Antibody specifically targeting CLDN6 and use thereof in preparation of car-t cell

Abstract

Provided are an antibody specifically targeting CLDN6 and a use thereof in the preparation of a CAR-T cell. The provided CLDN6 antibody specifically targets CLDN6, but does not recognize CLDN3, CLDN4, or CLDN9 or exhibits significantly different recognition methods. A sequence of the antibody specifically targeting CLDN6A can be used to construct a chimeric antigen receptor (CAR) and a bivalent antibody (bispecific antibody). The CAR constructed on the basis of the sequence of the antibody can be transduced into a T cell to create a CAR-T cell specifically targeting CLDN6, which can be used for treating solid tumors such as ovarian cancer.

Description

Antibodies specifically targeting CLDN6 and their application in CAR-T cell preparation Technical Field

[0001] This invention relates to antibodies that specifically target CLDN6 and their application in the preparation of CAR-T cells, and relates to the fields of genetic engineering and antibody technology. Background Technology

[0002] CLDN6 (Claudin-6) is a tumor-specific protein expressed in a variety of solid tumors, including ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer, but is almost not expressed in healthy adult tissues. This specific expression pattern makes CLDN6 a highly promising target for cancer 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, as well as various glandular tissues and type II alveolar epithelial cells. CLDN4 has an even wider expression range, being most abundant in the gastrointestinal tract, and also expressed in membranes in adipose tissue, adrenal glands, tonsils, appendix, basal ganglia, bone marrow, mammary glands, bronchi, cerebellum, cerebral cortex, cervical plexus, choroid plexus, colon, duodenum, and endometrium. In contrast, CLDN9 expression is relatively limited, mainly playing an important role in the cochlea of ​​the inner ear, participating in the regulation of inner ear ion balance and hearing function. CLDN6 shares high structural homology with CLDN3, CLDN4, and CLDN9, especially with only a 3-amino acid difference in the extracellular domain between CLDN6 and CLDN9. The widespread expression of these family proteins also affects the safety of applications related to CLDN6 antibodies.

[0004] Against this backdrop, there is an urgent need for an antibody that can specifically target CLDN6 without binding to CLDN3, CLDN4, and CLDN9, so as to treat tumors without affecting the normal physiological functions of other claudin protein family members and improve the safety of antibody use. Summary of the Invention

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

[0006] The first aspect of the present invention provides a single-domain antibody that specifically targets CLDN6, said single-domain antibody having amino acid sequences of three CDR regions shown in SEQ ID NO:1-3, SEQ ID NO:5-7, SEQ ID NO:9-11, SEQ ID NO:13-15 or SEQ ID NO:17-19, or having amino acid sequences of three CDR regions that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to those in SEQ ID NO:1-3, SEQ ID NO:5-7, SEQ ID NO:9-11, SEQ ID NO:13-15, and SEQ ID NO:17-19, respectively.

[0007] In some embodiments, the single-domain antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 or SEQ ID NO:20, or has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:20.

[0008] In the context of this invention, the term "antibody" is used in the broadest sense and explicitly covers single-domain antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two intact antibodies, provided they exhibit the desired biological activity.

[0009] In this invention, the modified antibody sequence also falls within the scope of protection of this invention. The term "modification" refers to any form of modification to an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with a different amino acid. The term "deletion" refers to reducing one or more amino acids in the original amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence. In this invention, modification preferably occurs in regions other than variable regions, such as the constant region or frame region of the antibody, and the modified antibody retains the desired functional characteristics of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties. The term "identity," also known as "homology," refers to an amino acid sequence that is at least 80% identical to the sequence provided in this invention. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0010] In this invention, the CDR area is defined according to the IMGT numbering system.

[0011] Unless otherwise stated, the antibodies described herein are isolated antibodies. The term "isolated" as used herein means nucleic acids or antibodies or fragments thereof that have been extracted from their natural environment. "Isolated" nucleic acids or antibodies or fragments thereof therefore 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 through recombinant expression in host cells, as well as chemically synthesized nucleic acids and / or antibodies.

[0012] The second aspect of the present invention provides a humanized CLDN6 single-domain antibody, wherein the humanized CLDN6 single-domain antibody is obtained by humanizing residues at key positions in the frame region of SEQ ID NO:16 or SEQ ID NO:20 with reference to a human germline frame.

