Anti-CLDN6 nano antibody and related application thereof

Abstract

The invention discloses an anti-CLDN6 nano antibody and related application thereof, and relates to the field of antibodies. The invention provides an anti-CLDN6 nano antibody, which comprises HCDR1, HCDR2 and HCDR3 in a heavy chain variable region of which the amino acid sequence is as shown in SEQ ID NO: 4, the nano antibody has excellent antigen binding activity, high specificity and good affinity, has relatively strong killing activity on cells with high expression of CLDN6, and can be used for preparing anti-CLDN6 drugs. The requirements of CLDN6 positive tumor immunotherapy on efficient, specific and high-penetrability antibody molecules are met, and a new strategy and means are provided for tumor therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular, to anti-CLDN6 nanobodies and related applications thereof. BACKGROUND

[0002] The Claudin family is a class of four-transmembrane structure proteins involved in maintaining epithelial cell polarity and barrier function, which plays a key role in cell-cell junction, signal transduction and cell differentiation regulation. As an important member of the family, Claudin-6 (CLDN6) has a unique expression pattern and biological function: in normal adult tissues, the expression level of CLDN6 is very low or almost undetectable, and its expression is mainly limited to epithelial tissues in the early stage of embryonic development, so it is classified as an "oncofetal antigen". This strict spatiotemporal expression characteristic makes it have natural tumor targeting potential.

[0003] However, in a variety of malignant tumors, CLDN6 can occur abnormal reactivation expression (ectopic expression), especially in reproductive system malignancies. Studies have shown that CLDN6 is highly expressed in tumor tissues such as ovarian cancer, testicular embryonal carcinoma, endometrial cancer and some gastric cancer, and its expression level is closely related to low tumor differentiation, enhanced invasiveness and poor prognosis of patients. This "tumor-specific high expression, low expression in normal tissues" feature makes CLDN6 an ideal target for tumor immunotherapy, which can effectively reduce damage to normal tissues during treatment and improve treatment safety.

[0004] Based on the tumor-specific expression characteristics of CLDN6, new immunotherapy strategies targeting this target have become a research hotspot, including CAR-T cell therapy, bispecific antibodies (BiTE), antibody drug conjugates (ADC) and cancer vaccines, etc. For example, the CLDN6-CAR-T therapy (BNT211) developed by BioNTech combined with RNA vaccine (CARVac) has shown good safety and preliminary efficacy in clinical studies of recurrent ovarian cancer and testicular cancer, further verifying the clinical value of CLDN6 as a tumor-associated antigen.

[0005] Although CLDN6-targeted therapy has broad prospects, there are still many challenges in the existing technology: traditional antibody molecules have large molecular weight, limited tissue penetration, and insufficient enrichment efficiency in solid tumors; the affinity and specificity of some antibodies need to be improved, which may lead to off-target effects; in addition, there is still a lack of efficient, stable and tumor-penetrating antibody molecules targeting CLDN6, which limits its application in various treatment strategies.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The present application aims to provide anti-CLDN6 nanobodies and related applications thereof.

[0008] The present application is achieved in the following manner: In a first aspect, the embodiments of the present application provide an anti-CLDN6 nanobody, comprising: HCDR1, HCDR2 and HCDR3 in a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 4.

[0009] In a second aspect, the embodiments of the present application provide an antibody, comprising: the anti-CLDN6 nanobody according to the preceding embodiments.

[0010] In a third aspect, the embodiments of the present application provide an antibody conjugate, comprising: the anti-CLDN6 nanobody according to the preceding embodiments or the antibody according to the preceding embodiments.

[0011] In a fourth aspect, the embodiments of the present application provide a reagent or kit, comprising: the anti-CLDN6 nanobody according to the preceding embodiments, the antibody according to the preceding embodiments or the antibody conjugate according to the preceding embodiments.

[0012] In a fifth aspect, the embodiments of the present application provide an immunoconjugate or a pharmaceutical composition, comprising: the anti-CLDN6 nanobody according to the preceding embodiments or the antibody according to the preceding embodiments.

[0013] In a sixth aspect, the embodiments of the present application provide uses of the anti-CLDN6 nanobody according to the preceding embodiments, the antibody according to the preceding embodiments, the antibody conjugate according to the preceding embodiments or the immunoconjugate or the pharmaceutical composition according to the preceding embodiments in the manufacture of a product for preventing or treating a CLDN6-positive tumor, in the manufacture of a product for detecting CLDN6 or in the detection of CLDN6 for non-disease treatment or diagnosis purposes.

