Search

CN-122011207-A - CAR-T cell over-expressing H2A.Z as well as preparation method and application thereof

CN122011207ACN 122011207 ACN122011207 ACN 122011207ACN-122011207-A

Abstract

The invention discloses a CAR-T cell over-expressing H2A.Z, a preparation method and application thereof. In particular, the invention provides chimeric antigen receptors comprising the h2a.z gene or variants functionally identical to the h2a.z gene. The chimeric antigen receptor also includes a signal peptide, an antigen binding domain that targets CLDN18.2, a hinge region, a transmembrane region, a costimulatory domain, and an intracellular signaling domain. The CLDN18.2-CAR-T cell over-expressing the H2AZ.1 gene has a treatment effect obviously superior to that of a CLDN18.2-CAR-T control group not over-expressing the H2AZ.1 gene in a C57BL/6 mouse model inoculated with CLDN18.2 positive tumor cells MC38-CLDN18.2 and KPC.

Inventors

  • ZHU MINGZHAO
  • XU LILI
  • Lv Mengjie

Assignees

  • 中国科学院微生物研究所

Dates

Publication Date
20260512
Application Date
20260106

Claims (10)

  1. 1. The chimeric antigen receptor is characterized in that the chimeric antigen receptor comprises an h2a.z gene or a variant functionally identical to the h2a.z gene, optionally, the h2a.z gene is selected from an h2az.1 gene and an h2az.2 gene, optionally, the h2az.1 gene is derived from a human, a mouse, a rat, a rabbit, a zebra fish, a cow, a chicken, a dog, a goat, a pig, a monkey or a camel, optionally, the h2az.1 gene encodes an amino acid sequence having an amino acid sequence as shown in SEQ ID No. 7, or encodes an amino acid sequence having at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% identity with an amino acid sequence as shown in SEQ ID No. 7, or encodes an amino acid sequence consisting of an amino acid sequence as shown in SEQ ID No. 7, optionally, the h2az.1 gene has a nucleotide sequence as shown in SEQ ID No. 8, or has a nucleotide sequence having at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% identity with a nucleotide sequence as shown in SEQ ID No. 8, or the nucleotide sequence as shown in SEQ ID No. 19, or the nucleotide sequence having at least 37 amino acid sequence as shown in SEQ ID No. 19, or the nucleotide sequence as shown in SEQ ID No. 19.
  2. 2. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor targets one or more of the following antigens: B Cell Maturation Antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLADR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC, CD19, WT-l, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha Fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor ）、CD28、CD30（TNFRSF8）、CD33、CD4、CD40、CD44 v6、CD51、CD52、CD56、CD74、CD80、CEA、CNT0888、CTLA-4、DR5、EGFR、EpCAM、CD3、FAP、 fibronectin extra domain-B, folic acid receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human dispersing factor receptor kinase, IGF-l receptor, GPI-NMB, human dispersing factor receptor kinase IGF-I, igGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5B1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin Canag, N-glycolylneuraminic acid, NPC-1C, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-B, TRAIL-R1, TRAILR2, tumor antigen CTAA.88, VEGF-A, VEGFR-l, VEGFR2 and vimentin, carcinoembryonic antigen (CEA), beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), enterocarboxylesterase, mut hsp70-2, M-CSF, prostase, prostate Specific Antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, her2/neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2 (ephrinB 2), CD22, insulin Growth Factor (IGF) -I, IGF-II, IGF-I receptor, mesothelin, claudin18.2, preferably the chimeric antigen receptor is a chimeric antigen receptor targeting CLDN 18.2.
  3. 3. The chimeric antigen receptor according to claim 1 or 2, further comprising, optionally, a self-cleaving sequence, optionally, a 2A peptide, optionally, the 2A peptide is selected from the group consisting of P2A, T2A, E a and F2A peptides, optionally, the P2A peptide has, or consists of, the amino acid sequence shown in SEQ ID No. 6, optionally, the P2A peptide is encoded by the nucleotide sequence shown in SEQ ID No. 9.
  4. 4. The chimeric antigen receptor according to claim 2 or 3, further comprising or consisting of a signal peptide, an antigen binding domain targeting CLDN18.2, a hinge region, a transmembrane region, a co-stimulatory domain and an intracellular signal transduction domain, optionally the signal peptide having or consisting of an amino acid sequence as shown in SEQ ID NO:1, optionally the hinge region and transmembrane region having or consisting of an amino acid sequence as shown in SEQ ID NO:3, optionally the co-stimulatory domain having or consisting of an amino acid sequence as shown in SEQ ID NO:4, optionally the intracellular signal transduction domain having or consisting of an amino acid sequence as shown in SEQ ID NO:5, optionally the antigen binding domain targeting CLDN18.2 comprises or consists of a light chain CDR1 as shown in SEQ ID NO:11, a light chain CDR2 as shown in SEQ ID NO:12, a light chain CDR3 as shown in SEQ ID NO:13, a heavy chain CDR 15 as shown in SEQ ID NO:15, a heavy chain CDR1 as shown in SEQ ID NO:16, and optionally a heavy chain CDR2 as shown in SEQ ID NO:14, and optionally a heavy chain comprising a heavy chain as shown in SEQ ID NO: 16.
  5. 5. An isolated nucleic acid molecule, characterized in that it encodes a chimeric antigen receptor according to any one of claims 1 to 4.
  6. 6. Expression vector, characterized in that it comprises a nucleic acid molecule according to claim 5, optionally a retroviral vector, a lentiviral vector or an adenovirus-associated vector, preferably a retroviral vector.
  7. 7. Immune cells, characterized in that the immune cells comprise a chimeric antigen receptor according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5 or an expression vector according to claim 6, optionally selected from T cells, B cells, natural killer cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, hematopoietic stem cells and/or peripheral blood mononuclear cells, preferably the immune cells are T cells, preferably the immune cells are CD3 + T cells.
  8. 8. A method of preparing an immune cell, characterized in that the method comprises the step of introducing into the immune cell a nucleic acid molecule according to claim 5 or an expression vector according to claim 6.
  9. 9. Pharmaceutical composition, characterized in that it comprises a chimeric antigen receptor according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6 or an immune cell according to claim 7 and a pharmaceutically acceptable excipient, optionally further comprising a chemotherapeutic drug, optionally selected from cyclophosphamide, doxorubicine, paclitaxel, cisplatin, gemcitabine, nitrogen mustard, carmustine, nimustine, capecitabine, fluorouracil, tegafur, methotrexate, vincristine, carboplatin, oxaliplatin and irinotecan, preferably the chemotherapeutic drug is cyclophosphamide.
  10. 10. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6 or the immune cell according to claim 7 in the manufacture of a medicament for the prevention and/or treatment of a tumor, optionally a CLDN18.2 positive tumor, optionally a tumor selected from the group consisting of gastric cancer, colorectal cancer, pancreatic cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, prostate cancer and bladder cancer.

