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bcma antibodies, chimeric antigen receptors and drugs

A chimeric antigen receptor and antibody technology, which can be used in antibody mimics/stents, drug combinations, anti-tumor drugs, etc., and can solve problems such as few antibody categories

Active Publication Date: 2020-09-15
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are currently fewer classes of antibodies against BCMA

Method used

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  • bcma antibodies, chimeric antigen receptors and drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1 Phage library panning

[0061] Biotinylated hBCMA protein was used as the panning antigen for the fully human antibody library. First, block the phage antibody at room temperature with blocking solution (PBST / 5% skimmed milk powder) for 2 h, and the input amount of phage is 2×10 12 Phage, then add 10μg antigen, incubate at room temperature for 1h, add 50μl pre-blocked M-280Streptavidin magnetic beads, incubated at room temperature for 30min.

[0062] 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.

[0063] Gradually increase the screening intensity of each round, and stop panning when the enrichment degree reaches more than 100 times.

[0064] 2 Use phage Elisa to screen anti-BCMA single-ch...

Embodiment 2

[0093] Detect the binding force of the 4 BCMA antibodies obtained in Example 1, namely 2073, 2077, 2079 or 2082, to the hBCMA protein

[0094] Detection method:

[0095] (1) hBCMA protein coating, dilution from 1μg / ml, 3-fold serial dilution, a total of 8 gradients, the concentrations are 1μg / ml, 333ng / ml, 111ng / ml, 37ng / ml, 12.3ng / ml, 4.1 ng / ml, 1.37ng / ml, 0.45ng / ml, Costar-9018 microtiter plate was coated with 100μl hBCMA protein diluent, overnight at 4°C.

[0096] (2) After blocking with 3% BSA at room temperature for 2 hours, add the phage supernatant (10^10 pfu) corresponding to BCMA antibody 2073, 2077, 2079 or 2082, and incubate at room temperature for 2 hours.

[0097] (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.

[0098] (4) Read at 450nm with a microplate reader, see the results figur...

Embodiment 3

[0101] Construction of chimeric antigen receptor expression vector

[0102] Build method:

[0103] (1) Whole gene synthesis: signal peptide (nucleotide sequence SEQ ID NO.66, amino acid sequence SEQ ID NO.73), BCMA antibody light chain variable region (light chain variable region of 2073, 2077, 2079 or 2082), Linker (nucleic acid sequence SEQ ID NO.67, amino acid sequence SEQ ID NO.74), BCMA antibody heavy chain variable region (heavy chain variable region of 2073, 2077, 2079 or 2082), hinge region (hinge) (nucleic acid sequence SEQ ID NO.74) ID NO.68, amino acid sequence SEQ ID NO.75), CD8α transmembrane domain (TM) (nucleic acid sequence SEQ ID NO.69, amino acid sequence SEQ ID NO.76), 4-1BB co-stimulatory signal transduction region (nucleic acid sequence SEQ ID NO.70, amino acid sequence SEQ ID NO.77) and CD3ζ signaling domain (nucleotide sequence SEQ ID NO.71, amino acid sequence SEQ ID NO.78). The above sequences are sequentially connected. Four kinds of chimeric antig...

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PUM

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Abstract

The invention discloses a BCMA antibody, a chimeric antigen receptor and a drug, and relates to the technical field of cellular immunotherapy. The BCMA antibody provided by the invention has a heavy chain CDR region shown in SEQ ID NO. 6-8 and a light chain CDR region shown in SEQ ID NO. 14-16, or has a heavy chain CDR region shown in SEQ ID NO. 38-40 and a light chain CDR region shown in SEQ ID NO. 46-48. The BCMA antibody has the ability to specifically bind a BCMA protein, and a chimeric antigen receptor T cell prepared by using the BCMA antibody has a specific killing effect on a target cell positive for the BCMA protein and can be used to prepare drugs for treating or preventing tumors.

Description

technical field [0001] The invention relates to the technical field of cellular immunotherapy, in particular to a BCMA antibody, chimeric antigen receptor and medicine. Background technique [0002] Multiple myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells. Current MM treatments induce remission, but almost all patients eventually relapse and die. Although some monoclonal antibodies have shown promise in treating MM in preclinical studies and early clinical trials, there has been no consensus. Clearly, new immunotherapies for MM are clearly in great need, and the development of effective antigen-specific adoptive T cell therapy for this disease would be a major advance. T cells can be genetically modified to express a chimeric antigen receptor (CAR), which includes a fusion protein of an antigen recognition portion and a T cell activation domain. For B lineage malignancies, the most commonly used approach is the adoptive T ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/62C12N15/13C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/2878C07K2317/53C07K2317/55C07K2317/565C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 晁瑞华刘明耀杜冰盛涵樱
Owner EAST CHINA NORMAL UNIV
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