[0014] In a seventh aspect, the embodiments of the present application provide an isolated nucleic acid or a vector containing the nucleic acid, encoding the anti-CLDN6 nanobody according to the preceding embodiments or the antibody according to the preceding embodiments.

[0015] In an eighth aspect, the embodiments of the present application provide a recombinant cell containing the isolated nucleic acid or the vector containing the nucleic acid according to the preceding embodiments.

[0016] In a ninth aspect, the embodiments of the present application provide a method for preparing the anti-CLDN6 nanobody according to the preceding embodiments or the antibody according to the preceding embodiments, comprising: culturing the recombinant cell according to the preceding embodiments.

[0017] The present application has the following beneficial effects: The anti-CLDN6 nanobody provided by the application has excellent antigen binding activity, high specificity and good affinity, has strong killing activity on cells with high expression of CLDN6, meets the demand of CLDN6 positive tumor immunotherapy for high-efficiency, specific and high-penetration antibody molecules, and provides a new strategy and means for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 The supernatant of the nanobody screened in the embodiments of the application is used to determine the binding capacity by immunofluorescence; Figure 2 The binding capacity of the CLDN6 nanobody and the antigen is analyzed by indirect ELISA in the embodiments of the application; Figure 3 The binding specificity of the purified CLDN6 nanobody and CLDN6, CLDN3, CLDN4 and CLDN9 overexpressed cell lines is detected by immunofluorescence in the embodiments of the application; Figure 4 The construction of the gene fragment of the bispecific antibody (CLDN6xCD3); Figure 5 The killing efficiency of the bispecific antibody on the CLDN6 positive cell line is detected in the embodiments of the application; Figure 6 The survival period of the bispecific antibody on the mouse CDX transplantation model is analyzed in the embodiments of the application. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the application more clear, the technical solutions in the embodiments of the application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0021] The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, Second Ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0022] CLDN6 is a key factor in intracellular signaling pathways, with relatively complex structure and function, making it extremely difficult to screen specific nanobodies. Nanobodies, as a class of single-domain antibodies (VHH) derived from heavy chain antibodies of Camelidae, have unique advantages such as small molecular weight (about 15 kDa), strong tissue penetration, high stability, excellent affinity and easy engineering, and show great potential in tumor targeted therapy. The development of specific nanobodies against CLDN6 can overcome the shortcomings of traditional antibodies, provide key molecular tools for new therapies such as CLDN6-targeted CAR-T, ADC, and bispecific antibodies, and further promote the clinical translation of CLDN6-targeted tumor therapy.

[0023] The present application finally obtains specific anti-CLDN6 nanobodies through appropriate screening techniques and large-scale screening, constructs a bispecific antibody of the anti-CLDN6 nanobodies and anti-CD3 single-chain antibodies, and still maintains good antigen binding activity to CLDN6. In-vivo and in-vitro experiments prove that the bispecific antibody has strong killing activity to cells with high expression of CLDN6, effectively improves the treatment effect, and provides a key material for subsequent development of immunotherapy with high-efficiency and long-lasting anti-tumor activity.

[0024] NOMENCLATURE The term "antibody" herein is used in the broadest sense, and includes full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antigen binding fragments, as long as they exhibit the desired biological activity, such as specific binding to CLDN6 antigen or fragments thereof. An antigen binding fragment is a functional fragment of an antibody, and generally has the same binding specificity as the antibody from which it is derived. The antigen binding fragment includes any one selected from F(ab')2, Fab', Fab, Fv and scFv of an antibody. It is easy for those skilled in the art to understand that the functional fragment of the antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of splitting disulfide bonds by chemical reduction according to the content described in the present application. The antigen binding fragment can also be obtained by recombinant genetic engineering techniques known to those skilled in the art or by synthesis, for example, by an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems and the like.

[0025] The term "CDR" herein refers to "complementarity determining region", which refers to the highly variable regions of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding affinity of the antibody or antigen binding fragment to the antigen or epitope it recognizes.

