Anti-claudin 6 nanobodies and uses thereof

Abstract

The present application relates to the technical field of nanobody, in particular to anti-CLDN6 nanobody and its application. The present application provides a kind of anti-CLDN6 nanobody with high affinity and specificity. The nanobody can specifically recognize and bind to the CLDN6 target of various solid tumors, including ovarian cancer, liver cancer, endometrial cancer, testicular cancer and gastric cancer, etc. The nanobody of the present application can be used as a core recognition element to construct a variety of therapeutic and diagnostic platforms such as chimeric antigen receptor T cells, NK cells, bispecific antibodies, antibody drug conjugates or molecular imaging probes. The technical solution can solve the technical problem of lack of high specificity anti-CLDN6 nanobody in the prior art, and provide an effective means for the immunotherapy of CLDN6 positive solid tumors.

Description

Anti-CLDN6 nanobodies and their applications Technical Field

[0001] This invention relates to the field of nanobody technology, specifically to anti-CLDN6 nanobody and its applications. Background Technology

[0002] Claudin-6 (CLDN6), a transmembrane protein, exhibits abnormally high expression in various malignant tumors, making it a potential pan-cancer target. Specifically, CLDN6 is significantly upregulated in multiple cancer types, including ovarian cancer, testicular cancer, lung cancer, liver cancer, and gastric cancer.

[0003] A study analyzed 62 patients with ovarian cancer who underwent surgical treatment (DU H et al. Claudin6: Therapeutic prospects for tumors, and mechanisms of expression and regulation (Review) [J]. Molecular Medicine Reports, 2021, 24(3)), including 36 cases of papillary serous carcinoma of the ovary and 26 cases of ovarian serous adenoma. Immunohistochemistry was used to detect the expression of CLDN6. The results showed that the positive rate of CLDN6 was 69.4% (25 / 36) in papillary serous carcinoma of the ovary, while it was 34.6% (9 / 26) in ovarian serous adenoma. This indicates that CLDN6 expression is more significant in more malignant subtypes of ovarian cancer.

[0004] Another study on hepatocellular carcinoma (LU Y et al. The Expression of CLDN6 in Hepatocellular Carcinoma Tissue and the Effects of CLDN6 on Biological Phenotypes of Hepatocellular Carcinoma Cells [J]. Journal of Cancer, 2021, 12(18): 5454-63.) collected 48 human hepatocellular carcinoma samples, covering different degrees of differentiation (13 well-differentiated, 20 moderately differentiated, and 15 poorly differentiated). Immunohistochemical results revealed that the positive rate of CLDN6 in the 48 human hepatocellular carcinoma tissues was 79.17% (38 / 48), mainly located in the cell membrane and cytoplasm. This finding further confirms the widespread presence of CLDN6 in various types of cancer and its potential value as a diagnostic biomarker or therapeutic target.

[0005] A study on endometrial carcinoma (CAO X et al. Knockdown of CLDN6 inhibits cell proliferation and migration via PI3K / AKT / mTOR signaling pathway in endometrial carcinoma cell line HEC-1-B [J]. OncoTargets and Therapy, 2018, Volume 11: 6351-60.) included 82 patients with endometrial carcinoma (EC) and their matched non-tumor tissue samples. The expression level of CLDN6 was assessed using qRT-PCR. The results showed that CLDN6 was significantly upregulated in EC tissues compared to non-tumor tissues (P<0.001). Furthermore, the expression level of CLDN6 was closely related to patient age, histological type, clinical stage, and grade. Kaplan-Meier survival analysis showed that EC patients with high CLDN6 expression had shorter survival times, suggesting that CLDN6 may be an important prognostic indicator.

[0006] Despite the significant potential of CLDN6 as a therapeutic target, the high heterogeneity and complex immunosuppressive microenvironment of solid tumors limit the effectiveness of existing therapies. Traditional treatments such as small molecule drugs, monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs) each face their own challenges: small molecule and ADC drugs are prone to drug resistance; antibody drugs are limited by tumor heterogeneity. In contrast, cell therapy, as a "living" form of drug, possesses the ability to survive long-term in vivo and exert sustained effects. It can not only directly kill solid tumor cells but also secrete cytokines or antibodies, providing additional anti-tumor activity. In particular, novel cell therapies such as chimeric antigen receptor T cells (CAR-T) and chimeric antigen receptor natural killer cells (CAR-NK) hold unique advantages in combating solid tumors due to these characteristics.

[0007] In conclusion, given the widespread expression of CLDN6 in various cancers and its important role in tumor biology, developing next-generation therapeutic strategies based on the CLDN6 target has significant clinical implications and promising application prospects. However, these CLDN6-targeted therapeutic strategies urgently require recognition molecules with high specificity, strong affinity, and good tissue penetration to achieve precise targeting of tumor cells.

[0008] While traditional monoclonal antibodies have been widely used in targeted therapy, their large molecular weight, poor tissue penetration, high production costs, and difficulty in effectively accumulating in the solid tumor microenvironment limit their efficacy. In contrast, nanobodies, derived from the single-domain variable region (VHH) of camel heavy chain antibodies, offer advantages such as small molecular weight, structural stability, high affinity, ease of engineering, and strong penetration, making them particularly suitable for targeting membrane proteins such as CLDN6, which are highly expressed in solid tumors. Furthermore, nanobodies can serve as modular building blocks, flexibly conjugating drugs, toxins, radionuclides, or used to construct recognition domains for CAR-T / CAR-NK therapy, demonstrating unique potential in multimodal therapy. Therefore, developing high-affinity nanobodies targeting CLDN6 not only helps improve targeted delivery efficiency but also provides a key tool for subsequently constructing novel immunotherapies, molecular imaging, or combination therapy platforms, demonstrating significant technological necessity and application value. Summary of the Invention

[0009] The present invention aims to provide an anti-CLDN6 nanobody to solve the technical problem of the lack of anti-CLDN6 nanobodies targeting the CLDN6 protein in the prior art.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The amino acid sequence of the anti-CLDN6 nanobody is SEQ ID NO.1, the amino acid sequence of its CDR1 is SEQ ID NO.2, and the amino acid sequence of its CDR3 is SEQ ID NO.3;

[0012] Alternatively, the amino acid sequence of its CDR1 is SEQ ID NO.10, the amino acid sequence of its CDR2 is SEQ ID NO.11, and the amino acid sequence of its CDR3 is SEQ ID NO.12;

[0013] Alternatively, the amino acid sequence of its CDR1 is SEQ ID NO.19, the amino acid sequence of its CDR2 is SEQ ID NO.20, and the amino acid sequence of its CDR3 is SEQ ID NO.21;

[0014] Alternatively, the amino acid sequence of its CDR1 is SEQ ID NO.28, the amino acid sequence of its CDR2 is SEQ ID NO.29, and the amino acid sequence of its CDR3 is SEQ ID NO.30;

[0015] Alternatively, the amino acid sequence of its CDR1 is SEQ ID NO.37, the amino acid sequence of its CDR2 is SEQ ID NO.38, and the amino acid sequence of its CDR3 is SEQ ID NO.39.

[0016] Furthermore, the amino acid sequence of its CDR1 is SEQ ID NO.19, the amino acid sequence of its CDR2 is SEQ ID NO.20, and the amino acid sequence of its CDR3 is SEQ ID NO.21.

