CLDN6 single domain antibody and humanization thereof

Abstract

The invention provides a CLDN6 single domain antibody and a humanization method thereof. The CLDN6 single-domain antibody provided by the invention has targeted specificity to CLDN6, only recognizes the CLDN6 and does not recognize CLDN3, CLDN4 and CLDN9 of the same family, or the recognition modes are obviously different. The CLDN6 single-domain antibody 1H07 with targeting specificity provided by the invention can be used for constructing a chimeric antigen receptor (CAR) and a bivalent antibody (bispecific antibody). The CAR constructed based on the antibody sequence can be transduced into a T cell to create a CAR-T cell specifically targeting CLDN6, and the CAR-T cell can be used for treating solid tumors such as ovarian cancer.

Description

[0001] This application is a divisional application, and the parent case information is as follows: The name of the invention is "Antibodies specifically targeting CLDN6 and their use in the preparation of CAR-T cells"; the original application date is 2024.11.08, and the original application number is 2024115913181. Technical Field

[0002] The present invention relates to a CLDN6 single-domain antibody and humanization thereof, and relates to the fields of genetic engineering and antibody technology. Background Art

[0003] 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.

[0004] CLDN6 belongs to the claudin protein family, which has 24 members expressed in mammals. 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, primarily playing 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.

[0005] 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

[0006] 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:

[0007] In a first aspect, the present invention provides a single-domain antibody specifically targeting CLDN6, wherein the single-domain antibody comprises HCDR1-HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 16.

[0008] In some embodiments, the numbering scheme for the CDRs includes IMGT, Chothia, Kabat, AbM, 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] Furthermore, the amino acid sequences of HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 16 are shown in SEQ ID NOs: 13-15.

[0012] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16.

[0013] In some embodiments, the single-domain antibody includes a heavy chain variable region obtained by humanizing the framework region, and the heavy chain variable region obtained by humanizing the framework region is 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.

[0014] In certain embodiments, "humanization" as used herein generally refers to humanizing a non-human antibody to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Generally, the CDRs (or portions thereof) of a humanized antibody are derived from non-human antibody sequences, while the FRs (or portions thereof) are derived from human antibody sequences. Optionally, the humanized antibody also includes at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0015] The second aspect of the present invention provides an antibody derivative, which comprises any one of the following: 1) an antibody-marker conjugate, wherein the antibody-marker conjugate comprises the single-domain antibody described in the first aspect of the present invention and a detectable label conjugated thereto; 2) a chimeric antigen receptor comprising the heavy chain variable region of the single-domain antibody described in the first aspect of the present invention; 3) a pharmaceutical composition, wherein the pharmaceutical composition comprises the single-domain antibody described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0016] In some embodiments, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, a avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme.

[0017] Furthermore, the chimeric antigen receptor includes one or more of an extracellular hinge region, a transmembrane domain, an intracellular immunoreceptor tyrosine activation motif, and a co-stimulatory domain.

[0018] In some embodiments, the extracellular hinge region is selected from the extracellular hinge region 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.

[0019] Furthermore, the extracellular hinge region is CD8 Hinge.

[0020] Furthermore, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO: 34.

[0021] In some embodiments, 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.

[0022] Furthermore, the transmembrane domain is CD8 TM.

[0023] Furthermore, the amino acid sequence of the CD8 TM is shown in SEQ ID NO: 35.

[0024] In some embodiments, the intracellular immunoreceptor tyrosine-based activation motif is selected from the intracellular immunoreceptor tyrosine-based activation motifs 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.

[0025] Furthermore, the intracellular immunoreceptor tyrosine-based activation motif is a CD3ζ intracellular immunoreceptor tyrosine-based activation motif.

[0026] Furthermore, the amino acid sequence of CD3ζ is shown in SEQ ID NO:36.

[0027] In some embodiments, 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.

[0028] Furthermore, the costimulatory domain is 4-1BB.

[0029] Furthermore, the amino acid sequence of the 4-1BB is shown in SEQ ID NO: 37.

[0030] In some embodiments, the chimeric antigen receptor further comprises a suicide gene or a detectable tag.

[0031] Furthermore, the suicide gene or detectable tag includes EGFRt.

[0032] Furthermore, the amino acid sequence of the EGFRt is shown in SEQ ID NO: 38.

[0033] Furthermore, the suicide gene or detectable tag also includes a signal peptide of EGFRt.

[0034] Furthermore, the amino acid sequence of the EGFRt signal peptide is shown in SEQ ID NO: 39.

