Ulbp2 specific chimeric antigen receptor, car-t cell and application thereof

Abstract

The present application relates to the technical field of biology and medicine, and particularly relates to a ULBP2 specific chimeric antigen receptor, a CAR-T cell and application thereof. The ULBP2 specific chimeric antigen receptor comprises a ULBP2 antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the ULBP2 antigen binding domain comprises an amino acid sequence as shown in SEQ ID NO: 1. After the ULBP2 specific chimeric antigen receptor is transduced into lymphocytes, the killing ability of the lymphocytes on tumor cells is significantly enhanced, and the lymphocytes have a significant directional killing effect on tumor cells with high expression of ULBP2. The present application also relates to application of the ULBP2 specific CAR-T cell in combination with an anti-PD1 antibody for treating cancer.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, and in particular to a ULBP2-specific chimeric antigen receptor, CAR-T cells, and their applications. Background Technology

[0002] Gastric cancer is one of the major diseases threatening human life and health. Surgical resection is one of the most effective treatments; however, most patients are diagnosed at an advanced stage, making surgery unsuitable. In recent years, molecular targeted therapy combined with chemotherapy has been widely used in the treatment of advanced gastric cancer, but problems such as significant individual variability in efficacy and drug resistance still exist. Therefore, much current research focuses on exploring the underlying molecular mechanisms of gastric cancer to provide potential molecular markers for early diagnosis and clinical treatment.

[0003] UL16 binding protein-2 (ULBP2) is an immunological factor discovered during the development of proteomics. Studies have shown that ULBP2 is closely related to the occurrence and development of various tumors. ULBP2 expression in early-stage and advanced-stage gastric cancer tissues is significantly higher than in chronic gastritis. Simultaneously, serum ULBP2 concentrations are also significantly elevated in early-stage and advanced-stage gastric cancer. Compared with commonly used serum tumor markers such as CEA and CA199, serum ULBP2 has better diagnostic value for both early-stage and advanced-stage gastric cancer. Therefore, ULBP2 is a potential target for the diagnosis and treatment of gastric cancer.

[0004] Using autologous T cells expressing chimeric antigen receptors (CARs) for tumor cell immunotherapy is a promising strategy, as demonstrated by CD19-targeted CAR-T cell therapy for B-cell malignancies. A CAR is a synthetic receptor composed of an antigen-binding domain, typically a single-chain fragment variable (scFv) derived from a monoclonal antibody, linked via hinges or spacers to a transmembrane domain and an intracellular signaling domain from the T cell receptor complex, containing CD3 zeta (CD3ζ) and a co-stimulatory signaling domain. CAR-T cells mediate tumor cell killing by binding to specific target antigens on the surface of cancer cells via the CAR-T single-chain antibody, without presenting antigens on the major histocompatibility complex (MHC). Upon direct binding to a specific target antigen, CAR-T cells are activated through the function of their intracellular signaling domains. The scFv, acting as the antigen recognition domain, initiates and determines the intensity of T cell activation, providing specificity in a manner independent of the MHC. Generally, CAR-T cells with high affinity scFv exhibit stronger antitumor activity. Therefore, the preparation of high affinity scFv...

[0005] Currently, research and application of ULBP2 as a target in cancer treatment are still limited, especially its application in combination with chimeric antigen receptors, and whether and how it can be effectively used to treat tumors and cancers more efficiently. Further research is needed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a ULBP2-specific chimeric antigen receptor comprising a ULBP2 antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. The ULBP2 antigen-binding domain includes a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0007] Furthermore, the ULBP2 antigen-binding domain includes a heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO: 8.

[0008] Furthermore, the ULBP2 antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0009] Furthermore, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 10.

[0010] Furthermore, the intracellular signal transduction domain includes the 4-1BB co-stimulatory signaling molecule and the human CD3ζ signal transduction domain.

[0011] Furthermore, the 4-1BB co-stimulatory signaling molecule has the amino acid sequence shown in SEQ ID NO: 11.