[0026] The term "skeleton region" herein refers to "framework region" or "FR" region, which refers to the region of the heavy chain variable region of the antibody other than the CDR region; the heavy chain skeleton region can be further subdivided into adjacent regions (FR1, FR2, FR3 and FR4) separated by CDRs, wherein the heavy chain skeleton region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 skeleton regions. The heavy chain variable region is obtained by arranging the CDRs and FRs (arranged from the amino-terminal to the carboxy-terminal) as follows: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0027] The "multivalent antibody" herein is a polymer of monovalent antibodies that recognize the same epitope, and has higher antigen affinity than the corresponding monovalent antibody.

[0028] The "multispecific antibody" herein is a polymer of monovalent antibodies that bind to different targets or different binding regions on the same target, with stronger antigen recognition ability than the corresponding monovalent antibody.

[0029] The "chimeric antibody" herein is usually an antibody that fuses the variable region of a non-human antibody with the constant region or skeleton region of a human antibody, which can reduce the immune response induced by the non-human antibody.

[0030] The "fusion antibody" herein includes fusion nanobodies, including but not limited to new fusion molecules formed by combining other structures (such as BSA, IgG-Fc, etc.) through genetic engineering technology, such as enzymes that can prolong their half-life, antibacterial peptides, or imaging substances, etc.

[0031] The "treatment" in the present application includes preventing or reducing a certain condition, reducing the speed of the development of a certain condition, reducing the risk of developing a certain condition, preventing or delaying the development of symptoms related to a certain condition, reducing or terminating symptoms related to a certain condition, producing complete or partial reversal of a certain condition, curing a certain condition, or a combination thereof.

[0032] For cancer, "treatment" can mean inhibiting or slowing the growth, reproduction, or metastasis of tumor or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the development of the tumor, or some combination thereof.

[0033] In one aspect, the present application provides an anti-CLDN6 nanobody, which comprises: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 4.

[0034] In some embodiments, the HCDR1, HCDR2 and HCDR3 are defined by any one of Kabat, Chothia, IMGT, AbM or Contact systems.

[0035] In some embodiments, the antibody comprises: HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 1~3 in turn.

[0036] In some embodiments, the nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific antibody and a fusion nanobody. Nanobodies are small in molecular weight, encoded by a single gene, easy to genetically engineer, and can be polymerized by short connecting sequences to form multivalent or multispecific antibody structures.

[0037] In some embodiments, the heavy chain variable region further comprises a framework region.

[0038] In some embodiments, the amino acid sequence of the heavy chain variable region of the nanobody is set forth in SEQ ID NO: 4.

[0039] In another aspect, embodiments of the present application provide an antibody comprising the anti-CLDN6 nanobody of any of the preceding embodiments.

[0040] In some embodiments, the antibody comprises any one of a full-length antibody, a heavy chain antibody, a chimeric antibody, a multivalent antibody (bivalent, trivalent, tetravalent, pentavalent or hexavalent), a multispecific antibody and a fusion antibody.

[0041] In some embodiments, the multispecific antibody comprises any one of a bispecific antibody, a trispecific antibody and a tetraspecific antibody.

[0042] A bispecific antibody (BsAb) refers to an artificial antibody that can target two antigens or different epitopes of the same antigen simultaneously. A bispecific antibody can be IgG-like, i.e. a full-length bispecific antibody, or non-IgG-like bispecific antibody that is a non-full-length antibody construct. A full-length bispecific antibody typically retains the traditional monoclonal antibody (mAb) structure with two Fab arms and one Fc region, except that the two Fab sites bind different antigens. Non-full-length bispecific antibodies can lack the entire Fc region. These include chemically linked Fabs, composed only of Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFv). There are also fusion proteins that mimic the variable domains of two antibodies.

[0043] In some embodiments, the bispecific antibody comprises a bispecific T-cell engaging antibody (BiTE). The core function of a bispecific T-cell engaging antibody (BiTE) is to simultaneously target the CD3 molecule on the surface of T cells and a specific antigen on the surface of tumor cells through two different antigen binding domains, thereby directly "recruiting" T cells to the vicinity of tumor cells, activating the killing function of T cells, and achieving specific clearance of tumor cells.