[0035] In some embodiments, the chimeric antigen receptor further comprises a linker.

[0036] Furthermore, the linker includes 2A peptide and IRES.

[0037] Furthermore, the 2A peptide includes P2A, T2A, E2A and F2A.

[0038] Furthermore, the linker is T2A.

[0039] Further, the amino acid sequence of the T2A is shown as SEQ ID NO: 40.

[0040] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0041] Further, the amino acid sequence of the signal peptide is shown as SEQ ID NO: 41.

[0042] In some embodiments, the chimeric antigen receptor further comprises an Fc sequence.

[0043] Further, the Fc sequence is shown as SEQ ID NO: 32.

[0044] In some embodiments, the chimeric antigen receptor further comprises a membrane exit signal peptide.

[0045] Further, the sequence of the membrane exit signal peptide is shown as SEQ ID NO: 33.

[0046] In certain embodiments, the skilled person in the art can change the combination type and sequence of the signal peptide, extracellular hinge region and transmembrane domain, costimulatory domain and intracellular immunoreceptor tyrosine-based activation motif according to the actual situation or needs, no matter what form of change, as long as the chimeric antigen receptor has the CDR sequence of the heavy chain variable region or the heavy chain variable region sequence of the humanized antibody described above in the present application, which all belong to the protection scope of the present application. Most preferably, the chimeric antigen receptor is selected from any one of the following group: 1, the chimeric antigen receptor obtained in turn by the signal peptide, the single domain antibody of the first aspect of the present application, CD8 Hinge, CD8 TM, 4-1BB costimulatory domain, CD3ζ intracellular immunoreceptor tyrosine-based activation motif, T2A, EGFRt signal peptide; 2, the derivative chimeric antigen receptor formed by substituting, deleting or adding one or more amino acids on the basis of the amino acid sequence of the chimeric antigen receptor described in 1.

[0047] The third aspect of the present application provides a biological material, which comprises: (I) a nucleic acid molecule encoding the single domain antibody of the first aspect of the present application or the antibody derivative of the second aspect of the present application; or (II) a vector comprising the nucleic acid molecule of (I); or (III) a recombinant host cell comprising the nucleic acid molecule of (I) and / or the vector of (II); or (IV) a CAR-T cell comprising the chimeric antigen receptor in the antibody derivative of the second aspect of the present application.

[0048] 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.

[0049] In some embodiments, the nucleic acid molecule comprises a base sequence encoding HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:46.

[0050] Furthermore, the base sequences encoding HCDR1-HCDR3 are shown in SEQ ID NOs: 68-70.

[0051] In an optional embodiment, the nucleic acid molecule comprises a base sequence encoding a heavy chain variable region as shown in SEQ ID NO:46.

[0052] In an optional embodiment, the nucleic acid molecule comprises a base sequence encoding a heavy chain variable region as shown in SEQ ID NO:48.

[0053] In an optional embodiment, the nucleic acid molecule comprises a base sequence encoding a heavy chain variable region as shown in SEQ ID NO:49.

[0054] In an optional embodiment, the nucleic acid molecule comprises a base sequence encoding a heavy chain variable region as shown in SEQ ID NO: 50.

[0055] In an optional embodiment, the nucleic acid molecule comprises a base sequence encoding a heavy chain variable region as shown in SEQ ID NO: 51.

[0056] Furthermore, the nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 74.

[0057] Furthermore, the nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO: 75.

[0058] Furthermore, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO: 76.

[0059] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 77.

[0060] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:78.

[0061] Furthermore, the nucleic acid molecule also comprises a base sequence encoding 4-1BB as shown in SEQ ID NO: 79.

[0062] Furthermore, the nucleic acid molecule also comprises a base sequence encoding EGFRt as shown in SEQ ID NO:80.

[0063] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an EGFRt export signal as shown in SEQ ID NO: 81.

[0064] Furthermore, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 82.

[0065] Furthermore, the nucleic acid molecule further comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO: 83.

[0066] The fourth aspect of the present invention provides the use of the single-domain antibody described in the first aspect of the present invention, the chimeric antigen receptor in the antibody derivative described in the second aspect of the present invention, and / or the nucleic acid molecule in the biomaterial described in the third aspect of the present invention, wherein the use includes any one of the following: 1) use in preparing a product for detecting CLDN6 protein; 2) use in constructing a bivalent antibody targeting CLDN6; 3) use in constructing CAR-T cells specifically targeting CLDN6; 4) use in preparing a product for treating CLDN6-positive related diseases.

