Nanobodies that identify different epitopes of CLDN6 and their applications

Abstract

This invention relates to the fields of antibody engineering and tumor diagnosis and treatment technology, specifically to CLDN6 nanobodies that recognize different epitopes and their related applications. To develop more nanobodies that recognize CLDN6, this invention provides two nanobodies capable of recognizing different epitopes of CLDN6, each recognizing a different antigenic epitope of the CLDN6 protein, wherein the different epitopes are spatially independent or partially overlapping. These nanobodies can be used alone or in combination and are suitable for tumor targeted therapy, immunotherapy, in vivo or in vitro diagnostics, and molecular imaging. Compared with existing technologies, the CLDN6 nanobodies provided by this invention have the advantages of small molecular weight, high stability, and strong specificity. Through a multi-epitope recognition strategy, the risk of antigen escape can be significantly reduced, and the targeting binding ability and application flexibility can be improved, making them of significant application value in the diagnosis and treatment of CLDN6-positive tumors.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antibody engineering technology, specifically to a nanobody capable of recognizing different epitopes of CLDN6, and the use of the nanobody in tumor diagnosis, treatment and related biomedical applications, including but not limited to the construction of bispecific antibodies, immune cell retargeting therapy, antibody-drug conjugates and applications in nucleic acid or protein delivery systems. Background Technology

[0002] The tight junction protein (CLaudins) family are important transmembrane proteins that maintain epithelial cell polarity and barrier function. Claudin-6 (CLDN6) is highly expressed during embryonic development but almost entirely absent in normal adult tissues. However, numerous studies have shown that CLDN6 is abnormally re-expressed in ovarian cancer, testicular cancer, and various solid tumors, and is closely related to tumor development, progression, and poor prognosis. Therefore, CLDN6 is considered an ideal targeting molecule with high tumor specificity, and holds significant promise for applications in tumor immunotherapy and precision medicine.

[0003] Currently, targeting strategies for CLDN6 mainly focus on traditional monoclonal antibodies, antibody fragments, or T-cell-based immunotherapies. However, existing antibodies mostly focus on a single antigenic epitope of CLDN6 and are largely derived from conventional antibody engineering routes, which suffer from problems such as large molecular weight, limited tissue penetration, and insufficient engineering flexibility. Furthermore, due to the high homology of extracellular domains among CLDN family members, antibodies targeting CLDN6 also face potential cross-reactivity risks, limiting their safety and clinical application.

[0004] Nanobodies, derived from heavy-chain antibodies of camelids, consist of only a single variable domain and possess significant advantages such as small molecular weight, structural stability, high affinity, good solubility, and ease of modular engineering. Compared to traditional antibodies, nanobodies can more easily penetrate dense tumor tissue and are suitable for constructing multivalent or multispecific molecules. However, current reports on nanobodies targeting CLDN6 are limited, especially lacking nanobodies capable of systematically recognizing different epitopes of CLDN6 and clearly defining their binding characteristics and application value.

[0005] From a functional application perspective, nanobodies capable of simultaneously or separately recognizing different epitopes of CLDN6 offer unique advantages in various application scenarios. For example, nanobodies with different epitopes can be used to construct bispecific or multispecific antibody molecules to achieve efficient redirection of immune cells; they can also be used in combination diagnostics and therapy (theranostics) to improve the selectivity and signal intensity of tumor targeting; furthermore, the synergistic or complementary binding of different epitopes can help reduce the risk of tumor antigen escape and improve the durability and reliability of treatment.

[0006] However, to date, there is a lack of nanobodies that systematically identify and validate different epitopes of CLDN6, as well as systematic application plans for such nanobodies in tumor therapy and related biomedical fields. Especially at the engineering application level, existing technologies have not fully revealed the application potential of CLDN6 nanobodies with different epitopes in bispecific antibody construction, T-cell redirection therapy, and in vivo expression after nucleic acid delivery. For example, CLDN6 nanobodies with different epitopes can serve as functional modules for constructing bispecific T-cell connectors (BiTEs) or other multispecific immune molecules to improve immune cell recruitment efficiency and reduce the risk of antigen escape. Simultaneously, these nanobodies can also be delivered via nucleic acid forms such as mRNA or self-amplified RNA (saRNA) for sustained in vivo expression, overcoming the problems of short half-life and frequent administration required by traditional protein drugs. However, the existing background technologies have not yet established a complete technical system centered on "CLDN6 nanobodies with different epitopes" and capable of expanding to the aforementioned multiple therapeutic modalities. Therefore, there is an urgent need to provide a nanobody that can recognize different epitopes of CLDN6 and to systematically elucidate its technical solutions in various immunotherapy and nucleic acid delivery application scenarios, so as to lay the foundation for the development of related technologies in the future.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The technical problem to be solved by this invention is that existing targeted antibodies against CLDN6 are mostly concentrated on a single epitope, lack engineering flexibility, and are difficult to meet the diverse needs of tumor immunotherapy.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: two nanobodies capable of recognizing different epitopes of CLDN6 are provided, wherein the nanobodies capable of recognizing different epitopes of CLDN6 respectively recognize different antigenic epitopes of the CLDN6 protein.