[0017] Furthermore, the amino acid sequence of the anti-CLDN6 nanobody is SEQ ID NO.28 for CDR1, SEQ ID NO.29 for CDR2, and SEQ ID NO.30 for CDR3.

[0018] Furthermore, the amino acid sequence of the anti-CLDN6 nanobody is SEQ ID NO.37 for CDR1, SEQ ID NO.38 for CDR2, and SEQ ID NO.39 for CDR3.

[0019] Furthermore, the anti-CLDN6 nanobody has an amino acid sequence of FR1 that is any one of SEQ ID NO.4, 13, 22, 31, and 40; or, any one of an amino acid sequence that differs from the sequence of SEQ ID NO.4, 13, 22, 31, or 40 by 4, 3, 2, or 1 amino acid.

[0020] The amino acid sequence of its FR2 is any one of SEQ ID NO.5, 14, 23, 32 and 41; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.5, 14, 23, 32 or 41 by 4, 3, 2 or 1 amino acid.

[0021] The amino acid sequence of its FR3 is any one of SEQ ID NO. 6, 15, 24, 33 and 42; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO. 6, 15, 24, 33 or 42 by 4, 3, 2 or 1 amino acid;

[0022] The amino acid sequence of its FR4 is any one of SEQ ID NO.7, 16, 25, 34 and 43; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.7, 16, 25, 34 or 43 by 4, 3, 2 or 1 amino acid.

[0023] Furthermore, the anti-CLDN6 nanobody has an amino acid sequence of any one of SEQ ID NO. 8, 17, 26, 35 and 44.

[0024] This technical solution also provides an application of anti-CLDN6 nanobody in the preparation of products for treating ovarian cancer, liver cancer, and endometrial cancer.

[0025] This technical solution also provides a bispecific antibody, a multi-antibody, and an antibody-drug conjugate containing the aforementioned anti-CLDN6 nanobody.

[0026] From a molecular perspective, anti-CLDN6 nanobodies, as functionally active molecules that specifically bind to the CLDN6 target, possess advantages such as structural stability, strong targeting, and ease of genetic engineering modification of their antigen-binding domains. Nanobodies, with their small molecular weight and precise antigen-binding epitopes, can be fused or linked with other functional modules (such as another antibody fragment or cytotoxic drugs) through conventional biopharmaceutical techniques like gene splicing and conjugation modification, while maximizing the retention of their own CLDN6-targeting activity. While the application scenarios of single nanobodies are limited due to CLDN6 as a potential drug target, their therapeutic potential can be expanded by constructing bispecific antibodies, multi-antibodies, or antibody-drug conjugates (ADCs) (e.g., bispecific antibodies achieving synergistic effects on two targets, ADCs enhancing killing specificity, etc.). This transformational pathway aligns with the conventional development logic of biopharmaceuticals from single-active molecules to multifunctional derivatives, and numerous precedents of successfully constructing bispecific antibodies and ADCs with nanobodies demonstrate its feasibility. The bispecific antibodies, multi-antibodies, and antibody-drug conjugates formed using this approach all exhibit ideal therapeutic effects.

[0027] This technical solution provides an antibody against CLDN6, which contains an anti-CLDN6 nanobody.

[0028] Traditional antibodies, represented by IgG, consist of heavy and light chains, and their antigen recognition relies on the synergistic action of six complementarity-determining regions (CDRs) on the variable region (VH) of the heavy chain and the variable region (VL) of the light chain. Nanobodies, on the other hand, contain only the heavy chain variable region and are the smallest naturally occurring functional antigen-binding fragments. The nanobodies in this scheme can serve as heavy chain variable region components, assembling with other fragments to form traditional antibodies. The term "antibody" as used herein refers to traditional antibodies containing the nanobody chains described in this scheme.

[0029] This technical solution also provides a CAR-engineered immune cell, wherein the immune cell expresses a chimeric antigen receptor; the chimeric antigen receptor includes a CLDN6 nanobody; and the immune cell is a T cell or an NK cell.

[0030] Preferably, the chimeric antigen receptor comprises the following sequentially connected portions: a signal peptide region, a single-domain antibody region, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region.

[0031] The nucleotide sequence of the signal peptide region is SEQ ID NO.47; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.48; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.49; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.50; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.51; and the nucleotide sequence of the single-domain antibody region is any one of SEQ ID NO.9, 18, 27, 36, and 45.

[0032] This technical solution also provides a chimeric antigen receptor fusion protein, comprising the following sequentially connected parts: a signal peptide region, a single-domain antibody region, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region.

[0033] The nucleotide sequence of the signal peptide region is SEQ ID NO.47; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.48; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.49; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.50; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.51; and the nucleotide sequence of the single-domain antibody region is any one of SEQ ID NO.9, 18, 27, 36, and 45.

[0034] The technical principle of this technical solution is as follows:

[0035] Based on the high specific expression characteristics of CLDN6 protein in various malignant tumors and the structural and functional advantages of its nanobody, this invention constructs a highly specific recognition molecule targeting CLDN6 and derived therapeutic products.

[0036] (1) Specific recognition of anti-CLDN6 nanobody

[0037] CLDN6, a four-transmembrane protein, possesses tumor-specific antigenic epitopes in its extracellular region, serving as a key site for targeted recognition. The anti-CLDN6 nanobody of this invention originates from the single-domain variable region (VHH) of a camel heavy chain antibody. Its antigen-binding function is achieved through an antigen-binding pocket composed of three complementarity-determining regions (CDR1, CDR2, and CDR3). By precisely matching the spatial conformation of the CLDN6 extracellular region through specific amino acid sequence combinations of CDR1 (SEQ ID NO.1 / 10 / 19 / 28 / 37), CDR2 (SEQ ID NO.2 / 11 / 20 / 29 / 38), and CDR3 (SEQ ID NO.3 / 12 / 21 / 30 / 39), a highly specific antigen-antibody binding pair is formed. This pair can specifically recognize and bind to CLDN6-positive tumor cells without cross-reacting with CLDN6 that is poorly expressed or not expressed in normal tissues. Simultaneously, the framework regions (FR1-FR4) of the nanobody provide stable spatial support for the CDR regions, further ensuring the stability of antigen-binding activity.

[0038] (2) Further applications of anti-CLDN6 antibody

[0039] The nanobodies of this invention can be used as functional components to assemble with other antibody fragments to form conventional IgG-type antibodies. These assembled antibodies retain the CLDN6-specific binding domain of the nanobodies, while simultaneously utilizing the Fc fragment of IgG to mediate antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and other immune effects, thereby eliminating CLDN6-positive tumor cells through a dual action of "specific binding-immune killing".

[0040] (3) Targeted killing of CAR-engineered immune cells

[0041] This invention integrates an anti-CLDN6 nanobody as the recognition domain into a chimeric antigen receptor (CAR) to construct CAR-T / NK engineered immune cells. The CAR signal peptide region guides the CAR molecule to be correctly positioned on the immune cell membrane surface; the single-domain antibody region (corresponding to the nanobody encoded by nucleotide sequences such as SEQ ID NO. 9 and 18) specifically recognizes CLDN6 on the surface of tumor cells, initiating a conformational change in the CAR molecule. The CD8 hinge region ensures the spatial flexibility of the recognition domain, the CD28 transmembrane region enables the CAR molecule to be anchored on the cell membrane, and the CD28 intracellular region provides the first activation signal, which, together with the signal amplification effect of the CD3zeta intracellular region, activates the proliferation and activation program of immune cells, releasing killing molecules such as perforin and granzymes, specifically lysing CLDN6-positive tumor cells; at the same time, the activated immune cells can secrete cytokines (such as IL-2 and IFN-γ), remodeling the tumor immune microenvironment and inhibiting tumor growth and metastasis.

