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CLDN18.2-targeted chimeric antigen receptor and composition and application of CLDN18.2-targeted chimeric antigen receptor

A chimeric antigen receptor and antigen technology, applied in the field of biomedicine, can solve the problems of high mortality, poor prognosis, and limited treatment options for advanced or recurrent gastric cancer, and achieve good specificity, good animal tolerance, and inhibition The effect of tumor growth

Pending Publication Date: 2021-12-07
CHENGDU KELUN PRECISION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The core defects of existing drugs and unmet clinical needs make the treatment options for advanced or recurrent gastric cancer very limited, with extremely poor prognosis and high mortality

Method used

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  • CLDN18.2-targeted chimeric antigen receptor and composition and application of CLDN18.2-targeted chimeric antigen receptor
  • CLDN18.2-targeted chimeric antigen receptor and composition and application of CLDN18.2-targeted chimeric antigen receptor
  • CLDN18.2-targeted chimeric antigen receptor and composition and application of CLDN18.2-targeted chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0320] Example 1: Preparation of Antibody Targeting Human Claudin 18.2scFv

[0321] Two murine anti-CLDN18.2 monoclonal antibodies 1E9.2 and 2C6.9 were screened by hybridoma technology. The above murine monoclonal antibody was analyzed to obtain its CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VH, VL sequences (see sequence information table). Then carry out the above method of humanization to finally obtain the humanized antibody 1E9.2-HZ11 (its VH and VL are respectively shown in SEQ ID NO: 13 and 14); 2C6.9-HZ21 (its VH and VL are respectively shown in SEQ ID NO:16 and 17). The sequence of CDR-H2 of 2C6.9-HZ21 was further modified to the sequence shown in SEQ ID NOs:37 or 53.

[0322] The VH and VL of the above humanized antibody were linked by a linker (SEQ ID NO: 44) to obtain scFv, and the sequence information of each scFv is shown in the table below.

[0323] Table 2: Structure of scFv

[0324]

[0325]

Embodiment 2

[0326] Example 2: Construction and preparation of chimeric antigen receptor (CAR) lentiviral expression vector

[0327] 1) Construction of lentiviral plasmid:

[0328] Based on the scFv sequence in the above examples and the reference T scFv sequence (the reference T is derived from the document J NatlCancer Inst. 2019Apr 1; ​​111(4):409-418, which is hu8E5 in the document), further construct CAR lentiviral expression carrier. Using the intracellular domain of CD137 and the ITAM region of CD3Zeta as the activation signal, fuse with the above scFv, add signal peptide, CD8 hinge region, CD8 transmembrane region, use primer F (SEQ ID NO:54) and primer R (SEQ ID NO: 55), construct a chimeric antigen receptor expression vector, and the structure of the constructed chimeric antigen receptor is shown in Table 3 below.

[0329] Table 3: Structures of Chimeric Antigen Receptors

[0330]

[0331] 2) Virus packaging:

[0332] Add the above-constructed CAR lentiviral plasmid and tr...

Embodiment 3

[0333] Example 3: Construction and preparation of co-expression CAR lentiviral expression vector

[0334]On the basis of the CAR structure in Example 2, other biologically active molecules (such as PD-1 scFv, whose nucleotide sequence is shown in SEQ ID NO: 72, its upstream P2A self-cleavage peptide nucleotide sequence is SEQ ID NO: 69; or mIL-15, its nucleotide sequence is shown in SEQ ID NO: 73, its upstream P2A self-cleavage peptide nucleotide sequence The sequence is the sequence of SEQ ID NO: 70) to obtain co-expressed CAR. The lentiviral expression vector co-expressing CAR was prepared by the same method as in Example 2. When the above-mentioned co-expressed CAR is expressed in cells, it will be cleaved from the PGP in the P2A sequence, so that the linked biologically active molecules are secreted outside the CAR-T cell or chimerically expressed on the CAR-T cell membrane, synergistically exerting anti-tumor effects. effect. For example, since CLDN18.2 CAR-T specifica...

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PUM

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Abstract

The invention relates to the field of biological medicines, in particular to a single-chain antibody specifically bound with CLDN18.2 and a chimeric antigen receptor (CAR) containing the single-chain antibody. The invention also relates to reconstructed immune cells expressing the CAR, or co-expressing the CAR and a further bioactive molecule (e.g., a PD-1 antibody or mIL-15), nucleic acid molecules encoding such CAR or co-expressed molecules, and methods of preparing the reconstructed immune cells. The invention also relates to application of the CAR and the immune cells in preventing and / or treating cancers such as gastric cancer, gastric adenocarcinoma and pancreatic cancer and a method for preventing and / or treating the cancers such as the gastric cancer, the gastric adenocarcinoma and the pancreatic cancer.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular, the invention relates to a single-chain antibody specifically binding to CLDN18.2 and a chimeric antigen receptor (CAR) comprising the single-chain antibody. The present invention also relates to engineered immune cells expressing said CAR, or co-expressing said CAR and another biologically active molecule (such as PD-1 antibody or mIL-15), and nucleic acid molecules encoding such CAR or co-expressing molecules And a method for preparing said engineered immune cell. The present invention also relates to the use of these CARs and immune cells for preventing and / or treating cancers such as gastric cancer, gastric adenocarcinoma, and pancreatic cancer, and methods for preventing and / or treating cancers such as gastric cancer, gastric adenocarcinoma, and pancreatic cancer. Background technique [0002] Chimeric antigen receptor (CAR)-T cells are an emerging therapeutic approach with remarka...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K45/06A61P35/00A61P35/02
CPCC07K16/28C07K14/7051C07K14/70578C07K16/2818C07K16/2827C07K14/5443C12N15/86C12N5/0636A61K39/001102A61K39/001111A61K39/00114A61K45/06A61P35/00A61P35/02C07K2317/565C07K2317/56C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/828A61K2039/852
Inventor 常建辉朱雁林王江漫肖亮薛彤彤王晶翼
Owner CHENGDU KELUN PRECISION BIOTECHNOLOGY CO LTD
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