[0013] In some embodiments, the humanized CLDN6 single-domain antibody has the amino acid sequences of the three CDR regions shown in SEQ ID NO:13-15 or SEQ ID NO:17-19, or has the amino acid sequences of the three CDR regions that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to those in SEQ ID NO:13-15 and SEQ ID NO:17-19, respectively.

[0014] In some embodiments, the humanized CLDN6 single-domain antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, or has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.

[0015] In some embodiments, the humanized CLDN6 single-domain antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, or has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0016] A third aspect of the present invention provides a chimeric antigen receptor based on a single-domain antibody, wherein the chimeric antigen receptor comprises either the single-domain antibody described in the first aspect or the humanized CLDN6 single-domain antibody described in the second aspect.

[0017] In some embodiments, the chimeric antigen receptor further includes one or more of the following: an extracellular hinge region, a transmembrane domain, an intracellular immune receptor tyrosine activation motif, and a co-stimulatory domain.

[0018] In some embodiments, the chimeric antigen receptor further includes an extracellular signal peptide, EGFRt.

[0019] In some embodiments, the chimeric antigen receptor further includes the EGFRt signal peptide.

[0020] In some embodiments, the chimeric antigen receptor further includes a T2A linker.

[0021] In some embodiments, 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.

[0022] 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.

[0023] In some embodiments, the intracellular immune receptor tyrosine activation motif is selected from the intracellular immune receptor tyrosine 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.

[0024] 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.

[0025] In some embodiments, the extracellular hinge region is the CD8 extracellular hinge region.

[0026] In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

[0027] In some embodiments, the intracellular immune receptor tyrosine activation motif is the CD3ζ intracellular immune receptor tyrosine activation motif.

[0028] In some embodiments, the co-stimulation signaling domain is a 4-1BB co-stimulation signaling domain.

[0029] In some embodiments, those skilled in the art may change the combination of signal peptide, extracellular hinge region and transmembrane domain, co-stimulatory domain and intracellular immune receptor tyrosine activation motif 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 single-domain antibody or humanized CLDN6 single-domain antibody described in this invention, it falls within the protection scope of this invention.

[0030] In the most preferred embodiment, the chimeric antigen receptor is formed by sequentially connecting an extracellular signal peptide, the single-domain antibody described in the first aspect or the humanized CLDN6 single-domain antibody described in the second aspect, the CD8 extracellular hinge region, the CD8 transmembrane domain, the 4-1BB co-stimulatory signal domain, the CD3ζ intracellular immune receptor tyrosine activation motif, the T2A linker, the EGFRt signal peptide, and EGFRt.

[0031] The fourth aspect of the present invention provides an antibody derivative, which is an antibody-label conjugate, comprising any one of the single-domain antibody described in the first aspect or the humanized CLDN6 single-domain antibody described in the second aspect and a detectable label conjugated thereto.

[0032] In some embodiments, the detectable marker includes at least one of a radioactive isotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

[0033] The fifth aspect of the present invention provides a pharmaceutical composition comprising the single-domain antibody of the first aspect, the humanized CLDN6 single-domain antibody of the second aspect, the chimeric antigen receptor of the third aspect, or the antibody derivative of the fourth aspect.

[0034] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0035] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used non-toxic, pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0036] The pharmaceutical compositions of the present invention can also be used in combination with other drugs for treating solid tumors. These other compounds for treating solid tumors can be administered simultaneously with the main active ingredient (e.g., the antibody described in the first aspect of the invention), or even simultaneously in the same composition. Other therapeutic compounds can also be administered alone as a single composition or in a dosage form different from that of the main active ingredient.

[0037] In this invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not 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 skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0038] The sixth aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the single-domain antibody of the first aspect, the humanized CLDN6 single-domain antibody of the second aspect, or the chimeric antigen receptor of the third aspect.

[0039] In some embodiments, the nucleotide sequence is as shown in or is the same as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58. NO:58 has a nucleotide sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity.

[0040] In this 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 analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the antibody described in this invention is isolated, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0041] A seventh aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the sixth aspect.

[0042] In this invention, the terms "recombinant expression vector," "vector," and "expression vector" are used interchangeably to refer to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors may include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include lactoblastic multivacuolar viral vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, EB virus vectors, retroviral vectors, etc. The recombinant expression vector is suitable for in vitro or in vivo delivery systems, including viral vectors, lipid nanoparticles (LNPs), or other delivery vectors capable of delivering and expressing the nucleic acid molecules in a subject.