[0044] In some embodiments, when the antibody is a bispecific antibody (bispecific T-cell engaging antibody), the bispecific antibody further comprises an antibody against CD3. It has been verified that the bispecific antibody constructed by the nanobody against CLDN6 and the antibody against CD3 has good anti-tumor activity and can be used to prepare a drug for preventing, diagnosing and treating at least one of ovarian cancer, testicular cancer, endometrial cancer, gastric cancer and lung cancer.

[0045] In some embodiments, the anti-CLDN6 Nanobody and the anti-CD3 antibody are linked by a linker peptide.

[0046] In some embodiments, the anti-CD3 antibody comprises a single-chain antibody against CD3.

[0047] In some embodiments, the amino acid sequence of the anti-CD3 single-chain antibody is set forth in SEQ ID NO: 5.

[0048] In some embodiments, the linker peptide is (G4S)n, n is a non-zero natural number.

[0049] In some embodiments, the n is 1-20.

[0050] In some embodiments, the n is 3 or 4.

[0051] In another aspect, embodiments of the present application provide an antibody conjugate comprising: the anti-CLDN6 Nanobody of any of the preceding embodiments or the antibody of any of the preceding embodiments.

[0052] In some embodiments, the antibody conjugate further comprises a label, a purification tag and / or a solid support coupled to the anti-CLDN6 Nanobody or the antibody or antigen-binding fragment thereof.

[0053] In some embodiments, the label comprises a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

[0054] In some embodiments, the fluorescent dye comprises, but is not limited to, a fluorescein-based dye and its derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxycoumarin (FAM), tetra-chloro-fluorescein (TET), and the like or analogs thereof), a rhodamine-based dye and its derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or analogs thereof), a Cy series dye and its derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, and the like or analogs thereof), an Alexa series dye and its derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like or analogs thereof), and a protein-based dye and its derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0055] In some embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0056] In some embodiments, the radioisotope includes, but is not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu, and 18 F.

[0057] In some embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, metachromatic animal fluorescein and its derivatives, ruthenium bispyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lucigenin and its derivatives, and peroxymalate and its derivatives.

[0058] In some embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0059] In some embodiments, the solid support includes, but is not limited to, microspheres, plates, and membranes.

[0060] In some embodiments, the solid support includes any one or more of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microwell plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0061] In another aspect, embodiments of the present application provide a reagent or a kit comprising: the anti-CLDN6 Nanobody of any of the foregoing embodiments, the antibody of any of the foregoing embodiments, or the antibody conjugate of any of the foregoing embodiments.

[0062] In another aspect, embodiments of the present application provide an immunoconjugate or a pharmaceutical composition comprising the anti-CLDN6 Nanobody of any of the foregoing embodiments or the antibody of any of the foregoing embodiments. In some embodiments, the immunoconjugate further comprises a therapeutic agent.

[0063] In some embodiments, the therapeutic agent comprises at least one of: a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signal molecule, a PD-L1 inhibitor, and a PD-1 / PD-L1 monoclonal antibody drug.

[0064] In some embodiments, the relevant biomarkers for immune checkpoint inhibitor therapy include PD-L1, MSI / bMSI, TMB / bTMB, TNB, and EGFR mutation, ALK fusion, TP53 mutation, KRAS mutation.

[0065] In some embodiments, the above-mentioned chemotherapeutic drug is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, flufenamide, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, danusertib, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecin, and saquinavir.

[0066] In some embodiments, the above-mentioned photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or a derivative thereof; (2) a photosensitizing compound containing a tetrapyrrole ring; (3) a traditional Chinese medicine photosensitizer; or (4) a combination of ALA or a derivative thereof with a compound in (2) or (3), respectively.

[0067] In some embodiments, the above-mentioned photothermal agent is selected from IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, and IR-26.

[0068] In some embodiments, the inhibitory second signal molecule can be PD-1; CTLA-4; PD-1 and CTLA-4.

[0069] In some embodiments, the PD-1 / PD-L1 mAb drug is selected from at least one of the following group: Nivolumab, Pembrolizumab, Pidilizumab, Lambrolizumab, BMS-936559, Atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDR001, REGN2810, SHR1210, STIA110X, STIA1110, TSR042, BMS-936558, BGB-A317, BCD-100 and JS001.

[0070] In some embodiments, the therapeutic agent further comprises a cytotoxic agent.