[0042] The anti-CLDN6 nanobody and its derivatives of the present invention have the following significant advantages compared with the prior art:

[0043] (1) High specificity and high targeting

[0044] The nanobody of this invention, through a unique combination of CDR sequences, can precisely recognize the tumor-specific epitope of CLDN6, binding only to CLDN6-positive tumor cells such as ovarian cancer, liver cancer, and endometrial cancer, while not recognizing CLDN6 that is low-expressed or not expressed in normal tissues. This solves the problems of poor targeting and easy damage to normal tissues by traditional tumor therapeutic drugs. Especially in CAR-T / NK cell therapy, it can significantly reduce off-target effects and improve treatment safety.

[0045] (2) Excellent penetration ability of solid tumors

[0046] Nanobodies have smaller molecular weights and more compact spatial structures than traditional monoclonal antibodies. They can penetrate the dense stromal barrier of solid tumors and accumulate efficiently within the tumor tissue. This solves the technical pain points of traditional antibodies, such as large molecular weight, poor tissue permeability, and difficulty in effectively functioning in the microenvironment of solid tumors, providing a highly efficient targeted tool for the treatment of solid tumors.

[0047] (3) The structure is stable and easy to be modified for engineering purposes.

[0048] The nanobody of this invention has good thermal stability and acid-base tolerance, which facilitates industrial production and storage. At the same time, the nanobody can be used as a modular component to flexibly construct a variety of therapeutic products such as traditional antibodies, CAR-T / NK cells, and antibody-drug conjugates (ADCs), and is suitable for the treatment of various CLDN6 positive cancers such as ovarian cancer, liver cancer, and endometrial cancer, which significantly expands the application scenarios.

[0049] (4) CAR-engineered immune cells show significant therapeutic effects

[0050] The CAR-T / NK cells constructed using nanobodies in this invention can overcome the problems of the tumor immunosuppressive microenvironment. The CAR-T / NK cells constructed in this invention use nanobodies as the recognition domain, possessing both high targeting and strong activation signals (CD28+CD3zeta dual signaling domains), which can efficiently activate the killing function of immune cells. Simultaneously, the engineered immune cells can survive long-term in vivo and continuously exert a killing effect. They can also reshape the tumor immune microenvironment by secreting cytokines, overcoming the immunosuppressive characteristics of solid tumors. Compared with small molecule drugs and traditional antibody therapies, they have the advantages of long-lasting efficacy and low likelihood of developing drug resistance.

[0051] In summary, this approach fills the technological gap in CLDN6-targeting nanobodies and promotes the development of precision oncology. Existing technologies lack high-affinity nanobodies targeting CLDN6. This invention provides, for the first time, a well-defined and stable anti-CLDN6 nanobody, offering a crucial tool for basic research on the CLDN6 target. Furthermore, its derivative antibody drugs and CAR-T / NK cell products provide novel precision treatment strategies for CLDN6-positive tumors, addressing the limitations and poor efficacy of existing CLDN6-targeting therapies. This approach possesses significant clinical application value and industrialization potential. Attached Figure Description

[0052] Figure 1 shows the results of bacterial culture PCR (polymerase chain reaction) identification of the alpaca nanobody library in Example 2 (1-24 represent the 24 nanobody sequences N1-N24 obtained in this study).

[0053] Figure 2 is a schematic diagram of the CAR sequence structure in Example 4.

[0054] Figure 3 shows the statistical results of the 24-hour killing rate of five CAR-T cells targeting CLDN6 against the target cell OVCAR3 ovarian cancer cells in Example 4 (N6: CAR-T cells expressing N6 nanobodies; N11: CAR-T cells expressing N11 nanobodies; N13: CAR-T cells expressing N13 nanobodies; N14: CAR-T cells expressing N14 nanobodies; N16: CAR-T cells expressing N16 nanobodies; PC: positive control; UTD: negative control; mean ± standard deviation, n=3).

[0055] Figure 4 shows the representative flow cytometry results of nanobody N16 against BAF3-CLDN6 cells in Example 5 (FSC-A vs SSC-A scatter plot; FSC-A represents forward scattered light, and SSC-A represents side scattered light).

[0056] Figure 5 shows the representative flow cytometry results of nanobody N16 in Example 5 targeting BAF3-CLDN6 cells (SSC-A vs FITC-A scatter plot; FITC-A represents fluorescein isothiocyanate fluorescence signal, SSC-A represents side-scattered light; this is the result of the test).

[0057] Figure 6 shows the representative flow cytometry results of nanobody N16 in Example 5 targeting BAF3-CLDN6 cells (showing the fluorescence signal intensity of fluorescein isothiocyanate FITC and the statistical cell positivity rate).

[0058] Figure 7 shows the representative flow cytometry results of nanobody N13 in Example 5 targeting BAF3-CLDN6 cells (showing the fluorescence signal intensity of fluorescein isothiocyanate FITC and the statistical cell positivity rate).

[0059] Figure 8 shows the representative flow cytometry results of nanobody N14 in BAF3-CLDN6 cells in Example 5 (showing the fluorescence signal intensity of fluorescein isothiocyanate FITC and the statistical cell positivity rate).

[0060] Figure 9 shows the representative flow cytometry results of nanobody N11 in Example 5 targeting BAF3-CLDN6 cells (showing the fluorescence signal intensity of fluorescein isothiocyanate FITC and the statistical cell positivity rate).

[0061] Figure 10 shows the representative flow cytometry results of nanobody N6 in Example 5 against BAF3-CLDN6 cells (showing the fluorescence signal intensity of fluorescein isothiocyanate FITC and the statistical cell positivity rate). Detailed Implementation

[0062] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0063] To facilitate understanding of this technical solution, the relevant technical terms and concepts are explained in detail below:

[0064] Nanobodies (Nb) are a class of single-domain antibodies derived from camel-like animals (such as alpacas and camels), consisting solely of a heavy chain variable region (single-domain variable region, VHH). The VHH structure, cloned and expressed independently, possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody. It is the smallest known unit capable of binding to target antigens, with a molecular weight of 15 kDa and a volume only one-tenth that of a traditional antibody; hence, it is also called a nanobody.

[0065] Antibodies (also known as immunoglobulins, Ig) are proteins produced by the proliferation and differentiation of B lymphocytes after stimulation by antigens. They specifically recognize and bind to antigens and are core molecules in the humoral immune function of the immune system. Antibodies are generally composed of two heavy chains and two light chains linked by disulfide bonds, with a typical Y-shaped four-chain structure (represented by IgG), containing an antigen-binding domain (Fab region) and an effector domain (Fc region). Through the antigen-binding domain (CDR region of VH and VL), they specifically recognize antigens such as bacteria and viruses, subsequently mediating effects such as toxin neutralization, complement activation, and binding to immune cells to eliminate pathogens or abnormal cells. Based on the amino acid composition of the heavy chain constant region, they can be divided into five classes: IgG, IgA, IgM, IgD, and IgE.