[0067] Furthermore, the products for treating CLDN6-positive related diseases include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips.

[0068] Furthermore, the CLDN6-positive related diseases include solid tumors.

[0069] Furthermore, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer.

[0070] Furthermore, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors.

[0071] Furthermore, the ovarian epithelial tumor includes ovarian adenocarcinoma tumor.

[0072] Furthermore, the solid tumor is an ovarian adenocarcinoma tumor.

[0073] The fifth aspect of the present invention provides a method, which includes any one of the following: (1) an in vitro method for detecting CLDN6 in a sample for non-therapeutic purposes, the method comprising: contacting the sample for testing with the single-domain antibody described in the first aspect of the present invention or the antibody-marker conjugate in the antibody derivative described in the second aspect of the present invention, and detecting the formation of a complex between the single-domain antibody and CLDN6; (2) a method for producing the single-domain antibody described in the first aspect of the present invention, the method comprising: culturing the recombinant host cells in the biomaterial described in the third aspect of the present invention, and isolating the single-domain antibody described in the first aspect of the present invention from the culture; (3) an in vitro method for promoting apoptosis of ovarian adenocarcinoma cells for non-therapeutic purposes, the method comprising: co-culturing CAR-T cells in the biomaterial described in the third aspect of the present invention with ovarian adenocarcinoma cells.

[0074] Furthermore, the ovarian adenocarcinoma cell line is SKOV3.

[0075] 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 single-domain 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.

[0076] Furthermore, the CLDN6-positive related diseases include solid tumors.

[0077] Furthermore, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer.

[0078] Furthermore, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors.

[0079] Furthermore, the ovarian epithelial tumor includes ovarian adenocarcinoma tumor.

[0080] Furthermore, the solid tumor is an ovarian adenocarcinoma tumor.

[0081] Furthermore, the pharmaceutical composition comprises an effective amount of the single domain 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.

[0082] 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.

[0083] Advantages and beneficial effects of the present invention: The CLDN6 single-domain antibody 1H07 provided by the present invention has specific targeting for CLDN6, recognizing only CLDN6 and not its cognate CLDN3, CLDN4, and CLDN9, or with significantly different recognition patterns. 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

[0084] Figure 1 Diagram of the construction of a phage display library for alpaca immunization.

[0085] Figure 2 This is the result of FACS binding verification of CLDN6 transfection supernatant.

[0086] Figure 3 This figure shows the validation results of the binding of candidate antibodies to cells expressing CLDN6-related homologous proteins.

[0087] Figure 4 This is a diagram showing the binding verification results of the purified candidate antibodies and CLDN6 homologous proteins.

[0088] Figure 5 This is the result of affinity analysis of CLDN6 antibodies.

[0089] Figure 6 This is the FACS result of the 1H07 humanized sequence.

[0090] Figure 7 This is the affinity result diagram of the humanized sequence of 1H07.

[0091] Figure 8 Schematic diagram of CAR-T vector construction.

[0092] Figure 9 The figure shows the killing rate detection of CLDN6-2D11, humanized 2D11-HM4, 1H07, and humanized 1H07-HM2 CAR-T.

[0093] Figure 10 This is a graph showing the killing rate of candidate antibodies against SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9. DETAILED DESCRIPTION

[0094] 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.

[0095] In this application, 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) naturally lacking light chains and are found in camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. These antibodies contain two constant regions (CH2 and CH3), a hinge region, and a variable heavy chain domain (VHH). Single-domain antibodies containing only a single heavy chain variable domain, or VHH antibodies, are then cloned. VHH antibodies have a crystal structure of an oval, measuring 4 nm x 2.5 nm x 3 nm. Their molecular weight is only 1 / 10 that of a typical antibody, approximately 12-14 kDa. They are the smallest complete antigen-binding fragment and are therefore also called nanobodies. The term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen.

[0096] 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 other conventional binding assays known in the art. In specific embodiments of the present invention, specificity and targeting refer to the ability of the single-domain antibody 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 and thus can be clearly distinguished from CLDN6.

[0097] In the present invention, antibody sequences that have been modified to have an amino acid sequence identity of 80% or greater to the single-domain 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 terms "insertion" or "addition" refer to changes in an amino acid sequence that result 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 are able to determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0098] 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.

