times with PBS, PE-Goat anti-Human IgG Fc (Invitrogen, Cat#: 12-4998-82) (1:500 dilution) was used as the secondary antibody for 45 minutes at room temperature in the dark. The cells were then washed three times with PBS and resuspended in 200 μl of PBS for flow cytometry analysis. The results showed that the four purified antibodies, 1-D05, 1-G01, 1-H06, and 2-D11, showed little binding to CLDN3 and CLDN4. 1-H07 showed strong binding to CLDN4, but with significant differences to CLDN6. The three antibodies 2-D11, 1-H07, and 8-G02 were selected for further FACS binding detection (the concentrations of the candidate CLDN6 target antibodies were 30 μg / ml, 10 μg / ml, and 3 μg / ml, 100 μl / well). The results are as follows: Figure 3 As shown, 1-H07 and 2-D11 were found to be ideal. Among them, PC (731B2) is an antibody from US11345731 patent.

[0131] 3. Affinity verification of candidate antibodies

[0132] After all candidate antibodies were purified, they were tested with cells overexpressing CLDN6 and CLDN9 by gradient dilution (CLDN6 target candidate antibodies were diluted 3-fold starting from 30 μg / ml and 10 points). The results are as follows: Figure 4 、 Figure 5 As shown, only 1-H07 and 2-D11 basically did not bind to CLDN9, while the other antibodies all bound at high concentrations, but bound more strongly to CLDN6. Overall, 2-D11 had better binding specificity.

[0133] 4. CLDN6-2D11 cloning and humanization results

[0134] Based on the original antibody sequence information of 2D11, the homology model of the antibody was obtained by modeling and the CDRs were analyzed. A range of framework amino acids, these amino acid sites usually affect the conformation or antigen binding activity of CDR. The human germline was obtained by IMGT analysis. After splicing the selected human germline framework with the CDRs of the antibody, the framework region sequences of the designed humanized antibody and the original antibody were compared. By analyzing the homology modeling results of the parent antibody, amino acids similar to the surface residues of the human antibody were selected for replacement while maintaining the antibody activity and reducing heterology. The antibody humanized sequence 2D11-HM01~07 was designed, with a total of 7 sequences. The humanized antibodies designed above were gene synthesized separately and subcloned into the pcDNA3.4-IgG1Fc expression vector. Control cells CHO-S and 3×10 CHO-S cells corresponding to CLDN3 / 4 / 6 / 9 overexpression were used. 5 The concentrations of the candidate CLDN6 target antibodies were 30 μg / ml, 10 μg / ml and 3 μg / ml, and 100 μl / well were used for flow cytometry detection. The results were as follows: Figure 6 As shown; Based on the above results, humanized antibodies HM3, HM4 and HM7 were selected for FACS EC50 affinity detection (3-fold gradient dilution 10 points), and the results are shown as follows Figure 7 As shown, the candidate antibodies all had good binding results.

[0135] 5. CLDN6 1H07 clone humanization results

[0136] Based on the original antibody sequence information of 1HM07, the homology model of the antibody was obtained by modeling and the CDRs were analyzed. A range of framework amino acids, these amino acid sites usually affect the conformation or antigen binding activity of CDR. The human germline was obtained by IMGT analysis. After splicing the selected human germline framework with the CDRs of the antibody, the framework region sequences of the designed humanized antibody and the original antibody were compared. By analyzing the homology modeling results of the parent antibody, amino acids similar to the surface residues of the human antibody were selected for replacement while maintaining the antibody activity and reducing heterology. The antibody humanized sequences 1H07-HM01~04 were designed, a total of 4 sequences. The humanized antibodies designed above were gene synthesized separately and subcloned into the pcDNA3.4-IgG1Fc expression vector. Control cells CHO-S and 3×10 CHO-S cells corresponding to CLDN3 / 4 / 6 / 9 overexpression were used. 5 The concentrations of the candidate CLDN6 target antibodies were 30 μg / ml, 10 μg / ml and 3 μg / ml, and 100 μl / well were used for flow cytometry detection. The results were as follows: Figure 8 As shown; and affinity testing was performed by diluting 10 points in 3-fold gradient starting from 30 μg / ml. The results are shown Figure 9 shown.

[0137] Example 2 Construction of CAR-T cells using candidate antibody sequences and verification of killing ability

[0138] 1. Construction of CAR-T cells based on cloned sequences

[0139] The corresponding candidate antibody sequence and the positive control CAR-T antibody 731B2 sequence (from US11345731 patent) were synthesized to construct the pCDH-EF1α lentiviral expression plasmid. Figure 10 The structure is shown in the schematic diagram. The lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in the logarithmic growth phase, and the cell culture supernatant was harvested 48-72 hours after transfection. After concentration and filtration, the CAR lentivirus was obtained and stored at -80°C for later use. Peripheral blood mononuclear cells (PBMCs) were separated from human peripheral blood, and T cells were isolated using human CD3 / 28 magnetic beads. Viral transduction was performed within 72 hours of activation, and the medium was changed 24 hours after transduction and cultured until the eighth day. The cells were collected by centrifugation and resuspended in normal saline. Flow cytometry was used to identify EGFRt molecules on the surface of T cells to ensure that the positive rate was greater than 30%. If it was lower, EGFR-PE primary antibody plus PE magnetic beads were used for enrichment and sorting.

