Nectin4-targeting car-t cell and use thereof in cancer treatment

Abstract

The present invention relates to the field of immunotherapy technology, and specifically, to a Nectin4-targeting CAR-T cell and use thereof in preparing a medicament for treating cancer. The method for preparing the Nectin4-targeting CAR-T cell comprises: introducing a Nectin4-targeting CAR gene into a T cell via a lentiviral vector, such that the CAR gene is integrated into the genome of the T cell.

Description

CAR-T cell targeting Nectin4 and its application in cancer treatment

[0001] The present application claims the priority of the Chinese Invention Patent Application with the application date of February 26, 2025, the application number of 2025102170772, and the invention name of "CAR-T cell targeting Nectin4 and its application in cancer treatment". TECHNICAL FIELD

[0002] The present application belongs to the technical field of immunotherapy, and specifically relates to a CAR-T cell targeting Nectin4 and its application in cancer treatment. BACKGROUND

[0003] T cell adoptive immunotherapy is a promising method for cancer treatment. Immunotherapy methods enhance the specificity of isolated genetically modified human T cells to specific tumor-associated antigens. Genetic modification can involve the expression of chimeric antigen receptors or exogenous T cell receptors to graft antigen specificity onto T cells. In contrast to exogenous T cell receptors, the specificity of chimeric antigen receptors is derived from the variable domains of monoclonal antibodies. Therefore, T cells expressing chimeric antigen receptors (CAR-T cells) induce a tumor immune response in a major histocompatibility complex-unrestricted manner. T cell adoptive immunotherapy has been used as a clinical therapy for many cancers, including B-cell malignancies, multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, mesothelioma, melanoma, prostate cancer, and pancreatic cancer, etc.

[0004] Lectins Nectin1, Nectin2 and Nectin3 are widely expressed in normal adult tissues, but Nectin4 only exists during embryogenesis, and the expression amount in normal tissues decreases after adulthood. Nectin4, also known as PVRL4 (poliovirus receptor-like 4), is encoded by the Nectin4 gene and has a molecular weight of about 66 kDa. Nectin4 is overexpressed in a variety of tumor cells, such as lung cancer, breast cancer, pancreatic cancer, ovarian cancer, urothelial cancer, and bladder cancer, etc. Nectin4 is a type I single-pass transmembrane protein and belongs to the Ca 2+Nectin4 is an immunoglobulin-dependent molecule composed of an extracellular domain, a transmembrane region, and a cytoplasmic tail. The extracellular domain includes a distal IgV domain and two IgC domains, which can bind to various growth factor receptors, including EGFR and HER2, thereby influencing multiple signal transduction pathways and significantly impacting cell growth, migration, and apoptosis. It is closely related to tumorigenesis and metastasis. The cytoplasmic tail of Nectin4 binds to the adaptor molecule afadin via its C-terminal amino acid sequence. Subsequently, afadin binds to and recruits filamentous actin components (F-actin), thereby promoting cell-cell adhesion. Nectin4 promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / Akt pathway. Targeting Nectin4 could be an effective strategy for treating cancers with high Nectin4 expression.

[0005] To overcome the technical challenges of limited types of CAR-T cells targeting Nectin4 and poor therapeutic effects in existing technologies, developing new antibody sequences targeting this target in order to provide a more efficient treatment option has become a hot research topic. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a CAR-T cell targeting Nectin4 and its application in cancer treatment. This invention discovers a new antibody sequence targeting this target, and the designed CAR-T cells can be applied to the treatment of cancers such as ovarian cancer and breast cancer.

[0007] In a first aspect, an anti-Nectin4 antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions H-CDR 1-3 in the amino acid sequence of SEQ ID NO:15, and the light chain variable region comprises light chain complementarity-determining regions L-CDR 1-3 in the amino acid sequence of SEQ ID NO:16.

[0008] In one embodiment, H-CDR 1-3 respectively comprise the amino acid sequences of SEQ ID NO:17-19. In one embodiment, L-CDR 1-3 respectively comprise the amino acid sequences of SEQ ID NO:20-22.

[0009] In one embodiment, the anti-Nectin4 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:15. The light chain variable region comprises the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:16.

[0010] In one embodiment, the antigen-binding fragment is a single-chain Fv. In one embodiment, the single-chain Fv contains the amino acid sequence of SEQ ID NO:10.

[0011] In a second aspect, a polynucleotide is provided that comprises a nucleotide sequence encoding the anti-Nectin4 antibody or an antigen-binding fragment thereof described herein.

[0012] In a third aspect, a chimeric antigen receptor is provided, which comprises the anti-Nectin4 antibody or its antigen-binding fragment as described herein.

[0013] In one embodiment, the chimeric antigen receptor further comprises a CD8α transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain. Optionally, the chimeric antigen receptor further comprises a CD8 signal peptide.

[0014] In one embodiment, the chimeric antigen receptor comprises a structure of CD8 signal peptide-anti-Nectin4 scFv-CD8α transmembrane domain-CD28 costimulatory domain-4-1BB costimulatory domain-CD3ζ signaling domain.

[0015] In one embodiment, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:11. In one embodiment, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:12. In one embodiment, the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO:13. In one embodiment, the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:14. In one embodiment, the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:9.

[0016] In one embodiment, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:2.

[0017] In the fourth aspect, a polynucleotide is provided that encodes the chimeric antigen receptor described herein.

[0018] In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:1 or its degenerate sequence.

[0019] In the fifth aspect, a lentiviral vector containing the polynucleotides described herein is provided.

[0020] In a sixth aspect, a method for preparing CAR-T cells targeting Nectin4 is provided, characterized by comprising the following steps:

[0021] The CAR gene targeting Nectin4 was introduced into T cells via a lentiviral vector, allowing the CAR gene to integrate into the T cell genome.

[0022] The nucleotide sequence of the CAR gene targeting Nectin4 is the polynucleotide described herein, preferably the nucleotide sequence shown in SEQ ID NO:1, or the protein sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:2.

[0023] In one embodiment, the preparation method includes:

[0024] (1) Select cells with a passage number of no more than 3 generations, adjust the cell density according to the cell growth density and state, and plate 293T cells with a growth density of 80%.

[0025] (2) Once the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out;

[0026] (3) Use lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G, as well as constructed lentiviral plasmids containing CAR structures, and perform lentiviral plasmid preparation according to the plasmid instructions.

[0027] (4) Mix the plasmid mixture obtained in step (3) with the transfection reagent until homogeneous, let stand at room temperature, add it to the 293T cells after changing to fresh culture medium, mix well, and continue culturing.

[0028] (5) Collect the culture supernatant and filter it through a filter membrane;

[0029] (6) The collected viral fluid was concentrated and the viral titer was determined by infecting 293T cells and by flow cytometry to detect the CAR positivity of infected 293T cells for later use.

