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CN-116284446-B - Targeting CD200 chimeric antigen receptor and application thereof

CN116284446BCN 116284446 BCN116284446 BCN 116284446BCN-116284446-B

Abstract

The invention provides a targeting CD200 chimeric antigen receptor and application thereof, wherein the targeting CD200 chimeric antigen receptor comprises a signal peptide, a single-chain antibody ScFv, strepII, CD alpha range, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3 zeta chain which are spliced in sequence from the N end to the C end, and the single-chain antibody ScFv specifically recognizes a CD200 antigen on the surface of a tumor cell. The chimeric antigen receptor has remarkable effect in preparing the medicine for treating acute myelogenous leukemia.

Inventors

  • ZHANG TONGCUN
  • TIAN GAOHUI

Assignees

  • 武汉科技大学

Dates

Publication Date
20260508
Application Date
20230215

Claims (9)

  1. 1. A targeting CD200 chimeric antigen receptor is characterized by comprising a signal peptide, a single-chain antibody ScFv, strepII, CD alpha range, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3 zeta chain which are spliced in sequence from N end to C end, wherein the single-chain antibody ScFv specifically recognizes a CD200 antigen on the surface of a tumor cell; The amino acid sequence of the single-chain antibody ScFv is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.
  2. 2. A CD200 chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO.3 and the nucleotide sequence is shown in SEQ ID NO. 4.
  3. 3. The chimeric antigen receptor targeting CD200 as claimed in claim 1, wherein the amino acid sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.5 and the nucleotide sequence is shown in SEQ ID NO. 6.
  4. 4. Use of a targeted CD200 chimeric antigen receptor according to any one of claims 1-3 for the preparation of a medicament for the treatment of acute myeloid leukemia.
  5. 5. A recombinant chimeric antigen receptor gene vector, characterized in that a BRD-PTK-EF1 alpha vector is taken as a framework, and a lentivirus, retrovirus or transposon vector of the encoding nucleotide sequence of the chimeric antigen receptor according to any one of claims 1 to 3 is inserted.
  6. 6. The use of a recombinant chimeric antigen receptor gene vector according to claim 5 in the preparation of a medicament for the treatment of acute myeloid leukemia.
  7. 7. An immune cell expressing a chimeric antigen receptor, which is obtained by transfecting an immune cell with the coding nucleotide sequence of the chimeric antigen receptor according to any one of claims 1 to 3 or the recombinant chimeric antigen receptor gene vector according to claim 5, wherein the immune cell is selected from one of umbilical cord blood, peripheral blood, IPSC-derived T cells, NK cells, NKT cells, alpha beta T cells, gamma delta T cells, CD4+ T cells and CD8+ T cells.
  8. 8. The method for preparing immune cells expressing chimeric antigen receptor according to claim 7, wherein the method comprises the steps of activating isolated immune cells for 1 day, then infecting the immune cells with the recombinant chimeric antigen receptor gene vector according to claim 5 to obtain immune cells expressing chimeric antigen receptor, and detecting anti-tumor activity of the immune cells expressing chimeric antigen receptor, wherein the selected cell line is a cell membrane which is highly expressed or expresses CD200 protein.
  9. 9. The use of an immune cell expressing a chimeric antigen receptor according to claim 7 in the preparation of a medicament for the treatment of acute myeloid leukemia.

Description

Targeting CD200 chimeric antigen receptor and application thereof Technical Field The invention relates to the field of medical biology, in particular to a targeting CD200 chimeric antigen receptor and application thereof. Background The principle of the CAR-T cell is that an antibody single chain variable region (Scfv) capable of recognizing a certain tumor antigen and an intracellular region of a CD 3-zeta chain are coupled into a chimeric protein in vitro by a genetic engineering method, and the T cell of a patient cultured in vitro is transfected by a gene transduction method so as to express the Chimeric Antibody Receptor (CAR). After T cells of a patient are "reprogrammed," a large number of killer CAR-T cells are generated, enabling specific targeting of tumor cells. Compared with the traditional immunotherapy, the CAR-T has the remarkable advantages of more accurate treatment, more accurate targeting, wider tumor killing range, longer lasting effect and the like. Although the current method is continuously reported in the field of CAR-T tumor treatment, the CAR-T cell treatment process still faces the problems of off-target caused by high heterogeneity of tumors, treatment caused by singleness of targets and the like. Wherein the high heterogeneity of tumor cells directly leads to limitations in the course of treatment of CAR-T therapy, whereas the single nature of the target limits the broad spectrum of treatments. Thus, selecting membrane surface markers that are specifically expressed in tumor cells and have broad-spectrum expression in different tumors becomes a crucial step in the effectiveness of CAR-T therapy. CD200 (OX-2) is a cell surface glycoprotein that inhibits alloimmune and autoimmune responses through its receptor CD200R, and is highly expressed on many malignant tumors including Chronic Lymphocytic Leukemia (CLL), acute Myeloid Leukemia (AML), and cancer stem cells. The major obstacle in the treatment of AML is the elimination of Leukemia Stem Cells (LSCs), a drug resistant cell with long-term self-renewal capacity that is considered a source of relapse. The invention patent number CN114728048a discloses that CD200 receptor antagonist binding molecules can be used as medicaments for the treatment of solid, liquid or neuroendocrine tumor cancers. The invention patent No. CN102906115A discloses that the anti-CD 200 antibody can be used as a cancer therapeutic drug. The two patents do not disclose that chimeric antigen receptors targeting CD200 can be used as drugs for the treatment of AML. Disclosure of Invention In view of this, the present invention proposes a chimeric antigen receptor targeted by CD200 as a drug that can treat AML. The technical scheme of the invention is that firstly, the invention provides a target CD200 chimeric antigen receptor, which comprises a signal peptide, a single-chain antibody ScFv, strepII, CD alpha range, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3 zeta chain which are spliced from the N end to the C end in sequence, wherein the single-chain antibody ScFv specifically recognizes a CD200 antigen on the surface of a tumor cell. Based on the technical scheme, preferably, the amino acid sequence of the single-chain antibody ScFv is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2. Based on the above technical scheme, preferably, the amino acid sequence of the CD28 intracellular domain is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4. Based on the technical scheme, preferably, the amino acid sequence of the intracellular co-stimulatory domain 4-1BB is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6. The invention also provides an application of the chimeric antigen receptor taking CD200 as a target spot in preparing a medicament for treating acute myeloid leukemia. Thirdly, the invention provides a gene vector of a recombinant chimeric antigen receptor, which takes a BRD-PTK-EF1 alpha vector as a framework and inserts a lentivirus, retrovirus or transposon vector of a coding nucleotide sequence of the chimeric antigen receptor. Fourth, the invention provides an application of a recombinant chimeric antigen receptor gene vector in preparing a medicament for treating acute myeloid leukemia. Fifth, the invention provides an immune cell expressing a chimeric antigen receptor, which is obtained by transfecting an immune cell by a coding nucleotide sequence of the chimeric antigen receptor or a recombinant chimeric antigen receptor gene vector, wherein the immune cell is selected from one of umbilical cord blood, peripheral blood, IPSC-derived T cells, NK cells, NKT cells, alpha beta T cells, gamma delta T cells, CD4+ T cells and CD8+ T cells. The invention provides a preparation method of immune cells expressing chimeric antigen receptors, which comprises the following steps of activating the isolated immune cel