[0140] 2. Verification of CAR-T cell killing ability

[0141] 1) 2D11 and humanized 2D11-HM7, as well as control 731B2 CAR-T and negative control ctrl-T, killed target CHO-S cells (overexpressing CLDN3 / 4 / 6 / 9, a total of four groups) with an effector-target ratio of 5:1. The results are as follows Figure 11 As shown, the results showed that several groups of CAR-T can effectively recognize and kill CLDN6-CHO-S cells.

[0142] 2) Different donor negative controls WT-T, 2D11 and humanized 2D11-HM4, 1H07 and humanized 1H07-HM2 CAR-T cells killed target CHO-S cells (overexpressing CLDN3 / 4 / 6 / 9, a total of four groups), with effector-target ratios of 1:1 and 5:1. The results are as follows Figure 12 As shown, the results showed that several groups of CAR-T can effectively recognize and kill CLDN6-CHO-S cells.

[0143] 3) SKOV3 cell lines (adherent) overexpressing CLDN3 / 4 / 6 / 9 were constructed, and RTCA was used to monitor the killing of 731B2, 2D11, 2D11HM3, and 1H07HM2 CAR-T cells. Lysis was a positive control with the addition of lysis buffer, and SKOV3 was an untreated group. The specific experimental steps are as follows: 50 μL of culture medium was added to each well as a background measurement. Subsequently, the target cells were suspended in 100 μL of complete culture medium at an appropriate density (15,000 cells / well) and carefully added to each well of the E-Plate. The E-Plate was placed in the RTCA instrument, and the cell index (CI) value was continuously recorded for approximately 48 hours to monitor cell adhesion and growth. Among them, 12-24 hours after inoculation, pause the recording and remove the E-Plate, add 50μL T cell suspension (according to the effector-target ratio of 1:1 and 5:1) or lysis solution (positive control) to the target well, and quickly return the E-Plate to the instrument to continue monitoring. The cell index (CI) and corresponding killing results of SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9 are shown in the figure. Figure 13 As shown, it was demonstrated that the candidate antibody could effectively and specifically kill SKOV3 cells overexpressing CLDN6.

[0144] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. A single-domain antibody specifically targeting CLDN6, characterized in that: The single-domain antibody comprises HCDR1-HCDR3 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:

20.

2. The single domain antibody according to claim 1, characterized in that The numbering schemes for the CDRs include IMGT, Chothia, Kabat, AbM, Contact.

3. The single domain antibody according to claim 1, characterized in that The numbering scheme for the CDRs is IMGT.

4. The single domain antibody according to claim 1, characterized in that The amino acid sequences of HCDR1-HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 17-19.

5. The single domain antibody according to claim 1, characterized in that The single-domain antibody further comprises a heavy chain variable region in which some amino acids in the framework region of the heavy chain variable region as shown in SEQ ID NO: 20 are replaced with humanized amino acids.

6. The single domain antibody according to claim 5, characterized in that The heavy chain variable region in which some amino acids in the framework region are replaced with humanized amino acids is shown in any of the following groups: a) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25; b) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; c) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; d) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; e) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29; f) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30; g) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

31.

7. The single domain antibody according to claim 1, characterized in that The single domain antibody further comprises an Fc sequence.

8. The single domain antibody according to claim 7, characterized in that The Fc sequence is shown in SEQ ID NO:

32.

9. The single domain antibody according to claim 1, characterized in that The single domain antibody further comprises an export signal peptide.

10. The single domain antibody according to claim 9, characterized in that The sequence of the membrane export signal peptide is shown in SEQ ID NO:

33.

11. An antibody derivative, characterized in that The antibody derivative comprises any one of the following: a. an antibody-marker conjugate comprising the single domain antibody according to any one of claims 1 to 10 and a detectable marker conjugated thereto; b. A pharmaceutical composition comprising the single domain antibody according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier; c. A chimeric antigen receptor comprising the heavy chain variable region of the single domain antibody according to any one of claims 1 to 10.

12. The antibody derivative according to claim 11, characterized in that The detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme.

13. The antibody derivative according to claim 11, characterized in that The chimeric antigen receptor further includes one or more of an extracellular hinge region, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory domain.

14. The antibody derivative according to claim 13, characterized in that The extracellular hinge region is selected from the extracellular hinge regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

15. The antibody derivative according to claim 13, characterized in that The extracellular hinge region is CD8 Hinge.

16. The antibody derivative according to claim 15, characterized in that The amino acid sequence of the CD8 Hinge is shown in SEQ ID NO:

34.

17. The antibody derivative according to claim 13, characterized in that The transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

18. The antibody derivative according to claim 13, characterized in that The transmembrane domain is CD8 TM.

19. The antibody derivative according to claim 18, characterized in that The amino acid sequence of the CD8 TM is shown in SEQ ID NO:

35.