[0030] In one implementation, the method for preparing the lentiviral plasmid containing the CAR structure in step (3) is as follows:

[0031] S1. Insert the synthesized sequence into the vector plasmid, transform competent cells DH5α, and plate the bacterial culture onto agar plates containing ampicillin for culture; pick multiple clones from the agar plates and inoculate them into liquid LB medium containing ampicillin for constant temperature shaking culture;

[0032] S2. Extract plasmids according to the instructions, perform Sanger sequencing on each cloned plasmid, and select the bacterial culture with the correct sequence number from the clones based on the sequencing data for inoculation and shake flask culture.

[0033] S3. Extract the expression vector plasmid, measure its concentration and purity using a spectrophotometer, then verify the extracted expression vector plasmid by double enzyme digestion and agarose gel electrophoresis, and finally perform Sanger sequencing.

[0034] The transfection reagent mentioned in step (4) is lipofectamine 2000, and the amount added is 2 μL / μg plasmid; and / or the room temperature standing time mentioned in step (4) is 20 min.

[0035] In step (5), the culture supernatant was collected after 48h and 72h and filtered.

[0036] In the seventh aspect, CAR-T cells targeting Nectin4 are provided, which contain the chimeric antigen receptor, polynucleotide, or lentivirus described herein, or are prepared using the methods described herein.

[0037] In the eighth aspect, the application of Nectin4-targeted CAR-T cells in cancer treatment, as described in this article, is provided.

[0038] In one implementation, the cancer is a cancer that overexpresses Nectin4, preferably one or more of lung cancer, breast cancer, pancreatic cancer, ovarian cancer, urothelial carcinoma, and bladder cancer.

[0039] In a ninth aspect, a method for treating cancer in a patient is provided, comprising the step of administering CAR-T cells targeting Nectin4 as described herein to the patient. In one embodiment, the cancer is a cancer that overexpresses Nectin4, preferably one or more of lung cancer (such as non-small cell lung cancer), breast cancer, pancreatic cancer, ovarian cancer, urothelial carcinoma, and bladder cancer.

[0040] In a tenth aspect, CAR-T cells targeting Nectin4 as described herein are provided for use in the treatment of cancer. In one embodiment, the cancer is a cancer that overexpresses Nectin4, preferably one or more of lung cancer, breast cancer, pancreatic cancer, ovarian cancer, urothelial carcinoma, and bladder cancer. In one embodiment, the lung cancer is non-small cell lung cancer.

[0041] In the eleventh aspect, a pharmaceutical composition is provided comprising the antibody or CAR-T cells described herein. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0042] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0043] A method for preparing CAR-T cells targeting Nectin4 includes the following steps:

[0044] The CAR gene targeting Nectin4 was introduced into T cells via a lentiviral vector, allowing the CAR gene to integrate into the T cell genome.

[0045] The structure and sequence of the CAR gene targeting Nectin4 are as follows: CD8 signal peptide-anti-Nectin4scFv-CD8α transmembrane domain-CD28 co-stimulatory domain-4-1BB co-stimulatory domain-CD3ζ signal transduction domain; the nucleotide sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:1; the protein sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:2.

[0046] The specific steps are as follows:

[0047] (1) Select cells with a passage number of no more than 3 generations, adjust the cell density according to the cell growth density and state, and plate 293T cells with a growth density of 80%.

[0048] (2) Once the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out;

[0049] (3) Use lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G, as well as constructed lentiviral plasmids containing CAR structures, and perform lentiviral plasmid preparation according to the plasmid instructions.

[0050] (4) Mix the plasmid mixture obtained in step (3) with the transfection reagent until homogeneous, let stand at room temperature, add it to the 293T cells after changing to fresh culture medium, mix well, and continue culturing.

[0051] (5) Collect the culture supernatant and filter it through a filter membrane;

[0052] (6) The collected viral fluid was concentrated and the viral titer was determined by infecting 293T cells and by flow cytometry to detect the CAR positivity of infected 293T cells for later use.

[0053] The beneficial technical effects of this invention include:

[0054] (1) A novel antibody sequence targeting Nectin4 is provided, which binds to Nectin4 with high affinity.

[0055] (2) A CAR gene and CAR-T cells targeting Nectin4 were designed, which can be applied to the treatment of cancers such as ovarian cancer and breast cancer. The Nectin4-targeting CAR-T cells of this invention can specifically recognize the Nectin4 antigen, bind only to Nectin4-positive tissues, and not recognize other tissues and organs, thus eliminating the risk of off-target effects and exhibiting good targeting. In addition, the Nectin4-targeting CAR-T cells of this invention have excellent tumor-killing function and can effectively kill various Nectin4-positive tumor cells.

[0056] (3). This invention provides a process for preparing CAR-T cells targeting Nectin4, which achieves stable integration of the CAR gene through a lentiviral vector, thereby enhancing the targeted killing ability of T cells; by measuring the viral titer, the viral infection efficiency can be precisely controlled, thus optimizing the production process of CAR-T cells. Attached Figure Description

[0057] Figure 1 shows the agarose gel electrophoresis verification of the expression vector plasmid.

[0058] Figure 2 shows the positive rate of CAR-T cells detected by flow cytometry using Nectin4 antigen.

[0059] Figure 3 shows the detection results of CAR-T cells specifically killing human ovarian cancer cells SK-OV-3.

[0060] Figure 4 shows the detection results of CAR-T cells specifically killing A549 non-small cell lung cancer cells.

[0061] Figure 5 shows the detection results of CAR-T cells specifically killing human glioblastoma cells U-87MG.

[0062] Figure 6 shows the relationship between the concentration of anti-Nectin4 scFv and absorbance, indicating the binding affinity of anti-Nectin4 scFv to recombinant human Nectin4 protein. Detailed Implementation

[0063] The following definitions are provided to enable those skilled in the art to understand the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0064] definition

[0065] As used in this article, the term "Nectin4" is a transmembrane glycoprotein, short for Nectin Cell Adhesion Molecule 4, and belongs to the Nectin family. Nectin4 functions to mediate cell adhesion and signal transduction, and is a key target in research on various diseases, especially cancer.

[0066] As used herein, the term "antibody" refers to a naturally occurring or partially or wholly synthetic (e.g., recombinant) immunoglobulin. Antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies encompass a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, synthetic antibodies, recombinant antibodies, human antibodies, non-human antibodies (e.g., murine antibodies), humanized antibodies, chimeric antibodies, and intracellular antibodies. In this document, an antibody may be an anti-Nectin4 antibody.

[0067] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule of a non-intact antibody that binds to an antigen and / or epitope, including portions of the intact antibody. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-chain antibody molecules (e.g., scFv), etc. In this document, the antigen-binding fragment may be in the form of scFv.