[0099] In the present invention, the terms "nucleic acid" or "nucleic acid molecule" are intended to include polymeric forms of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, and / or their analogs, including DNA, RNA, and DNA / RNA hybrids. They also include DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, and the like. Once the coding sequence for an antibody of the present invention is isolated, recombinant techniques can be used to obtain the antibody in large quantities. An exemplary method is to clone the encoding gene into a vector, transfer it into cells, and then isolate it from the propagated host cells by conventional methods.

[0100] The vector of the present application refers to an artificial construct which is capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. The vector of the present application is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a gene of interest encoding the antibody or a precursor thereof of the present application, a promoter, a terminator, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. The known vectors include plasmid vectors, lentivirus vectors, adenovirus vectors, AAV virus vectors, etc.

[0101] In certain embodiments, the antibody of the present application is obtained by artificial synthesis. The method of artificially synthesizing an antibody is known in the art, for example, the antibody of the present application is obtained by direct synthesis of amino acids. In certain embodiments, the antibody of the present application is obtained by genetic engineering expression. The genetic engineering expression system includes a prokaryotic cell expression system, a eukaryotic cell expression system, and a cell-free system expression system. Among them, the prokaryotic cell expression system includes an E. coli expression system. The eukaryotic cell expression system includes a yeast expression system, an insect cell expression system, and a mammalian cell expression system.

[0102] In the present application, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present application can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical composition of the present application can contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In certain cases, a pharmaceutical acid, base or buffer can be used to adjust the pH of the formulation to improve the stability of the formulated compound or its administration 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 composition of the present application can be administered to a subject by any route as long as it reaches the target tissue.

[0103] The pharmaceutical composition of the present application can also be used in combination with other drugs for treating solid tumors. Other therapeutic compounds for treating solid tumors can be administered simultaneously with, even in the same composition as, the main active ingredient (e.g., the single-domain antibody of the first aspect of the present application). Other therapeutic compounds can also be administered separately in a separate composition or in a different dosage form from the main active ingredient.

[0104] In the present application, 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 that can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. Such carriers are well known in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids such as water, saline, glycerol and ethanol. There can also be auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like in such vehicles.

[0105] The present application will be further described in detail with reference to the following drawings and examples. The following examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials and reagents used in the following examples are all obtained from commercial channels unless otherwise specified. Simple improvements made to the present application according to its essence all fall within the scope of the present application.

[0106] Example 1 Construction of phage display library, panning of antibodies and humanization

[0107] 1. Construction of phage display library by immunizing alpaca

[0108] Two alpacas were immunized with 293F-CLDN6, with an immunization interval of 14 days. Peripheral blood was collected 7 days after the last immunization, and the serum was separated and detected for immunization titer by ELISA. After passing, PBMC was separated using lymphocyte separation medium. RNA was extracted, reverse transcribed using PrimeScript™ II 1st Strand cDNA Synthesis Kit to prepare cDNA, and VHH sequences were amplified from the cDNA sample using a single-domain antibody cloning primer combination, and subcloned into the phage display vector pDisplay, and electrotransformed into SS320 competent cells to construct a single-domain antibody phage display library. Two alpacas were mixed to construct the library, and 23 clones were sequenced. Two antibody sequences with one base missing and one peak were removed, and the remaining sequences were all antibody sequences with good diversity. The phage display library is shown in Figure 1 .

[0109] 2. Verification of specific recognition of candidate antibody clones obtained by panning

[0110] 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 was CLDN6 target transfection supernatant (100 μl / well), and the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#:12-4998-82) (1:500 dilution). PC was used as 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.

[0111] 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 incubated with a candidate CLDN6 target antibody (10 μg / ml, 100 μl / well) as the primary antibody at room temperature for 1 hour. 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 washed three times with PBS and resuspended in 200 μl of PBS for flow cytometric 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 marked differences from CLDN6. Three antibodies, 2-D11, 1-H07, and 8-G02, were selected for further FACS binding assay (the concentrations of the CLDN6 target candidate antibodies were 30 μg / ml, 10 μg / ml, and 3 μg / ml, 100 μl / well). The results are shown in the figure. Figure 3 As shown, 1-H07 and 2-D11 were found to be ideal. Among them, PC (731B2) is an antibody from US11345731 patent.

[0112] 3. Affinity verification of candidate antibodies

[0113] 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 to 10 points starting from 30 μg / ml). The results are as follows: Figure 4 、 Figure 5 As shown, only 1-H07 and 2-D11 had little binding to CLDN9.