[0071] In some embodiments, the pharmaceutical composition comprises at least one of a pharmaceutically acceptable excipient, carrier and diluent. The carrier is a pharmaceutically acceptable carrier, including but not limited to a filler, lubricant, disintegrant, binder, glidant, etc.

[0072] In some embodiments, the pharmaceutically acceptable carrier includes but is not limited to polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, one of color indigo or a combination thereof.

[0073] In another aspect, embodiments of the present application provide use of the anti-CLDN6 Nanobody of any of the preceding embodiments, the antibody of any of the preceding embodiments, the antibody conjugate of any of the preceding embodiments or the immunoconjugate or pharmaceutical composition of any of the preceding embodiments in the manufacture of a product for preventing or treating a CLDN6-positive tumor, in the manufacture of a product for detecting CLDN6 or in detecting CLDN6 for non-disease treatment or diagnostic purposes.

[0074] In some embodiments, the CLDN6-positive tumor comprises at least one of ovarian cancer, testicular cancer, endometrial cancer, gastric cancer and lung cancer.

[0075] In some embodiments, the product comprises at least one of an immune cell, a reagent, a kit, a drug and a pharmaceutical composition.

[0076] In another aspect, the embodiments of the present application provide an isolated nucleic acid or a vector comprising the nucleic acid, which encodes the anti-CLDN6 Nanobody of any of the preceding embodiments or the antibody of any of the preceding embodiments.

[0077] In consideration of the degeneracy of codons, the gene sequence encoding the above antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody, or the modified gene can be artificially synthesized according to the codon bias of the host expressing the antibody to improve the expression efficiency of the antibody.

[0078] In some embodiments, the vector comprising the nucleic acid is a recombinant vector. The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and can refer to any recombinant polynucleotide construct that can directly introduce a DNA fragment of interest into a host cell by transformation, transfection or transduction for expression of the gene of interest.

[0079] In some embodiments, the recombinant vector is a plasmid or a virus; the virus is an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus or an oncolytic virus.

[0080] In another aspect, the embodiments of the present application provide a recombinant cell comprising the isolated nucleic acid or the vector comprising the nucleic acid of any of the preceding embodiments.

[0081] In some embodiments, the recombinant cell comprises a prokaryotic cell or a eukaryotic cell. The prokaryotic cell includes but is not limited to a bacterial cell such as Escherichia coli; the eukaryotic cell includes but is not limited to a yeast cell, an insect cell, an animal cell or a plant cell; the yeast cell can be but is not limited to a Pichia pastoris cell or a Saccharomyces cerevisiae cell. Alternatively, the recombinant cell can be a 293 cell, a 293T cell, a 293FT cell, a CHO cell or a Per6 cell. The 293 series of cells, the Per6 cell and the CHO cell are commonly used mammalian cells for producing antibodies or recombinant proteins, which are well known to those skilled in the art.

[0082] In addition, the embodiments of the present application provide a method for preparing the anti-CLDN6 Nanobody of any of the preceding embodiments or the antibody of any of the preceding embodiments, which comprises culturing the recombinant cell of any of the preceding embodiments.

[0083] The present application does not have specific limitations on the culture conditions of the host cell, and the culture conditions capable of allowing the host cell to express the antibody can be obtained based on conventional technical knowledge.

[0084] The features and properties of the present application are further described in detail below in combination with the embodiments.

[0085] All animal experiments were conducted in accordance with the animal ethics guidelines and were approved by the Animal Ethics Committee of the corresponding institution.

[0086] Example 1 Construction, panning and ELISA primary screening of phage nanobody library (1) Immunization of Bactrian camel Take 2 mg of CLDN6 extracellular domain recombinant protein, add 2 mL Freund's complete adjuvant, and emulsify with an emulsifier. Immunize the Bactrian camel by subcutaneous injection at multiple points on the neck, then immunize every two weeks (2 mg of protein), and use Freund's incomplete adjuvant, a total of 4 times. After the last immunization, collect peripheral blood to test the titer. Peripheral blood from the Bactrian camel is collected one week after the booster immunization to isolate lymphocytes.

