Chimeric antigen receptor capable of secreting bispecific antibody as well as expression vector and application thereof

A chimeric antigen receptor and bispecific antibody technology, applied in the field of tumor immunotherapy, can solve problems such as antigen escape, loss of CD19 expression, tumor cell cancer recurrence, etc., and achieve the effect of reducing the risk of recurrence

Active Publication Date: 2020-04-10
华夏源细胞工程集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although CD19 CAR-T cells have achieved great success, as many as two-thirds of patients who receive CD19 CAR-T cell therapy will experience loss of CD19 expression, leading to antigen escape and tumor cell persistence. Growth that manifests as cancer recurrence

Method used

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  • Chimeric antigen receptor capable of secreting bispecific antibody as well as expression vector and application thereof
  • Chimeric antigen receptor capable of secreting bispecific antibody as well as expression vector and application thereof
  • Chimeric antigen receptor capable of secreting bispecific antibody as well as expression vector and application thereof

Examples

Experimental program
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Embodiment 1

[0026] An embodiment of the present invention provides a chimeric antigen receptor that secretes a bispecific antibody, such as figure 1 As shown, the chimeric antigen receptor includes: sequentially connected signal peptide (human CD8α signal peptide, CD8α leader), single-chain antibody targeting CD19 (CD19 scFv), lengthened CD8α hinge region (human CD8α hinge region, CD8α Hinge ), transmembrane region (human CD8α transmembrane region, CD8α transmembrane), co-stimulatory factor, intracellular signal peptide (human CD3ζ intracellular signal peptide, CD3ζ signal), P2A connecting peptide, IL2 signal peptide (IL2 leader), targeting CD20 single-chain antibody (CD20 scFv), CD3-targeting single-chain antibody (CD3scFv) and tag protein, the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1 in the sequence listing, CD19-targeting single-chain antibody The nucleotide sequence of the CD8α hinge region is shown in SEQ ID NO: 2 in the sequence listing, the nucleotide sequ...

Embodiment 2

[0035]The present invention provides an expression vector, which includes the vector and the chimeric antigen receptor provided in Example 1. Specifically, the vector may be a lentiviral vector, a retroviral vector, an electrotransduction vector or a Sleeping Beauty transposon vector. In this embodiment, the vector is a lentiviral vector.

[0036] The following briefly introduces the preparation method of the expression vector, as follows:

[0037] Amplify and obtain the CART-BiTEs 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 CART-BiTEs gene sequence, obtain Digested product, and the lentiviral vector plasmid pCDH-EF1-MCS-T2A-copGFP were subjected to double enzyme digestion reaction with Xba I and EcoR I, respectively, to obtain the restriction fragment containing CART-BiTEs and pCDH-EF1-MCS- Digested fragment of T2A-copGFP. The enzyme digestion reaction co...

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Abstract

The invention discloses a chimeric antigen receptor capable of secreting a bispecific antibody as well as an expression vector and an application thereof, which belong to the field of tumor immune drugs. The chimeric antigen receptor comprises a signal peptide, a CD19-targeting single-chain antibody, a lengthened CD8 alpha hinge region, a transmembrane region, a costimulatory factor, an intracellular signal peptide, a P2A connecting peptide, an IL2 signal peptide, a CD20-targeting single-chain antibody, a CD3-targeting single-chain antibody and a tag protein which are connected in sequence. The chimeric antigen receptor is the bispecific antibody capable of specifically targeting CD19 positive tumor cells and secreting targeting CD20 and targeting CD3 at the same time, the non-conductive Tcells can be targeted and activated, so that the tumor antigen CD20 is targeted, and the purpose that the CAR-T cells effectively kill malignant blood tumor cells of a B cell line in vivo is achieved. Meanwhile, the T cells of the chimeric antigen receptor can prevent CD19 positive tumor cells with low abundance expression from generating immune escape, so that the recurrence risk of malignant blood tumors of a B cell line is reduced.

Description

technical field [0001] The present invention relates to the field of tumor immune drugs, in particular to a chimeric antigen receptor secreting a bispecific antibody, 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. In the past, most of them were middle-aged and elderly people, and gradually spread to young patients. This may be related to factors such as modern people's high life pressure, increased environmental pollution, and unhealthy living habits. [0003] Chimeric antigen receptor (CAR) technology is a cell therapy technology that has developed very rapidly in recent years. At present, although CD19 CAR-T cells have achieved great success, as many as two-thirds of patients who receive CD19 CAR-T cell therapy will experience loss of CD19 expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/00A61P35/00A61P35/02
CPCC07K14/7051C07K16/2887C07K16/2809C07K16/2896A61K39/001112A61K39/001124A61P35/00A61P35/02C07K2319/02C07K2319/03A61K2039/5156A61K2039/804
Inventor 张坤
Owner 华夏源细胞工程集团股份有限公司
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