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Preparation method and application of newcastle disease oncolytic virus expressing PD-L1 single chain antibody

A Newcastle disease virus, PD-L1 technology, applied in the field of Newcastle disease oncolytic virus, can solve problems such as reduced stability

Inactive Publication Date: 2019-04-16
南京昂科利医药科技创新研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the original antibody, ScFV has little change in its ability to bind to the antigen, but its stability is significantly reduced and it is easy to degrade

Method used

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  • Preparation method and application of newcastle disease oncolytic virus expressing PD-L1 single chain antibody
  • Preparation method and application of newcastle disease oncolytic virus expressing PD-L1 single chain antibody
  • Preparation method and application of newcastle disease oncolytic virus expressing PD-L1 single chain antibody

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Experimental program
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Effect test

Embodiment

[0026] 1) Preparation of pBRN-FL (112-RRQRRF-117)

[0027] By mutation PCR, the amino acid residues 112-117 of the F protein of pBRN-FL full-length clone were mutated to 112-R-R-Q-R-R-F-117: using pBRN-FL as a template, PCR was performed with the primers in Table 1, and the reaction system was:

[0028]

[0029] ddH 2 O make up to 20ul

[0030] The reaction program is: ①98°C, 5min; ②98°C, 20sec; ③68°C, 90sec; ④Steps 2-3 cycled 18 times; ⑤68°C, 15min;

[0031] After the reaction is completed, take 9ul of the reaction product, add 1ul of DNA restriction endonuclease DpnI, and react at 37°C for 2h; after the reaction, take 100ul of competent DH5αE. Then heat shock at 42°C for 90sec; then ice bath for 2min, then add 500ul antibiotic-free LB medium, recover in a 37°C constant temperature shaking incubator at 180rpm for 30min; apply to LB solid medium plate containing ampicillin antibiotic, cultivate overnight in an incubator at 37°C, pick Take a single clone to 5ml containing...

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Abstract

The invention discloses a preparation method and application of a newcastle disease oncolytic virus expressing PD-L1 single chain antibody and an application thereof. The amino acid residues at sits of 112-117 of F protein of mutant recombinant LaSota strain newcastle disease virus is mutated into 112-R-R-Q-R-F-117, so that the newcastle disease oncolytic virus has independent infection capability; a PD-L1 single chain antibody gene is inserted between a P gene and M gene of a full-length clone of the newcastle disease virus LaSota strain through homologous recombination to obtain a finally modified full-length clone pBRN-FL (112-RRQRRF-117)-PDL1(ScFV); the recombinant virus is purified by chick embryo proliferation and ultracentrifugation. The rNDV-LaSota (112-RRQRRF-117)-PDL1(ScFV) recombinant virus has safety comparable to that of a LaSota original strain, and also has tumor killing capability and immune checkpoint inhibition function of a virulent strain.

Description

technical field [0001] The invention relates to a Newcastle disease oncolytic virus expressing a PD-L1 single-chain antibody for treating lung cancer, belonging to the field of biomedicine. Background technique [0002] Oncolytic viruses can infect tumor cells, replicate and proliferate in tumor cells, and eventually lead to the death of host tumor cells. Oncolytic viruses are released to continue to infect and kill other tumor cells, and play a role in eliminating tumors. Existing oncolytic viruses include herpes simplex virus, adeno-associated virus, avian reovirus, and Newcastle disease virus. These four types of viruses have their own advantages and disadvantages, but there are still many problems in the ability of existing oncolytic viruses to kill tumors, such as narrow tumor-tropic spectrum, weak oncolytic ability, and easy host immune clearance. Therefore, researchers seek various methods to modify the oncolytic virus to develop strong oncolytic ability, small side ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N15/90C12N7/01A61K35/768A61P35/00A61P11/00C12R1/93
CPCA61K35/768A61P11/00A61P35/00C07K16/2827C07K2317/622C12N7/00C12N15/85C12N15/907C12N2760/18121C12N2760/18152C12N2800/107
Inventor 廖亚金
Owner 南京昂科利医药科技创新研究院有限公司
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