[0087] (2) Construction of nanobody library When the camel reaches a certain immune titer, it is given a final booster immunization. Seven days later, 200 mL of peripheral blood is collected using a blood collection bag to isolate lymphocytes. The isolated lymphocytes are removed and RNA extraction is performed according to the steps of the Promega RNA extraction kit. Immediately after RNA extraction from the lymphocytes, reverse transcription cDNA is performed using the TaKaRa reverse transcription kit. Subsequently, the VHH gene is amplified using nested PCR. The amplified VHH gene is inserted into the pMECS phage display vector, and the TG1 competent cells are electroporated. The culture broth after electroporation is diluted by 10 times using LB / Amp-GLU medium, and then 100 μL of the diluted broth is spread on LB / Amp-GLU plates. The plates are incubated at 37°C for 8 h, and the colony counts at different dilutions are counted to calculate the library capacity, which is 6.56 x 1011. At the same time, 50 colonies with similar morphology and size are randomly selected and cultured for 4-6 h for bacterial liquid PCR to identify the positive rate of the library, i.e., the insertion rate of the library reaches 97%.

[0088] (3) Screening of CLDN6 nanobody First, the helper phage is prepared, concentrated, and rescued from the phage library. The nanobody phage library panning steps are as follows: Antigen coating: Dilute CLDN6-mFc recombinant protein with PBS, 20 μg per well (the antigen coating amount for the subsequent two rounds of panning is 10 μg / well and 5 μg / well, respectively), and coat in a 96-well enzyme-labeled plate at 4°C overnight. Washing: After overnight coating, discard the liquid in the wells, and wash each well with 200 μL of PBST for 5 times. Blocking: Add 200 μL of 5% skimmed milk powder to each well, and incubate at 37°C for 1 h. ​Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST; Incubation of recombinant phage: Dilute the recombinant phage with 5% skim milk powder to 5× Add 100 µL of pfu / mL to each well and incubate at room temperature for 2 h. Washing: Discard the liquid in the wells and wash each well 15 times with 200 μL PBST. Add 100 µL of freshly prepared 0.1M triethylamine to each well, incubate at room temperature for 10 min, transfer the eluent to a 1.5 mL centrifuge tube, and quickly add an equal volume of 1M Tris-HCl (pH=7.4) to neutralize. Recombinant phage titer determination: Collect the neutralized phage solution and determine the phage titer; infect 2 mL of TG1 in the logarithmic growth phase with the remaining phage solution and incubate at 37℃ for 30 min; add 8 mL of 2×YT / Amp GLU medium and incubate at 37℃ and 220 rpm until the logarithmic growth phase. Rescue: Add 8 mL of 2×YT ampicillin-resistant medium, add 4% glucose, and incubate at 37℃ and 220 rpm; bacteriophage concentration; Repeat the above steps - Then, a second and third round of screening will be carried out.

[0089] (4) Detection of specific recombinant phage enrichment Antigen Coating: Dilute the two antigens with PBS and coat each well with 400 ng of the antigen into a 96-well ELISA plate overnight at 4°C. Washing: After overnight coating, discard the liquid in the wells and wash each well three times with 200 μL PBST. Blocking: Add 200 μL of 5% skim milk powder to each well and block at 37°C for 1 h. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Incubation of Recombinant Phage: Dilute the phage concentrate (1:10) and add 100 μL to each well, incubate at 37°C for 1 h. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Secondary Antibody: Dilute HRP-labeled mouse anti-M13 secondary antibody 1:2000, add 100 μL / well, and incubate at 37°C for 1 h. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Development: Add 100 μL of TMB development solution to each well and incubate at room temperature in the dark for 10-15 min. Termination and reading: After development, add 2M... Add 50 μL to terminate the reaction; read the absorbance at 450 nm. Analyze the data.

[0090] (5) Sequencing analysis of specific nanobodies Clones with a value more than three times the negative value were identified as positive by ELISA detection and sent for bacterial culture sequencing and comparative analysis. Finally, the anti-CLDN6 nanobody was obtained and named CLDN6-Nb-7. The sequence of the nanobody is shown in Table 1.

[0091] Table 1. Amino acid sequences of nanobodies

[0092] (6) Specificity analysis of anti-CLDN6 protein nanobodies The recombinant supernatant of the screened nanobodies was used as primary antibody and co-incubated with CLDN6-overexpressing HeLa and wild-type HeLa, respectively. The results were detected using 594 anti-M13 antibody. Figure 1 As shown, the obtained nanobody can bind to CLDN6-overexpressing HeLa, but not to wild-type HeLa, indicating that the nanobody has good specific binding activity.

