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Anti-GD2 antibody, CAR containing same, CAR combination and application thereof

A chimeric antigen receptor, CD8 technology, applied in the fields of molecular biology, cell biology and immuno-oncology, can solve the problems of inability to completely remove tumor cells, immune escape, etc., to enhance the killing function, avoid escape, prolong The effect of survival time

Active Publication Date: 2022-07-29
UTC THERAPEUTICS (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the heterogeneity of tumors, the main problem in this field is that a single CAR-T cannot completely eliminate tumor cells in the lesion, resulting in immune escape

Method used

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  • Anti-GD2 antibody, CAR containing same, CAR combination and application thereof
  • Anti-GD2 antibody, CAR containing same, CAR combination and application thereof
  • Anti-GD2 antibody, CAR containing same, CAR combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation and function of transiently expressing CAR-T cells targeting IL13RA2 chimeric antigen receptor and LACO

[0068] Preparation of CAR elements targeting IL13RA2 and LACO: This patent obtained the scFv sequence targeting IL13RA2 (ie H08) from WO2019178078 A1, and screened out a more specific scFv sequence targeting IL13RA2 (#5) by phage display. The lymphocyte-antigen-presenting cell costimulatory molecule LACO sequence was obtained from the company's patent WO2022037520A1, and H08 and #5 were connected to the CD8 signal peptide, CD8 hinge region, and CD8 transmembrane region by recombinant PCR, and then connected in series 4-1BB and CD3ζ intracellular signaling domain, prepared into a CAR structure targeting IL13RA2 ( figure 1 A and B); while the sequence of LACO contains CD8 signal peptide, anti-CD40 scFv, CD8 hinge region, CD28 transmembrane region and CD28 intracellular signal domain ( figure 1 C).

[0069] Constructed a vector for the productio...

Embodiment 2

[0075] Example 2 Preparation and function of CAR-T cells stably expressing targeting IL13RA2 chimeric antigen receptor and LACO

[0076] Lentiviral plasmid preparation: In order to verify the ability of targeting IL13RA2 CAR in combination with LACO to remove tumors for a long time, we used the original IL13RA2 CAR ( Figure 5 On the basis of A and B), an element capable of expressing IL13RA2 CAR and LACO at the same time was prepared. We connected LACO and two CARs in series through F2A to prepare a CAR element expressing LACO and H08: AH ( Figure 5 C), and CAR elements expressing LACO and #5: A#5 ( Figure 5 D). The four elements were cloned into the lentiviral vector pTRPE. After successful sequencing and comparison, the resulting plasmids were transformed into X-Blue strains, plated and screened. After overnight, kanamycin-positive single clones were selected and inoculated into 200 ml. In LB medium, shake the bacteria overnight and then use the QIAGEN plasmid extractio...

Embodiment 3

[0082] Example 3 Preparation and function of transiently expressing IL13RA2, GD2 CAR and IL13RA2, GD2 CAR, and LACO CAR T cells

[0083] Preparation of CAR vector transiently targeting GD2: same as above, GD2 scFv is connected with CD8 signal peptide, CD8 hinge region, CD8 transmembrane region by recombinant PCR method, followed by 4-1BB and CD3ζ intracellular signal domain in series, Prepared as a CAR structure targeting GD2 ( Figure 11 ). The CAR element was cloned into a pDA vector that could be transcribed in vitro, and the CAR mRNA targeting GD2 was also obtained by in vitro transcription.

[0084] 5 μg GD2 CAR mRNA was electroporated into T cells as GD2 CAR-T group, 5 μg H08 CAR mRNA was electroporated into T cells as IL13RA2 CAR-T group; 5 μg GD2 CAR mRNA and 5 μg H08 CAR mRNA were electroporated into T cells were used as GD2+IL13RA2 group; 5 μg GD2 CAR mRNA, 5 μg H08 CAR mRNA and 5 μg LACO mRNA were electroporated into T cells as GD2+IL13RA2+LACO group, and the elec...

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Abstract

The invention discloses an anti-GD2 antibody, a CAR containing the anti-GD2 antibody, a CAR combination and application of the anti-GD2 antibody. The chimeric antigen receptor combination comprises a CAR IL13RA2 molecule, a CAR GD2 molecule and an LACO fusion protein as shown in SEQ ID NO: 13, the CAR IL13RA2 molecule comprises scFv IL13RA2 with the amino acid sequence as shown in SEQ ID NO: 12, and the CAR GD2 molecule comprises scFvGD2 with the amino acid sequence as shown in SEQ ID NO: 9. The CAR combination provided by the invention can be used for enhancing the killing capability of CAR-T cells on a neuroglioma cell line with high expression of IL13RA2; after the LACO fusion protein is introduced to the H08 CAR, the tumor removal capability of the H08 CAR-T can be promoted, and meanwhile, the CAR combination can destroy the heterogeneity of U87 tumor cells, so that the escape of tumors is avoided.

Description

technical field [0001] The present invention belongs to the fields of molecular biology, cell biology and immuno-oncology. The present invention relates to an anti-GD2 antibody, a chimeric antigen receptor (CAR) containing the same, a CAR combination and applications thereof. Background technique [0002] Malignant gliomas (MG), including glioblastoma multiforme and glioblastoma, account for 20,000 new cases each year in the United States. According to the American Brain Tumor Association, as of 2010, 140,000 people in the United States had malignant brain tumors. Although MG is a rare disease, its malignancy and lethality are very high. Existing standard treatments are very limited, and the five-year survival rate after surgery and radiotherapy is very low. There are also very few new treatment options for patients who relapse after surgery. Therefore, the development of new targets and new treatment methods is an urgent need for the majority of patients. Due to the he...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2866C07K16/3084C07K16/2878C07K14/7051C12N15/86C12N5/0636A61K39/001119A61K39/001117A61K39/001171A61P35/00C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C07K2319/00C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156
Inventor 赵阳兵刘晓军訾振国
Owner UTC THERAPEUTICS (SHANGHAI) CO LTD
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