[0066] Heavy chain (H chain): Two identical polypeptide chains in an antibody molecule, with a molecular weight of approximately 50 kDa (twice that of the light chain). The type determines the antibody class, which is divided into five classes: γ, α, μ, δ, and ε, corresponding to IgG, IgA, IgM, IgD, and IgE, respectively. Each heavy chain contains an N-terminal variable region (VH) and a C-terminal constant region (CH). The constant region mediates antibody effector functions (such as activating complement and binding to immune cells).

[0067] Light chain (L chain): Two identical small polypeptide chains in the antibody molecule, with a molecular weight of approximately 25 kDa. Each light chain contains an N-terminal variable region (VL) and a C-terminal constant region (CH). The VL and VH work together to form the antigen binding site.

[0068] VH (Variable Region of Heavy Chain): A variable structural domain at the N-terminus of the heavy chain. It is one of the core units of antigen binding and must bind to VL to form a functional antigen-binding pocket. It contains 3 complementarity-determining regions (CDR1-CDR3) and 4 framework regions (FR1-FR4). The CDR regions directly participate in antigen recognition.

[0069] VL (Variable Region of Light Chain): A variable domain at the N-terminus of the light chain. Together with VH, it forms the antigen-binding region of the antibody, enhancing the specificity and affinity of antigen binding. Its structure is similar to VH, containing 3 CDR regions and 4 FR regions, assisting VH in recognizing antigenic epitopes.

[0070] CDR (Complementarity Determining Region): Highly variable regions in the VH and VL sequences, with three CDRs in each class (CDR1, CDR2, and CDR3). These six CDRs together form an antigen-binding pocket, directly binding complementary to the antigen epitope and determining antibody specificity.

[0071] FR (Framework Region): A conserved sequence between the CDR regions in VH and VL, with four FRs (FR1-FR4) in each type of variable region. It provides structural support for the CDR region, ensuring its proper exposure and binding of the antigen.

[0072] Fab (Antigen-binding Fragment): A fragment produced after an antibody is digested with papain. Each antibody molecule can produce two identical Fab fragments, containing complete VH, VL, CL, and CH1 domains. These fragments are solely responsible for antigen binding (preserving antibody specificity) and have no Fc-related effector functions.

[0073] Fc (crystallizable fragment): A fragment produced after antibody digestion with papain. It consists of CH2 and CH3 domains of two heavy chains, is crystallizable, and has no antigen-binding activity. Its core function is to mediate antibody effects, such as binding to Fc receptors on immune cells, activating complement, and determining antibody half-life.

[0074] VHH (Variable Domain of Heavy Chain-only Antibody) is the only antigen-binding domain in heavy chain antibodies (HCAbs) from camels (alpacas, camels, etc.), and is the smallest naturally occurring functional antigen-binding fragment. It consists of a framework region (FR1-FR4) and a complementarity-determining region (CDR1-CDR3).

[0075] M13KO7 helper phage is a specialized helper phage modified from wild-type M13 filamentous phage. It provides structural proteins and packaging signals required for phage particle assembly to support the formation of recombinant phages.

[0076] CAR-T cells (Chimeric Antigen Receptor T-Cells) are a novel immunocellular therapy that uses genetic engineering to modify T lymphocytes to express chimeric antigen receptors (CARs). In other words, they are T cells that express chimeric antigen receptors. CARs consist of an antigen-binding domain (e.g., monoclonal antibody scFv, nanobody VHH), a transmembrane domain, and an intracellular signaling domain. CARs can specifically recognize tumor cell surface antigens, activate T cell killing activity, and achieve precise targeted elimination of tumor cells.

[0077] CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells) are novel engineered immune cells obtained by modifying natural killer cells (NK cells) using genetic engineering technology. The core feature is the introduction of chimeric antigen receptors (CARs) into the NK cell genome, enabling them to specifically recognize target cells (such as tumor cells) and kill them efficiently, while retaining the natural immune advantages of NK cells.

[0078] PBMCs (Peripheral Blood Mononuclear Cells) are a population of cells with single nuclei isolated from peripheral venous blood. They mainly consist of lymphocytes (T cells, B cells, NK cells), monocytes, a small number of dendritic cells, and do not contain erythrocytes or multinucleated granulocytes. They can be used for immune cell research (e.g., T cell isolation, starting material for CAR-T cell preparation), cytokine detection, and immune function assessment.

[0079] 293T cells (Human Embryonic Kidney 293T Cell): An adherent cell line derived from human embryonic kidney 293 cells, obtained through transfection with the SV40 large T antigen gene. They exhibit adherent growth, rapid proliferation, and extremely high transfection efficiency (commonly transfected via liposomes and viruses). They stably express the SV40 large T antigen, promoting the amplification of plasmids containing the SV40 origin of replication, facilitating the production of recombinant proteins or viral vectors (such as lentiviruses). Their main applications include recombinant protein expression, viral vector packaging (commonly used in CAR-T cell preparation), and gene function validation.

[0080] 293F cells (Human Embryonic Kidney 293F Cell): A suspension-derived cell line of 293 cells. It exhibits high growth density, good compatibility with serum-free culture, and is suitable for large-scale production of recombinant proteins (such as antibodies and cytokines). Its main applications are industrial-grade recombinant protein expression and protein raw material production in biopharmaceuticals.

[0081] H929 cells are a human multiple myeloma cell line, officially named NCI-H929. They are widely used in the research of hematologic malignancies, especially in the pathogenesis, drug screening, and treatment strategy development of multiple myeloma.

[0082] Bispecific antibodies (Biantibodies): These are artificially designed antibodies that can simultaneously and specifically bind to two different antigenic epitopes (which can be different epitopes of the same antigen or epitopes of different antigens). In this protocol, an anti-CLDN6 nanobody can be fused with another antibody fragment with a specific function (such as an antibody fragment that binds to molecules on the surface of immune cells or other tumor-related targets) to form a bispecific antibody. This bispecific antibody can target CLDN6-positive cells and also mediate synergistic functions such as immune killing and signal pathway blocking, thereby improving therapeutic specificity and efficacy.

[0083] Multi-antibodies (multispecific antibodies): Compared to bispecific antibodies, they can specifically bind to three or more different antigenic epitopes, making them more structurally complex artificial antibody molecules. In this approach, the anti-CLDN6 nanobody is used as the core module, combined with multiple antibody domains with different functions, to achieve multi-target synergistic targeting, multiple immune regulation, and other functions, adapting to more complex disease treatment scenarios and overcoming the limitations of single-target drugs.

[0084] Antibody-drug conjugates (ADCs) are targeted drugs composed of an antibody (or antibody fragment), a linker, and a cytotoxic drug (such as a chemotherapy drug or toxin). In this regimen, the anti-CLDN6 nanobody serves as a targeting carrier. Through the linker, it conjugates with the cytotoxic drug, leveraging the nanobody's specific binding ability to CLDN6 to precisely deliver the cytotoxic drug to CLDN6-positive target cells. This approach kills target cells while minimizing damage to normal cells, significantly reducing drug toxicity and improving therapeutic efficacy.