[0093] Example 2: Expression, purification, and reactivity with antigen of anti-CLDN6 nanobody A Nanobody-hFc fusion protein expression platform was constructed based on the pcDNA3.1 eukaryotic expression vector, and the CLDN6-Nb-7 antibody sequence was cloned into the pcDNA3.1-hFc-His vector. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE results showed that high-purity Nanobody-hFc fusion protein was obtained after affinity chromatography purification, with band sizes all around 55 kDa.

[0094] To assess the reactivity of the recombinant nanobody-hFc fusion protein with the antigen, 200 ng / well of CLDN6 recombinant protein was pre-coated onto an ELISA plate, incubated overnight at 4°C, and then the plate was blocked. Different amounts of recombinant nanobody were then added (dilution: ~10µg / mL), add secondary antibody, wash, develop color, terminate the reaction, and use an ELISA reader to measure the optical density at 450 nm (…). The binding affinity was determined by measuring the binding properties using a four-parameter nonlinear regression curve. The results showed that the anti-CLDN6 nanobody and the recombinant CLDN6 protein both exhibited high specificity binding. Figure 2 ).

[0095] Example 3: Immunofluorescence detection of the binding of recombinant nanobodies to CLDN6 positive cells HeLa cells were infected with lentiviruses containing the full-length genes of CLDN6, CLDN3, CLDN4, and CLDN9, and high-purity, stably CLDN6-expressing cells were obtained by flow cytometry sorting. Hela cells, and then the recombinant Nanobody prepared in Example 2 was added to the cells and incubated at 37°C for 40 min. After washing with PBS for 3 times, the APC@goat anti-human secondary antibody was added to the cells and incubated at 37°C for 40 min. After washing with PBS for 3 times, the cells were detected by flow cytometry. The results are shown in Fig. 2, which show that the recombinant Nanobody (Nanobody-hFc fusion protein) can bind to the above cells well. Figure 3

[0096] Example 4 Preparation of anti-CLDN6 and CD3 bispecific antibody The Nanobody CLDN6-Nb-7 in the present application was connected in series with the single-chain antibody of the anti-CD3 molecule (see Table 1) to a eukaryotic expression vector, transfected into HEK293T cells for expression, and purified by a Ni column. The construction is shown in Figure 4 The purified bispecific antibody (CLDN6-BiTE) was added to the co-culture system of T cells and OV-90 and OVCAR-3, and incubated for 24 h. The killing activity was evaluated by detecting the fluorescence intensity. The results are shown in Figure 5 Fig. 3, which show that the killing ability of the bispecific antibody and T cell group on tumor cells is significantly enhanced compared with the T cell group only. This indicates that the bispecific antibody can promote the killing ability of T cells.

[0097] Example 5 Anti-tumor effect verification of bispecific antibody in mouse CDX model The anti-tumor effect of the bispecific antibody (CLDN6-BiTE) was verified in a mouse CDX model (cell line-derived xenograft). The main purposes were to evaluate the in vivo anti-tumor activity, T cell-dependent mechanism of action, and dose-effect relationship. The following is a standard experimental procedure, which is suitable for in vivo pharmacodynamic verification of BiTE drugs targeting CD3 × TAA (such as CLDN6): To evaluate the in vivo anti-tumor activity of the bispecific T cell engaging antibody (BiTE) targeting (CLDN6 × CD3), a mouse CDX model was constructed. Human tumor cells expressing target antigens (such as OVCAR-3, OV-90) were cultured under standard conditions to the logarithmic growth phase, and then mixed with Matrigel at a ratio of 1:1, and the concentration was adjusted to 1 × cells / mL. Then, 6–8-week-old female NOD / SCID or NSG mice were subcutaneously inoculated with 100 µL of cell suspension (1 × cells in total) on the right side of the back. The tumor size was measured every 2–3 days during the tumor growth using a caliper.

[0098] When the tumor volume grew to about 80–100 mm ​At that time, participants were randomly assigned to either a control group or a low-dose BiTE group (0.1 mg / kg). In the humanized model, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. T cells (1 × BiTE (cells / mouse) was administered via tail vein injection 24 hours before or simultaneously with the first BiTE administration. BiTE antibody was administered intraperitoneally every other day for 2–3 weeks. Mouse body weight and tumor volume were continuously monitored throughout the treatment period. Tumor tissue and peripheral blood were collected for subsequent analysis after treatment or upon reaching the ethical endpoint. In terms of survival, mice in the BiTE-injected experimental group had a significantly longer survival time compared to the control group, as detailed in the following figures. Figure 6 As shown.