[0010] Furthermore, the different antigenic epitopes are spatially independent epitopes on the CLDN6 molecule; even further, the different antigenic epitopes are partially overlapping but not completely identical epitopes; even further, the antigenic epitopes are located in the extracellular domain of CLDN6.

[0011] Furthermore, the heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO:1 or SEQ ID NO:4, the amino acid sequence of CDR2 is shown in SEQ ID NO:2 or SEQ ID NO:5, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3 or SEQ ID NO:6.

[0012] Furthermore, in the aforementioned nanobodies capable of recognizing different epitopes of CLDN6, the CDR1 amino acid sequence of the heavy chain variable region of nanobodies 1 is shown in SEQ ID NO:1, the CDR2 amino acid sequence is shown in SEQ ID NO:2, and the CDR3 amino acid sequence is shown in SEQ ID NO:3; the CDR1 amino acid sequence of the heavy chain variable region of nanobodies 2 is shown in SEQ ID NO:4, the CDR2 amino acid sequence is shown in SEQ ID NO:5, and the CDR3 amino acid sequence is shown in SEQ ID NO:6.

[0013] Among them, the nanobodies that can recognize different epitopes of CLDN6 also include a backbone region, and the structure of the heavy chain variable region of the backbone region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0014] Among them, the amino acid sequences of the nanobodies that can recognize different epitopes of CLDN6 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0015] Among the nanobodies that can recognize different epitopes of CLDN6, the nanobodies are at least one of monovalent nanobodies, multivalent nanobodies, multispecific antibodies, and fusion nanobodies.

[0016] The "multivalent nanobodies" in this invention are polymers of monovalent antibodies that recognize different epitopes of the same antigen, exhibiting higher antigen affinity than monovalent antibodies. The "multispecific antibodies" are polymers of monovalent antibodies that recognize different antigenic epitopes, capable of binding to different epitopes of different targets, and possessing higher antigen recognition capabilities than monovalent antibodies. Fusion nanobodies include, but are not limited to, new fusion molecules formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering techniques, such as enzymes, antimicrobial peptides, or imaging substances that can extend their half-life.

[0017] In a second aspect, the present invention also provides an engineered construct comprising the above-described nanobodies capable of recognizing different epitopes of CLDN6. The engineered construct is at least one of a multivalent antibody, a multispecific antibody, or an antibody fusion protein.

[0018] Furthermore, the engineered construct is a bispecific antibody, which is constructed by tandemly linking the above-mentioned nanobodies capable of recognizing different epitopes of CLDN6.

[0019] Furthermore, the engineered construct is a multispecific antibody, with one end containing at least one nanobody capable of recognizing different epitopes of CLDN6, and the other end containing a binding unit capable of binding to molecules on the surface of immune effector cells, the two being linked by a linker peptide. Even further, the other end is an anti-CD3 single-chain antibody, with the amino acid sequence shown in SEQ ID NO:9.

[0020] In some embodiments, the linker peptide is (G4S)n, where n is a non-zero natural number.

[0021] In some embodiments, n is 1 to 20.

[0022] In some embodiments, n is 3 or 4.

[0023] This engineered construct can be used to mediate the recognition and killing of CLDN6-positive target cells by immune cells.

[0024] In a third aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned nanoantibody that recognizes different epitopes of CLDN6 or the above-mentioned engineered construct.

[0025] Furthermore, the nucleic acid molecule is in the form of DNA, mRNA, or self-amplified RNA. It is used for the expression of corresponding nanobodies or engineered constructs in vivo or in vitro.

[0026] In a fourth aspect, the present invention also provides a pharmaceutical composition comprising the above-described nanobody, engineered construct, or nucleic acid molecule encoding the above-described nanobody or engineered construct, and a pharmaceutically acceptable carrier, excipient, or delivery system.

[0027] In a fifth aspect, the present invention provides the use of the above-described nanobody, engineered construct, or nucleic acid molecule that recognizes different epitopes of CLDN6 in the preparation of a medicament for the prevention or treatment of tumors or in the preparation of a reagent for the diagnosis of tumors. The tumor is a CLDN6-positive tumor. Further, the tumor is at least one of the following: ovarian cancer, testicular cancer, endometrial cancer, gastric cancer, embryonal tumors, glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, soft tissue sarcoma, acute and chronic leukemia, Hodgkin's and non-Hodgkin's lymphomas, and head and neck tumors.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] This invention provides, for the first time, a class of nanobodies capable of systematically recognizing different epitopes of CLDN6, enriching the types of CLDN6-targeting antibodies and providing new molecular tools for subsequent antibody engineering and immunotherapy. Due to their small molecular weight and structural stability, the nanobodies described in this invention exhibit excellent tissue penetration and engineerable adaptability, making them suitable for the construction of multivalent, multispecific antibodies and other fusion molecules. This invention screened two nanobodies recognizing different epitopes; their tandem combination can reduce the risk of tumor antigen escape during treatment, improving the stability and durability of CLDN6-targeted therapy. Furthermore, the nanobodies described in this invention can be delivered in protein or nucleic acid form, and are particularly suitable for use in conjunction with mRNA or self-amplified RNA delivery platforms, thereby achieving sustained in vivo expression and improving therapeutic efficacy. This invention provides a highly flexible and scalable technical solution for the diagnosis and treatment of CLDN6-positive tumors, possessing significant scientific research value and potential clinical application prospects. Attached Figure Description

[0030] To facilitate the explanation of the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings. However, the scope of protection of the present invention is not limited to the embodiments shown in the following drawings.

