Chimeric antigen receptor (CAR) targeting bispecific sites and application thereof

A bispecific, single-chain antibody technology, applied in the field of genetic engineering, can solve the problems that CAR vectors are not easy to transduce T cells, different efficacy, different virus preparation capabilities, etc., to reduce the probability of immune escape, reduce tumor recurrence rate, Good anti-tumor effect

Pending Publication Date: 2021-10-12
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the heterogeneity of tumors, especially solid tumors, more than one target antigen is often expressed on the surface of tumor cells, and with the progress of research, researchers have also found that a small number of patients treated with CAR-T cells have down-regulated or mutated tumor cells and The CAR-T target antigen expressed on its surface escapes (Robbie G.Majzner et al. (2018) TumorAntigen Escape from CAR T-cell Therapy), which eventually leads to poor therapeutic effect and recurrence
Therefore, it is necessary to design a CAR structure targeting multiple targets. If two CARs with diff

Method used

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  • Chimeric antigen receptor (CAR) targeting bispecific sites and application thereof
  • Chimeric antigen receptor (CAR) targeting bispecific sites and application thereof
  • Chimeric antigen receptor (CAR) targeting bispecific sites and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 plasmid construction

[0050] Design the CAR-structure with anti-CD19 ScFv light chain or anti-CD22 ScFv light chain in front, the nucleic acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:2, use double enzyme digestion to cut and recover the fragments respectively, and connect the gene fragments , transformed and picked a single clone, and the obtained vector numbers were 19(5'-SP-VL(ScFv1)-Linker1-VH(ScFv2)-Linker2-VL(ScFv2)-Linker1-VH(ScFv1)-CD8 hinge-8TM- CD137-CD3ζ-3'), 20(5'-SP-VH(ScFv2)-Linker1-VL(ScFv1)-Linker2-VH(ScFv1)-Linker1-VL(ScFv2)-CD8 hinge-8TM-CD137-CD3ζ- 3'). The CAR structural element comprises: heavy chain nucleotide sequence such as SEQ ID NO:3, amino acid sequence such as SEQ ID NO:14, light chain nucleotide sequence such as SEQ ID NO:4, amino acid sequence such as SEQ ID NO:13 target ScFv to CD19; heavy chain nucleotide sequence such as SEQ ID NO:5, amino acid sequence such as SEQ ID NO:15, light chain nucleotide sequence such as S...

Embodiment 2

[0051] Example 2 Target cell preparation and target antigen detection

[0052] K562 was used as model cells to construct target cells with high exogenous expression of CD19, CD22, and co-expression of CD19 and CD22, and the expression of antigens on the surface of target cells was detected by anti-CD19 antibody and anti-CD22 antibody. see results figure 2 Shown: K562-CD19 is only CD19 positive cells, K562-CD22 is only CD22 positive cells, K562-CD19-CD22 is CD19 and CD22 double positive cells.

Embodiment 3

[0053] Example 3 Preparation of lentivirus and infection of T lymphocytes

[0054] In this example, the calcium phosphate method is used to package the lentivirus, specifically: 293T cells are cultured to a better state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and expression plasmid Add the ratio column to the centrifuge tube of 1.5, add CaCl 2 and 2×HBS, mix well, let it stand at room temperature, and then add it to the treated 293T cell culture medium. After 3-5h, change the medium again to 10mL DMEM medium containing 10% FBS, and collect the cell supernatant after 48h or 72h. Viruses were purified and titered.

[0055] Table 1. Titer determination

[0056]

[0057] The prepared lentivirus was used to infect CHO cells, and the CD19 CAR-T, CD22 CAR-T, and double CAR-T (19 and 20) infected with CHO cells were labeled with CD19-FC and CD22-His flow cytometry reagents to detect the double CAR-T. The expression of targeting CD19CAR a...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a bispecific single-chain antibody, a CAR structure containing the bispecific single-chain antibody, an expression vector, an immune cell and application. In the single-chain antibody, the amino acid sequence of a Linker1 structure is shown as SEQ ID NO:17 or a functional variant thereof; the amino acid sequence of a Linker2 structure is shown as SEQ ID NO:18 or a functional variant thereof; the Linker1 is connected with a heavy chain variable region of ScFv1 and a light chain variable region of ScFv2 or is connected with a heavy chain variable region of the ScFv2 and a light chain variable region of the ScFv1; and the Linker2 is connected with the light chain variable region of the ScFv2 and the heavy chain variable region of the ScFv2. The construction of the CAR structure overcomes the technical effect defect of a double-target CAR in the prior art; two different tumour antigens can be targeted at the same time; the killing rate of tumour cells is improved; and the immune escape probability of the tumour cells is reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a bispecific single-chain antibody, a CAR structure comprising the bispecific single-chain antibody, an expression vector, immune cells and applications. Background technique [0002] CAR-T (Chimeric Antigen Receptor T) cell therapy has achieved remarkable results in the treatment of hematological malignancies. However, due to the heterogeneity of tumors, especially solid tumors, more than one target antigen is often expressed on the surface of tumor cells, and with the progress of research, researchers have also found that a small number of patients treated with CAR-T cells have down-regulated or mutated tumor cells and The CAR-T target antigen expressed on its surface escapes (Robbie G. Majzner et al. (2018) TumorAntigen Escape from CAR T-cell Therapy), which eventually leads to poor therapeutic effect and recurrence. Therefore, it is very necessary to d...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/70578C07K14/7051C12N15/86C12N5/0636A61K39/001112A61K39/001113A61P35/00A61P35/02C07K2317/31C07K2317/622C07K2317/56C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/804A61K2039/884A61K2039/82A61K2039/812A61K2039/892A61K2039/852A61K2039/86A61K2039/844A61K2039/876
Inventor 陈雪娇黄霞赵永春陈军赵文旭齐亚男徐艳敏单娟娟
Owner CHONGQING PRECISION BIOTECH CO LTD
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