[0043] The eighth aspect of the present invention provides a recombinant host cell expressing the single-domain antibody of the first aspect, the humanized CLDN6 single-domain antibody of the second aspect, or the chimeric antigen receptor of the third aspect.

[0044] The recombinant host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. The host cell includes any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. Preferably, the recombinant host cell includes prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; even more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cell is a T cell.

[0045] In some embodiments, the recombinant host cell comprises the recombinant expression vector described in the seventh aspect.

[0046] In some embodiments, the recombinant host cell comprises a recombinant immune cell.

[0047] In some embodiments, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages.

[0048] In some embodiments, the recombinant immune cells are T cells.

[0049] The ninth aspect of the present invention provides any of the following applications, the applications including:

[0050] 1) The use of the single-domain antibody described in the first aspect, the humanized CLDN6 single-domain antibody described in the second aspect, or the antibody derivative described in the fourth aspect in the detection of CLDN6 protein or its antigen fragments.

[0051] 2) The use of the single-domain antibody described in the first aspect, the humanized CLDN6 single-domain antibody described in the second aspect, or the antibody derivative described in the fourth aspect in the preparation of products for detecting CLDN6 protein or its antigen fragments.

[0052] 3) The use of the single-domain antibody described in the first aspect, the humanized CLDN6 single-domain antibody described in the second aspect, the chimeric antigen receptor described in the third aspect, the nucleic acid molecule described in the sixth aspect, the recombinant expression vector described in the seventh aspect, or the recombinant host cell described in the eighth aspect in the preparation of drugs for treating CLDN6 positive tumors.

[0053] 4) The application of the nucleic acid molecule described in the sixth aspect or the recombinant expression vector described in the seventh aspect in the preparation of recombinant host cells, wherein the recombinant host cells are the recombinant host cells described in the eighth aspect.

[0054] 5) The application of the chimeric antigen receptor described in the third aspect in the construction of recombinant immune cells.

[0055] In some embodiments, the CLDN6-positive tumor is ovarian adenocarcinoma.

[0056] In some embodiments, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages.

[0057] In some embodiments, the recombinant immune cells are T cells.

[0058] The tenth aspect of the present invention provides any one of the following methods, the method comprising:

[0059] 1) A method for detecting CLDN6 protein or an antigen fragment thereof, the method comprising: contacting a sample to be tested with the single-domain antibody of the first aspect, the humanized CLDN6 single-domain antibody of the second aspect, or the antibody derivative of the fourth aspect, and detecting the formation of a complex of the antibody and CLDN6.

[0060] 2) A method for producing any one of the single-domain antibody of the first aspect, the humanized CLDN6 single-domain antibody of the second aspect, and the chimeric antigen receptor of the third aspect, the method comprising: culturing the recombinant host cell of the eighth aspect.

[0061] 3) A method for preparing recombinant host cells according to the eighth aspect, the method comprising: introducing the recombinant expression vector according to the seventh aspect into host cells.

[0062] 4) A method for treating a subject with a CLDN6-positive tumor, the method comprising: administering to the subject any one of the following: the single-domain antibody of the first aspect, the humanized CLDN6 single-domain antibody of the second aspect, the chimeric antigen receptor of the third aspect, recombinant immune cells constructed from the chimeric antigen receptor of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0063] In some embodiments, the administration method includes recombinant host cells prepared by ex vivo, immune cell reinfusion, or in vivo delivery expression system.

[0064] 5) A method for promoting apoptosis in ovarian adenocarcinoma cells, the method comprising: co-culturing any one of the chimeric antigen receptors described in the third aspect with ovarian adenocarcinoma cells.

[0065] In some embodiments, the ovarian adenocarcinoma cells are SKOV3.

[0066] In some embodiments, the subject includes mammals; in a specific embodiment of the invention, the subject is preferably a human.