[0068] As used herein, the term "scFv fragment" refers to an antibody fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH) covalently linked in any order via a peptide linker. In one embodiment, the scFv may comprise the three LCDRs and three HCDRs described herein. In one embodiment, the scFv may comprise the heavy chain variable region and the light chain variable region described herein. The single-chain Fv may comprise the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:10.

[0069] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. An antibody contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, those skilled in the art will readily identify the CDRs as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art. The anti-Nectin4 antibody described herein may contain 3 H-CDRs and 3 L-CDRs. The H-CDRs are CDR-H1: DYYMH (SEQ ID NO:17); CDR-H2: NFHPYNDDTKYNEKFKG (SEQ ID NO:18); CDR-H3: SYGNYPWFAY (SEQ ID NO:19); and the L-CDRs are CDR-L1: KASQDIKSYLS (SEQ ID NO:20); CDR-L2: YATSLAD (SEQ ID NO:21); and CDR-L3: QQFTSSPFT (SEQ ID NO:22). The anti-Nectin4 antibody may also contain the following frame region sequences: FR-H1: QVKLQESGAELVRSGASVKLSCTASGFNIK (SEQ ID NO:23); FR-H2: WVKQRPEQSLEWIG (SEQ ID NO:24); FR-H3: KAKLTADKSSSTAYMQLSSLTSEDSAVYYCAR (SEQ ID NO:25); FR-H4: WGQGTTVTVSS (SEQ ID NO:26); FR-L1: DIELTQSPSSMYASLGERVTITC (SEQ ID NO:27); FR-L2: WYQQKPWKSPKTLIY (SEQ ID NO:28); FR-L3: GVPSRFSGSGSGQDYSLTISSLESDDTATYYC (SEQ ID NO:29); and FR-L4: FGSGTKLELK (SEQ ID NO:30). In one embodiment, the light chain variable region of the anti-Nectin4 antibody comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16.In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15.

[0070] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D "Affinity constant" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The antibody affinity constant Kaff (Affinity Constant) is a core quantitative indicator measuring the binding strength between an antibody and an antigen epitope; a larger value indicates a stronger binding ability between the antibody and the antigen. The antibody affinity constant Kaff is related to the equilibrium dissociation constant K... D It is the reciprocal relationship describing the antibody-antigen binding ability; the two are essentially two different ways of expressing the same interaction.

[0071] As used herein, the term "chimeric antigen receptor" or "CAR" generally refers to a group of peptides, typically two in the simplest embodiment, which, when in immune effector cells, provide cell-to-target cell specificity (typically cancer cells) and generate intracellular signaling. In some embodiments, the CAR comprises at least one extracellular antigen-binding domain (such as scFv), a transmembrane domain, and a cytoplasmic signaling domain, which includes functional signaling domains derived from stimulatory and / or co-stimulatory molecules.

[0072] As used herein, the term "polynucleotide molecule" or "nucleic acid" refers to a chain-like compound composed of nucleotides. Nucleotide monomers can be nucleotides or deoxynucleotides. Polynucleotide molecules can encompass both DNA and RNA molecules. The nucleic acid described herein can be a nucleic acid encoding anti-Nectin4 scFv or a nucleic acid encoding a chimeric antigen receptor. When referring to a specific nucleotide sequence encoding anti-Nectin4 scFv or a nucleic acid encoding a chimeric antigen receptor, it should be understood that this encompasses a degenerate sequence that can encode the same amino acid sequence.

[0073] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0074] As used herein, the term "host cell" refers to a cell that can be used to deliver a vector, including but not limited to prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or other human cells. Host cells can include single cells or populations of cells. Host cells can include immune cells. Immune cells include T cells, B cells, NK cells, monocytes, macrophages, or dendritic cells, or any combination thereof. Immune cells can express the chimeric antigen receptors described herein to form immune cells for cancer treatment (e.g., CAR-T cells).

[0075] Vector introduction into host cells can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0076] As used herein, the term “pharmaceuticalally acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0077] As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonging survival compared to the expected survival (if no treatment was received).

[0078] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease associated with high Nectin4 expression.

[0079] As used herein, the terms "cancers overexpressing Nectin4" and "diseases associated with high Nectin4 expression" refer to a condition in a subject's diseased cells, such as cancer cells, where the expression level of Nectin4 is higher than that in the same subject's normal healthy cells. In some cases, "cancers overexpressing Nectin4" and "diseases associated with high Nectin4 expression" may be used interchangeably. In specific implementations, for example, cancers overexpressing Nectin4 may include lung cancer, breast cancer, pancreatic cancer, ovarian cancer, urothelial carcinoma, and bladder cancer.

[0080] As used herein, the percentage sequence identity between two sequences (amino acid or nucleotide sequences) is a function of the number of common positions shared by the sequences relative to the length of the sequences being compared (i.e., % identity = number of common positions / total number of positions compared x 100). This calculation takes into account the number of any gaps and the length of each gap, which are introduced to maximize the degree of sequence identity between the two sequences. Sequence comparisons and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art (see, for example, US 8008449, etc.). When referring to sequence identity, any point value and any range between 80% and 99% can be used, including at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

[0081] As used herein, “human T cell” or “T cell” refers to a T cell isolated from a donor (especially a human donor). T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions.

[0082] Chimeric antigen receptor

[0083] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T Immunotherapy) is a novel cellular immunotherapy technology that has rapidly developed in recent years. Based on the theory of immune system recognition and activation, it uses genetic engineering to artificially overexpress single-chain antibody variable region gene fragments on the surface of T cells that recognize specific tumor surface antigens. This allows T cells to recognize specific antigens and kill target cells expressing those antigens. The core theoretical basis of CAR-T immunotherapy is the recognition and activation of T lymphocytes. It primarily involves different scFvs recognizing different specific antigens on tumor cells, and then transmitting signals through the hinge and transmembrane region of the CD8 molecule to the CD28 or 4-1BB and TCR co-stimulatory activation region within the T lymphocyte membrane. This activates the body's own T lymphocytes, enabling them to specifically attack and kill the recognized tumor cells. Furthermore, because CAR-T cells use an antibody-based antigen recognition model, they are not subject to MHC restrictions.