[0031] Figure 1 The schematic diagram shows the structure of the CLDN6 protein, including the location distribution of its transmembrane and extracellular domains, to illustrate the region where the CLDN6 epitope recognized by the nanobody of the present invention is located.

[0032] Figure 2 In this embodiment of the invention, immunofluorescence was used to detect the binding specificity of the purified CLDN6 nanobody to the CLDN6 overexpressing cell line.

[0033] Figure 3 This invention demonstrates the binding characteristics of two types of nanobodies that recognize CLDN6, which cross-recognize CLDN6 derived from human, mouse, or non-human primates.

[0034] Figure 4 The illustration schematically shows the binding characteristics of two types of nanobodies that recognize CLDN6 to the extracellular domain 2 of CLDN6 in this invention.

[0035] Figure 5 This is used to illustrate the differences in spatial binding properties between the two types of nanobodies.

[0036] Figure 6 A schematic diagram of a T-cell connector incorporating the nanobody of the present invention is shown, wherein the nanobody is used in the molecule as a CLDN6 binding module.

[0037] Figure 7 The diagram illustrates the expression of T-cell connector nucleic acid molecules constructed from nanobodies based on different epitopes of CLDN6. The nucleic acid molecules are in the form of self-amplifying RNA (saRNA) and are used to express the corresponding nanobodies or engineered constructs in vivo or in vitro.

[0038] Figure 8 This demonstrates how T-cell connectives constructed from nanobodies based on different CLDN6 epitopes, delivered in the form of saRNA, effectively mediate the killing effect of immune effector cells on CLDN6-positive target cells. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0041] CLDN6 is a key factor in intracellular signaling pathways, with a relatively complex structure and function, making the screening of specific nanobodies extremely difficult. Nanobodies, as a class of single-domain antibodies (VHHs) derived from camel heavy chain antibodies, possess unique advantages such as small molecular weight (approximately 15 kDa), strong tissue penetration, high stability, excellent affinity, and ease of engineering, demonstrating great potential in targeted tumor therapy. Developing specific nanobodies targeting CLDN6 can overcome the shortcomings of traditional antibodies, providing key molecular tools for novel CLDN6-targeted therapies such as CAR-T, ADC, and bispecific antibodies, further promoting the clinical translation of CLDN6-targeted tumor therapy.

[0042] This invention, through appropriate screening techniques and large-scale screening, ultimately obtained specific anti-CLDN6 nanobodies. Furthermore, it was discovered that the two anti-CLDN6 nanobodies obtained in this invention are nanobodies that recognize different epitopes of CLDN6. These different epitopes are located in different extracellular regions of CLDN6, and there is no significant epitope competition or only partial competition between the nanobodies in space.

[0043] The anti-CLDN6 nanobody and anti-CD3 single-chain antibody obtained by screening in this invention were used to construct a bispecific antibody that still maintains good antigen-binding activity against CLDN6. In vivo and in vitro experiments confirmed that this bispecific antibody has strong killing activity against cells with high CLDN6 expression, effectively improving the therapeutic effect and providing a key material for the subsequent development of immunotherapies with highly efficient and durable anti-tumor activity.

[0044] Definition of noun

[0045] The term "antibody" as used herein is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antigen-binding fragments, provided they exhibit the desired biological activity, such as specific binding to the CLDN6 antigen or fragments thereof. An antigen-binding fragment, also known as a functional fragment of an antibody, typically has the same binding specificity as the antibody from which it originates. Antigen-binding fragments include any one selected from F(ab')2, Fab', Fab, Fv, and scFv of antibodies. Those skilled in the art will readily understand from the description of this invention that the aforementioned functional fragments of antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. The aforementioned antigen-binding fragments can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by synthesis, for example, by automated peptide synthesizers, such as those sold by Applied BioSystems.

[0046] In this article, the term "CDR" stands for "complementarity-determining region," which refers to the highly variable region of the heavy and light chains of an immunoglobulin, specifically the region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0047] The term "backbone region" in this article, synonymous with "framework region" or "FR region," refers to the region of the antibody's heavy chain variable region excluding the CDR region. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), including the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0048] The “multivalent antibody” in this article is a polymer of a monovalent antibody that recognizes the same epitope and has a higher antigen affinity than the corresponding monovalent antibody.

[0049] The "multispecific antibody" in this article is a polymer of monovalent antibodies that bind to different targets or different binding regions on the same target, and has a stronger antigen recognition ability than the corresponding monovalent antibody.

[0050] The “chimeric antibody” mentioned in this article is usually an antibody formed by fusing the variable region of a non-human antibody with the constant region or backbone region of a human antibody, which can reduce the immune response induced by non-human antibodies.

[0051] The term "fusion antibody" in this article includes fusion nanobodies, including but not limited to those formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering techniques to form new fusion molecules, such as enzymes, antimicrobial peptides, or imaging substances that can extend their half-life.