[0067] In some embodiments, the antibodies of the present invention are obtained through artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention are obtained through direct amino acid synthesis. In some embodiments, the antibodies of the present invention are obtained through 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 enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0068] Advantages and beneficial effects of the present invention: The CLDN6 antibody provided by the present invention specifically targets CLDN6, and does not recognize CLDN3, CLDN4, or CLDN9, or recognizes them in significantly different ways. The antibody sequence specifically targeting CLDN6 can be used to construct chimeric antigen receptors (CARs) and bivalent antibodies (bispecific antibodies). CARs constructed based on the antibody sequence can be transduced into T cells to create CAR-T cells specifically targeting CLDN6, which can be used to treat solid tumors such as ovarian cancer. Attached Figure Description

[0069] Figure 1 shows the phage display library constructed by alpaca immunity.

[0070] Figure 2 shows the FACS binding verification results of CLDN6 transfection supernatant.

[0071] Figure 3 shows the results of the validation of the binding of the candidate antibody to cells expressing CLDN6-related homologous proteins.

[0072] Figure 4 shows the results of the validation of the binding of the purified candidate antibody to the CLDN6 homologous protein.

[0073] Figure 5 shows the results of the affinity analysis of the CLDN6 antibody.

[0074] Figure 6 shows the FACS results of the 2D11 humanized sequence.

[0075] Figure 7 shows the affinity test results of the 2D11 humanized antibody.

[0076] Figure 8 shows the FACS results of the 1H07 humanized sequence.

[0077] Figure 9 shows the affinity results of the 1H07 humanized sequence.

[0078] Figure 10 is a schematic diagram of CAR-T vector construction.

[0079] Figure 11 shows the kill rate of CLDN6-2D11 and humanized antibody 2D11-HM7 CAR-T cells.

[0080] Figure 12 shows the CAR-T kill rate detection graphs of CLDN6-2D11, humanized 2D11-HM4, 1H07, and humanized 1H07-HM2.

[0081] Figure 13 shows the killing rate of candidate antibodies against SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9. Detailed Implementation

[0082] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified. Simple modifications to the present invention based on its essence are all within the scope of protection claimed by the present invention.

[0083] Example 1: Construction of phage display library, antibody screening and humanization

[0084] 1. Alpaca immune phage display library construction

[0085] Two alpacas were immunized with 293F-CLDN6 at 14-day intervals. Seven days after the last immunization, peripheral blood was collected, and serum was separated for ELISA titer determination. After passing the titer test, peripheral blood cells (PBMCs) were isolated using lymphocyte separation medium. RNA was extracted and analyzed using PrimeScript. TM II. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit to prepare cDNA. The VHH sequence was amplified from the cDNA sample using a single-domain antibody cloning primer combination and subcloned into the phage display vector pDisplay. The resulting cDNA was then electrotransformed into SS320 competent cells to construct a single-domain antibody phage display library. A mixed library was constructed using two alpacas. Twenty-three clones were sequenced. Two antibody sequences with a one-base deletion and one overridden peak were removed. The remaining sequences were all antibody sequences with good diversity. The phage display library is shown in Figure 1.

[0086] 2. Select candidate antibody clones for specific identification and verification.

[0087] CLDN6-VLP and CHO-S-Lenti-CMV-hCLDN6 cells were used for panning, with CHO-S-Lenti-CMV-hCLDN6 cells used for negative screening. Based on phage ELISA, eight different antibody sequences were obtained through solid-phase and cell panning. After PCR, the sequences were subcloned into the pcDNA3.4-IgG1Fc expression vector via SfiI digestion and then used for FACS binding verification experiments on the CLDN6 transfection supernatant. The experiments were conducted using 3 × 10⁻⁶ cells. 5 Each well corresponds to either overexpressing or blank control cells. The primary antibody is 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). The PC (AB89A) used in Figure 2 is a positive control (clone number: AB89A, reference patent: CN111875703A). The experimental results are shown in Figure 2, indicating that candidate clones 1-D05 (SEQ ID NO:4), 1-G01 (SEQ ID NO:8), 1-H06 (SEQ ID NO:12), 1-H07 (SEQ ID NO:16), 2-D11 (SEQ ID NO:20), and 3-D12 bind more strongly to CHO-S-CLDN6 cells.