20. The antibody derivative according to claim 13, characterized in that The intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and variants thereof.

21. The antibody derivative according to claim 13, characterized in that The intracellular signaling domain is the CD3ζ intracellular signaling domain.

22. The antibody derivative according to claim 21, characterized in that The amino acid sequence of CD3ζ is shown in SEQ ID NO:

36.

23. The antibody derivative according to claim 13, characterized in that The costimulatory domain is selected from the costimulatory domains of the following molecules: 4 1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof.

24. The antibody derivative according to claim 13, characterized in that The costimulatory domain is 4-1BB.

25. The antibody derivative according to claim 24, characterized in that The amino acid sequence of 4-1BB is shown in SEQ ID NO:

37.

26. The antibody derivative according to claim 11, characterized in that The chimeric antigen receptor also includes a suicide gene or a detectable tag.

27. The antibody derivative according to claim 26, characterized in that The suicide gene or detectable tag includes EGFRt.

28. The antibody derivative according to claim 27, characterized in that The amino acid sequence of EGFRt is shown in SEQ ID NO:

38.

29. The antibody derivative according to claim 26, characterized in that The suicide gene or detectable tag also includes a signal peptide of EGFRt.

30. The antibody derivative according to claim 29, characterized in that The amino acid sequence of the EGFRt signal peptide is shown in SEQ ID NO:

39.

31. The antibody derivative according to claim 11, characterized in that The chimeric antigen receptor further includes a linker.

32. The antibody derivative according to claim 31, characterized in that The linker includes 2A peptide and IRES.

33. The antibody derivative according to claim 32, characterized in that The 2A peptides include P2A, T2A, E2A and F2A.

34. The antibody derivative according to claim 31, characterized in that The linker is T2A.

35. The antibody derivative according to claim 34, characterized in that The amino acid sequence of T2A is shown in SEQ ID NO:

40.

36. The antibody derivative according to claim 11, characterized in that The chimeric antigen receptor also includes a signal peptide.

37. The antibody derivative according to claim 36, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO:

41.

38. A biomaterial comprising: (I) a nucleic acid molecule encoding the single domain antibody of any one of claims 1 to 10 or the chimeric antigen receptor of any one of claims 11 to 37; or (II) a vector comprising the nucleic acid molecule described in (I); or (III) a recombinant host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II); or (IV) A CAR-T cell comprising the chimeric antigen receptor of the antibody derivative according to any one of claims 11 to 37.

39. The biomaterial according to claim 38, characterized in that The nucleic acid molecule comprises a base sequence encoding HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NOs: 47, 52-58.

40. The biomaterial according to claim 39, characterized in that The base sequences encoding HCDR1-HCDR3 are shown in SEQ ID NOs: 71-73.

41. The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO:

74.

42. The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO:

75.

43. The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO:

76.

44. The biomaterial according to claim 38, characterized in that The nucleic acid molecule also comprises a base sequence encoding CD8 TM as shown in SEQ ID NO:

77.

45. The biomaterial according to claim 38, characterized in that The nucleic acid molecule also comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:

78.

46. ​​The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO:

79.

47. The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO:

80.

48. The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding an EGFRt export signal as shown in SEQ ID NO:

81.

49. The biomaterial according to claim 38, characterized in that The nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO:

82.

50. The biomaterial according to claim 38, wherein The nucleic acid molecule further comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO:

83.

51. Use of the single domain antibody according to any one of claims 1 to 10, the chimeric antigen receptor according to any one of claims 11 to 37, and / or the nucleic acid molecule according to any one of claims 38 to 50, characterized in that: The application includes any of the following: Application in the preparation of products for detecting CLDN6 protein; Application in constructing bivalent antibodies targeting CLDN6; Application in the construction of CAR-T cells specifically targeting CLDN6; Application in the preparation of products for treating ovarian adenocarcinoma.

52. The use according to claim 51, characterized in that The product for treating ovarian adenocarcinoma includes a pharmaceutical composition; The products for detecting CLDN6 protein include a kit, a nucleic acid chip, and a nucleic acid membrane strip.

53. A method comprising any of the following: (1) An in vitro method for detecting CLDN6 in a sample for non-therapeutic purposes, the method comprising: contacting a test sample with the single domain antibody according to any one of claims 1 to 10 or the antibody-marker conjugate according to claim 11, and detecting formation of a complex between the single domain antibody and CLDN6; (2) A method for producing the single domain antibody according to any one of claims 1 to 10, the method comprising: culturing the recombinant host cell in the biomaterial according to claim 11, and isolating the single domain antibody according to any one of claims 1 to 10 from the culture; (3) A method for promoting apoptosis of ovarian adenocarcinoma cells in vitro for non-therapeutic purposes, characterized in that the method comprises: co-culturing the CAR-T cells in the biomaterial according to claim 38 with ovarian adenocarcinoma cells.

54. The method according to claim 53, wherein The ovarian adenocarcinoma cell line is SKOV3.

Images