[0052] The term "treatment" in this invention includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0053] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0054] On the one hand, the anti-CLDN6 nanobody provided in the embodiments of the present invention includes: CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO:7 or SEQ ID NO:8.

[0055] In some embodiments, CDR1, CDR2, and CDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

[0056] In some embodiments, the antibody comprises: CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO:1 to 6 in sequence.

[0057] In some embodiments, the nanobody is at least one of monovalent nanobody, multivalent nanobody, multispecific antibody, and fusion nanobody. Nanobodies, due to their small molecular weight, are encoded by a single gene, are easily genetically engineered, and multiple nanobodies can be aggregated through short linker sequences to form multivalent or multispecific antibody structures.

[0058] In some embodiments, the heavy chain variable region further includes a skeleton region.

[0059] In some embodiments, the amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO:7-8.

[0060] On the other hand, embodiments of the present invention provide an antibody comprising: the anti-CLDN6 nanobody described in any of the foregoing embodiments.

[0061] In some embodiments, the antibody includes any one of the following: full-length antibody, heavy chain antibody, chimeric antibody, multivalent antibody (bivalent, trivalent, tetravalent, pentavalent or hexavalent), multispecific antibody and fusion antibody.

[0062] In some embodiments, the multispecific antibody includes any one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.

[0063] Bispecific antibodies (BsAbs) are artificial antibodies that can simultaneously target two antigens or different epitopes of the same antigen. Bispecific antibodies can be IgG-like (full-length bispecific antibodies) or non-IgG-like bispecific antibodies that are non-full-length antibody constructs. Full-length bispecific antibodies typically retain the structure of a traditional monoclonal antibody (mAb) with two Fab arms and an Fc region, but the two Fab sites bind to different antigens. Non-full-length bispecific antibodies may lack the entire Fc region. These include chemically linked Fabs, Fab regions alone, and various types of bivalent and trivalent single-chain variable fragments (scFvs). Fusion proteins that mimic the variable domains of two antibodies also exist.

[0064] In some embodiments, bispecific antibodies include bispecific T-cell conjugating antibodies (BiTEs). The core function of bispecific T-cell conjugating antibodies (BiTEs) is to simultaneously target CD3 molecules on the surface of T cells and specific antigens 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.

[0065] In some embodiments, when the antibody is a bispecific antibody (bispecific T-cell conjugating antibody), the bispecific antibody further includes an anti-CD3 antibody. It has been verified that the bispecific antibody obtained by constructing an anti-CLDN6 nanobody and an anti-CD3 antibody has good anti-tumor activity and can be used to prepare drugs for the prevention, diagnosis, and treatment of at least one of ovarian cancer, testicular cancer, endometrial cancer, gastric cancer, and lung cancer.

[0066] In some embodiments, the anti-CLDN6 nanobody and the anti-CD3 antibody are linked by a linker peptide.

[0067] In some embodiments, the anti-CD3 antibody comprises a single-chain antibody against CD3.

[0068] In some embodiments, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:9.

[0069] In some embodiments, the linker peptide is (G4S)n, where n is a non-zero natural number.

[0070] In some embodiments, n is 1 to 20.

[0071] In some embodiments, n is 3 or 4.

[0072] On the other hand, embodiments of the present invention provide the use of anti-CLDN6 nanobodies, antibodies, antibody conjugates, or immunoconjugates or pharmaceutical compositions as described in any of the foregoing embodiments 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.

[0073] In some embodiments, the CLDN6 positive tumor includes at least one of ovarian cancer, testicular cancer, endometrial cancer, gastric cancer, and lung cancer.

[0074] In some embodiments, the product includes at least one of immune cells, reagents, kits, drugs, and drug compositions.

[0075] On the other hand, embodiments of the present invention provide an isolated nucleic acid or a carrier containing said nucleic acid, which encodes the anti-CLDN6 nanobody or the antibody described in any of the foregoing embodiments.

[0076] Considering the degeneracy of codons, the gene sequence encoding the above antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0077] In some embodiments, the vector containing the nucleic acid includes a recombinant vector. The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can directly introduce the target DNA fragment into the host cell for target gene expression via transformation, transfection, or transduction.

[0078] In some embodiments, the nucleic acid molecule may be in the form of DNA, mRNA, or saRNA, and may be used to express the corresponding nanobodies or engineered constructs in vivo or in vitro.

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

[0080] On the other hand, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid described in any of the foregoing embodiments or a vector containing the nucleic acid.

[0081] In some embodiments, the recombinant cells include prokaryotic or eukaryotic cells. The prokaryotic cells include, but are not limited to, bacterial cells, such as *Escherichia coli*; the eukaryotic cells include, but are not limited to, yeast cells, insect cells, animal cells, or plant cells; the yeast cells may be, but are not limited to, *Pichia pastoris* or *Saccharomyces cerevisiae* cells. Optionally, the recombinant cells may be 293 cells, 293T cells, 293FT cells, CHO cells, or Per6 cells. The 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0082] Furthermore, embodiments of the present invention provide a method for preparing anti-CLDN6 nanobodies or antibodies as described in any of the foregoing embodiments, comprising: culturing recombinant cells as described in any of the foregoing embodiments.

