A bispecific chimeric antigen receptor molecule and its application in tumor therapy

一种嵌合抗原受体、特异性的技术，应用在抗肿瘤药、癌抗原成分、抗体医疗成分等方向，达到防止再次复发、强特异性、增强靶向性的效果

Active Publication Date: 2021-04-27
CELLYAN THERAPEUTICS WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the patient received autologous hematopoietic stem cell transplantation, the effect of CAR-T cell therapy does not exclude the role of ASCT

Method used

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  • A bispecific chimeric antigen receptor molecule and its application in tumor therapy
  • A bispecific chimeric antigen receptor molecule and its application in tumor therapy
  • A bispecific chimeric antigen receptor molecule and its application in tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Construction of human BCMA stable expression cell line

[0061] 1.1 Construction of plasmid vector

[0062] The vector system used in this example belongs to the third-generation self-inactivating lentiviral vector system. There are three plasmids in this system, namely the packaging plasmid psPAX2 encoding the protein Gag / Pol and the Rev protein; and the envelope plasmid pMD2 encoding the VSV-G protein. .G and the recombinant plasmid pCDH-CMV-huCD19-EF1-GFP-T2A of encoding human BCMA extracellular region and transmembrane region based on the empty vector pCDH-CMV-MCS-EF1-GFP-T2A-Puro (purchased from Addgene) - Puro.

[0063] According to the human BCMA sequence provided by Genbank accession number NM_001192.2, using the gene synthesis method based on PCR bridging, synthesize the signal peptide, human BCMA extracellular region, transmembrane region, and intracellular region, through the primer pair

[0064] SEQ ID NO: 187(huBCMA-F):5>ATGTTGCAGATGGCTGGGCAG...

Embodiment 2

[0077] Example 2 Screening and verification of specific single-chain antibody (scFv) in conjunction with human BCMA

[0078] 2.1 Screening of anti-human BCMA-specific single chain antibody based on phage display

[0079] Using the single-chain antibody phage display technology, the single-chain antibody sequence that specifically binds to human BCMA was screened from the directional engineered human single-chain antibody phage library.

[0080] To achieve this purpose, inoculate 400ml of 2×YT / ampicillin culture medium with glycerol bacteria (self-built library) displaying the natural library of fully human single-chain antibody on phage, so that the cell density reaches OD600=0.1, at 37°C and 200rpm Culture with shaking until the cell density reaches OD600=0.5. use The M13KO7 helper phage (purchased from ThermoFisher) was infected, and incubated at 30° C. and 50 rpm for 30 minutes. After adding 50mg / L kanamycin, shake and culture at 37°C and 200rpm for 30 minutes, separa...

Embodiment 3

[0097] Embodiment 3, preparation and activity analysis of the antibody of anti-BCMA

[0098] 3.1 Construct light chain and heavy chain eukaryotic expression vectors for the selected single-chain antibody, transfect HEK293F to induce recombinant expression and purify. The light chain and heavy chain in the single-chain antibody sequence obtained in Example 1 were respectively constructed into the monoclonal antibody expression plasmid pCMV-V5-Fc, and the plasmid was prepared in large quantities after the sequence was confirmed to be correct. The expression plasmids of the above light chain and heavy chain were mixed in an appropriate ratio and then transfected into well-growing HEK-293F cells at 37°C, 5% CO 2 , continuously cultivated on a shaker at 125rpm for 7 days, centrifuged at 4000rpm for 10min, removed the precipitate, collected the supernatant, and used Membrane filtration, the processed sample was affinity-purified with a Protein A (purchased from GE) affinity colu...

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Abstract

The invention provides a bispecific chimeric antigen receptor, which consists of a signal peptide, two specific antigen-binding fragments, an extramembrane spacer, a transmembrane region, and an intracellular co-stimulatory signal region. The specific antigen-binding The first antigen recognized and bound by the fragment is CD19, CD20, CD22, CD33, CD269, CD138, CD79a, CD79b, CD23, ROR1, CD30, B cell surface antibody light chain, CD44, CD123, LewisY, CD7 or CD46 One: the second antigen recognized and bound by the specific antigen-binding fragment is CD38, and the two specific antigen-binding fragments are connected by a linking peptide, and the bispecific chimeric antigen receptor is receptored by constructing a low-affinity chimeric antigen receptor The body and high-affinity chimeric antigen receptors can recognize two tumor-associated antigens respectively with very strong specificity. In addition, the present invention also provides the application of the above-mentioned bispecific chimeric antigen receptor in the treatment of tumors.

Description

technical field [0001] The invention relates to the field of cell immunotherapy, in particular to a bispecific chimeric antigen receptor molecule and its application in tumor treatment. Background technique [0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. Chimeric antigen receptor CAR consists of a tumor-associated antigen-binding region, an extracellular hinge region, a transmembrane region, and an intracellular signal transduction region. CAR-T cell therapy uses exogenous gene transfection technology to express fusion proteins of single-chain fragment variable (scFv) that recognizes tumor-associated antigens and T cell activation sequences on the surface of T cells, enabling specific recognition of tumor-associated antigens. The scFv of the antigen is coupled to the activation and proliferation signal domain in T cells through t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N7/01A61K35/17A61P35/00A61P37/02A61P31/12
CPCA61P31/12A61P35/00A61P37/02C12N7/00C07K14/7051C07K16/2878C07K16/2896C07K2317/92C07K2317/622C07K2319/33C07K2319/02C12N2740/15021C12N2510/00A61K39/4631A61K2239/29A61K39/464426A61K2239/48A61K2239/31A61K39/4611A61K2239/38A61K39/4644A61K39/464417C07K2317/31C07K2317/21C07K2319/03C07K2317/73A61K2039/505A61K38/00C07K16/2815A61K35/545A61K47/65A61K35/17A61K45/06C07K14/70514C07K14/70578A61K38/177C07K19/00A61K38/1774
Inventor 金丹李璐李国龙华权高
Owner CELLYAN THERAPEUTICS WUHAN CO LTD
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