[0085] Example 1: Preparation of CLDN6 antigen and alpaca anti-CLDN6 serum

[0086] (1) Preparation of CLDN6 antigen

[0087] Based on the CLDN6 protein and gene sequence information from bioinformatics websites (sequence identifier P56747 in the UniProtKB / Swiss-Prot database), an AAV9 (adeno-associated virus 9) expression plasmid expressing human CLDN6 protein (i.e., CLDN6-AAV9 expression plasmid) was constructed. Expression was validated by transiently transfecting 293F cells. After successful validation, the CLDN6-AAV9 virus was packaged, purified, and concentrated.

[0088] One day before transfection, 293T cells were seeded to a density of 60%-80% and cultured at 37°C in a 5% CO2 incubator. Using a standard transfection kit and the constructed CLDN6-AAV9 expression plasmid, cells were transfected according to the manufacturer's instructions. After transfection, the cells were cultured, and the released CLDN6-AAV9 viral particles were collected. The purification and concentration process of the CLDN6-AAV9 viral particles was as follows: After culturing 293T cells transfected with the CLDN6-AAV9 expression plasmid, PEG8000 and NaCl were added to the virus-containing culture supernatant, mixed well, and incubated overnight at 4°C. The next day, the supernatant incubated overnight at 4°C was centrifuged (3500g, 4°C, 30 min). After centrifugation, the supernatant was discarded, and the viral pellet was collected. The viral pellet was resuspended in 8-10 mL of serum-free DMEM medium and collected in a 50 mL tube for later use. Resuspend the cell pellet in 8-10 mL of serum-free DMEM medium, mix well, and freeze-thaw 3-5 times. After freeze-thaw, centrifuge (3000 g, 4 °C, 10 min) and collect the supernatant and pellet (cell debris) separately. Prepare cesium chloride solutions of different concentrations and add the treated virus solution to the top layer; perform ultracentrifugation (48000 rpm, 2 h 30 min). After centrifugation, puncture the bottom of the ultrafiltration tube with a needle and collect the adenovirus layer into a 50 mL tube. Dilute the collected virus solution 3-fold and filter through a 0.2 μm filter membrane; place the filtered liquid in a 50 mL ultrafiltration tube and concentrate by centrifugation at 2500 g for 30 min. After ultrafiltration and centrifugation 2-3 times, the liquid in the ultrafiltration tube is concentrated before proceeding to subsequent steps. Repeatedly pipette the remaining liquid in the ultrafiltration tube into a virus storage tube and store at -80 °C.

[0089] (2) Alpaca immunity and the acquisition of antiserum

[0090] Immunization dosage: 1×10⁻⁶ CLDN6-AAV9 virus per injection 12 vg.

[0091] First immunization: The sample was emulsified by mixing Freund's complete adjuvant with the sample at a volume ratio of 1:1 and then injected subcutaneously at multiple points.

[0092] Immunizations 2, 3, 4, and 5: Samples were emulsified with Freund's incomplete adjuvant at a 1:1 volume ratio and then injected subcutaneously. Booster immunizations were administered on days 21, 35, 49, and 63 using CLDN6-AAV9 virus and Freund's incomplete adjuvant at a 1:1 volume ratio. After the fifth immunization, blood was collected to determine the antiserum titer, and 100 mL of the titer-compliant blood was used for the construction of a phage antibody library.

[0093] Serum titers were determined using ELISA (Enzyme-Linked Immunosorbent Assay): On day 1, the test antigen was diluted to 1.0 μg / mL with antigen dilution buffer, and then 100 μL of antigen (Claudin 6 VLP protein, Abcam, catalog number ab316078) was added to each well. After sealing, the wells were incubated overnight at 4°C. On day 2, the supernatant was discarded; 200 μL of blocking buffer was added to each well and incubated at 37°C for 30 min, ready for use (the supernatant was discarded before use). Serum and negative control samples were serially diluted 10-fold using a 10-fold serial dilution method. 6 Prepare 10 times. 2 Up to 10 6 Five dilutions of samples were prepared. 100 μL of sample and negative control were added to appropriate wells, sealed, and incubated at 37°C for 1 h. The supernatant was discarded, and the sample was washed three times with washing buffer. 100 μL of HRP (horseradish peroxidase)-labeled goat anti-alpaca antibody IgG H&L (H&L refers to heavy and light chains, product number: SPAB02) was added to each well. This antibody was diluted 1:15000 before use. The sample was incubated at 37°C for 40 min, the supernatant was discarded, and the sample was washed five times with washing buffer. 100 μL of TMB (tetramethylbenzidine) one-step substrate reagent (mixed solutions A and B) was added to each well. The sample was incubated at room temperature for 15 min with gentle shaking. 50 μL of stop solution was added to each well, and the reading was taken quickly at 450 nm. The antigen dilution buffer and blocking buffer were standard reagents for serum titer ELISA testing and were commercially available; details are omitted here.

[0094] Principles for calculating serum positive titers: At the same dilution factor, the sample OD... 450 A positive result was defined as having an absorbance value (optical density at 450 nm) ≥ 2.1 times that of the negative control. If the absorbance value of the negative control was < 0.05, it was calculated as 0.05. The results are shown in Table 1. After 5 immunizations, the effective titer (serum titer) of the antiserum was 10. 4 Therefore, this antigen can induce alpacas to produce high-titer antiserum specifically targeting the human CLDN6 antigen protein.

[0095] Table 1: ELISA results of total serum IgG in alpacas after five immunizations

[0096]

[0097] Example 2: Alpaca phage library and screening

[0098] 100 mL of peripheral blood was collected from immunized alpacas. Peripheral blood mononuclear cells (PBMCs) were obtained by separating the blood using lymphocyte separation medium. RNA (ribonucleic acid) was extracted from the PBMCs according to the instructions of the QIAGEN Plus MiniRNA Extraction Kit. cDNA (complementary deoxyribonucleic acid) reverse transcription was performed on the extracted RNA according to the instructions of the Invitrogen Super III First-Strand Synthesis System Extraction Kit.

[0099] Next, the anti-CLDN6 nanobody was amplified. The reverse cDNA library was amplified using a gene template (two rounds of amplification), followed by the construction of the anti-CLDN6 nanobody display vector. The PCR product was double-digested with Pst-I-HF and Not-I-HF enzymes, then cloned into the pMECS phage display vector. The digested products were recovered using a PCR product recovery kit according to the manufacturer's instructions, and the concentration of the recovered product was measured for subsequent experiments.

[0100] Next, the enzyme digestion and ligation products were transformed into TG1 competent cells, and a phage antibody library was obtained. Then, VHH (nanobody) phage antibody library rescue was performed. After determining the recombinant phage titer, the recombinant phages were screened. The screening process consisted of three rounds, as detailed below:

[0101] Antigen coating: Dilute CLDN6 antigen protein to 4 μg / mL with PBS buffer. Take a 96-well microplate, select 3 replicates, add 100 μL (400 ng / well) to each well, and coat overnight at 4°C. Use PBS as a negative control.

[0102] Blocking: Discard the coating solution, add 150 μL of 2% skim milk powder to each well, and block at room temperature for 1 hour.

[0103] Incubate bacteriophages: Wash four times with PBST (phosphate-buffered saline containing Tween-20), and dilute the bacteriophage solution to 5 × 10⁻⁶ with 2% skim milk powder. 11 Add pfu / mL to the microplate, 100 μL / well, and incubate at room temperature for 2 h.