[0084] CN116589584A discloses the design of a monoclonal antibody or fragment thereof targeting human Nectin4 protein. The monoclonal antibody or fragment thereof targeting human Nectin4 protein comprises a heavy chain and a light chain. The heavy chain includes a heavy chain variable region (VH), and the light chain includes a light chain variable region (VL). The variable region VH includes three heavy chain complementarity-determining regions 1 to 3 (CDR-H1, CDR-H2, and CDR-H3), and the variable region VL includes three light chain complementarity-determining regions 1 to 3 (CDR-L1, CDR-L2, and CDR-L3). Any amino acid sequence in the three heavy chain complementarity-determining regions can be paired with any amino acid sequence in the three light chain complementarity-determining regions for use in a double-stranded configuration. Alternatively, any amino acid sequence in either the three heavy chain complementarity-determining regions or any amino acid sequence in either the three light chain complementarity-determining regions can be used as a single chain. The amino acid sequences of the corresponding complementarity-determining regions in the heavy and light chain variable regions of the monoclonal antibody are paired and used to express a humanized antibody. After constructing the expression cassette of the humanized antibody gene plasmid, the expression cassette is transfected into 293T cells via a delivery system for antibody expression. The delivery system can be one of lentivirus, retrovirus, conventional plasmid vector, episome vector, nanodelivery system, electrotransduction, or transposon. The recombinant cell line is an immune cell, which can be any type of immune cell including T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, or TCR-T cells. When immune cells express a chimeric antigen receptor (CAR), NK cells, NKT cells, TIL cells, and gamma-delta T cells are equivalent to T cells (or T cells can replace NK cells). Monoclonal antibodies targeting human Nectin4 protein can be formulated into pharmaceutically acceptable carriers, diluents, or excipients for use in biomaterials and / or biologics, exhibiting good shaping effects while maintaining good efficacy. When monoclonal antibodies are used in biological agents, the biological agent contains expression cassettes (such as gene expression frames), recombinant vectors (such as plasmids), recombinant proteins (such as fusion proteins, antibody proteins, etc.), recombinant microorganisms (such as Escherichia coli, bacteriophages, etc.), or recombinant cell lines (such as immune cells, CHO cells, etc.) constructed from the aforementioned nucleic acid sequences or amino acid sequences, and these nucleic acid sequences or amino acid sequences are derived from the aforementioned monoclonal antibodies. Recombinant vectors include gene recombinant expression vectors and chimeric antigen receptors. When monoclonal antibodies are used in biological agents, they exist as a component of the biological agent formulation. This biological agent component also contains reagents for detecting the concentration of human Nectin4 protein, reagents for detecting the degree of expression of human Nectin4 protein on the surface of tumor cells, antibody-coupled toxins for killing human Nectin4-positive cells, antibody-coupled with other antibodies to form polyclonal antibodies targeting human Nectin4 and other antigens, and antibody-coupled with other proteins to form functional recombinant proteins targeting human Nectin4.The chimeric antigen receptor (CAR) of this technology comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes an antigen-binding domain. The intracellular domain includes a co-stimulatory signaling region, an intracellular region of a cytokine receptor, and a portion of the CD3ζ chain. The co-stimulatory signaling region refers to a portion of the intracellular domain containing the co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen. The extracellular domain of the CAR provided by this technology includes the variable regions of the heavy and light chains of a humanized antibody targeting Nectin4 as the antigen-binding domain. When expressed in T cells, the CAR can recognize antigens based on antigen-binding specificity or protein receptor binding. The antigen-binding domain is fused with intracellular domains derived from the co-stimulatory molecule and the CD3ζ chain. The antigen-binding domain is also fused with intracellular domains combining CD28, 4-1BB, ICOS signaling domains, and CD3ζ signaling domains, respectively. Therapeutic applications are performed using cells transduced with a lentiviral vector encoding a nucleic acid construct of the present invention. Transduced T cells can elicit a CAR-mediated T cell response. The injected cells can kill the recipient's tumor cells, and the CAR-T cells can replicate in vivo, producing long-term persistence that leads to sustained tumor control. The CAR-T cell membrane can express a humanized antibody scFv structural antigen chimeric receptor targeting Nectin4, specifically killing Nectin4-positive target cells without off-target risk. The teachings of CN116589584A regarding monoclonal antibodies or fragments thereof and chimeric antigen receptors also apply to this application.

[0085] The chimeric antigen receptor described herein comprises at least an extracellular ligand-binding domain or portion thereof, a transmembrane domain, and an intracellular domain comprising one or more signal transduction domains and / or co-stimulatory domains. The extracellular ligand-binding domain or portion thereof may be an antibody or an antibody fragment. In this paper, the antibody fragment may be an scFv antibody fragment targeting Nectin4. The chimeric antigen receptor described herein may also include a signal peptide.

[0086] carrier

[0087] Constructs or expression cassettes can be delivered using known transfection and / or transduction vectors, including but not limited to lentiviral vectors, adeno-associated viruses, etc. Lentiviral vectors are a preferred vector type, capable of delivering large amounts of viral nucleic acid into host cells. Lentivirals are characterized by their unique ability to infect / transduce non-dividing cells, and after transduction, lentiviruses integrate their nucleic acid into the host cell's chromosome, but they themselves are not replicable. Lentivirals have three major genes encoding packaging proteins: gag, pol, and vsv-g, as well as one regulatory gene, rev.

[0088] Lentiviral vector systems or lentivirus particles

[0089] Lentiviral virions (particles) are expressed by a vector system encoding essential viral proteins to produce non-replicating lentiviral virions (viral particles). At least one vector exists containing a nucleic acid sequence encoding a lentiviral pol protein essential for reverse transcription and integration, operatively linked to a promoter. For example, the pol protein is expressed by multiple vectors. Vectors containing a nucleic acid sequence encoding a lentiviral gag protein, essential for forming a viral capsid operatively linked to a promoter, may also exist. This gag nucleic acid sequence may be located on a vector different from at least some of the pol nucleic acid sequences. The gag nucleic acid may be located on a vector separate from all the pol nucleic acid sequences encoding the pol protein.

[0090] The gag-pol, rev, and vsv-g vectors contain nucleotides of the lentiviral genome that package lentiviral RNA, called the lentiviral packaging sequence. As described above, lentiviral vector systems typically include at least two or three helper plasmids containing at least one of the gag, pol, or rev genes. Each of the gag, pol, and rev genes can be provided on a separate plasmid, or one or more genes can be provided together on the same plasmid. The gag, pol, and rev genes are provided on the same or separate plasmids, while vsv-g is provided on a single plasmid.

[0091] T cells

[0092] The T cells of the present invention can be prepared by the following method, which includes introducing the polynucleotide molecules, vectors, or viral particles described herein into the T cells. Specifically, the method includes one or more of the following steps: 1) viral vector construction; 2) lentiviral packaging using 293T cells; 3) PBMC isolation; 4) T cell sorting and activation; 5) lentiviral transfection of sorted and activated T cells; 6) culture, expansion, and flow cytometry detection of CAR-T cell positivity and CD3 positivity after infection; 7) cell killing assay of CAR-T cells obtained after infection; and 8) detection of the inhibitory effect of CAR-T cells on solid tumors in a mouse CDX model.