[0088] To verify the expression, purification, and binding of five candidate antibodies to cells overexpressing CLDN3 / 4 / 6 / 9, control cells CHO-S and 3 × 10⁶ CHO-S cells overexpressing CLDN3 / 4 / 6 / 9 were used. 5Cells were incubated with CLDN6 target candidate antibody (10 μg / ml, 100 μl / well) as primary antibody at room temperature for 1 hour. After washing the cells three times with PBS, they were incubated with PE-Goat anti-Human IgG Fc (invitrogen, Cat#:12-4998-82) (1:500 dilution) as secondary antibody at room temperature in the dark for 45 minutes. After washing the cells three times with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry. The results showed that the four purified antibodies 1-D05, 1-G01, 1-H06, and 2-D11 did not bind to CLDN3 and CLDN4 at all. 1-H07 bound strongly to CLDN4, but its binding was significantly different from that of CLDN6. Three antibodies, 2-D11, 1-H07, and 8-G02, were selected for further FACS binding assays (CLDN6 target candidate antibody concentrations were 30 μg / ml, 10 μg / ml, and 3 μg / ml, 100 μl / well). The results are shown in Figure 3, indicating that 1-H07 and 2-D11 were the most suitable. PC (731B2) used in Figure 3 served as a positive control (clone number: 731B2, reference patent: US11345731).

[0089] 3. Affinity verification of candidate antibodies: After purifying all candidate antibodies, they were serially diluted with cells overexpressing CLDN6 and CLDN9 (CLDN6 target candidate antibodies were serially diluted 3-fold at 10 points starting from 30 μg / ml). The results are shown in Figures 4 and 5. Only 1-H07 and 2-D11 did not bind to CLDN9. The other antibodies all bound at high concentrations, but the binding to CLDN6 was stronger. Overall, 2-D11 had better binding specificity.

[0090] 4. Results of humanization of CLDN6-2D11 clone

[0091] Based on the original 2D11 antibody sequence information, a homology model of the antibody was obtained through modeling, and CDRs were analyzed. The framework amino acids within a specific range typically influence the conformation of the CDR or its antigen-binding activity. Human germlines were obtained through IMGT analysis. The selected human germline framework was spliced ​​with the antibody's CDRs, and the framework region sequences of the designed humanized antibody were compared with those of the original antibody. Based on homology modeling results of the parent antibody, amino acids similar to those on the surface of the human antibody were selected for replacement while maintaining antibody activity and reducing heterology. Humanized antibody sequences 2D11-HM01~07 (amino acid sequences are shown in SEQ ID NO:25-31), totaling 7 sequences, were designed. The designed humanized antibodies were then genetically synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. Control cells CHO-S and CHO-S cells overexpressing CLDN3 / 4 / 6 / 9 were used. 5 Flow cytometry was performed on CLDN6 target candidate antibodies at concentrations of 30 μg / ml, 10 μg / ml, and 3 μg / ml, at 100 μl / well. The results are shown in Figure 6. Based on the above results, humanized antibodies HM3 (SEQ ID NO:27), HM4 (SEQ ID NO:28), and HM7 (SEQ ID NO:31) were selected for affinity testing using FACS EC50 (3-fold serial dilution at 10 spots). The results are shown in Figure 7, indicating that all candidate antibodies showed good binding results.

[0092] 5. Results of humanization of CLDN6 1H07 clone

[0093] Based on the original antibody sequence information of 1HM07, a homology model of the antibody was obtained through modeling, and CDRs were analyzed. The framework amino acids within a specific range typically influence the conformation of the CDR or its antigen-binding activity. Human germlines were obtained through IMGT analysis. The selected human germline framework was spliced ​​with the antibody's CDRs, and the framework region sequences of the designed humanized antibody were compared with those of the original antibody. Based on the homology modeling results of the parent antibody, amino acids similar to those on the surface of the human antibody were selected for replacement while maintaining antibody activity and reducing heterology. Humanized antibody sequences 1H07-HM01-04 (amino acid sequences are shown in SEQ ID NO: 21-24), totaling four sequences, were designed. The designed humanized antibodies were then genetically synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. Control cells CHO-S and CHO-S cells overexpressing CLDN3 / 4 / 6 / 9 were used. 5Flow cytometry was performed on CLDN6 target candidate antibody at concentrations of 30 μg / ml, 10 μg / ml, and 3 μg / ml, at 100 μl / well. The results are shown in Figure 8. Affinity was then measured at 10 wells using a 3-fold serial dilution starting from 30 μg / ml. The results are shown in Figure 9.

