CD22 protein targeting antibody, chimeric antigen receptor and drug

A chimeric antigen receptor and protein technology, which is applied in the fields of antibodies targeting CD22 protein, chimeric antigen receptors and drugs, can solve the problems of few types of CD22 antibodies, unsatisfactory tumor cell specific binding ability and lethality, etc.

Active Publication Date: 2020-08-04
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are currently few types of antibodies against CD22, and the existing CD22-specific chimeric antigen receptor T cells are not ideal for specific binding and killing of tumor cells

Method used

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  • CD22 protein targeting antibody, chimeric antigen receptor and drug
  • CD22 protein targeting antibody, chimeric antigen receptor and drug
  • CD22 protein targeting antibody, chimeric antigen receptor and drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1. Phage library panning

[0040] Biotinylated hCD22 protein was used as the panning antigen for the fully human antibody library. First, block the phage antibody with blocking solution (PBST / 5% skimmed milk powder) at room temperature for 2 hours. The amount of phage input is 1012 pfu, then add 10 μg antigen, incubate at room temperature for 1 hour, and add 50 μl pre-blocked bacteriophage after incubation. M-280Streptavidin magnetic beads, incubated at room temperature for 30min.

[0041] First wash off the unbound phages with PBST, then elute the phages bound to the magnetic beads with 0.1M HCl-Glycine, then neutralize the eluate with Tris-HCl, and take part of the phages to infect the large intestine in the logarithmic growth phase Bacillus TG1, the collected phages were used for the next round of panning.

[0042] Gradually increase the screening intensity of each round, and stop panning when the enrichment degree reaches more than 100 times.

[0043] 2. Use phage...

Embodiment 2

[0059] Detect the binding force of the CD22 antibody obtained in Example 1 and hCD22 protein

[0060] Detection method:

[0061] (1) For hCD22 protein coating, dilute from 1ìg / ml, 3-fold serial dilution, a total of 7 gradients, coat Costar-9018 microtiter plate with 100ìl hCD22 protein dilution, overnight at 4°C.

[0062] (2) After blocking with 3% BSA at room temperature for 2 hours, add the phage supernatant (1010 pfu) corresponding to CD22 antibody, and incubate at room temperature for 2 hours.

[0063] (3) After washing away the unbound phage, add Ml3 Bacteriophage antibody (HRP), and incubate at 4°C for 1 hour. After washing, TMB chromogenic solution was added to develop color, and 2M HCl was used to terminate the reaction.

[0064] (4) Read at 450nm with a microplate reader, see the results figure 1 .

[0065] from figure 1 From the results, it can be seen that the phage expressing the CD22 antibody has a good binding ability to the hCD22 protein, showing an S curve...

Embodiment 3

[0067] Construction of chimeric antigen receptor expression vector

[0068] Build method:

[0069] (1) Whole gene synthesis: signal peptide, CD22 antibody light chain variable region, linker, CD22 antibody heavy chain variable region, hinge region (hinge), CD8α transmembrane domain (TM), 4-1BB co-stimulatory signal transduction region and the CD3ζ signaling domain. The above sequences were sequentially connected to obtain the CD22 chimeric antigen receptor expression cassette, which was named: CD22 chimeric antigen receptor expression cassette, and the Kozac sequence was introduced at the front end of the expression cassette. The expression cassette structure is as follows: figure 2 shown.

[0070] The sequence of each element of the CD22 chimeric antigen receptor expression cassette is as follows:

[0071] The amino acid sequence of the signal peptide (Leader) is shown in SEQ ID NO.1, specifically as follows:

[0072] MLLLVTSLLLCELPHPAFLLIP.

[0073] Its corresponding n...

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Abstract

The invention provides a CD22 protein targeting antibody, a chimeric antigen receptor and a drug, and relates to the technical field of cellular immunotherapy. The chimeric antigen receptor has an antigen binding domain aiming at a tumor surface antigen CD22, and the amino acid sequence of a heavy chain variable region of the antigen binding domain is shown as SEQ ID NO.3; the amino acid sequenceof a light chain variable region of the antigen binding domain is shown as SEQ ID NO. 7. The chimeric antigen receptor can target a CD22 protein; T cells prepared from the chimeric antigen receptor can specifically act on CD22 positive target cells, have very good specificity and sustainability, are high in killing capacity and small in side effect on healthy tissues, and can be used for preparingmedicines for treating or preventing CD22 positive tumors.

Description

technical field [0001] The invention relates to the technical field of cellular immunotherapy, in particular to an antibody targeting CD22 protein, a chimeric antigen receptor and a drug. Background technique [0002] Multiple myeloma (multiple myeloma, MM) is the most common type of malignant plasma cell disease, with a high degree of malignancy, characterized by massive proliferation of clonal plasma cells. The current clinical treatment of MM can induce remission, but almost all patients will eventually relapse or even die. Although some monoclonal antibodies have shown promise in the treatment of MM in preclinical studies and early clinical trials, they have not been consistently approved due to insufficient efficacy and safety. Clearly, there is a great need to find new immunotherapies for MM, and the development of effective antigen-specific adoptive T cell therapies for the treatment of this disease will be a top priority. T cells can be genetically modified to expr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/28C12N15/62C12N5/10A61K39/00A61P35/00
CPCA61K39/0011A61P35/00A61K2039/804C07K14/7051C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N2510/00
Inventor 田颖李妍霜陶勇
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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