[0093] Methods and uses

[0094] The T cells of this invention can be used to treat various cancers. Those skilled in the art can readily determine the type of cancer for CAR-T cell therapy based on the chimeric antigen receptor expressed by the T cells. Cancers include, but are not limited to, lung cancer (such as non-small cell lung cancer), breast cancer, glioblastoma, pancreatic cancer, ovarian cancer, urothelial carcinoma, and bladder cancer. The treatment methods of this invention may include administering CAR-T cells to cancer patients. This invention also provides the use of CAR-T cells in the preparation of pharmaceuticals or kits for treating cancers, such as one or more of the cancers listed above.

[0095] The nucleotide and amino acid sequences involved in this invention

[0096] (1) The nucleotide sequence of the CAR gene targeting Nectin4 is as follows:

[0097] (2) The protein sequence of the CAR gene targeting Nectin4 is as follows:

[0098] (3) The nucleotide sequence of the CD8 signal peptide is as follows:

[0099] (4) The protein sequence of the CD8 signal peptide is as follows:

[0100] (5) The nucleotide sequence of anti-Nectin4 scFv is as follows:

[0101] (6) The protein sequence of anti-Nectin4 scFv is as follows:

[0102] The heavy chain variable region sequence for anti-Nectin4 scFv is as follows:

[0103] The light chain variable region sequence for anti-Nectin4 scFv is as follows:

[0104] The following are the amino acid sequences of CDR and FR.

[0105] FR-H1:QVKLQESGAELVRSGASVKLSCTASGFNIK(SEQ ID NO:23)

[0106] CDR-H1: DYYMH (SEQ ID NO:17)

[0107] FR-H2:WVKQRPEQSLEWIG(SEQ ID NO:24)

[0108] CDR-H2:NFHPYNDDTKYNEKFKG(SEQ ID NO:18)

[0109] FR-H3:KAKLTADKSSSTAYMQLSSLTSEDSAVYYCAR(SEQ ID NO:25)

[0110] CDR-H3:SYGNYPWFAY(SEQ ID NO:19)

[0111] FR-H4:WGQGTTVTVSS(SEQ ID NO:26)

[0112] FR-L1:DIELTQSPSSMYASLGERVTITC(SEQ ID NO:27)

[0113] CDR-L1: KASQDIKSYLS(SEQ ID NO:20)

[0114] FR-L2:WYQQKPWKSPKTLIY(SEQ ID NO:28)

[0115] CDR-L2:YATSLAD(SEQ ID NO:21)

[0116] FR-L3:GVPSRFSGSGSGQDYSLTISSLESDDTATYYC(SEQ ID NO:29)

[0117] CDR-L3: QQFTSSPFT(SEQ ID NO:22)

[0118] FR-L4:FGGSGTKLELK(SEQ ID NO:30)

[0119] (7) The nucleotide sequence of the CD8α transmembrane domain is as follows:

[0120] (8) The protein sequence of the CD8α transmembrane domain is as follows:

[0121] (9) The nucleotide sequence of the CD28 co-stimulatory domain is as follows:

[0122] (10) The protein sequence of the CD28 co-stimulatory domain is as follows:

[0123] (11) The nucleotide sequence of the 4-1BB co-stimulatory domain is as follows:

[0124] (12) The protein sequence of the 4-1BB co-stimulatory domain is as follows:

[0125] (13) The nucleotide sequence of the CD3ζ signal transduction domain is as follows:

[0126] (14) The protein sequence of the CD3ζ signaling domain is as follows:

[0127] Preparation method of lentiviral plasmids containing CAR structure

[0128] The preparation method of lentiviral plasmids containing CAR structures may include the following specific steps:

[0129] S1. Insert the synthesized sequence into the vector plasmid, transform competent cells DH5α, and plate the bacterial culture onto agar plates containing ampicillin for culture; pick multiple clones from the agar plates and inoculate them into 5 mL of liquid LB medium containing ampicillin, and culture them in a constant temperature shaker at 37℃ and 250 rpm for 12-16 h.

[0130] S2. Extract plasmids according to the instructions, perform Sanger sequencing on each cloned plasmid to verify the accuracy of the inserted sequence, and select the clone with the correct sequence for inoculation and shake flask culture based on the sequencing data.

[0131] S3. Extract the expression vector plasmid, measure its concentration and purity using a spectrophotometer, then verify the extracted expression vector plasmid by double enzyme digestion and agarose gel electrophoresis, and perform Sanger sequencing to verify the accuracy of the inserted sequence.

[0132] Further, in step (4), the transfection reagent is lipofectamine 2000, the amount added is 2 μL / μg plasmid, and the time for standing at room temperature is 20 min; in step (5), the culture supernatant after 48 h and 72 h is collected and filtered.

[0133] Example

[0134] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0135] Three packaging plasmids, pPACKH1-GAG, pPACKH1-REV, and pVSV-G, were extracted using an endotoxin-free plasmid extraction kit from MN (purchased from SBI, catalog number LV550A-1). Concentration and purity were measured using a spectrophotometer. Lentiviral packaging was performed using a four-plasmid packaging system. The four plasmids were a lentiviral expression plasmid containing a CAR structure, and the lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV, and pVSV-G (purchased from SBI, catalog number LV550A-1).

[0136] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0137] Example 1: Screening and activity determination of anti-Nectin4 scFv

[0138] S1, Animal Immunization

[0139] Mice were immunized using a commercially available recombinant Nectin4 antigen (Recombinant Human Nectin-4 (C-6His), nearshore protein, Cat. No. CJ19) as an immunogen. The specific procedure was as follows:

[0140] Preparation of Nectin4 protein emulsion: Dissolve 0.1 mg of protein in 350 μL of PBS buffer, then add the solution to a 1 mL syringe. Add an equal volume of Freund's adjuvant to another 1 mL syringe. Expel air, connect the two syringes using a Luer connector, and repeatedly inject the solution for 15-30 minutes until complete emulsification. To test successful emulsification, gently drop a drop of the emulsion onto the surface of water; if it remains intact for more than 10 minutes, emulsification is considered successful.

[0141] Three 6-8 week old female BALB / c mice were marked and immunized subcutaneously at multiple sites. Each mouse received a primary immunization with 30 μg of Nectin4 protein (200 μL emulsion). Fourteen days later, a booster immunization was performed using the same dose, with Nectin4 protein in PBS solution and Freund's incomplete adjuvant mixed thoroughly at a 1:1 (v / v) ratio. A second booster immunization was performed 14 days later, for a total of three immunizations. This resulted in the production of anti-human Nectin4 antibodies in the mice, which were then used for the construction of the scFv antibody library.

