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Programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof

A programmed death and PD-L1 technology, applied in the biological field, can solve problems such as loss of antibody activity, low drug concentration at tumor sites, and complex structure, and achieve the effects of good peptide-specific affinity, enhanced immunity, and low production costs

Active Publication Date: 2021-08-10
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, immune checkpoint blockade therapy targeting PD-1 / PD-L1 immune checkpoint has achieved great success in the field of tumor immunotherapy, among which PD-1 / PD-L1 monoclonal antibody inhibitors have been used in Significant curative effect in clinical tumor treatment (Liu B, Song Y, et al.Recent development in clinical applications of PD-1 and PD-L1 antibodies for cancer immunotherapy. Journal of Hematology&Oncology, 2017, 10(1): 174), including the observation The tumor shrunk significantly and produced a durable immune response in tumor patients. However, as a large four-polypeptide chain protein (molecular weight ~150kDa), monoclonal antibody drugs have inherent disadvantages related to their complex molecular structure: ①Complicated structure , the preparation is more difficult, the production cost increases, resulting in high treatment costs and limited wide application
② Poor stability, highly sensitive to external conditions, some common external disturbances will lead to loss of antibody activity, special transportation and storage conditions are required to ensure its activity
③The molecule is relatively large, and has poor ability to penetrate tumor tissue, and cannot effectively transport to the inside of tumor tissue. The drug concentration at the tumor site is very low, and the treatment effect on solid tumors is not good.

Method used

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  • Programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof
  • Programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof
  • Programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Panning and identification of PD-L1 specific binding peptides.

[0025] 1. Mutant construction and single clone selection

[0026]Aiming at the 9th, 10th, 11th, 13th, 14th, 17th, 18th, 24th, 25th, 27th, 28th, 32nd and 35th amino acid residues of the immunoglobulin binding region polypeptide of staphylococcal protein A, the abbreviation with NNK as the codon is designed and primers, using degenerate primers to obtain the coding gene of the affinity body polypeptide library by over-lapPCR. The gene encoding the polypeptide mutant was cloned into a phage display vector, and the recombinant plasmid was electrotransformed into TG1 host cells to obtain a storage capacity of 5×10 7 Affinity body polypeptide mutant library. Use the helper phage to display the mutants of the affinity body polypeptides, and then obtain the phage display library of the polypeptide mutants.

[0027] The PD-L1 ectodomain protein (purchased from Sino Biological, catalog number 10084-H08H) was used...

Embodiment 2

[0034] Expression and purification of PD-L1 specific binding polypeptide.

[0035] The 10 Affibody polypeptides that specifically bind to PD-L1 identified in Example 1 were used as objects for further research, and a prokaryotic expression system was used for mass expression of the polypeptides. The 10 kinds of Affibody coding genes were cloned into the expression vector to select pET-21b (preserved in the laboratory), and the recombinant expression vector was confirmed to be successfully constructed by double-enzyme digestion combined DNA sequencing technology. The expression host was E.coli BL21(DE3) (preserved in our laboratory), and the sequenced correct recombinant expression vector was transformed into E.coli BL21(DE3), and the single clone was picked and transferred to 5mL LB / A medium (1g tryptone, 0.5 g yeast extract, 1g sodium chloride to 100mL, autoclaved; then add ampicillin at a final concentration of 100μg / mL), culture overnight at 37°C, 180rpm; inoculate the cult...

Embodiment 3

[0038] Analysis of binding properties of PD-L1 specific binding peptides.

[0039] In this experiment, the eight kinds of PD-L1 specific binding Affibody polypeptides expressed and purified in Example 2 were used as further research objects, and their affinity properties with PD-L1 were detected by ELISA method, and BSA was selected as the negative control of irrelevant antigens. The specific operation is as follows:

[0040] Dilute PD-L1 / BSA to 10 μg / mL with pH 9.6 carbonate buffer, coat 96-well ELISA plate, overnight at 4°C; block with 3% skimmed milk powder-PBS, 37°C for 2h; add HA-labeled Z PD-L1 A2 ,Z PD-L1 D3 ,Z PD-L1 E3 ,Z PD-L1 GA2 ,Z PD-L1 GA2 ,Z PD-L1 GE2 ,Z PD-L1 P2 ,Z PD-L1 P10 ,Z PD-L1 P9 Recombinant protein, incubated at 37°C for 2h; added diluted HA antibody (purchased from Sangon Bioengineering Co., Ltd., catalog number D191044), incubated at 37°C for 2h; added diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Sangon Bioengineeri...

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Abstract

The invention relates to a programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof, and belongs to the technical field of biology. The polypeptide is obtained by mutating 1-20 amino acid residues from a staphylococcus protein A (SPA) immunoglobulin binding region (the sequence of the polypeptide is shown in SEQ ID: 2-5), and the polypeptide can specifically bind to PD-L1 and block PD-1 / PD-L1 interaction. According to the invention, a PD-L1 specific binding polypeptide coding sequence is obtained by panning from a total synthesis affinity small body polypeptide molecular library through four rounds of enrichment, a prokaryotic expression system is utilized to prepare the obtained polypeptide in a large scale, and the polypeptide is specifically bound with PD-L1, has no obvious affinity with BSA protein, and can be used for PD-L1 detection. The polypeptide has the PD-1 / PD-L1 interaction blocking capability at the same time, and the PD-1 / PD-L1 interaction blocking capability of the polypeptide is enhanced along with the increase of the concentration.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and specifically relates to a polypeptide specifically binding to programmed cell death-Ligand 1 (PD-L1), or a variant thereof, or a functional fragment thereof, and the Use of polypeptides, or variants thereof, or functional fragments. Background technique [0002] Immune checkpoint (immune checkpoint) is a signaling pathway in the immune system that regulates the strength of T cell immune response through co-signaling (co-stimulation or co-inhibition) molecules, and is crucial for maintaining autoimmune tolerance under normal physiological conditions. Programmed cell death 1 (PD-1) is an important negative regulator of immune cell activation, which can play an immunosuppressive role by binding to its ligand. PD-1 is mainly expressed on the surface of activated effector T cells, regulatory T cells, B cells, monocytes, and natural killer T cells, not on resting T cells, but can be detected w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12G01N33/68A61K39/395A61P37/04
CPCC07K16/1271G01N33/6872A61P37/04C07K2317/76G01N2333/70532
Inventor 刁爱坡赵青李玉银张晓涵
Owner TIANJIN UNIV OF SCI & TECH
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