[0114] 4. CLDN6 1H07 clone humanization results

[0115] Based on the sequence information of the original antibody 1HM07, the homology model of the antibody was obtained by modeling, and the framework amino acids within 5Å of the CDRs were analyzed. These amino acid sites usually affect the conformation or antigen binding activity of the 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, totaling 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 the flow cytometry test was performed in 100 μl / well. The results were as follows. Figure 6 As shown; and affinity test was performed by 3-fold gradient dilution starting from 30 μg / ml and 10 points, the results are shown Figure 7 shown.

[0116] Example 2 Construction of CAR-T cells using candidate antibody sequences and verification of killing ability

[0117] 1. Construction of CAR-T cells based on cloned sequences

[0118] 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, such as Figure 8The structure is shown in the schematic diagram. Lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in the logarithmic growth phase. Cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered to obtain the CAR lentivirus, which was stored at -80°C until further use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and transduced with the virus within 72 hours of activation. After 24 hours of transduction, the medium was changed and cultured until day 8. The cells were harvested by centrifugation and resuspended in saline. Flow cytometry was used to identify EGFRt molecules on the T cell surface, ensuring a positive rate of ≥30%. If the positive rate was lower, the cells were enriched and sorted using an EGFR-PE primary antibody and PE magnetic beads.

[0119] 2. Verification of CAR-T cell killing ability

[0120] 1) Different donor negative control WT-T, 2D11 and humanized 2D11-HM4, 1H07 and humanized 1H07-HM2 CAR-T killing target cells CHO-S (overexpressing CLDN3 / 4 / 6 / 9, a total of four groups), the effector-target ratio was 1:1 and 5:1, the results are as follows Figure 9 As shown, the results showed that several groups of CAR-T can effectively recognize and kill CLDN6-CHO-S cells.

[0121] 2) Adherent SKOV3 cell lines overexpressing CLDN3 / 4 / 6 / 9 were constructed and monitored for cytotoxicity by 731B2, 2D11, 2D11HM3, and 1H07HM2 CAR-T cells using RTCA. Lysis buffer served as a positive control, while SKOV3 cells were untreated. The specific experimental steps were as follows: 50 μL of culture medium was added to each well for background measurement. Target cells were then suspended in 100 μL of complete culture medium at an appropriate density (15,000 cells / well) and carefully added to each well of an E-Plate. The E-Plate was placed in the RTCA instrument, and cell index (CI) values ​​were continuously recorded for approximately 48 hours to monitor cell attachment and growth. 12-24 hours after seeding, recording was paused, the E-Plate was removed, and 50 μL of T cell suspension (at effector-to-target ratios of 1:1 and 5:1) or lysis buffer (positive control) was added to the target wells. The E-Plate was then quickly returned to the instrument for continued monitoring. The cell index (CI) and corresponding killing results of SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9 are shown in Figure 2. Figure 10 As shown, it was demonstrated that the candidate antibody could effectively and specifically kill SKOV3 cells overexpressing CLDN6.

[0122] Table 1. Sequence list of antibodies specifically targeting CLDN6

[0123] 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 in the heavy chain variable region as shown in SEQ ID NO: 16; Preferably, the numbering scheme of the CDR includes IMGT, Chothia, Kabat, AbM, Contact; Preferably, the numbering scheme of the CDRs is IMGT; Preferably, the amino acid sequences of HCDR1-HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 16 are shown in SEQ ID NOs: 13-15.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16; Preferably, the single-domain antibody comprises a heavy chain variable region obtained by humanizing the framework region, and the heavy chain variable region obtained by humanizing the framework region 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.

3. An antibody derivative, characterized in that The antibody derivative comprises any one of the following: 1) an antibody-marker conjugate, comprising the single domain antibody according to any one of claims 1 to 2 and a detectable marker conjugated thereto; 2) a chimeric antigen receptor comprising the heavy chain variable region of the single domain antibody according to any one of claims 1 to 2; 3) A pharmaceutical composition comprising the single domain antibody according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier; Preferably, 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.