[0083] The present invention does not specifically limit the culture conditions of the host cells; culture conditions that enable the host cells to express and produce the antibody can be obtained based on conventional technical knowledge.

[0084] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0085] All animal experiments are conducted in accordance with animal ethics guidelines and are carried out only after being approved by the relevant institution's animal experiment ethics committee.

[0086] Example 1: Construction, panning, and preliminary ELISA screening of phage nanobody libraries

[0087] (1) Bactrian camel immunization

[0088] Two mg of recombinant CLDN6 extracellular domain protein (CLDN6 protein structure shown in Figure 1) was added to 2 mL of Freund's complete adjuvant and emulsified thoroughly using an emulsifier. This protein was then injected subcutaneously at multiple sites in the neck of Bactrian camels for immunization. Subsequent immunizations (2 mg protein) were administered every two weeks for a total of four immunizations. Peripheral blood was collected after the final immunization to determine the titer. One week after the initial immunization, peripheral blood was collected from Bactrian camels to separate lymphocytes.

[0089] (2) Construction of nanobody library

[0090] Once the camels reached a certain immune titer, they underwent a final shock immunization. Seven days later, 200 mL of peripheral blood was collected using a blood collection bag for lymphocyte isolation. The isolated lymphocytes were then subjected to RNA extraction according to the Promega RNA extraction kit. Immediately after RNA extraction, cDNA was reverse transcribed using the TaKaRa reverse transcription kit, followed by nested PCR amplification of the VHH gene. The amplified VHH gene was inserted into the pMECS phage display vector and electroporated into TG1 competent cells. The electroporated culture was serially diluted (10-fold) using LB / Amp-GLU medium, and then 10... -4 10 -5 10 -6 10 -7 100 µL of the dilution buffer was spread onto LB / Amp-GLU plates and incubated upside down at 37°C for 8 hours. The colony counts at different dilutions were then used to calculate the antibody library capacity, which was 6.56 × 10⁻⁶. 9 Simultaneously, 50 colonies of similar morphology and size were randomly selected and cultured for 4-6 hours, followed by bacterial PCR to identify the library positivity rate, i.e., the insertion rate of the library reached 97%.

[0091] (3) Screening of CLDN6 nanobodies

[0092] First, the preparation, concentration, and phage library rescue of helper phages were performed. The panning steps for the nanobody phage library are as follows: ① Antigen coating: After diluting the CLDN6-mFc recombinant protein with PBS, 20 μg of antigen was coated per well (the antigen coating amounts for the subsequent two rounds of panning were 10 μg / well and 5 μg / well, respectively) into a 96-well ELISA plate and incubated overnight at 4°C; ② Washing: After overnight coating, the liquid in the wells was discarded, and each well was washed 5 times with 200 μL PBST; ③ Blocking: 200 μL of 5% skim milk powder was added to each well and the plate was incubated at 37°C for 1 h; ④ Washing: The liquid in the wells was discarded, and each well was washed 3 times with 200 μL PBST; ⑤ Incubation of recombinant phages: The recombinant phages were diluted with 5% skim milk powder to a concentration of 5 × 10⁻⁶. 11 Add 100 µL of pfu / mL to each well and incubate at room temperature for 2 h; ⑥ Wash: 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) for neutralization; ⑦ 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; ⑧ Rescue: Add 8 mL of 2×YT ampicillin-resistant medium, add 4% glucose, and incubate at 37℃ and 220 rpm; ⑨ Phage concentration; ⑩ Repeat steps ①-⑨ above for the second and third rounds of screening.

[0093] (4) Detection of specific recombinant phage enrichment

[0094] 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. Color development: Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 10-15 min. Termination and reading: After color development, add 50 μL of 2M H₂SO₄ to each well to terminate the reaction; read the absorbance at 450 nm. Analyze the data.

[0095] (5) Sequencing analysis of specific nanobodies

[0096] Clones with a negative value more than three times the negative value were identified as positive by ELISA test results and sent for bacterial culture sequencing and comparative analysis. Finally, anti-CLDN6 nanobody was obtained and named CLDN6-Nb-1. The sequence of CLDN6-Nb2 nanobody is shown in Table 1.

[0097]

[0098] Example 2: Identification of the specificity and species recognition ability of anti-CLDN6 nanobodies

[0099] (1) Specificity analysis of anti-CLDN6 protein nanobodies

[0100] 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-hFc antibody. Figure 2 As shown, the two obtained nanobodies can bind to CLDN6-overexpressing HeLa, but not to wild-type HeLa, indicating that both nanobodies have good specific binding activity.

[0101] (2) Species identification analysis of anti-CLDN6 protein nanobodies

[0102] The recombinant supernatant of the screened nanobodies was used as primary antibody and co-incubated with CLDN6-overexpressing HeLa from human, mouse, and cynomolgus monkeys, as well as wild-type HeLa. The results were detected using APC anti-hFc antibody. Figure 3As shown, the two obtained nanobodies can bind to HeLa overexpressed CLDN6 from human, mouse, or cynomolgus monkey sources, but do not bind to wild-type HeLa, indicating that the nanobodies have good specific binding activity and the ability to cross-recognize CLDN6 from human, mouse, or cynomolgus monkey sources.