[0104] Elution: Discard the phage sample, wash 10 times with PBST, then wash 5 times with PBS (phosphate buffer), add 100 μL of freshly prepared 0.1 M triethylamine to each well, let stand at room temperature for 10 min, aspirate the eluent and quickly neutralize with an equal volume of 1 M Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride buffer, pH 7.4.

[0105] Eluent phage titer determination: The enrichment effect of specific VHH recombinant phage was evaluated by detecting the titer of recombinant phage in each round of elution. The enrichment effect was assessed by counting enriched clones (Table 2).

[0106] Infection: Take 400 μL of eluent and infect 4 mL of logarithmic TG1 cells. Shake well and incubate at 37°C for 30 min. Add 16 mL of 2×YT medium (containing ampicillin and glucose) and culture at 37°C and 200 rpm until OD500. 600 It reaches 0.6-0.8.

[0107] Rescue: Add 20 μL of M13KO7 helper phage to the culture medium, shake well, let stand at room temperature for 1 h, centrifuge at 2800g for 10 min, discard the supernatant, resuspend the cells in 100 mL of 2×YT medium (containing ampicillin and kanamycin), and incubate at 37℃ and 225 rpm for 14 h.

[0108] Next, the phage particles were concentrated and purified for the next round of screening. This process was repeated twice to complete the second and third rounds of screening. The results of the nanobody library screening are detailed in Table 2. The O / I ratio represents the ratio of the number of output phages to the number of input phages, used to quantify the enrichment efficiency of specifically binding phages in each round of screening.

[0109] Table 2: Screening Results of Nanobody (VHH) Library

[0110]

[0111] To further validate the positive phage rate binding to human CLDN6-VHH (i.e., anti-CLDN6 nanobody) protein in the enriched library, 96 × 2 clones were randomly selected from the third-round enriched library for single phage ELISA detection. The results showed that most phage clones in the third-round library were positive, and the OD... 450 The P / N ratio was greater than 3.0 for both values ​​and PBS controls (Tables 3-1 and 3-2, underlined to indicate positive), indicating that the highly binding CLDN6-VHH phage library was successfully enriched by screening for human CLDN6 antigen protein.

[0112] Table 3-1: ELISA detection of phage library enrichment effect (first 96-well plate)

[0113]

[0114] Table 3-2: ELISA detection of phage library enrichment effect (second 96-well plate)

[0115]

[0116] Next, prokaryotic expression of nanobodies was performed: 100 μL of LB medium (containing ampicillin and glucose) was added to each well of a 96-well cell culture plate. Simultaneously, 96 single colonies were randomly picked from the plates used in the third round of titer determination and added to the medium. The plates were incubated at 37°C and 200 rpm for 6 hours. 1 mL of TB medium (high-quality broth) was added to each well of four 24-well cell culture plates. Single-clone bacterial cultures were transferred to the plates at a 1:100 ratio and incubated at 37°C and 200 rpm until the logarithmic growth phase. IPTG was added to each well to a final concentration of 1 mM, and incubated overnight at 37°C and 200 rpm. The cell culture plates were then centrifuged at 4°C and 12,000 rpm for 2 minutes. The supernatant was discarded, and the cells were frozen at -80°C for 30 minutes before being removed. Once the bacterial cells have returned to room temperature, add 500 μL of PBS to each well to resuspend the cells. Then centrifuge at 4°C and 12,000 rpm for 2 minutes and collect the supernatant. The supernatant is the crude extracted nanobody VHH.

[0117] Sequencing of nanobodies: Fractional bacterial cultures of each of the above-mentioned monoclonal strains were sequenced. Forty-two clones were randomly selected for sequencing, and the results showed a diversity of 50%. Alignment results showed that most differential sequences were located in the CDR binding region. The constructed human CLDN6-VHH phage antibody library had a library size of 4.21 × 10⁻⁶. 10 The positive rate was 50%, the sequence diversity was 50%, and the effective insertion rate was 100%.

[0118] Figure 1 shows the results of bacterial culture PCR identification of 24 clones out of 42 randomly selected clones. The nanobody sequences corresponding to these 24 clones are named N1-N24 respectively (where lanes 1-24 represent N1-N24 respectively, for amplification of the VHH fragment of the heavy chain variable region). Although the remaining clones are not shown, all of them achieved successful insertion of nanobody sequences (effective insertion rate of 100%).

[0119] Of these sequences (N1-N24), the in vitro killing effects of N6, N11, N13, N14, and N16 have been experimentally studied. Details can be found in Example 4 below, which presents the in vitro killing data of CAR-T cells prepared using the aforementioned nanobodies. The effects of the remaining nanobodies require further investigation. The N6, N11, N13, N14, and N16 nanobodies are located in lanes 6, 11, 13, 14, and 16 in Figure 1, and are the nanobodies used in this protocol. Specific sequence information is provided in Tables 4, 5, 6, 7, and 8.

[0120] Table 4: Sequence information of nanobody N6

[0121]

[0122] Table 5: Sequence information of nanobody N11

[0123]

[0124] Table 6: Sequence information of nanobody N13

[0125]

[0126] Table 7: Sequence information of nanobody N14

[0127]

[0128] Table 8: Sequence information of nanobody N16

[0129]

[0130] Example 3: Construction of a eukaryotic expression library of nanobodies and expression of nanobodies

[0131] To construct the recombinant expression plasmid, the nanobody sequence determined in Example 2 was combined with the eukaryotic expression plasmid pcDNA3.4 (a widely used mammalian expression vector provided by Thermo Fisher Scientific) and recombined into a eukaryotic protein expression plasmid using conventional molecular cloning techniques, followed by transfection into 293F cells.

[0132] One day before transfection, count the normally cultured 293F cells, aspirate an appropriate amount of cell suspension and centrifuge (1000 rpm, 5 min), discard the supernatant, and resuspend in fresh culture medium (specifically Gibco). TM Expi293 TM Expression medium (containing 6 mM L-glutamine) resuspended to 1 × 10⁻⁶ 6Cells / mL (total volume 50mL), continue incubation overnight at 37°C, 5% CO2, 125rpm. The next day, prepare the transfection complex: add 2.5mL of culture medium and 50μg of plasmid DNA (eukaryotic protein expression plasmid) to a 15mL sterile centrifuge tube and vortex to mix. Add 2.5mL of culture medium and 150μL of liposome transfection reagent (Lipofectin) to the diluted DNA solution in another 15mL sterile centrifuge tube and vortex again to mix. After incubation at room temperature for 5 min, add the transfection complex to 45mL of 293F culture medium and continue incubation at 37°C, 5% CO2, 125rpm. After 4-5 days, centrifuge to collect the expressing cells and supernatant (1000rpm, 5min) for protein purification.

[0133] Purification was performed using nickel column affinity chromatography. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) showed that the CLDN6 nanobody was successfully purified, yielding five eukaryotic expression antibodies: N6-VHH-Fc, N11-VHH-Fc, N13-VHH-Fc, N14-VHH-Fc, and N16-VHH-Fc (formed by N6, N11, N13, N14, or N16 nanobody + Fc sequence + His tag). The Fc sequence + His tag sequence is (SEQ ID NO. 46; the Fc sequence + His tag sequence is directly linked to the C-terminus of the VHH of the N6, N11, N13, N14, or N16 nanobody):

[0134] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

[0135] Example 4: Preparation and efficacy study of CAR-T

[0136] The original lentiviral expression vector (empty vector) used was pALD (a product of ALDEVRON). GenScript was commissioned to synthesize the entire genome, including the chimeric antigen receptor CAR sequence and downstream co-stimulatory signal domain, which were then inserted into the pALD plasmid to obtain the pALD-EF1aCORE-anti-CLDN6-28z expression vector. Sequencing confirmed its correctness.