Description

CAR-T cell over-expressing H2A.Z as well as preparation method and application thereof Technical Field The invention belongs to the field of cellular immunotherapy, in particular relates to engineering immune cell transformation, and is applied to tumor immunotherapy, and more particularly relates to a CAR-T cell over-expressing H2A.Z, and a preparation method and application thereof. Background Adoptive cell transfer therapy has been widely used in the field of tumor immunotherapy. The method comprises the core steps of amplifying a large number of T cells with tumor-associated antigen specificity in vitro, and then reinjecting the T cells back into a patient, thereby inducing the immune killing effect of the T cells on tumors. CAR-T, TCR-T, TILs, etc. all belong to adoptive cell transfer therapies, where CAR-T therapy is a T cell genetically engineered to express a tumor antigen receptor such that it recognizes and clears cells expressing a specific target antigen. When CAR-T binds to a target antigen expressed on the surface of tumor cells, independent of Major Histocompatibility Complex (MHC), this property can trigger potent T cell activation and produce a powerful anti-tumor effect. Up to now CAR-T against the CD19 target has achieved great success in the treatment of hematological tumors. Since CAR-T therapy is only viable when highly expressed, specifically expressed and stably expressed proteins on the surface of tumor cells are identified, it is very critical to select the appropriate tumor-associated antigen for solid tumors. Claudin 18.2/CLDN18.2 is a tight junction protein, is highly selectively expressed in non-malignant gastric mucosa, is positioned at the top end of a paracellular space, is a key component for forming a tight junction complex, and is highly expressed in malignant tumors such as gastric cancer, pancreatic cancer and the like, so that the CLDN18.2 becomes an important target point in tumor immunotherapy, and the CAR-T aiming at the CLDN18.2 target point has been proved to have safety and effectiveness in clinical trials for treating gastrointestinal cancers at present. Nevertheless, CAR-T therapy is challenging in treating solid tumors due to factors such as the immunosuppressive environment of solid tumors, insufficient ability of CAR-T cells to "home to, survive and proliferate," and so on, and therefore, to achieve the goal of precisely and strongly striking solid tumors, it is desirable to prepare a CAR-T cell that is more effective, better in tumor infiltration, and free of immunosuppression in the tumor microenvironment. Disclosure of Invention In order to obtain CAR-T cells with better effect and better tumor infiltration and for relieving immunosuppression in tumor microenvironment, the invention takes CLDN18.2-CAR-T cells as an example, and the H2AZ.1 gene is overexpressed in the CAR-T cells so as to improve the tumor inhibition effect of the CAR-T cells. Specifically, the present invention provides the following technical solutions. In one aspect, the invention provides chimeric antigen receptors comprising the h2a.z gene or a variant functionally identical to the h2a.z gene. In some embodiments, the h2a.z gene is selected from the group consisting of h2az.1 gene and h2az.2 gene. In some embodiments, the h2az.1 gene is derived from a human, mouse, rat, rabbit, zebra fish, cow, chicken, dog, goat, pig, monkey, or camel. In some embodiments, the h2az.1 gene encodes an amino acid sequence having an amino acid sequence set forth in SEQ ID No.7, or encodes an amino acid sequence having at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% identities with an amino acid sequence set forth in SEQ ID No.7, or encodes an amino acid sequence consisting of an amino acid sequence set forth in SEQ ID No. 7. In some embodiments, the h2az.1 gene has a nucleotide sequence as set forth in SEQ ID No. 8, or has a nucleotide sequence having at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% identities with a nucleotide sequence as set forth in SEQ ID No. 8, or the nucleotide sequence of the h2az.1 gene is set forth in SEQ ID No. 8. In some embodiments, the h2az.2 gene encodes an amino acid sequence having an amino acid sequence set forth in SEQ ID No. 18, or encodes an amino acid sequence having at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% identities with an amino acid sequence set forth in SEQ ID No. 18, or encodes an amino acid sequence consisting of an amino acid sequence set forth in SEQ ID No. 18. In some embodiments, the h2az.2 gene has a nucleotide sequence as set forth in SEQ ID No. 19, or has a nucleotide sequence having at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99