[0103] Example 3: Epitope Localization Analysis of Nanobody with Interchangeable CLDN6 and CLDN9 Domains

[0104] This embodiment further confirms the epitope location of the anti-CLDN6 nanobody of the present invention by constructing a chimeric protein with domain interchange between CLDN6 and its family member CLDN9, clarifying whether it recognizes the extracellular domain and specific extracellular segment of CLDN6. This embodiment is only used to illustrate the technical effects of the present invention and does not constitute a limitation on the scope of protection of the present invention.

[0105] CLDN6 and CLDN9 belong to the Claudin family and share a highly consistent overall topology, both containing four transmembrane domains, two extracellular domains (ECL1 and ECL2), and intracellular N-terminal and C-terminal regions. However, they differ significantly in the amino acid sequences of their extracellular domains. By substituting the extracellular or intracellular domains of CLDN6 and CLDN9 while maintaining their overall transmembrane topology, we can determine whether nanobody binding depends on a CLDN6-specific extracellular region. Using the full-length coding sequences of CLDN6 and CLDN9 as templates, the following chimeric protein expression vectors were constructed:

[0106] (1) CLDN6-ECL1(9): The corresponding CLDN6 ECL1 is replaced by the first extracellular domain (ECL1) of CLDN9 on the CLDN6 backbone;

[0107] (2) CLDN6-ECL2(9): The corresponding CLDN6 ECL2 is replaced by the second extracellular domain (ECL2) of CLDN9 on the CLDN6 backbone;

[0108] (3) CLDN6-ECL1(9)-ECL2(9): The corresponding extracellular domain of CLDN6 is replaced by the first and second extracellular domains of CLDN9 on the CLDN6 backbone.

[0109] The chimeric protein expression vectors described above were transfected into mammalian cells for expression, and the chimeric proteins were confirmed to be correctly located on the cell membrane surface by immunofluorescence or flow cytometry.

[0110] Flow cytometry (FACS) and immunostaining were used to detect the binding of the anti-CLDN6 nanobody of the present invention to cells expressing the above-mentioned chimeric proteins, and the results were compared with wild-type CLDN6 and CLDN9 as controls. Figure 4 As shown.

[0111] The results showed that the recognition of the anti-CLDN6 nanobody of the present invention depends on the CLDN6-specific second extracellular domain. Different nanobodies were confirmed to recognize the second extracellular domain of CLDN6, thus verifying the specificity and reliability of their epitope localization from the perspective of homology family comparison. This embodiment provides strong experimental evidence for the specific recognition ability of the nanobody described in this invention in a high homology target background.

[0112] Example 4: Amino acid level epitope analysis of nanobody based on the difference in the second extracellular domain of CLDN6 and CLDN9

[0113] Building upon the foregoing embodiments that confirmed the anti-CLDN6 nanobody of this invention primarily recognizes the second extracellular domain (ECL2) of CLDN6, this embodiment further utilizes sequence difference analysis and site-directed mutagenesis verification between CLDN6 and its homologous family member CLDN9 in the ECL2 region to precisely locate the binding epitopes of the nanobody at the amino acid level. This demonstrates that at least two nanobodies recognize different epitopes in the CLDN6 ECL2 domain. This embodiment is merely for illustrating the technical effects of the present invention and does not constitute a limitation on the scope of protection of the present invention.

[0114] CLDN6 and CLDN9 are highly identical in overall structure and transmembrane topology, but their second extracellular domain (ECL2) exhibits non-conserved differences at several amino acid sites. By comparing the ECL2 amino acid sequences of CLDN6 and CLDN9, potential differential sites involved in antibody binding can be screened. Furthermore, by replacing individual amino acid residues in the CLDN6 ECL2 with corresponding residues in CLDN9, or replacing corresponding residues in the CLDN9 ECL2 with CLDN6 residues, the role of these sites in nanobody recognition can be determined, thereby achieving precise epitope analysis.

[0115] Multiple sequence alignment was performed on the amino acid sequences of the second extracellular domain of human CLDN6 and CLDN9 to screen for amino acid sites located in the ECL2 region that differ between CLDN6 and CLDN9. Residues located in the predicted surface exposure region were preferably selected as candidate mutation sites.

[0116] Using the full-length coding sequence of CLDN6 as a template, multiple site-directed mutants of ECL2 were constructed, in which specific amino acid residues of CLDN6 were replaced with amino acids corresponding to those of CLDN9. Simultaneously, reverse mutants were constructed using CLDN9 as a template, replacing corresponding sites with amino acid residues of CLDN6. These mutants were expressed in mammalian cells to maintain the native conformation of the protein. Flow cytometry (FACS) and immunofluorescence staining were used to detect the binding of different nanobodies to wild-type CLDN6, wild-type CLDN9, and the aforementioned site-directed mutants, with the unmutated constructs serving as controls. Results are as follows: Figure 5 As shown.

[0117] Experimental results show that different nanobodies exhibit significant differences in their sensitivity to site-directed mutagenesis in CLDN6 ECL2:

[0118] (1) The binding ability of CLDN6-Nb-1 was significantly affected after a different group of amino acid residues were replaced, but the site replacement of the second nanobody was not significantly affected.

