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Anti-PD-L1 nano antibody and application thereof

A technology of PD-L1 and nano-antibody, which is applied in the field of biomedicine, can solve the problem that the affinity has not reached the ideal state, and achieve the effect of high affinity, weak immunogenicity and significant effect

Active Publication Date: 2021-03-30
QUREBIO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] However, in existing mAbs, the affinity is not ideal and the immunogenicity is strong due to the large size

Method used

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  • Anti-PD-L1 nano antibody and application thereof
  • Anti-PD-L1 nano antibody and application thereof
  • Anti-PD-L1 nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Alpaca immunization and construction of phage immune library

[0062] Alpaca immunization: immunize a healthy alpaca (vicugna pacos, alpaca, lama pacos) with PD-L1 extracellular region recombinant protein (Shenzhou, 10084-H08H), 0.11mg protein per immunization, a total of 5 times, the first time Freund's complete adjuvant CFA was used, and Freund's incomplete adjuvant IFA was used for the second to fifth times to emulsify and mix with the antigen, and then subcutaneously inject multiple points.

[0063] Building a library: Take 50ml of peripheral blood to separate peripheral blood mononuclear cells (PBMC), extract PBMC total RNA, reverse transcribe into cDNA by RT-PCR, design primers for PCR amplification of VHH genes, and build an immune library. Peripheral blood lymphocytes were separated, and 50 mL of peripheral blood was collected. PBMCs were separated according to the instructions of the lymphocyte separation medium, and total RNA of PBMCs was extracted ...

Embodiment 2

[0064] Example 2: Screening and Identification of Nanobody Immune Library

[0065] Two rounds of screening were performed with the recombinant protein in the extracellular region of PDL1.

[0066] The first round of panning: PDL1-hFc (Protein No. QP004) and hFc protein were respectively coated in immunotubes, 50 nM, 1 ml, overnight at 4°C. Blocking: Block the immunotube with 2% Milk / PBS at 37°C for 1 hour. Subtraction: Pour off the blocking solution, add 900 μl 2% Milk / PBS to the hFc-coated immunotube, then add 100ul phage immune library, and rotate at room temperature for 1 hour. Binding: transfer the phage immune library to the immune tube coated with PDL1-hFc, and rotate at room temperature for 1 hour. Washing: Wash the immunotube 5 times with 1×PBST, and 5 times with 1×PBS. Elution: 800 μl 100 mM TEA, 10 min at room temperature. Neutralization: Transfer the eluate to a 1.5ml EP tube and add 400μl 1M pH 7.4 Tris. Infection: The eluted phages after neutralization were a...

Embodiment 3

[0077] Example 3: Nanobody construction of FC fusion protein, cloning, expression, and purification of protein

[0078] Cloning design and construction: 20 clones were transformed into PD-L1-FC fusion protein whose C-terminus is human IgG1 FC. The reconstituted plasmid was expressed in HEK293 cells, purified by protein A affinity chromatography, and a total of 20 candidate PD-L1 VHH-FC fusion proteins QP326-QP345 were obtained, the sequences of which were shown in SEQ ID NO: 1-20. The rear end of the antibody is connected to the human IgG1 Fc segment, such as the sequence SEQ ID NO: 61. The numbering of the Fc fusion protein of the corresponding Nanobody is formed by adding the suffix Fc to the corresponding Nanobody.

[0079] In addition, QP322 is used as a reference in the examples, and the patent CN201910567277.5 can be referred to, and its anti-PD-L1 VHH sequence is:

[0080] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCA...

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Abstract

The present invention provides an anti-PDL1 nanometer antibody and uses thereof, the anti-PDL1 nanometer antibody at least comprises a VHH fragment, and the VHH fragment comprises three amino acid fragments such as CDR1, CDR2 and CDR3. The anti-PDL1 nano antibody and the Fc fusion protein of the anti-PDL1 nano antibody are strong in specificity, high in affinity and weak in human immunogenicity, and have a remarkable anti-tumor effect.

Description

[0001] Cross References to Related Applications [0002] This application claims the priority of the patent application number CN201911235055.X and the title of the invention "a trifunctional fusion protein containing TGF-β inhibitor and its application" filed on December 5, 2019, the entire content of which is by reference Incorporated into this article. technical field [0003] The present invention belongs to the field of biomedicine, and more specifically, the present invention relates to an anti-PD-L1 nanobody and its Fc fusion protein, and uses of the anti-PD-L1 nanobody and its Fc fusion protein. Background technique [0004] In the classical theory of immune surveillance, the immune system can recognize tumor antigens and eliminate them. If the immune system is able to completely eliminate tumor cells, immune clearance can proceed steadily. If tumor cells evade clearance by the immune system through mutations, the immune system will rebalance. During this process,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/46A61K39/395A61P35/00A61P35/02A61P37/02
CPCC07K16/2827C07K14/71A61K47/55A61P35/00A61P37/02A61P31/00C07K2317/565C07K2317/569C07K2317/622C07K2317/76C07K2319/00C07K2319/30A61K2039/505A61K38/00A61P35/02C07K2317/22C07K2317/24C07K2317/52C07K2317/64C07K2317/74C07K2317/92
Inventor 屈向东
Owner QUREBIO LTD
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