[0012] Furthermore, the human CD3ζ signal transduction domain has an amino acid sequence as shown in SEQ ID NO: 12.

[0013] Furthermore, the chimeric antigen receptor also includes a hinge region connecting the ULBP2 antigen-binding domain and the transmembrane domain.

[0014] Furthermore, the hinge region has an amino acid sequence as shown in SEQ ID NO: 13.

[0015] Furthermore, the chimeric antigen receptor also contains a signal peptide at its N-terminus.

[0016] Furthermore, the signal peptide has the amino acid sequence shown in SEQ ID NO: 14.

[0017] Furthermore, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 15.

[0018] The present invention also provides an isolated nucleic acid molecule that encodes the ULBP2-specific chimeric antigen receptor as described herein;

[0019] Furthermore, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 16.

[0020] The present invention also provides an expression vector comprising nucleic acid molecules as described herein.

[0021] Furthermore, the vector includes a plasmid vector or a viral vector.

[0022] Furthermore, the viral vector includes one or more combinations of lentiviral vectors, retroviral vectors, or adenoviral vectors.

[0023] The present invention also provides a recombinant lentivirus, which is obtained by co-transfecting a viral vector expressing a chimeric antigen receptor as described herein with a packaging helper plasmid into mammalian cells.

[0024] Furthermore, the packaging aid plasmids are pSPAX2 and pMD2.G.

[0025] Furthermore, the mammalian cells are 293T cells, Lenti-X 293T, or 293T(GP2).

[0026] The present invention also provides a host cell that expresses the ULBP2-specific chimeric antigen receptor as described herein, or that contains the expression vector as described herein.

[0027] Furthermore, the host cells include immune cells.

[0028] Furthermore, the immune cells include T cells, B cells, NK cells, monocytes, macrophages, or dendritic cells, or any combination thereof.

[0029] The present invention also provides the use of chimeric antigen receptors, nucleic acid molecules, expression vectors or host cells as described herein in the preparation of medicaments for the prevention and / or treatment of cancer.

[0030] Furthermore, the cancer is a cancer that overexpresses ULBP2.

[0031] Furthermore, the cancer in question is stomach cancer.

[0032] The inventors also surprisingly discovered that targeting ULBP2 can significantly improve the role of PD-1 inhibitors in the treatment of gastric cancer.

[0033] As used herein, a ULBP2 inhibitor can be any agent that inhibits ULBP2. Preferably, the ULBP2 inhibitor is an anti-ULBP2 antibody as described above, a ULBP2-specific chimeric antigen receptor (CAR), or a cell expressing a ULBP2-specific chimeric antigen receptor or containing an expression vector expressing a ULBP2-specific chimeric antigen receptor. As used herein, a PD-1 inhibitor can be any agent that inhibits PD-1. Preferably, the PD-1 inhibitor is an anti-PD-1 antibody.

[0034] Therefore, the present invention also provides the use of a ULBP2-specific chimeric antigen receptor, a nucleic acid molecule expressing a ULBP2-specific chimeric antigen receptor, an expression vector containing said nucleic acid molecule, or a host cell expressing a ULBP2-specific chimeric antigen receptor or containing an expression vector expressing a ULBP2-specific chimeric antigen receptor in combination with a PD-1 inhibitor in the preparation of a medicament for the prevention and / or treatment of cancer.

[0035] Furthermore, the cancer is a cancer that overexpresses ULBP2.

[0036] Furthermore, the cancer in question is stomach cancer;

[0037] Furthermore, the ULBP2-specific chimeric antigen receptor is as defined herein.

[0038] definition

[0039] As used herein, the term "chimeric antigen receptor" or "CAR" generally refers to a group of peptides, typically two in the simplest embodiments, that, 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 VHH, scFv, or a portion thereof), a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") containing functional signaling domains derived from stimulatory and / or co-stimulatory molecules.