[0119] (2) After a specific group of amino acid residues in CLDN6 ECL2 is replaced, the binding ability of CLDN6-Nb-2 to CLDN6 is significantly reduced or disappears, while it is not sensitive to mutations at other sites.

[0120] The above results indicate that although both nanobodies recognize the second extracellular domain of CLDN6, the specific sets of amino acid residues they bind to are different, suggesting that the two nanobodies recognize different epitopes in CLDN6 ECL2. This result further demonstrates the diversity and complementarity of the nanobodies in epitope recognition at the amino acid level, providing strong evidence for their synergistic application in the construction of multivalent or multispecific antibodies and in immunotherapy.

[0121] Example 5: Verification of the effect of T cell connectives constructed from CLDN6 epitopes delivered in the form of saRNA on the killing of CLDN6-positive target cells by immune effector cells.

[0122] Based on the previously obtained nanobodies that recognize different epitopes of CLDN6, this embodiment constructs a T-cell connector composed of the nanobodies and delivers it in the form of self-amplified RNA (saRNA) to verify its ability to mediate the killing effect of immune effector cells on CLDN6-positive target cells under in vitro conditions.

[0123] (1) Construction of saRNA-encoded T-cell adaptor

[0124] Two nanobodies that have been confirmed to recognize different epitopes of CLDN6 were selected as CLDN6 binding domains and engineered to bind to a binding domain capable of binding to CD3 molecules on the surface of T cells to construct T cell adaptor molecules, such as... Figure 6 As shown. The coding sequence of the T cell connector is inserted into a self-amplifying RNA vector, which contains RNA replication-related sequences and initiation elements for driving the expression of the target protein, such as... Figure 7 As shown.

[0125] (2) saRNA in vitro transcription and delivery

[0126] 2.1 Plasmid Synthesis and Extraction

[0127] The plasmids were synthesized by General Biotech Co., Ltd., and plasmid extraction was performed using an endotoxin-free plasmid extraction kit (purchased from Tiangen Biotech Co., Ltd.). The extracted plasmids were then digested with restriction endonucleases to form linearized plasmids, which were used as transcription templates. The specific enzymatic digestion steps for in vitro transcription to prepare saRNA are as follows.

[0128] 2.2 Preparation of linearized templates

[0129] Take 100 μg of the above-mentioned EGFP-saRNA recombinant plasmid carrying the nsP2 mutation, and prepare a 100 µL BspQ-1 restriction endonuclease (100 U) digestion system to linearize 10 μg of the plasmid. Incubate for 2 h.

[0130] 2.3 Purification of linearized plasmid DNA by phenol-chloroform precipitation method

[0131] After incubation, add 100 µL of phenol:chloroform to the system, vortex, centrifuge at 13000 rpm for 1 min, and transfer the supernatant to a new nuclease-free centrifuge tube. Add another 100 µL of nuclease-free water to the original system, vortex, centrifuge at 13000 rpm for 1 min, discard the supernatant, and transfer it to the same centrifuge tube. Add 200 µL of chloroform to the EP tube, vortex, and centrifuge at 13000 rpm for 5 min. Transfer 200 µL of the supernatant to a new nuclease-free centrifuge tube. Then add 1 / 10 volume of 3M pH 5.2 NaAc (20 µL) and 3 volumes of pre-chilled 100% ethanol (600 µL), and mix gently. Precipitate at -20°C for 20 minutes or overnight. Afterward, centrifuge at 13000 rpm for 15 min at 4°C, carefully remove the supernatant, and gently wash the precipitate with 1 mL of 75% ethanol. Centrifuge again at 13,000 rpm for 15 min at 4°C, and remove all supernatant. Open the tube cap and let stand for about 5-10 minutes to evaporate residual ethanol. Finally, add 15 µL of nuclease-free water to dissolve the precipitate, and determine the DNA concentration using Nanodrop. This is used for subsequent in vitro transcription.

[0132] 2.4 In vitro transcription

[0133] For the linearized template DNA sequence, vortex all components except for the T7 RNA Polymerase Mix, briefly centrifuge to collect the residue at the bottom of the tube, and store on ice for later use. Prepare the system according to Table 2. Here, "Template" refers to the purified linearized template described in 1.2. Use T7 RNA polymerase to generate mRNA on the linearized plasmid (the transcription system for the template DNA is shown in Table 2). Gently mix each component with a pipette, briefly centrifuge to collect the residue, and incubate at 37°C for 3 h. Add 1 μL LDNase I to the reaction system and incubate at 37°C for 15 min to digest the transcribed DNA template. Purify using conventional methods, such as lithium chloride precipitation, to obtain the corresponding mRNA.

[0134]

[0135] 2.5 Capping and purifying saRNA

[0136] The 5' cap is essential for mRNA to bind to the ribosome to initiate protein synthesis; the mRNA cap also helps protect mRNA from degradation by nucleases. In enzymatic capping, RNA from IVT is capped using specialized enzymes. saRNA is also capped enzymatically, specifically using vaccinia virus capping enzyme (VCE) and 2'O-methyltransferase. All saRNAs were analyzed by agarose gel electrophoresis after capping and stored frozen at -80°C.