[0142] Construction and panning of S2 and Nectin4 scFv phage antibody libraries

[0143] Screening process for anti-Nectin4 scFv: Mouse spleen cells were collected, and an antibody cDNA library was generated through RNA isolation, PCR amplification, and cloning into a phage display vector. Specifically, RNA was extracted from spleen cells using Trizol (Cat. No. 15596026, Thermo), and the RNA was reverse transcribed using a reverse transcription kit (Cat. No. RR086A, Takara) to obtain the cRNA sequence. The library fragments were obtained by PCR using library construction primers (VH-RP: AGGTSMARCTGCAGSAGTCWGG, SEQ ID NO:33; VH-FP: TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC, SEQ ID NO:34; VL-RP: GACATTGAGCTCACCCAGTCTCCA, SEQ ID NO:35; VL-FP1: CCGTTTGATTTCCAGCTTGGTGCC, SEQ ID NO:36; VL-FP2: CCGTTTTATTTCCAGCTTGGTCCC, SEQ ID NO:37; VL-FP3: CCGTTTTATTTCCAACTTTGTCCC, SEQ ID NO:38; VL-FP4: CCGTTTCAGCTCCAGCTTGGTCCC, SEQ ID NO:39) (primer synthesis company: Sangon Biotech). The reaction program was as follows: PCR reaction program: Step 1: 98℃, 2 min; Step 2: 94℃, 10 s; Step 3: 56℃, 30 s; Step 4: 68℃, 15 s; Step 5: Step 2-4 cycles 35 times; Step 6: 68℃, 10 min. The library fragments were double-digested with sfiI (Cat.No.R0123V, NEB) and NotI-HF (Cat.No.R3189V, NEB). The digested fragments were then ligated to the sfiI and NotI double-digested backbone of plasmid pCANTAB5E (Cat.No.PMV13001-SN, Bio-ViewShine). The ligated plasmid was integrated into TG1 competent cells (Cat.No.60502-2, Lucigen) by electroporation, thus obtaining the phage infection library. The phage infection library was then subjected to multiple rounds of panning.

[0144] The specific screening process is as follows: A phage infection library was used to harvest phage solutions. Phages were screened using ELISA plates coated with commercially available recombinant Nectin4 antigen (Recombinant Human Nectin-4 (C-6His), nearshore protein, Cat. No. CJ19), with a coating volume of 100 μL per well. Three screenings were performed with coating concentrations of 50 μg / mL, 20 μg / mL, and 10 μg / mL of recombinant Nectin4 antigen, using PBS as the buffer. Positive phages binding to the recombinant Nectin4 antigen were obtained and sent to a sequencing company (Sangon Biotech) for sequencing to obtain the nucleotide and amino acid sequences of anti-Nectin4 scFv.

[0145] The obtained anti-Nectin4 scFv nucleotide sequence was spliced ​​with the human Fc nucleotide sequence, and the protein was produced by a protein production company (Nearshore Protein) to obtain human Fc-anti-Nectin4 scFv for subsequent verification experiments.

[0146] The human Fc amino acid sequence (A233-K449) used in this embodiment is an intrinsic sequence, obtained from GenBank: XTI96198.1. The human Fc amino acid sequence (A233-K449) used in this patent is as follows:

[0147] The corresponding nucleotide sequences are as follows:

[0148] S3. ELISA method to measure the binding affinity of human Fc-Nectin4 scFv to human Nectin4.

[0149] Coating antigen reagent: Commercial recombinant Nectin4 antigen (Recombinant Human Nectin-4 (C-6His), nearshore protein, Cat. No. CJ19) was diluted with PBS to 6 concentrations (5 μg / mL-0.156 μg / mL, see Table 1 for details) and added sequentially to microplates, 100 μL per well, one concentration per group, 30 wells per group, 100 μL per well. Controls were set up with 1% BSA and 1×PBS. Incubated overnight at 4°C.

[0150] Blocking and washing: Pour out the Nectin4 protein working solution from the microplate, add washing buffer (PBST > 250 μL) to each well, gently shake, discard the liquid, pat dry on absorbent paper, and wash at least 4 times. Then add 100 μL of 1x blocking buffer for blocking and incubate at 37°C for one hour. The blocking buffer is prepared as follows: First, prepare 1x buffer (Cat. No. PM5090-50x2L, Coolaber) using ddH2O; then prepare 1x buffer (i.e., 1x blocking buffer) containing 1% BSA using BSA (Cat. No. 36101ES60, Yisheng Biotechnology).

[0151] Preparation and incubation of human Fc-Nectin4 scFv working solution: Wash the ELISA plate four times. Dilute the human Fc-Nectin4 scFv obtained by screening with PBS to 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.313 μg / mL, and 0.156 μg / mL respectively. Then, add 100 μL of human Fc-Nectin4 scFv working solution to the coated human Nectin4 antigen at a volume of 200 rpm for 2 h at room temperature.

[0152] Secondary antibody (goat anti-human IgG1 Fc antibody, HRP-labeled, specifically recognizing human Fc tags) incubation: Pour out human Fc-Nectin4 scFv working solution, wash the ELISA plate 4 times, dilute the secondary antibody (Cat.No.10702-MM01T-H, Sino) to the working concentration (1:5000) with PBS, then add the secondary antibody working solution to the ELISA plate at a volume of 100 μL / well, and incubate at room temperature for 1 h.

[0153] Color development and detection: Discard the secondary antibody working solution, wash the microplate four times, and use the TMB colorimetric kit (Cat.No.C520026-0500, Sangon) to add 100 μL of TMB solution to each well. Incubate at room temperature in the dark for 10-15 minutes, then add 50 μL of stop solution to each well. Read the data from the microplate at 450 nm using a microplate reader and perform calculations and analysis.

[0154] S4, Results

[0155] Sequencing results: The nucleotide sequence of Nectin4 scFv obtained by sequencing company (Sangon Biotech) is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.

[0156] Binding activity results: Table 1 shows the binding affinity assay data of the screened Nectin4 scFv, and Table 2 shows that the antibody affinity constant Kaff of Nectin4 scFv is approximately 9.93E+08L / mol.

[0157] According to the Kabat numbering method, the CDR sequence of Nectin4 scFv is determined as follows:

[0158] CDR-H1: DYYMH (SEQ ID NO:17)

[0159] CDR-H2:NFHPYNDDTKYNEKFKG(SEQ ID NO:18)

[0160] CDR-H3:SYGNYPWFAY(SEQ ID NO:19)

[0161] CDR-L1: KASQDIKSYLS(SEQ ID NO:20)

[0162] CDR-L2:YATSLAD(SEQ ID NO:21)

[0163] CDR-L3: QQFTSSPFT (SEQ ID NO:22).

[0164] Table 1: OD450 values ​​at different antigen and antibody concentrations

[0165] Table 2: Mean Kafl (M⁻¹) of Nectin4 scFv-Human IgG Fc Note: OD50 is the minimum antigen concentration required for antibody binding to reach half of the maximum OD value.

[0166] The data in Table 2 and Figure 6 show that the screened anti-Nectin4 scFv exhibits binding affinity to recombinant human Nectin4 protein, with an antibody affinity constant Kaff of approximately 9.93E+08L / mol.

