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CD22 single domain antibody, nucleotide sequence, reagent kit, CAR-T virus vector and CAR-T cell

A nucleotide sequence, single-domain antibody technology, applied in nucleic acid vectors, DNA/RNA fragments, viruses/phages, etc., can solve the problems of difficult to achieve effective structure of CAR-T cells, low binding force, low affinity, etc. To achieve the effect of good killing of tumor cells, low production cost and high expression

Active Publication Date: 2020-06-02
SHENZHEN PREGENE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional CD22 antibody has a large molecular weight, low binding force and low affinity to the antigen, is not easy to modify and has poor stability, and requires single-chain modification, so it is difficult to achieve it with traditional CD22 antibodies (such as monoclonal antibodies, etc.) Effective construction of CAR-T cells

Method used

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  • CD22 single domain antibody, nucleotide sequence, reagent kit, CAR-T virus vector and CAR-T cell
  • CD22 single domain antibody, nucleotide sequence, reagent kit, CAR-T virus vector and CAR-T cell
  • CD22 single domain antibody, nucleotide sequence, reagent kit, CAR-T virus vector and CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Anti-CD22 Antigen Specific Antibody Library Construction

[0033] It should be noted that the CD22 antigen used in this example was purchased from Beijing Sino Biological, the article number is 11958-H08H1. The QIAGEN kit is the QIAamp RNA Blood Mini Kit (50) from Beijing Yaanda Biotechnology Co., Ltd., Cat. No. 52304. The cDNA synthesis kit is MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0 from TAKARA Company.

[0034] 1. Immunization experiment.

[0035] 1) Select a healthy alpaca as the immune target;

[0036] 2) Using 2 mg CD22 antigen mixed with an equal volume of Freund's adjuvant to immunize a healthy alpaca, a total of 6 immunization experiments were carried out;

[0037] 3) After the 4th immunization experiment, the alpaca serum was sampled, and its antigen immune titer was determined, until the measured immune titer of the alpaca serum reached more than 10,000 times, 100ml of alpaca whole blood was taken, and isolate lymphocytes;

[0038] 4) lys...

Embodiment 2

[0067] The screening of embodiment 2 specific phage

[0068] 1) Preliminary preparation. Prepare 5*10 CD22-negative control cell lines and modified CD22-positive cell lines respectively 6 , use CPBSH or 2% BSA-PBS to incubate on a drum at room temperature for 2 hours, and centrifuge for later use.

[0069] 2) Screening. The phage (5*10 11 ~1*10 12 ) into negative cells, CPBS or 2% BSA-PBS to 0.5ml, incubate on a drum at room temperature for 2 hours; take supernatant, add to positive cells, CPBS or 2% BSA-PBS to 0.5ml , incubate on a drum at room temperature for 2 hours; so that the outer shell of the phage can specifically secrete CD22 antibody and bind to CD22 antigen; wash 5 times with PBST, wash 3 times with PBS, centrifuge, and discard the supernatant, so that no binding The phages were washed away.

[0070] 3) Titer determination. The above cell-bound phages were resuspended, and 2ml of TG1 in the logarithmic growth phase was taken, infected, and allowed to stand a...

Embodiment 3

[0076] Example 3 Screening of specific positive monoclonal antibodies

[0077] 1) Screen the highly expressed recombinant plasmids.

[0078] The specific CD22 antibody gene fragment obtained in Example 2 was amplified by PCR to obtain PCR products with restriction endonucleases BbsI and BamHI sites; the above PCR products and pSJF2 were treated with restriction endonucleases BbsI and BamHI respectively Vector; T4 ligase is used to connect the above-mentioned digested gene fragments and recombine them to obtain a recombinant plasmid sdAb-pSJF2 capable of high-efficiency expression in Escherichia coli.

[0079] 2) Screen CD22 positive cloned antibodies.

[0080] Pick a single colony from the agar plate where the colony grows, inoculate it in a 96-well deep-well culture plate containing Amp-containing 2YT liquid medium, and cultivate it for 4 hours; inoculate the single clones one by one in the numbered cells separated by small grids On the LB solid plate containing Amp; add IP...

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Abstract

The invention discloses a CD22 single domain antibody, a nucleotide sequence, a reagent kit, a CAR-T virus vector and a CAR-T cell. The amino acid sequences of the CD22 single domain antibody sequentially comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions, wherein the FR1, FR2, FR3 and FR4 regions are framework region amino acid sequences, the CDR1, CDR2 and CDR3 regions are variable regionamino acid sequences, and the amino acid sequences are shown as table 1. According to the technical scheme, through a method combining genetic engineering, the single domain antibody of specific binding CD22 protein is obtained. The antibody specificity combination CD22 protein is high in specific binding capacity and high in affinity.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and antibody drugs, in particular to a CD22 single domain antibody, nucleotide sequence, kit, CAR-T virus vector and CAR-T cells. Background technique [0002] CD22, also known as sialic acid-binding immunoglobulin-like lectin 2, is a type II transmembrane protein of B cells. There are two different subtypes, α and β, with a molecular weight of about 135Kd. CD22 is a member of the immunoglobulin superfamily and is a B lymphocyte-restricted antigen. CD22 is expressed on the cell membrane of mature B cells, and the expression level increases when the cells are activated. In addition, CD22 is also abundantly expressed in B-cell lymphoma cells, so it is an ideal target for the treatment of B-cell non-Hodgkin's lymphoma. [0003] Chimeric antigen receptor T cells, also known as CAR-T, are a new type of immune cell therapy based on genetically modified T cells to achieve specific ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/62C12N15/86C12N5/10A61P35/00
CPCA61P35/00C07K16/2803C07K2317/22C07K2317/24C07K2317/565C07K2317/567C07K2317/569C07K2317/92C07K2319/00C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2800/107C12N2800/22
Inventor 张继帅包朝乐萌李莹莹苏红昌宋宗培蔡清华丁怡瑾栗红建
Owner SHENZHEN PREGENE BIOPHARMA CO LTD
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