[0040] As used herein, the transmembrane domains used in chimeric antigen receptors are not limited and can be any transmembrane domain well known in the art for constructing chimeric antigen receptors. As an example, the transmembrane domain can have the amino acid sequence shown in SEQ ID NO: 10. As used herein, the intracellular signaling domains used in chimeric antigen receptors are not limited and can be any intracellular signaling domain well known in the art for constructing chimeric antigen receptors. As an example, the intracellular signaling domain can comprise a 4-1BB co-stimulatory signaling molecule and a human CD3ζ signaling domain. Further, the 4-1BB co-stimulatory signaling molecule can have the amino acid sequence shown in SEQ ID NO: 11. Further, the human CD3ζ signaling domain can have the amino acid sequence shown in SEQ ID NO: 12. Further, the chimeric antigen receptor can also include a hinge region connecting the extracellular antigen-binding domain and the transmembrane domain. As used herein, the hinge region used in a chimeric antigen receptor is not limited and can be any hinge region well known in the art for connecting an extracellular antigen-binding domain and a transmembrane domain in a chimeric antigen receptor. As an example, the hinge region can have the amino acid sequence shown in SEQ ID NO: 13. Further, the chimeric antigen receptor also includes a signal peptide at its N-terminus. As used herein, the signal peptide used in a chimeric antigen receptor is not limited and can be any signal peptide well known in the art for use in chimeric antigen receptors. As an example, the signal peptide can have the amino acid sequence shown in SEQ ID NO: 14.

[0041] 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.

[0042] 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 "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0043] 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.

[0044] As used herein, the term "host cell" refers to a cell that can be used to introduce 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, HEK 293 cells, or other human cells. Host cells can include single cells or cell populations. Host cells can include immune cells. Immune cells include T cells, B cells, NK cells, monocytes, macrophages, or dendritic cells, or any combination thereof.

[0045] 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.

[0046] 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.

[0047] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a disease associated with high ULBP2 expression) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are 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 prolonged survival compared to expected survival (if no treatment was received).

[0048] 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 ULBP2 expression.

[0049] As used herein, the terms "cancer overexpressing ULBP2" and "diseases associated with high ULBP2 expression" refer to a condition in a subject's diseased cells, such as cancer cells, where the expression level of ULBP2 is higher than that in the same subject's normal healthy cells. In some cases, "cancer overexpressing ULBP2" and "diseases associated with high ULBP2 expression" may be used interchangeably. In specific implementations, for example, cancer overexpressing ULBP2 may include gastric cancer, etc. Attached Figure Description

[0050] Figure 1 The results show the ELISA detection results demonstrating the binding of the ULBP2-specific antibody to the recombinant ULBP2 protein.

[0051] Figure 2 A schematic diagram of the structure of a chimeric antigen receptor (CAR) plasmid targeting ULBP2 is shown.

[0052] Figure 3 The results of the ULBP2-CAR-T cell killing assay on gastric cancer cell lines were shown.

[0053] Figure 4 The diagram shows the killing effect of ULBP2-CAR-T cells on SNU-216 cells.

[0054] Figure 5 The image shows the effect of ULBP2-CAR-T cells killing gastric cancer organoids.

[0055] Figure 6 The graph shows the killing efficiency of ULBP2-CAR-T cells in killing gastric cancer organoids.

[0056] Figure 7 The in vivo tumor-killing effect of ULBP2-CAR-T in a CDX model constructed from MKN-45 cells was demonstrated.

[0057] Figure 8 The tumor growth inhibitory effect of ULBP2-CAR-T in a CDX model constructed from MKN-45 cells was demonstrated. Detailed Implementation

[0058] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0059] Example 1: Screening for ULBP2-specific antibodies

[0060] ULBP2 Recombinant Protein Expression and Purification: The full-length ULBP2 gene CDS sequence was amplified from the human ULBP2 gene CDS sequence template and cloned into the pET-28a vector (provided by Professor Sun Hui of the Second Hospital of Lanzhou University) to obtain the recombinant plasmid pET-28a-ULBP2 (full-length). The recombinant plasmid was transformed into BL21 competent cells, and after culturing to OD600≈0.6-0.8, protein expression was induced by IPTG. The bacterial cells were sonicated to disrupt the cell structure, inclusion bodies were collected, and the inclusion bodies were washed, dissolved, dialyzed for refolding, and purified using Ni-NTA protein to obtain the ULBP2 recombinant protein.