[0167] Example 2: Construction of lentiviral plasmids containing CAR structures

[0168] S1. The nucleotide sequence of the CAR gene targeting Nectin4 (SEQ ID NO:1) was commercially synthesized (Shanghai Sangon Biotech Co., Ltd.) to obtain the synthetic sequence. The synthetic sequence was inserted into the pLenti-MCS-EF1-GFP plasmid (purchased from ALSTEM, catalog number: LV010), transformed into *E. coli* competent cells DH5α, and plated onto agar plates containing ampicillin. Multiple clones were picked from the agar plates and inoculated into 5 mL of liquid LB medium (containing ampicillin), and cultured on a shaker at 37°C and 250 rpm for 12–16 h.

[0169] S2. Plasmids were extracted according to the instructions of the plasmid miniprep kit (catalog number: DP103-03) purchased from Tiangen Biotech Co., Ltd. Each cloned plasmid was sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequences. Based on the sequencing data provided by Shanghai Sangon Biotech Co., Ltd., the bacterial culture of the clone with the correct sequence was selected for mass inoculation and shake-flask culture.

[0170] S3. The expression vector plasmid was extracted using the MN endotoxin-free plasmid large-scale extraction kit. The concentration and purity were measured using a spectrophotometer. The extracted expression vector plasmid was then double-digested with AgeI-HF (NEB, catalog number: R3552S) and BsrGI (NEB, catalog number: R3575S) and verified by agarose gel electrophoresis (Figure 1). Finally, the expression vector plasmid was sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequence.

[0171] Example 3: Preparation of CAR-T cells targeting Nectin4

[0172] (1) Plating within 24 hours before transfection: Select 293T cells with a passage number of no more than 3 times, adjust the cell density according to the cell growth density and state, and plate 293T cells with a growth density of 80%.

[0173] (2) Once the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out;

[0174] (3) Use lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G, as well as constructed lentiviral plasmids containing CAR structures, and perform lentiviral plasmid preparation according to the plasmid instructions.

[0175] (4) Mix the plasmid mixture obtained in step (3) with the transfection reagent evenly, let it stand at room temperature for 20 minutes, add it to the 293T cells after changing the fresh culture medium, mix well, and continue culturing; wherein, the transfection reagent is lipofectamine 2000 (stored at 4℃), and the amount added is 2μL / μg plasmid;

[0176] (5) Collect the culture supernatant after 48h and 72h respectively, and filter it through a 0.45μm filter membrane;

[0177] (6) The collected viral fluid was concentrated using the PEG8000 concentration method, and the viral titer was determined by infecting 293T cells and by subsequent flow cytometry detection of the CAR positivity of infected 293T cells. The virus was stored at -80℃ for later use.

[0178] Example 4: PBMC isolation, T cell activation, lentiviral infection and flow cytometry detection

[0179] 1. PBMC separation

[0180] 1) Collect 6 mL of human peripheral blood (for research purposes);

[0181] 2) Dilution: Add an equal volume of PBS at room temperature and gently mix by pipetting.

[0182] 3) Sample addition: Take a 50mL centrifuge tube, add 6mL of Ficoll (lymphocyte separation solution) into the centrifuge tube (the volume ratio of Ficoll to the blood before dilution is 1:1), tilt the centrifuge tube at 45°, and slowly add the diluted blood about 1cm above the Ficoll liquid surface along the tube wall.

[0183] 4) Centrifugation: Centrifuge at 18-20℃ and 2000rpm for 30min with a speed of 4 / 4. After centrifugation, the liquid will separate into four layers from the bottom of the tube to the surface: red blood cells and granulocytes, layered liquid, mononuclear cells, and plasma.

[0184] 5) Recovery: Insert the pipette directly into the cloud layer (or first aspirate the upper layer of plasma), gently aspirate the cloud layer, and place it into a new centrifuge tube;

[0185] 6) Washing: Add at least 3 times the volume of PBMCs (peripheral blood mononuclear cells) of PBS, centrifuge at 18-20℃ and 1400 rpm for 10 min, and repeat twice;

[0186] 7) Cell Counting: Discard the supernatant, add 1 mL of lymphocyte culture medium, mix well by pipetting, and prepare a PBMC cell suspension. Count using a hemocytometer: Mix one drop of PBMC suspension with one drop of 2% trypan blue staining solution and add to a hemocytometer. Count the total number of cells within 4 large squares under a microscope. Cell count / mL = Total number of cells in 4 large squares / 4 × 10⁻⁶ 4 ×2 (dilution factor).

[0187] 2. T cell activation, lentiviral infection, and cell expansion

[0188] Day 1: Cell resuscitation: Take PBMC cells from liquid nitrogen and resuscitate them;

[0189] PBMC plating: Collect PBMC cells, count them, and finally adjust the concentration to 2×10⁻⁶. 6 Cells / mL, magnetic beads (purchased from Miltenyi, catalog number: 130-128-758) were used to activate PBMC cells, 2 × 10⁶ cells / mL. 6 Add 5 μL of magnetic beads to each cell, mix the magnetic beads with the PBMC cells, and then add 500 μL of cell suspension to each well of a 24-well plate, i.e., add 1 × 10⁶ cells per well. 6 One cell;

[0190] Day 2: Viral infection: Infection was performed with MOI=5. 1 mL of virus culture medium suspension was prepared and added to a 24-well plate.

[0191] Day 4: Transfer all cells from the 24-well plate to a 75cm culture medium containing 20mL of culture medium. 2 Observe the cell state in the culture flask;

[0192] Day 8: Cell status and number were observed. Cells were centrifuged and resuspended, and the positivity rate of CAR-T cells targeting Nectin4 was detected by flow cytometry using biotin-Nectin4 antigen (purchased from ACRO, catalog number: NE4-H82E7). The results are shown in Figure 2, with a positivity rate of CAR-T cells targeting Nectin4 of 61.88%.

[0193] Example 5: Real-time cell killing detection using RIMI (Real-Time Intelligent Monitoring Instrument for Cell Growth)

[0194] 1) Taking human ovarian cancer cells SK-OV-3, non-small cell lung cancer cells A549, and human astrocytoma cells U-87MG as examples (SK-OV-3 and A549 are Nectin4 target positive cells, and U-87MG are Nectin4 target negative cells), after digestion, cell suspensions were prepared, and after mixing by pipetting, cell counting was performed.

[0195] 2) Dilute the cell suspension to 4×10⁻⁶. 4 Cells / mL concentration, keep on ice for later use;

[0196] 3) Take out the RIMI (purchased from Six Beans, model: CM100-α) detection plate and add 50 μL of culture medium;

[0197] 4) Compile the built-in test program of the RIMI detector for this test in the RIMI detector program;

[0198] 5) Place the RIMI detection plate into the detector and observe whether the Mesege item in the program is normal. After it is normal, start the experimental program.