[0094] Example 2: Construction of CAR-T cells from candidate antibody sequences and verification of their killing ability

[0095] 1. Constructing CAR-T cells based on cloning sequences

[0096] The corresponding candidate antibody sequence and the positive control CAR-T antibody PC(731B2) were synthesized to construct the pCDH-EF1α lentiviral expression plasmid, as shown in the structural diagram in Figure 10. The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated, filtered, and the CAR lentivirus was stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads, and viral transduction was performed within 72 hours of activation. After 24 hours of transduction, the medium was changed, and the cells were cultured until day 8. Cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify EGFRt molecules on the surface of T cells, ensuring a positive rate greater than 30%. If the positive rate was lower, EGFR-PE primary antibody with PE magnetic beads was used for enrichment and sorting.

[0097] 2. Validation of CAR-T cell killing ability

[0098] 1) 2D11 and humanized 2D11-HM7, as well as control PC(731B2) CAR-T and negative control ctrl-T killed target cells CHO-S (four groups overexpressing CLDN3 / 4 / 6 / 9), with an effector-to-target ratio of 5:1. The results are shown in Figure 11. The results show that all groups of CAR-T can effectively recognize and kill CLDN6-CHO-S cells.

[0099] 2) Different donor negative controls WT-T, 2D11 and humanized 2D11-HM4, 1H07 and humanized 1H07-HM2 CAR-T killed target cells CHO-S (overexpressing CLDN3 / 4 / 6 / 9 in four groups), with effector-to-target ratios of 1:1 and 5:1. The results are shown in Figure 12. The results show that all groups of CAR-T can effectively recognize and kill CLDN6-CHO-S cells.

[0100] 3) SKOV3 cell lines overexpressing CLDN3 / 4 / 6 / 9 (adherent) were constructed, and RTCA was used to monitor the killing effect of PC(731B2), 2D11, 2D11HM3, and 1H07HM2 CAR-T cells. The lysis group served as a positive control with added lysis buffer, while SKOV3 was the untreated group. The specific experimental steps were as follows: 50 μL of culture medium was added to each well as background measurement. Then, the target cells were suspended at an appropriate density (15,000 cells / well) in 100 μL of complete culture medium 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. During the inoculation process, recording was paused 12-24 hours after inoculation, and the E-Plate was removed. 50 μL of T cell suspension (at an effector-to-target ratio 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. Figure 13 shows the cell index (CI) and corresponding killing results for SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9, demonstrating that the candidate antibody can effectively and specifically kill SKOV3 cells overexpressing CLDN6.

[0101] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A single-domain antibody specifically targeting CLDN6, characterized in that, The single-domain antibody has the amino acid sequences of the three CDR regions shown in SEQ ID NO:1-3, SEQ ID NO:5-7, SEQ ID NO:9-11, SEQ ID NO:13-15 or SEQ ID NO:17-19, or has the amino acid sequences of the three CDR regions that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to those in SEQ ID NO:1-3, SEQ ID NO:5-7, SEQ ID NO:9-11, SEQ ID NO:13-15, and SEQ ID NO:17-19, respectively. Preferably, the single-domain antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 or SEQ ID NO:20, or has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:

20.

2. A humanized CLDN6 single-domain antibody, characterized in that, The humanized CLDN6 single-domain antibody was obtained by humanizing residues at key positions in the frame region of SEQ ID NO:16 or SEQ ID NO:20 with reference to a human germline frame. Preferably, the humanized CLDN6 single-domain antibody has the amino acid sequences of the three CDR regions shown in SEQ ID NO:13-15 or SEQ ID NO:17-19, or has the amino acid sequences of the three CDR regions that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to those in SEQ ID NO:13-15 and SEQ ID NO:17-19, respectively. Preferably, the humanized CLDN6 single-domain antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, or has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:

24. Preferably, the humanized CLDN6 single-domain antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31, or has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:

31.