[0061] Mouse immunization: 200 µg of purified, BCA-quantified ULBP2 recombinant protein (with an appropriate amount of natural adjuvant added to a final concentration of 1 mg / mL) was administered intraperitoneally for the first immunization of mice. A second immunization was performed 14 days later with 100 µg of full-length ULBP2 recombinant protein, followed by a third immunization 28 days later with 50 µg of full-length ULBP2 recombinant protein. The total immunization period was 42 days, using the same immunization method as before. Blood samples from immunized mice were collected 30 days after immunization for ELISA to determine serum antibody titers.

[0062] Microfluidic sorting to construct a positive B cell library: After immunization, mice were sacrificed and their spleens were harvested. B lymphocytes were enriched and separated using magnetic bead sorting technology. Positive B cells expressing high-affinity antibodies were enriched and separated using microfluidic technology.

[0063] Screening for high-affinity and high-specificity ULBP2 scFv: Based on a self-optimized single-cell reverse transcription technology, the mRNA of enriched positive B cells was reverse transcribed. Nested PCR, temperature gradient PCR, and falling PCR techniques were then used to amplify the antibody variable region sequence of B cells. The antibody sequence was cloned into the Lenti-cmv-DP3 display vector to prepare the scFv library. The ULBP2 antibody sequence plasmid was packaged into 293T cells using a lentiviral packaging system to obtain a ULBP2 single-chain antibody cell display library. Based on flow cytometry and enzyme-linked immunosorbent assay (ELISA), a high-affinity and high-specificity antibody against ULBP2 was screened. Its amino acid sequence is shown in SEQ ID NO: 9, and its nucleotide sequence is shown in SEQ ID NO: 17.

[0064] Example 2: Preparation of ULBP2-specific antibody

[0065] (1) Cell culture: 293T cells in the logarithmic growth phase were digested with Trypsin and diluted to 4×10⁻⁶. 5The cells were seeded at a density of 1 / mL in 2L roller flasks; 200ml of DMEM high glucose medium (containing 3% fetal bovine serum) was added to each roller flask, and the roller speed was adjusted to 20 rpm to ensure that the cells adhered evenly to the surface of the roller flask. After about 24 hours, when the cells had reached 90% confluence on the wall surface, transfection was performed.

[0066] (2) Transient transfection: The nucleotide sequence of the ULBP2-specific antibody identified in Example 1 was cloned into the pcDNA3 vector and transiently transfected into 293T cells;

[0067] (3) Harvesting supernatant: On the 5th day after transfection, the cell expression supernatant was collected. The supernatant was centrifuged at 4500 rpm for 15 min, then filtered through a 0.45 µm filter membrane. The filtrate was stored at 4 °C and prepared for purification.

[0068] (4) Antibody purification:

[0069] Connect a 1 mL Protein A HP column to the purification pump and set the flow rate to approximately 1 mL / min;

[0070] Wash the purification column with 20 mL of elution buffer and discard the washing solution;

[0071] Wash the purification column with 20 mL of binding buffer and discard the washing solution;

[0072] Place the inlet tube into the filtrate collected in (3), and collect the filtrate after it flows through the purification column. Repeat this step to pass the collected filtrate through the purification column a second time.

[0073] Wash the purification column with 20 mL of binding buffer and use Coomassie Brilliant Blue to detect the presence of contaminating proteins in the wash buffer (to ensure that non-specifically bound proteins are completely eluted in this step), then discard the wash buffer.

[0074] The elution buffer was slowly passed through the purification column at a flow rate of 1 mL / min, and the eluent was collected. During the elution process, the protein concentration in the eluent was monitored using Coomassie Brilliant Blue reagent to ensure complete elution of the target antibody protein. Finally, all the collected eluent was added to neutralization buffer at a ratio of 10:1 and then dialyzed and ultrafiltered to concentrate the solution. The concentration of the concentrate was measured, and a portion of the antibody concentrate was taken for verification after labeling. The remainder was stored at -20°C.