[0199] 6) After the internal program 1 of the detector is completed, take out the detection plate and add 50 μL of tumor cell suspension to the corresponding well. Mix the cell suspension in each tube before adding.

[0200] 7) After adding the cell suspension, place the detection plate in the incubator and let it stand for 30 minutes to allow the cells to settle naturally;

[0201] 8) After 30 minutes, place the test plate into the tester and run the internal program 2 of the tester;

[0202] 9) Observe the cell growth curve after 24 hours. When the cells are in the logarithmic growth phase, prepare to add effector T cells.

[0203] 10) Remove effector T cells from the culture flask, centrifuge, wash, count, and prepare effector group cell concentrations according to different effector-target ratios;

[0204] 11) Pause the program, remove the detection plate, add 100 μL of effector cells to the corresponding position, put it back into the detector, continue the program, and observe daily.

[0205] Figure 3-5 shows the results after the RIMI program was completed, demonstrating that CAR-T cells effectively killed Nectin4-target positive tumor cells (SK-OV-3, A549), but had no killing effect on Nectin4-target negative tumor cells (U-87MG). This shows that the Nectin4-targeted CAR-T cells of the present invention have excellent anti-tumor function, good specificity, and high expected safety. In Figure 3-5, 2.5:1 indicates that the ratio of effector cells to target cells is 2.5:1; "culture medium" indicates that no effector cells were added, only tumor target cells; uninfected T cells indicate that the effector cells are uninfected T cells.

[0206] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An anti-Nectin4 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the heavy chain complementarity-determining region H-CDR 1-3 in the amino acid sequence of SEQ ID NO:15, and the light chain variable region comprises the light chain complementarity-determining region L-CDR 1-3 in the amino acid sequence of SEQ ID NO:16; Optionally, H-CDR 1-3 respectively contain the amino acid sequences of SEQ ID NO:17-19; L-CDR 1-3 respectively contain the amino acid sequences of SEQ ID NO:20-22.

2. The anti-Nectin4 antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the amino acid sequence of SEQ ID NO:

16.

3. The anti-Nectin4 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antigen-binding fragment is a single-chain Fv; preferably, the single-chain Fv contains the amino acid sequence of SEQ ID NO:

10.

4. A polynucleotide comprising a nucleotide sequence encoding an anti-Nectin4 antibody or an antigen-binding fragment thereof according to any one of claims 1-3.

5. A chimeric antigen receptor comprising, as an antigen-binding domain, an anti-Nectin4 antibody or an antigen-binding fragment thereof according to any one of claims 1-3.

6. The chimeric antigen receptor according to claim 5, further comprising a CD8α transmembrane domain, a co-stimulatory domain, and a CD3ζ signal transduction domain, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain; optionally, the chimeric antigen receptor further comprises a CD8 signal peptide.

7. The chimeric antigen receptor according to claim 5 or 6, comprising a structure of CD8 signal peptide-anti-Nectin4 scFv-CD8α transmembrane domain-CD28 costimulatory domain-4-1BB costimulatory domain-CD3ζ signal transduction domain.

8. The chimeric antigen receptor according to claim 6 or 7, wherein the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11; the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 12; the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 13; the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO: 14; and the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO:

9.

9. The chimeric antigen receptor according to any one of claims 5-8, comprising the amino acid sequence of SEQ ID NO:

2.

10. A polynucleotide encoding a chimeric antigen receptor according to any one of claims 5-9.

11. The polynucleotide of claim 10, comprising the nucleotide sequence of SEQ ID NO:1 or a degenerate sequence thereof.

12. A lentiviral vector comprising the polynucleotides according to claim 10 or 11.

13. A method for preparing CAR-T cells targeting Nectin4, characterized in that, Includes the following steps: The CAR gene targeting Nectin4 was introduced into T cells via a lentiviral vector, allowing the CAR gene to integrate into the T cell genome. The nucleotide sequence of the CAR gene targeting Nectin4 is the polynucleotide according to claim 10, preferably the nucleotide sequence shown in SEQ ID NO:1, or the protein sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:

2.

14. The method for preparing Nectin4-targeting CAR-T cells according to claim 13, characterized in that, The specific steps include: (1) Select cells with a passage number of no more than 3 generations, adjust the cell density according to the cell growth density and state, and plate 293T cells with a growth density of 80%. (2) Once the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out; (3) Use lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G, as well as constructed lentiviral plasmids containing CAR structures, and perform lentiviral plasmid preparation according to the plasmid instructions. (4) Mix the plasmid mixture obtained in step (3) with the transfection reagent until homogeneous, let stand at room temperature, add it to the 293T cells after changing to fresh culture medium, mix well, and continue culturing. (5) Collect the culture supernatant and filter it through a filter membrane; (6) The collected viral fluid was concentrated and the viral titer was determined by infecting 293T cells and by flow cytometry to detect the CAR positivity of infected 293T cells for later use.

15. The method for preparing Nectin4-targeting CAR-T cells according to claim 14, characterized in that, The method for preparing the lentiviral plasmid containing the CAR structure mentioned in step (3) is as follows: S1. Insert the synthesized sequence into the vector plasmid, transform competent cells DH5α, and plate the bacterial culture onto agar plates containing ampicillin for culture; pick multiple clones from the agar plates and inoculate them into liquid LB medium containing ampicillin for constant temperature shaking culture; S2. Extract plasmids according to the instructions, perform Sanger sequencing on each cloned plasmid, and select the bacterial culture with the correct sequence number from the clones based on the sequencing data for inoculation and shake flask culture. S3. Extract the expression vector plasmid, measure its concentration and purity using a spectrophotometer, then verify the extracted expression vector plasmid by double enzyme digestion and agarose gel electrophoresis, and perform Sanger sequencing. and / or The transfection reagent mentioned in step (4) is lipofectamine 2000, and the amount added is 2 μL / μg plasmid; and / or the standing time at room temperature mentioned in step (4) is 20 min; and / or In step (5), the culture supernatant was collected after 48h and 72h and filtered.

16. CAR-T cells targeting Nectin4, comprising a chimeric antigen receptor according to any one of claims 5-9, a polynucleotide according to claim 10 or 11, or a lentivirus according to claim 12, or prepared by the method according to any one of claims 13-15.

17. The application of Nectin4-targeting CAR-T cells according to claim 16 in cancer treatment.

18. The application according to claim 17, wherein the cancer is a cancer that overexpresses Nectin4, preferably one or more of lung cancer, breast cancer, pancreatic cancer, ovarian cancer, urothelial carcinoma and bladder cancer, optionally, the lung cancer is non-small cell lung cancer.

19. A pharmaceutical composition comprising an antibody according to any one of claims 1-3 or CAR-T cells according to claim 16.

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