3. A chimeric antigen receptor based on a single-domain antibody, characterized in that, The chimeric antigen receptor comprises any one of the single-domain antibody of claim 1 or the humanized CLDN6 single-domain antibody of claim 2; preferably, the chimeric antigen receptor further comprises one or more of the following: an extracellular hinge region, a transmembrane domain, an intracellular immune receptor tyrosine activation motif, and a co-stimulatory domain. Preferably, the chimeric antigen receptor further includes an extracellular signal peptide and EGFRt; Preferably, the chimeric antigen receptor further includes the EGFRt signal peptide; Preferably, the chimeric antigen receptor further includes a T2A linker; More 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; More 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; More preferably, the intracellular immune receptor tyrosine activation motif is selected from the intracellular immune receptor tyrosine 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 their variants; More preferably, the co-stimulatory domain is selected from the co-stimulatory 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; Most preferably, the extracellular hinge region is the CD8 extracellular hinge region; Most preferably, the transmembrane domain is a CD8 transmembrane domain; Most preferably, the intracellular immune receptor tyrosine activation motif is the CD3ζ intracellular immune receptor tyrosine activation motif. Most preferably, the co-stimulation signal domain is a 4-1BB co-stimulation signal domain; Most preferably, the chimeric antigen receptor is any one of the following: an extracellular signal peptide, the single-domain antibody of claim 1 or the humanized CLDN6 single-domain antibody of claim 2, a CD8 extracellular hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signal domain, a CD3ζ intracellular immune receptor tyrosine activation motif, a T2A linker, an EGFRt signal peptide, and EGFRt, sequentially connected in series.

4. An antibody derivative, characterized in that, The antibody derivative is an antibody-label conjugate, which includes any one of the single-domain antibody of claim 1 or the humanized CLDN6 single-domain antibody of claim 2 and a detectable label conjugated thereto. Preferably, the detectable marker includes at least one of the following: radioactive isotopes, metal nanomaterials, fluorescein, biotin, avidin, biotin / avidin protein complex, biotin / avidin protein complex, chromophores, electron-dense substances, and enzymes.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, the chimeric antigen receptor of claim 3, or the antibody derivative of claim 4. Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, or the chimeric antigen receptor of claim 3; Preferably, the nucleotide sequence is as shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58, or is the same as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:

58. NO:58 has a nucleotide sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 6.

8. A recombinant host cell, characterized in that, The recombinant host cell expresses the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, or the chimeric antigen receptor of claim 3. Preferably, the recombinant host cell comprises the recombinant expression vector of claim 7; Preferably, the recombinant host cell comprises recombinant immune cells; More preferably, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages; Most preferably, the recombinant immune cells are T cells.

9. Any of the following applications, characterized in that, The applications include: 1) The use of the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, or the antibody derivative of claim 4 in the detection of CLDN6 protein or its antigen fragment; 2) The use of the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, or the antibody derivative of claim 4 in the preparation of products for detecting CLDN6 protein or its antigen fragments; 3) The use of the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, the chimeric antigen receptor of claim 3, the nucleic acid molecule of claim 6, the recombinant expression vector of claim 7, or the recombinant host cell of claim 8 in the preparation of a drug for treating CLDN6 positive tumors; 4) The use of the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7 in the preparation of recombinant host cells, wherein the recombinant host cell is the recombinant host cell of claim 8; 5) The application of the chimeric antigen receptor as described in claim 3 in the construction of recombinant immune cells; Preferably, the CLDN6-positive tumor is ovarian adenocarcinoma; Preferably, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages; Most preferably, the recombinant immune cells are T cells.

10. The following method, characterized in that, The method includes: 1) A method for detecting CLDN6 protein or its antigen fragment, the method comprising: contacting a sample to be tested with the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, or the antibody derivative of claim 4, and detecting the formation of a complex of the antibody and CLDN6; 2) A method for producing any one of the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, and the chimeric antigen receptor of claim 3, the method comprising: Cultivate the recombinant host cells as described in claim 8; 3) A method for preparing recombinant host cells according to claim 8, the method comprising: introducing the recombinant expression vector according to claim 7 into host cells; 4) A method for treating a subject with a CLDN6-positive tumor, the method comprising: administering to the subject the single-domain antibody of claim 1, the humanized CLDN6 single-domain antibody of claim 2, the chimeric antigen receptor of claim 3, recombinant immune cells constructed from the chimeric antigen receptor of claim 3, or the pharmaceutical composition of claim 5; 5) A method for promoting apoptosis of ovarian adenocarcinoma cells, the method comprising: co-culturing any one of the chimeric antigen receptors described in claim 3 with ovarian adenocarcinoma cells; Preferably, the ovarian adenocarcinoma cells are SKOV3.

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