[0099] The above experiments collectively demonstrate that the anti-CLDN6 nanobody screened in this invention can specifically bind to the CLDN6 target, exhibiting high affinity, and can be widely applied to the prevention and treatment of tumors or autoimmune diseases, providing a new option for the prevention or treatment of tumors or autoimmune diseases.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanobody against CLDN6, characterized in that, It includes: The amino acid sequences are HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:

4.

2. The nanobody according to claim 1, characterized in that, The HCDR1, HCDR2, and HCDR3 are defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact. Optionally, the antibody comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1~3 in sequence; Optionally, the nanobody is at least one of monovalent nanobody, multivalent nanobody, multispecific antibody and fusion nanobody; Optionally, the amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO:

4.

3. An antibody, characterized in that, It includes: The anti-CLDN6 nanobody according to claim 1 or 2; Optionally, the antibody includes any one of the following: full-length antibody, heavy chain antibody, chimeric antibody, multivalent antibody, multispecific antibody, and fusion antibody; Optionally, the multispecific antibody includes any one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies; Optionally, the bispecific antibody includes a bispecific T-cell conjugating antibody; Optionally, when the antibody is a bispecific antibody, the bispecific antibody further includes: an anti-CD3 antibody; Optionally, the anti-CLDN6 nanobody and the anti-CD3 antibody are linked by a linker peptide; Optionally, the anti-CD3 antibody includes a single-chain antibody against CD3; Optionally, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:5; Optionally, the linker peptide is (G4S)n, where n is a non-zero natural number; Optionally, n is 1 to 20; Optionally, n is 3 or 4.

4. An antibody conjugate, characterized in that, It includes: The anti-CLDN6 nanobody according to claim 1 or 2, or the antibody according to claim 3; Optionally, the antibody conjugate may further include a label, purification tag, and / or solid-phase carrier conjugated to the anti-CLDN6 nanobody or the antibody or its antigen-binding fragment.

5. A reagent or kit, characterized in that, It includes: The anti-CLDN6 nanobody according to any one of claims 1 to 2, the antibody according to claim 3, or the antibody conjugate according to claim 4.

6. An immunoconjugate or pharmaceutical composition, characterized in that, It includes the anti-CLDN6 nanobody as described in claim 1 or 2, or the antibody as described in claim 3; Optionally, the immune conjugate further includes a therapeutic agent; Optionally, the therapeutic agent includes at least one of the following: chemotherapeutic drugs, radionuclides, photosensitizers, photothermal agents, immune checkpoint inhibitors, toxins, factors, kinase inhibitors, antibodies against inhibitory second signaling molecules, PD-L1 inhibitors, and PD-1 / PD-L1 monoclonal antibody drugs; Optionally, the pharmaceutical composition may further include at least one of a pharmaceutical excipient, a carrier, and a diluent.

7. The use of the anti-CLDN6 nanobody as described in any one of claims 1 to 2, the antibody as described in claim 3, the antibody-drug conjugate as described in claim 4, or the immunoconjugate or pharmaceutical composition as described in claim 6 in the preparation of products for the prevention or treatment of CLDN6-positive tumors, the preparation of products for the detection of CLDN6, or the detection of CLDN6 for non-disease treatment or diagnostic purposes; Optionally, the CLDN6 positive tumor includes at least one of ovarian cancer, testicular cancer, endometrial cancer, gastric cancer, and lung cancer; Optionally, the product includes at least one of: immune cells, reagents, kits, drugs, and drug compositions.

8. An isolated nucleic acid or a vector containing said nucleic acid, characterized in that, It encodes the anti-CLDN6 nanobody as described in any one of claims 1 to 2 or the antibody as described in claim 3.

9. A recombinant cell, characterized in that, It contains the isolated nucleic acid as described in claim 8 or a vector containing said nucleic acid.

10. A method for preparing the anti-CLDN6 nanobody according to any one of claims 1 to 2 or the antibody according to claim 3, characterized in that, It includes: Cultivate the recombinant cells as described in claim 9.

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