[0137] The chimeric antigen receptor CAR (CLDN6 CAR) is constructed based on the CLDN6 antibody sequence. In addition to the CLDN6 nanobody sequence proposed in this protocol, the remaining sequences are conventional chimeric antigen receptor-related sequences in the prior art, including the following parts, in order from extracellular to intracellular: signal peptide (SEQ ID NO.47), single-domain antibody region (one of the five nanobodies provided in this protocol, namely: SEQ ID NO.9, SEQ ID NO.18, SEQ ID NO.27, SEQ ID NO.36, or SEQ ID NO.45), CD8 hinge region (SEQ ID NO.48), CD28 transmembrane region (CD28 TM, SEQ ID NO.49), CD28 intracellular region (CD28 cyto, SEQ ID NO.50), and CD3zeta intracellular region (CD3zeta cyto, CD3ζ, SEQ ID NO.51). The above sequences are linked together from the 5' end to the 3' end (sequence information is detailed in Table 9), which constitutes the CAR sequence of this embodiment (the nucleotide sequence corresponding to the CAR fusion protein, structural diagram is detailed in Figure 2). This sequence is integrated into the pALD plasmid using conventional molecular cloning methods to obtain the expression vector. This expression vector expresses the CAR fusion protein after being transfected into T cells. The CAR fusion protein is a transmembrane protein that can guide T cells to kill target cells. This protocol tested the efficacy of five CAR fusion proteins: CAR fusion protein with anti-CLDN6 nanobody N6, CAR fusion protein with anti-CLDN6 nanobody N11, CAR fusion protein with anti-CLDN6 nanobody N13, CAR fusion protein with anti-CLDN6 nanobody N14, and CAR fusion protein with anti-CLDN6 nanobody N16.

[0138] Table 9: Chimeric antigen receptor CAR sequence information (see Tables 3-5 for single-domain antibody information).

[0139]

[0140] First, lentiviruses were prepared and seeded into 293T cells (day 0) in T25 culture flasks. On day 1, virus packaging was performed. In a DNA tube, 4.8 µg of transfer plasmid (pALD-EF1aCORE-anti-CLDN6-28z expression vector), 2.7 µg of helper plasmid pLP1, 1.8 µg of helper plasmid pLP2, and 2.7 µg of helper plasmid pLP-VSVG were added, totaling 12 µg, and Opti-MEM medium was added to bring the volume to 100 µL. In a PEI (polyethyleneimine) tube, 36 µg of PEI was added, and Opti-MEM was added to bring the volume to 100 µL, mixing well with the DNA. The supernatant in the T25 culture flask was discarded, and 4.8 mL of fresh culture medium and the DNA-PEI complex (obtained by mixing the contents of the DNA and PEI tubes) were added to the flask. The flask was gently shaken back and forth and side to side to mix. The flask was then incubated at 37°C in a 5% CO2 incubator for 6 hours, after which the medium was changed. On day 3, after 48 hours of culture, the virus was harvested. The viral supernatant was collected (temporarily stored at 4°C), and cultured further with added culture medium. On day 4, the viral supernatant was collected again after 72 hours and combined with the viral supernatant after 48 hours. After centrifugation at 1300g for 15 minutes (4°C), the mixture was filtered through a 0.45µm filter. 3mL of Takara Lenti-X lentiviral concentrate was added to the supernatant and incubated overnight at 4°C. Centrifugation was then performed again at 1300g for 15 minutes (4°C). The supernatant was discarded, the precipitate was resuspended in PBS, and aliquoted to obtain lentivirus containing the expression vector (stored at -80°C).

[0141] Next, CAR-T cell preparation was performed. On day 0, PBMCs were resuscitated and initially activated. PBMCs were removed from liquid nitrogen and rapidly resuscitated in a 37°C water bath, centrifuged at 350g for 5 min, washed once with PBS, and incubated with anti-CD3 magnetic beads for 20 min. CD3 cells were then separated using a magnetic separation column. + T cells were resuspended and counted using CAR-T medium (Lonza X-vivo 15). Cells were seeded in T75 flasks, topped with 17 mL of CAR-T medium, and T cell activation and expansion reagent (T Cell TransAct) was added. TM170 μL of CAR-T cells were collected from T75 culture flasks and transferred to 50 mL centrifuge tubes. The cells were centrifuged at 350 g for 5 min, the supernatant was discarded, and the cells were resuspended in 6 mL of CAR-T medium. 1 mL of CAR-T medium was added to each well of a 24-well plate (6 wells total), with 200 μL of lentivirus and 8 μg / mL polybrene added to each well. The cells were centrifuged at 1500 g for 1.5 h at 32 °C. The cells were then transferred to T25 culture flasks, the medium was brought to 5 mL, and interleukin-2 (IL-2, 100 U / mL) was added. The transfected cells were expanded and cultured, and the cells were used for cell killing experiments. The above procedure for preparing CAR-T cells is a conventional method in existing technology. The only difference between this protocol and existing technologies is the sequence of the CAR fusion protein, which yields T cells expressing a specific CAR fusion protein, enabling effective recognition and killing of target cells. This step yields CAR-T cells, of which there are five types: CAR-T cells expressing N6 nanobodies, CAR-T cells expressing N11 nanobodies, CAR-T cells expressing N13 nanobodies, CAR-T cells expressing N14 nanobodies, and CAR-T cells expressing N16 nanobodies.

[0142] A co-incubation system was established using an E:T ratio (effect-to-target ratio). OVCAR3 cells (ovarian cancer cells expressing luciferase) were used as target cells, and CAR-T cells as effector cells. Positive controls (PC: CAR-T cells with a positive control sequence), negative controls (UTD: only unmodified T cells), and a single-target cell group (containing only OVCAR3 cells) were also included. After 24 hours of co-culture, the cell culture plates were removed from the incubator and incubated at room temperature for 30 minutes to allow the temperature to equilibrate. An equal volume of Bio-Lite assay reagent, equilibrated to room temperature, was added. Cells were incubated at room temperature for at least 3 minutes to allow for complete lysis before detection. Detection was performed using a microplate reader in chemiluminescence mode. The cell kill rate was calculated as: Kill rate % = (1 - RLU of the co-incubation group / RLU of the single-target cell group) × 100%, where RLU is the relative intensity of the fluorescence signal output by the detection device.

[0143] The experimental results are detailed in Figure 3. The killing rate of CAR-T cells expressing N6 nanobodies was 36.85%, that of CAR-T cells expressing N11 nanobodies was 26.41%, that of CAR-T cells expressing N13 nanobodies was 64.27%, that of CAR-T cells expressing N14 nanobodies was 64.97%, and that of CAR-T cells expressing N16 nanobodies was 58.93%. The killing rate of the positive control (a single-chain variable region fragment scfv antibody sequence targeting CLDN6 consistent with existing technology) was 39.48%, and the killing rate of the negative control was 9.71%. Therefore, when N13 and N14 nanobodies are used in the preparation of CAR-T cells, the in vitro tumor-killing effect of CAR-T cells is the most ideal, followed by N16, N6, and N11 nanobodies.