4. The antibody derivative according to claim 3, characterized in that The chimeric antigen receptor comprises one or more of an extracellular hinge region, a transmembrane domain, an intracellular immunoreceptor tyrosine activation motif, and a co-stimulatory domain; Preferably, 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; Preferably, the extracellular hinge region is CD8 Hinge; Preferably, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO: 34; Preferably, 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; Preferably, the transmembrane domain is CD8 TM; Preferably, the amino acid sequence of the CD8 TM is as shown in SEQ ID NO: 35; Preferably, the intracellular immunoreceptor tyrosine-based activation motif is selected from the intracellular immunoreceptor tyrosine-based activation motifs 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; Preferably, the intracellular immunoreceptor tyrosine-based activation motif is a CD3ζ intracellular immunoreceptor tyrosine-based activation motif; Preferably, the amino acid sequence of CD3ζ is shown in SEQ ID NO: 36; Preferably, 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; Preferably, the costimulatory domain is 4-1BB; Preferably, the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 37; Preferably, the chimeric antigen receptor further comprises a suicide gene or a detectable tag; Preferably, the suicide gene or detectable tag comprises EGFRt; Preferably, the amino acid sequence of EGFRt is shown in SEQ ID NO: 38; Preferably, the suicide gene or detectable tag further comprises a signal peptide of EGFRt; Preferably, the amino acid sequence of the EGFRt signal peptide is shown in SEQ ID NO: 39; Preferably, the chimeric antigen receptor further comprises a linker; Preferably, the linker comprises a 2A peptide, an IRES; Preferably, the 2A peptide includes P2A, T2A, E2A and F2A; Preferably, the linker is T2A; Preferably, the amino acid sequence of T2A is shown in SEQ ID NO: 40; Preferably, the chimeric antigen receptor further comprises a signal peptide; Preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 41; Preferably, the chimeric antigen receptor further comprises an Fc sequence; Preferably, the Fc sequence is as shown in SEQ ID NO: 32; Preferably, the chimeric antigen receptor further comprises a membrane export signal peptide; Preferably, the sequence of the export signal peptide is shown as SEQ ID NO:

33.

5. Biomaterial comprising: (I) a nucleic acid molecule encoding the single domain antibody according to any one of claims 1 to 2 or the antibody derivative according to any one of claims 3 to 4; 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 3 to 4.

6. The biomaterial according to claim 5, 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 NO: 46; Preferably, the base sequences encoding HCDR1-HCDR3 are shown in SEQ ID NOs: 68-70; Preferably, the nucleic acid molecule comprises the base sequence encoding the heavy chain variable region as shown in SEQ ID NO: 46; Preferably, the nucleic acid molecule comprises the base sequence encoding the heavy chain variable region as shown in SEQ ID NO: 48; Preferably, the nucleic acid molecule comprises the base sequence encoding the heavy chain variable region as shown in SEQ ID NO: 49; Preferably, the nucleic acid molecule comprises the base sequence encoding the heavy chain variable region as shown in SEQ ID NO: 50; Preferably, the nucleic acid molecule comprises the base sequence encoding the heavy chain variable region as shown in SEQ ID NO:

51.

7. The biomaterial according to claim 5, characterized in that The nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 74; Preferably, the nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO: 75; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO: 76; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 77; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD3ζ as shown in SEQ ID NO: 78; Preferably, the nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO: 79; Preferably, the nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO: 80; Preferably, the nucleic acid molecule further comprises a base sequence encoding an EGFRt export signal as shown in SEQ ID NO: 81; Preferably, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 82; Preferably, the nucleic acid molecule further comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO:

83.

8. Use of the single domain antibody according to any one of claims 1 to 2, the chimeric antigen receptor in the antibody derivative according to any one of claims 3 to 4, and / or the nucleic acid molecule in the biomaterial according to any one of claims 5 to 7, characterized in that: The application includes any of the following: 1) Application in the preparation of products for detecting CLDN6 protein; 2) Application in the construction of bivalent antibodies targeting CLDN6; 3) Application in the construction of CAR-T cells specifically targeting CLDN6; 4) Application in the preparation of products for the treatment of CLDN6-positive related diseases.

9. The use according to claim 8, characterized in that The products for treating CLDN6-positive related diseases include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips; Preferably, the CLDN6-positive related diseases include solid tumors; Preferably, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer; Preferably, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors; Preferably, the ovarian epithelial tumor comprises an ovarian adenocarcinoma tumor; Preferably, the solid tumor is an ovarian adenocarcinoma tumor.

10. A method comprising any one 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 2 or the antibody-marker conjugate in the antibody derivative according to any one of claims 3 to 4, 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 2, the method comprising: culturing the recombinant host cell in the biomaterial according to any one of claims 5 to 7, and isolating the single domain antibody according to any one of claims 1 to 2 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 any one of claims 5 to 7 with ovarian adenocarcinoma cells; Preferably, the ovarian adenocarcinoma cell is SKOV3.

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