T cell antigen coupler targeting CD19, expression vector and application thereof
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A cell antigen and expression vector technology, applied in receptors/cell surface antigens/cell surface determinants, peptides containing localization/targeting motifs, applications, etc., can solve the problems of adverse reactions of cytokine storm and patient death , to achieve good application prospects, strong anti-tumor effect, and the effect of reducing cytokine storm
Active Publication Date: 2020-01-14
华夏源细胞工程集团股份有限公司
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At present, although CAR-T cells can effectively kill tumor cells, it also brings adverse reactions of cytokine storm, which can lead to death of patients in severe cases.
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Embodiment 1
[0020] The present invention provides a CD19-targeted T cell antigen coupler, comprising: a signal peptide (human CD8α signal region, CD8α leader), a CD19-targeted single-chain antibody (aCD19), and a linker sequence (L) connected in sequence , single-chain antibody targeting CD3, mutated CD8α hinge region (human CD8α hinge region with cysteine 164 and 181 mutated to serine), CD8α transmembrane region (human CD8α transmembrane region), intracellular region, P2A connecting peptide, IL2 signal peptide and anti-PD1 single-chain antibody secretion region, the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence of the single-chain antibody targeting CD19 is shown in the sequence listing Shown in SEQ ID NO: 2, the nucleotide sequence of the connecting sequence is shown in SEQ ID NO: 3 in the sequence listing, the nucleotide sequence of the mutated CD8α hinge region is shown in SEQ ID NO: 4 in the sequence listing, ...
Embodiment 2
[0027] The invention provides an expression vector, which includes a vector and a T cell antigen coupler. The vector can be a lentiviral vector.
[0028] The following briefly introduces the preparation method of the expression vector, as follows:
[0029] Amplify and obtain the CD19-TAC gene sequence by PCR (Polymerase Chain Reaction, polymerase chain reaction), add restriction site Xba I and restriction site EcoR I respectively at the two ends of the CD19-TAC gene sequence, obtain Digested product, and the lentiviral vector plasmid pCDH-EF1-MCS-T2A-c opGFP were subjected to double digestion reaction of Xba I and EcoR I, respectively, to obtain the restriction fragment containing CD19-TAC and the fragment containing pCDH-EF1-MCS - Restricted fragment of T2A-copGFP. The enzyme digestion reaction conditions are as follows: the enzyme digestion temperature is 37° C., and the enzyme digestion time is 30 min. The digestion system (total volume 50 μL) includes: 5 μL of 10×buffer...
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Abstract
The invention discloses a T cell antigen coupler targeting CD19 and an expression vector and application thereof, and belongs to the field of tumor immune drugs. The T cell antigen coupler targeting CD19 includes a signal peptide, a single-chain antibody targeting CD19, a catenative sequence, a single-chain antibody targeting CD3, a mutant CD8 alpha hinge region, a CD8 alpha transmembrane domain,an intracellular region, a P2A linker peptide, an IL2 signal peptide, and an anti-PD1 single-chain antibody secretion region, wherein the signal peptide, the single-chain antibody targeting CD19, thecatenative sequence, the single-chain antibody targeting CD3, the mutant CD8 alpha hinge region, and the CD8 alpha transmembrane domain, the intracellular region, the P2A linker peptide, the IL2 signal peptide, and the anti-PD1 single-chain antibody secretion region are connected in sequence, the nucleotide sequence of the signal peptide is shown as SEQ ID NO:1in a sequence table, the nucleotide sequence of the single-chain antibody targeting CD19 is shown as SEQ ID NO:2 in the sequence table, the nucleotide sequence of the catenative sequence is shown as SEQ ID NO:3 in the sequence table, andthe nucleotide sequence of the mutant CD8 alpha hinge region is as shown as SEQ ID NO:4 in the sequence table. According to the T cell antigen coupler targeting CD19, the cytokine storm produced during the treatment process of T cells can be reduced, meanwhile anti-PD1 single-chain antibody is secreted, and thus the immunosuppressive signal between PD-1:PD-L1 / PD-L2 is closed.
Description
technical field [0001] The invention relates to the field of tumor immunity medicine, in particular to a CD19-targeting T cell antigen coupler and its expression vector and application. Background technique [0002] Malignant tumors are one of the diseases that seriously threaten human health. According to incomplete statistics, the annual incidence of malignant tumors in China has reached more than 4 million cases, among which the incidence of lymphoma, leukemia, multiple myeloma and other hematological tumors remains high. . Chimeric antigen receptor (CAR) technology is a cell therapy technology that has developed very rapidly in recent years and is used to treat malignant tumors. At present, although CAR-T cells can effectively kill tumor cells, they also bring adverse reactions of cytokine storm, which can lead to death of patients in severe cases. Therefore, there is an urgent need for a safe T cell receptor for the treatment of malignant tumors. Contents of the inv...
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