[0144] Example 5: Affinity Study of Nanobodies

[0145] Flow cytometry was used to verify the affinity of BAF3-CLDN6, BAF3-CLDN9, and 293F-CLDN9 cells with anti-CLDN6 nanobodies (N13, N14, N16, N6, and N11, sequences shown in Tables 4-8). All three cell lines were genetically engineered and stably express specific Claudin proteins. BAF3-CLDN6 cells, a mouse leukemia pre-B cell line expressing the exogenous CLDN6 gene, were used to verify whether anti-CLDN6 antibodies could specifically bind. BAF3-CLDN9 cells, a mouse leukemia pre-B cell line expressing the exogenous CLDN9 gene, were used to verify whether anti-CLDN9 antibodies could specifically bind. 293F-CLDN9 cells, a human embryonic kidney-derived suspension cell line (HEK293F), expressing the exogenous CLDN9 gene, were used to evaluate the binding activity of anti-CLDN9 antibodies in a human cell background. Nanobodies N13, N14, N16, N6, and N11 were used as primary antibodies to incubate cells. Then, the cells were labeled with their corresponding rabbit anti-cameloid VHH antibodies (FITC-labeled, i.e., secondary antibodies) and detected by flow cytometry. The specific procedure is as follows: After cell counting, 8 × 10⁸ cells were directly collected. 7 Take 500 μL of cells and transfer them to a flow cytometry tube as a blank control. Add 1 μg of primary antibody to a centrifuge tube, and add 5 × 10⁻⁶ cells to each tube. 6 Cells (500 μL final volume), three replicates per group, incubated at 4°C in the dark for 30 min with intermittent mixing, washed twice with PBS, and used as experimental group samples. Secondary antibody was diluted 1:2000 and added to the experimental and control group samples, incubated at 4°C in the dark for 20 min with intermittent mixing, and washed twice with PBS. Then, flow cytometry validation was performed.

[0146] The experimental results are detailed in Table 10, and some representative flow cytometry images are shown in Figures 4-10.

[0147] Table 10: Results of nanobody affinity experiment (the positive rate of the blank control was 0%, and it is not shown in the table).

[0148]

[0149] This study systematically evaluated the affinity and specificity of five anti-CLDN6 nanobodies (N13, N14, N16, N6, and N11) using flow cytometry on three genetically engineered cell lines stably expressing specific Claudin proteins. A three-layer validation system was set up: BAF3-CLDN6 cells served as CLDN6 target-positive cells to directly verify the binding ability of the nanobodies to CLDN6; BAF3-CLDN9 cells served as mouse-derived control cells of the same CLDN9 family to rule out cross-reactivity of the nanobodies to CLDN family homologous proteins; and 293F-CLDN9 cells served as CLDN9-positive cells with a human background to further verify the performance of cross-reactivity in the human cell system. A blank control (0% positivity rate) was also included to exclude non-specific binding interference.

[0150] Affinity to the CLDN6 target: In target cells BAF3-CLDN6, the five nanobodies showed varying degrees of affinity for the target cells. N14, N16, and N6 exhibited strong affinity, while N13 showed significantly higher binding activity than the negative level, and N11 also demonstrated some affinity activity.

[0151] Cross-reactivity assessment for CLDN9: In mouse-derived BAF3-CLDN9 cells, the positive rates of all nanobodies were significantly lower than those for BAF3-CLDN6. Among them, the average positive rate of N11 was 0.05%, with almost no cross-reactivity; the average positive rate of N13 was 3.15%, with extremely weak cross-reactivity; the average positive rates of N16, N14, and N6 were 7.93%, 9.04%, and 10.51%, respectively. Although there was weak cross-reactivity, the positive rates were far lower than the binding activity to the target CLDN6, demonstrating good target specificity.

[0152] In human 293F-CLDN9 cells: cross-reactivity showed subtle differences from mouse cells, but the overall trend was consistent. N11 (mean 0.02%) and N6 (mean 0.20%) showed almost no cross-reactivity, while N13 (mean 8.54%) showed a weak cross-reactivity. N16 (mean 17.83%) and N14 (mean 20.63%) showed slightly higher cross-reactivity than mouse cells, which is speculated to be related to differences in CLDN9 expression conformation and cell surface modification in the human cell background. However, the positive rate was still significantly lower than that for CLDN6 binding, and did not affect target specificity. Notably, N6 showed no cross-reactivity in human CLDN9 cells, suggesting that its specificity for CLDN6 is superior in the human system.

[0153] In summary, this experiment clarified the affinity and specificity characteristics of five nanobodies: N14, N16, N6, and N13 are high / relatively strong affinity anti-CLDN6 nanobodies, with N6 exhibiting the best specificity in a human cell background. All effectively bound nanobodies showed only weak cross-reactivity with CLDN9, demonstrating excellent target specificity and meeting the core requirements for subsequent biopharmaceutical development.

[0154] The experimental results provide key data support for the development schemes of chimeric antigen receptor cells, bispecific antibodies, multi-antibodies, and ADCs mentioned above. The highly specific and high-affinity nanobodies screened can be used as core targeting modules for genetic engineering or drug conjugation. Their good targeting ability can ensure that the derivatives can accurately bind to CLDN6 positive cells, reduce off-target effects, and lay an experimental foundation for improving the efficacy of drug therapy and reducing toxic side effects.

[0155] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An anti-CLDN6 nanobody, characterized in that, The amino acid sequence of its CDR1 is SEQ ID NO.1, the amino acid sequence of its CDR2 is SEQ ID NO.2, and the amino acid sequence of its CDR3 is SEQ ID NO.

3.

2. The anti-CLDN6 nanobody according to claim 1, characterized in that, Its amino acid sequence is SEQ ID NO.

8.

3. The application of the anti-CLDN6 nanobody according to claim 1 or 2 in the preparation of products for treating ovarian cancer, characterized in that, The product is CAR-engineered immune cells; the immune cells express a chimeric antigen receptor; the immune cells are T cells or NK cells; the chimeric antigen receptor comprises the following sequentially connected parts: a signal peptide region, a single-domain antibody region, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region; the nucleotide sequence of the signal peptide region is SEQ ID NO.47; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.48; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.49; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.50; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.51; and the nucleotide sequence of the single-domain antibody region is SEQ ID NO.

9.

4. A CAR-engineered immune cell, characterized in that, The immune cells express a chimeric antigen receptor; the immune cells are T cells or NK cells; the chimeric antigen receptor comprises the following sequentially connected parts: a signal peptide region, a single-domain antibody region, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region; the nucleotide sequence of the signal peptide region is SEQ ID NO.47; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.48; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.49; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.50; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.51; and the nucleotide sequence of the single-domain antibody region is SEQ ID NO.

9.

5. A chimeric antigen receptor fusion protein, characterized in that, It includes the following sequentially connected regions: signal peptide region, single-domain antibody region, CD8 hinge region, CD28 transmembrane region, CD28 intracellular region, and CD3zeta intracellular region; the nucleotide sequence of the signal peptide region is SEQ ID NO.47; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.48; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.49; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.50; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.51; and the nucleotide sequence of the single-domain antibody region is SEQ ID NO.9.

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