[0075] Example 3: ELISA detection of the binding of ULBP2 specific antibody to recombinant ULBP2 protein

[0076] (1) Antigen coating: Dilute the antigen (ULBP2 recombinant protein prepared in Example 1) to 10µg / mL with 1× carbonate coating buffer (Na2CO3 (1.59g) + NaHCO3 (2.93g, adjust the pH value and bring the volume to 50mL). Take 50µL of the diluted antigen and coat a 96-well plate with 0.5µg / 200µL (set up a positive control group, a negative control group and an experimental group, with 3 replicates for each group).

[0077] (2) Blocking: Discard the liquid in the well, wash 3 times with washing buffer (prepared by adding Tween-20 to 1×PBS to make a 0.05% solution), add 100μL of blocking solution (skim milk powder (1g) + 1×TBST (20mL)) to each well, and incubate at 37℃ for 1h;

[0078] (3) Antibody incubation: The 96-well plate was washed 5 times in a plate washer. Different concentrations of antibody were added according to different groups. The plate was placed at 37°C for 1 hour. The liquid in the wells was discarded. The plate was washed with washing buffer on a shaker for 3 minutes. The washing was repeated three times.

[0079] (6) Incubation with enzyme-labeled secondary antibody: Add 100 μL of diluted enzyme-labeled secondary antibody (1:5000 dilution) to each well of a 96-well plate and incubate at 37°C in the dark for 30 min;

[0080] (7) Color development: The 96-well plate is washed 6-7 times in a plate washer, and 100 TMB color development solution is added to each well. At this time, the liquid in the well turns blue. The plate is developed at 37°C in the dark for 5-15 minutes.

[0081] (8) Detect absorbance: Add 50 μL of stop solution to each well (the liquid in the well turns yellow), and detect the absorbance at 450 nm using an ELISA reader.

[0082] The results are as follows Figure 1 As shown, the results indicate that the binding activity of the ULBP2-specific antibody obtained in Example 1 to the ULBP2 protein is essentially equivalent to that of the commercially available ULBP2 monoclonal antibody. The antibody's ability to recognize other antigens was also tested using ELISA; the results showed that the ULBP2-specific antibody could not recognize other antigens, demonstrating good specificity.

[0083] Example 4: Construction of a CAR plasmid targeting ULBP2

[0084] The chimeric antigen receptor (CAR) comprises an scFv antibody, a transmembrane domain of human CD8, a 4-1BB co-stimulatory signaling molecule, and a human CD3ζ signaling domain. It was constructed based on the pCDH-EF1α-hCAR-T2A-GFP plasmid. Using the ULBP2scFv sequence (SEQ ID NO: 9), it was assembled according to the CAR construction pattern diagram using the Gibson assembly system. Figure 2The ULBP2-CAR plasmid was successfully constructed.

[0085] To verify the accuracy of the constructed CAR plasmid gene sequence, we first designed sequencing primers based on the ULBP2-CAR plasmid structure and sent the successfully identified CAR plasmid to Beijing Qingke Bio-Sequencing Co., Ltd. for Sanger sequencing. After obtaining the sequencing sequence, we compared it and selected the clone number with the correct sequence. The sequencing results showed that the ULBP2-CAR plasmid was completely correct. Furthermore, the correctly sequenced ULBP2-CAR plasmid was transiently transfected into 293T cells. Cells were collected 24 hours later, and the expression of the ULBP2-CAR plasmid was verified using flow cytometry. The results showed that the ULBP2 expression rate in 293T cells transfected with the ULBP2-CAR plasmid reached 76.5%.

