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Bispecific T cell agonist CD3-FAP/nanoBiTE, preparation and application thereof in tumor resistance

A CD3-FAP, bispecific technology, applied in the direction of anti-tumor drugs, application, recombinant DNA technology, etc., can solve the problem of nano antibody construction that has not been reported, and achieve the effect of enhancing the anti-hepatic cancer effect

Pending Publication Date: 2022-01-18
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the previous scientific research, in order to optimize the water solubility, stability and increase the production expression of BiTE to improve the functional effect, the predecessors have done a lot of exploration, but the use of nanobodies to construct BiTE has not been reported.
No one has explored the choice of two specific Nanobodies and linker peptides

Method used

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  • Bispecific T cell agonist CD3-FAP/nanoBiTE, preparation and application thereof in tumor resistance
  • Bispecific T cell agonist CD3-FAP/nanoBiTE, preparation and application thereof in tumor resistance
  • Bispecific T cell agonist CD3-FAP/nanoBiTE, preparation and application thereof in tumor resistance

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preparation example Construction

[0041] The present invention also provides a method for preparing a polynucleotide comprising bispecific T cell agonist CD3-FAP / nanoBiTE, the preparation method comprising the following steps:

[0042] (1). Amplify the coding sequence of Nanobody anti-CD3 Nb;

[0043] (2). Amplify the coding sequence of Nanobody anti-FAP Nb;

[0044] (3). The coding sequence obtained in step (1) is connected with the coding sequence obtained in step (2) by the coding sequence of (Gly4Ser)3.

[0045] The present invention also provides a polypeptide comprising a bispecific T cell agonist CD3-FAP / nanoBiTE, the polypeptide comprising a CD3 nanobody and a FAP nanobody linked together by a (Gly4Ser)3 linking peptide.

[0046] In one embodiment, the amino acid sequence of the polypeptide comprising the bispecific T cell agonist CD3-FAP / nanoBiTE of the present invention is SEQ ID No.1.

[0047] The present invention also provides a method for preparing the bispecific T cell agonist CD3-FAP / nanoBiTE...

Embodiment 1

[0061] 1.1 Construction of recombinant expression vector

[0062] The two nanobodies anti-CD3 Nb and anti-FAP Nb required for this group of experiments were prepared by phage antibody library technology, and the corresponding gene sequences were obtained by sequencing.

[0063] For ligation product construction, cloning and expression was performed using a method comprising the following steps:

[0064] (1) Use Primer Premier to design a pair of primers: the upstream primer is: 5′-CGCGGATCCATGGGCACTGCTACCGTTGAG-3′, which introduces the restriction site KpnI; the downstream primer is 5′-CCAAGCTGGTACAAGAGCTGCTTTGCTTGTG-3′, which introduces the restriction site HindⅢ.

[0065] (2) After PCR amplification using the recombinant plasmid PUC57-CD3-FAP / nanoBiTE as a template, the amplification conditions are: 92°C pre-denaturation: 2 minutes, the cycle is set to 92°C denaturation for 30s, 56°C for 30s, 72°C Extend for 90s, 30 cycles, and finally extend for 10 minutes at 72°C.

[006...

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Abstract

The present invention relates to a polypeptide comprising a bispecific T cell agonist CD3-FAP / nanoBiTE, a polynucleotide encoding the polypeptide, a preparation method of the polypeptide and polynucleotide, and application of the polypeptide. The bispecific T cell agonist CD3-FAP / nano BiTE is a novel bispecific T cell engager (BiTE)-CD3-FAP / nano BiTE based on a nano antibody, the bispecific T cell agonist CD3-FAP / nano BiTE induces T cells to target tumor cells, so that the tumor resistant effect is exerted, and a new strategy is provided for tumor cell immune targeted therapy of BiTE.

Description

technical field [0001] The present invention relates to a biological material for treating tumors, in particular to a bispecific T cell agonist, a preparation method of the bispecific T cell agonist, and an application of the agonist in antitumor. Background technique [0002] BiTE (bispecific T cell engager) is a bispecific antibody that uses single-chain antibody technology to connect the light and heavy chain variable regions of two different antibodies through a polypeptide chain, which can effectively activate effector T cells and precisely guide T cell specificity. killing target cells. Compared with other bispecific antibodies, the biggest difference between BiTE is its structure. It uses single-chain antibody technology to connect the variable regions of two different antibodies through a short peptide. Compared with traditional antibodies, it lacks the Fc segment, has a smaller molecular weight, lower immunogenicity, and stronger tissue penetration. BiTEs connect ...

Claims

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Application Information

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IPC IPC(8): C12N15/13C07K16/46C12N15/70A61K39/395A61P35/00
CPCC07K16/2809C07K16/40C12N15/70A61P35/00C07K2317/569C07K2317/31C07K2317/73
Inventor 卢小玲谢深霞施维杨晓梅李翠
Owner GUANGXI MEDICAL UNIVERSITY