CD38 antibody, chimeric antigen receptor and drug

A chimeric antigen receptor and antibody technology, which can be used in antibody mimics/scaffolds, drug combinations, anti-tumor drugs, etc., and can solve the problem of few types of CD38 antibodies.

Active Publication Date: 2019-01-25
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently fewer classes of antibodies against CD38

Method used

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  • CD38 antibody, chimeric antigen receptor and drug
  • CD38 antibody, chimeric antigen receptor and drug
  • CD38 antibody, chimeric antigen receptor and drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1 Phage library panning

[0056] Biotinylated hCD38 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.

[0057] 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.

[0058] Gradually increase the screening intensity of each round, and stop panning when the enrichment degree reaches more than 100 times.

[0059] 2 Use phage Elisa to screen anti-CD38 single-ch...

Embodiment 2

[0088] Construction of chimeric antigen receptor expression vector

[0089] Build method:

[0090](1) Whole gene synthesis: signal peptide (nucleotide sequence SEQ ID NO.66, amino acid sequence SEQ ID NO.73), CD38 antibody light chain variable region (light chain variable region of 2427, 2430, 2446 or 2453), Linker (nucleic acid sequence SEQ ID NO.67, amino acid sequence SEQ ID NO.74), CD38 antibody heavy chain variable region (heavy chain variable region of 2427, 2430, 2446 or 2453), 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).

[0091] The above-mentioned element sequences were connected sequentially to ...

Embodiment 3

[0099] Preparation of Strain Containing Chimeric Antigen Receptor Expression Vector

[0100] method:

[0101] (1) Take out DH5α competent cells from -80°C refrigerator and thaw on ice.

[0102] (2) Add 5ng of plasmid to the competent medium, mix gently, and place on ice for 5 minutes.

[0103] Plasmids are: pCDH-EF1-CAR-CD38-2427-copGFP, pCDH-EF1-CAR-CD38-2430-copGFP, pCDH-EF1-CAR-CD38-2446-copGFP, or pCDH-EF1-CAR-CD3-2453 -copGFP.

[0104] (3) Heat shock at 42°C for 90 seconds, and place on ice for 30 minutes.

[0105] (4) Add 0.5ml non-resistant LB, and incubate at 37°C, 180rpm for 30 minutes.

[0106] (5) Spread on an ampicillin-resistant plate.

[0107] (6) Invert overnight culture at 37°C.

[0108] (7) Pick a single clone and culture it in ampicillin-resistant LB at 37°C and 200 rpm for 9-12 hours.

[0109] (8) Glycerin was added to the bacterial solution, and the final concentration of glycerol was 10%, and the bacterial strain was stored in a refrigerator at -80°...

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PUM

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Abstract

The invention discloses a CD38 antibody, a chimeric antigen receptor and a drug, and relates to the technical field of cellular immunotherapy. The CD38 antibody provided by the invention 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, or has a heavy chain CDR region shown in SEQ ID NO. 54-56 and a light chain CDR region shown in SEQ IDNO. 62-64. The antibody has the ability to specifically bind a CD38 protein, and a chimeric antigen receptor T cell prepared by using the antibody has a specific killing effect on a target cell positive for the CD38 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 CD38 antibody, chimeric antigen receptor and medicine. Background technique [0002] Multiple myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells. It is the most common type of malignant plasma cell disease. It often occurs in middle-aged and elderly people. Patients are often accompanied by bone pain and hypercalcemia syndrome, anemia, kidney damage. Although the application of traditional chemotherapy, hematopoietic stem cell transplantation and targeted drugs to treat myeloma can induce remission, almost all patients will eventually relapse and die. One of the important reasons is that as the disease progresses, the normal immunoglobulin levels The production is inhibited, so it is prone to various bacterial infections. Although some monoclonal antibodies have shown promise in treating MM in preclinical studies and...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K19/00C12N15/62C12N15/13C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/2896C07K2317/53C07K2317/55C07K2317/565C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 晁瑞华刘明耀杜冰盛涵樱
Owner EAST CHINA NORMAL UNIV
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