[0086] Example 5: Preparation of CAR-T cells targeting ULBP2

[0087] Peripheral blood was collected from healthy donors and lymphocytes were isolated. After 48 hours of stimulation with CD3 / CD28 magnetic beads, T cells were activated and expanded to obtain high-purity T cells. The CAR lentiviral plasmid constructed in this invention was co-transfected with packaging plasmids pSPAX2 and pMD2.G into HEK293T cells. After 48 hours, the culture supernatant was collected, filtered through a 0.45μm filter, and concentrated by ultracentrifugation to obtain CAR lentiviral. The obtained CAR lentiviral plasmid was then used to infect and stimulate activated T cells to prepare CAR-T cells.

[0088] The expression efficiency of CAR targeting ULBP2 on the surface of T cells was detected by flow cytometry, and the results showed that the expression efficiency of CAR was 78.53%.

[0089] Example 6: CAR-T cell killing experiment on gastric cancer cells

[0090] To detect the ULBP2 targeting of ULBP2-CAR-T cells in killing gastric cancer cells, a gastric cancer cell killing assay was performed using standard lactate dehydrogenase (LDH). MKN-45 and SNU-216 cells in logarithmic growth phase with high ULBP2 expression, as well as ULBP2-knockout SNU-216 cells (2 × 10⁻⁶ cells), were used. 3CAR-T cells were co-cultured with different effector cell to target cell (E:T) ratios in 96-well cell culture plates. After 8 hours, 50 μl of supernatant from each well was transferred to a fresh 96-well flat-bottomed clear plate, and 50 μl of CytoTox 96 reagent (Promega, G1780) was added. The plate was incubated at room temperature for 30 minutes, and 50 μl of stop solution was added to each well. The absorbance signal at 490 nm was measured using a plate reader. Maximum LDH release was determined by adding 0.2% Triton X-100, and cytotoxicity (%) was calculated. Results are as follows. Figure 3 As shown, ULBP2-CAR-T significantly killed MKN-45 and SNU-216 cells with high ULBP2 expression, but had no killing effect on SNU-216 cells with ULBP2 knockout. Figure 4 This image shows the killing effect of ULBP2-CAR-T cells on SNU-216 cells with high ULBP2 expression, as observed under a microscope. It demonstrates the killing effect of CAR-T cells on SNU-216 cells at different potency ratios.

[0091] For organoid killing assays, diffuse gastric cancer (DGC) organoids were seeded onto Matrigel layers and incubated with ULBP2-CAR-T cells. In short, a 96-well standard culture plate was first moistened with culture medium. Then, 15 μl of undiluted Matrigel was evenly coated onto each well and allowed to solidify at room temperature for 15 minutes. The confluent organoids were collected, mechanically sheared, precipitated, and then seeded into pre-coated 96-well plates (approximately 2.5 × 10⁶ cells per well). 3 ULBP2-CAR-T cells were added at different E:T ratios and co-cultured with organoids for 8 hours. Cytotoxicity was determined using the LDH assay described above. Microscopic images of the killing effect and the calculated killing efficiency are shown below. Figure 5 and Figure 6 As shown, ULBP2-CAR-T cells have a killing effect on DGC organoids.

[0092] Example 7: In vivo tumor-killing activity of ULBP2-CAR-T

[0093] A CDX model of hPBMC-derived NSG mice was established. In short, MKN-45 cells stably expressing ULBP2 and firefly luciferase (MKN-45-ULBP2-T2A-Luc) were generated, suspended in 100 μl PBS, mixed with 100 μl Matrigel, and then injected into 6-week-old NSG mice (3 × 10⁶ cells per mouse). 6 (Subcutaneous injection). Mice received ULBP2-CAR-T cells once at 7 dpi (2 × 10⁶ cells per mouse).6 Adoptive metastasis (via intravenous injection) was performed. The combination therapy group received anti-PD-1 (10 mg / kg, intraperitoneally) every 5 days starting at 8 dpi. Untransduced human T cells served as a negative control. Tumor volume was measured using digital calipers, with body weight monitored every other day starting at 7 dpi. Tumor volume was calculated using the following formula: Tumor volume = (long axis) × (short axis)² / 2. The experimental endpoint was defined as death or a tumor size reaching 1500 mm. 3 The result is as follows Figure 7 As shown, ULBP2-CAR-T cells can significantly kill CDX cells constructed from MKN-45 cells, and the combination with PD-1 antibody significantly improves the tumor killing effect.

