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Anti-PD-L1 nano antibody as well as Fc fusion protein and application thereof

A PD-L1 and nanobody technology, applied in the field of biomedicine, can solve the problem of unsatisfactory affinity, and achieve the effect of high affinity, weak immunogenicity and strong specificity

Active Publication Date: 2020-12-29
QUREBIO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] However, the affinity of the existing monoclonal antibody has not reached the ideal state, and due to its large size, it has strong immunogenicity

Method used

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  • Anti-PD-L1 nano antibody as well as Fc fusion protein and application thereof
  • Anti-PD-L1 nano antibody as well as Fc fusion protein and application thereof
  • Anti-PD-L1 nano antibody as well as Fc fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Preparation method of PD-L1 nanobody Fc fusion protein:

[0065] 1. Screening of nanobodies against PD-L1

[0066] 1.1 Library Construction

[0067] a) The PD-L1-his fusion protein of the immunized camel was obtained by purifying on a nickel column, the sequence of which is shown in SEQ ID NO.43. One Xinjiang Bactrian camel was selected for subcutaneous multi-point immunization, and 50 mL of peripheral blood was collected after four immunizations to separate PBMCs;

[0068] b) Total RNA was extracted with TRIzol reagent. After electrophoresis to identify sufficient RNA purity, use Super IIIFirst-Strand Synthesis System for RT-PCR transcribed 8ug RNA, and after 2 rounds of nested PCR, recovered and purified the target nucleic acid fragments with a DNA product purification kit.

[0069] c) The phage vector pComb3XSS and the target fragment were respectively digested with sfiI, digested overnight at 50°C, and then the target fragment was recovered by tapping t...

Embodiment 2

[0161] Example 2 Humanization of Anti-PD-L1 Nanobodies

[0162] By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy and light chain variable regions with high homology to QP1162 (SEQ ID No.35) and QP1166 (SEQ ID No.39) were selected respectively. The region germline gene was used as a template, and the CDRs of the mouse antibody were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues for back mutation combination. The amino acid residues are identified and annotated by the Kabat numbering system.

[0163] Primer PCR was designed to build the VH gene fragments of each humanized antibody, and then homologous recombination was performed with the expression vector pQD with signal peptide and constant region gene (FC) fragments to construct the full-length expression vector VH-FC...

Embodiment 3

[0192] Example 3 Activity identification of humanized anti-PD-L1 nanobody

[0193] Construct and design anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecule QP3711461, in the form of Figure 8 shown.

[0194]

[0195] 1. In vitro activity identification of PD-L1 function (mixed lymphocyte reaction MLR)

[0196] Prepare DC (donor1) cells: recover PBMC, use EasySep TM Human Monocyte Isolation Kit (Stemcell 19359) was used to isolate mononuclear cell monocytes, add rhGM-CSF (1000U / ml) and rhIL4 (500U / ml), and culture the cells at 37°C for 6 days to induce iDCs; every 2-3 days half change medium, Supplement rhGM-CSF (1000U / ml) and rhIL4 (500U / ml) at the same time; collect the cells and centrifuge at 300xg for 5 minutes, resuspend with the medium added with rhGM-CSF (1000U / ml) and rhIL4 (500U / ml), and add LPS at the same time (1 μg / ml), continue to culture the cells at 37°C for 1 day to induce mature DCs; collect the cells and count them for later use.

[0197] Prepare T(do...

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Abstract

The invention provides an anti-PDL1 nano antibody as well as an Fc fusion protein and application thereof. The anti-PDL1 nano antibody and the Fc fusion protein thereof are strong in specificity, highin affinity and weak in human immunogenicity. The stability is high, and the obvious anti-tumor effect is achieved.

Description

technical field [0001] The invention relates to an anti-PD-L1 nanobody, the invention also relates to an Fc fusion protein of an anti-PD-L1 nanobody, the invention also relates to the application of an anti-PD-L1 nanobody, and the invention also relates to an anti-PD-L1 The application of the Fc fusion protein of the nanobody belongs to the field of biomedicine. Background technique [0002] 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, the immunogenicity of tumor cells gradually decreases. The ability of tumor cells to proliferate is weakened under the pressure of the immune system, making the detection of tumor cells more difficult. [0003] The activation of oncogenes cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00A61K39/395A61P35/00A61P31/00A61P37/00
CPCC07K16/2827A61P35/00A61P31/00A61P37/00C07K2319/30C07K2317/569C07K2317/565C07K2317/24A61K2039/505C07K16/28A61P37/02C07K2317/92C07K2319/00C07K2317/52C07K2317/76C07K2317/31C07K2317/35C07K2317/60C07K2317/70Y02A50/30C07K16/46C07K19/00
Inventor 屈向东潘琴金后聪都业杰郑翰
Owner QUREBIO LTD
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