[0137] 2.6 Encapsulation of saRNA-LNP

[0138] The aRNA prepared above was conjugated with lipid nanoparticles (LNPs) for delivery. Lipid ethanol solutions were prepared by dissolving cationic lipids (SM-102), auxiliary lipids (DSPC), cholesterol, and PEGylated lipids (such as DMG-PEG2000) in ethanol according to the molar percentages shown in Table 3.

[0139] The saRNA was diluted with citrate-sodium citrate buffer (pH=4.5) for later use. Further, using microfluidic technology, the organic phase containing lipid molecules (SM-102, DSPC, cholesterol, DMG-PEG2000) and the aqueous phase containing nucleic acid molecules (diluted with citrate-sodium citrate buffer (pH=4.5)) were thoroughly mixed, with an N:P ratio of 6 for the LNP and mRNA mixture. The organic solvent was then removed by ultrafiltration, resulting in LNP-saRNA.

[0140] Table 3. Components and proportions of LNP

[0141] Components mole percentage SM102 50% DMG-PEG2000 1.5% DSPC 10% cholesterol 38.5%

[0142] The saRNA encoding the T cell adaptor was delivered into mammalian cells via lipid nanoparticles (LNPs) to achieve in vitro expression of the T cell adaptor.

[0143] 2.7 Co-culture system of immune effector cells and target cells

[0144] Tumor cells expressing CLDN6 were selected as CLDN6-positive target cells, and human peripheral blood mononuclear cells (PBMCs) were selected as immune effector cells and co-cultured with the target cells at a predetermined ratio.

[0145] T-cell connectives expressed after saRNA delivery were added to a co-culture system, or a culture system treated with control saRNA (the control saRNA encoding a T-cell connective constructed from an unrelated nanobody) was added, to compare the differences in the effects of immune effector cells on target cells under different treatment conditions. Figure 8 As shown.

[0146] The results showed that, under the condition of delivering and expressing T-cell connectives constructed from nanobodies based on different CLDN6 epitopes in the form of saRNA, the killing effect of immune effector cells on CLDN6-positive target cells was significantly enhanced; while the killing effect on CLDN6-negative target cells was significantly reduced or not significantly increased. This embodiment further verifies the application potential of the technical solution of the present invention in tumor immunotherapy.

Claims

1. Two nanobodies capable of recognizing different epitopes of CLDN6, characterized in that: The nanobodies that can recognize different epitopes of CLDN6 can respectively recognize different antigenic epitopes of the CLDN6 protein.

2. The nanobody capable of recognizing different epitopes of CLDN6 according to claim 1, characterized in that: The heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO:1 or SEQ ID NO:4, the amino acid sequence of CDR2 is shown in SEQ ID NO:2 or SEQ ID NO:5, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3 or SEQ ID NO:

6.

3. The nanobody capable of recognizing different epitopes of CLDN6 according to claim 1, characterized in that: It also includes a skeleton region, the structure of which is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

4. The nanobody capable of recognizing different epitopes of CLDN6 according to claim 1, characterized in that: The amino acid sequences of the nanobody are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

5. An engineered construct comprising a nanobody capable of recognizing different epitopes of CLDN6 as described in any one of claims 1 to 4.

6. The engineered construct according to claim 5, characterized in that: The engineered construct is a bispecific antibody, constructed by tandemly linking the above-mentioned nanobodies capable of recognizing different epitopes of CLDN6; or the engineered construct is a multispecific antibody, one end of which contains at least one nanobodies capable of recognizing different epitopes of CLDN6, and the other end of which contains a binding unit capable of binding to molecules on the surface of immune effector cells, the two being linked by a linker peptide; preferably, the other end is a single-chain antibody against CD3, the amino acid sequence of which is shown in SEQ ID NO:

9.

7. A nucleic acid molecule encoding a nanobody that recognizes different epitopes of CLDN6 as described in any one of claims 1 to 4, or an engineered construct as described in claim 5 or 6.

8. The nucleic acid molecule according to claim 7, characterized in that: The nucleic acid molecule is in the form of DNA, mRNA, or self-amplifying RNA.

9. A pharmaceutical composition, characterized in that: It includes the nanobody that recognizes different epitopes of CLDN6 as described in any one of claims 1 to 4, the engineered construct as described in claim 5 or 6, or the nucleic acid molecule as described in claim 7 or 8, and a pharmaceutically acceptable carrier, excipient, or delivery system.

10. The use of the nanobody for recognizing different epitopes of CLDN6 according to any one of claims 1 to 4, the engineered construct according to claim 5 or 6, or the nucleic acid molecule according to claim 7 or 8 in the preparation of a medicament for the prevention or treatment of tumors or in the preparation of a reagent for the diagnosis of tumors; preferably, the tumor is a CLDN6 positive tumor; preferably, the tumor is at least one of ovarian cancer, testicular cancer, endometrial cancer, gastric cancer, embryonal tumors, glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, soft tissue sarcoma, acute and chronic leukemia, Hodgkin and non-Hodgkin lymphoma, and head and neck tumors.

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