[0094] Bionic imaging of CDX mice was performed using VISQUE Invivo Smart-LF (Vieworks, Korea) at specified time points, and tumors were harvested and weighed at the experimental endpoint. Results are as follows: Figure 8 As shown, tumor growth in the ULBP2-CAR-T treatment group was significantly inhibited, and the combination with PD-1 antibody significantly improved the tumor suppression effect.

[0095] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, they should all fall within the protection scope of the present invention.

Claims

1. A ULBP2-specific chimeric antigen receptor, characterized in that, It contains the ULBP2 antigen-binding domain, transmembrane domain, and intracellular signal transduction domain. The ULBP2 antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively.

2. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The ULBP2 antigen-binding domain includes a heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO:

8.

3. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The ULBP2 antigen-binding domain contains the amino acid sequence shown in SEQ ID NO:

9.

4. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The transmembrane domain has an amino acid sequence as shown in SEQ ID NO:

10.

5. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The intracellular signal transduction domain includes the 4-1BB co-stimulatory signaling molecule and the human CD3ζ signal transduction domain.

6. The ULBP2-specific chimeric antigen receptor according to claim 5, characterized in that, The 4-1BB co-stimulatory signaling molecule has the amino acid sequence shown in SEQ ID NO:

11.

7. The ULBP2-specific chimeric antigen receptor according to claim 5, characterized in that, The human CD3ζ signal transduction domain has an amino acid sequence as shown in SEQ ID NO:

12.

8. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor further includes a hinge region connecting the ULBP2 antigen-binding domain and the transmembrane domain.

9. The ULBP2-specific chimeric antigen receptor according to claim 8, characterized in that, The hinge region has an amino acid sequence as shown in SEQ ID NO:

13.

10. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor also contains a signal peptide at its N-terminus.

11. The ULBP2-specific chimeric antigen receptor according to claim 10, characterized in that, The signal peptide has the amino acid sequence shown in SEQ ID NO:

14.

12. The ULBP2-specific chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:

15.

13. An isolated nucleic acid molecule, characterized in that, Its encoding is the ULBP2-specific chimeric antigen receptor according to any one of claims 1-12.

14. The nucleic acid molecule according to claim 13, characterized in that, The nucleic acid molecule contains a nucleotide sequence as shown in SEQ ID NO:

16.

15. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 13 or 14.

16. The expression vector according to claim 15, characterized in that, The vector includes a plasmid vector or a viral vector; the viral vector includes one or more combinations of lentiviral vectors, retroviral vectors, or adenoviral vectors.

17. A recombinant lentivirus, characterized in that, It is obtained by co-transfecting a viral vector expressing the ULBP2-specific chimeric antigen receptor according to any one of claims 1-12 into mammalian cells with a packaging helper plasmid.

18. A host cell, characterized in that, It expresses the ULBP2-specific chimeric antigen receptor according to any one of claims 1-12, or contains the expression vector according to claim 15 or 16.

19. The host cell according to claim 18, characterized in that, The host cells include immune cells.

20. The host cell according to claim 19, characterized in that, The immune cells include T cells, B cells, NK cells, monocytes, macrophages, or dendritic cells, or any combination thereof.

21. Use of the chimeric antigen receptor according to any one of claims 1-12, the nucleic acid molecule according to claim 13 or 14, the expression vector according to claim 15 or 16, or the host cell according to any one of claims 18-20 in the preparation of a medicament for the prevention and / or treatment of cancer. The cancer in question is stomach cancer.

22. The use of the chimeric antigen receptor according to any one of claims 1-12, the nucleic acid molecule according to claim 13 or 14, the expression vector according to claim 15 or 16, or the host cell according to any one of claims 18-20, in combination with a PD-1 inhibitor, in the preparation of a medicament for the prevention and / or treatment of cancer. The cancer in question is stomach cancer.

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