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Anti-VEGF/PD-L1 bispecific antibody and application thereof

A bispecific antibody, PD-L1 technology, applied in the field of biomedicine, can solve the problems of evaluation model, low expression level, large difference in quality control, etc., and achieve good thermal stability

Pending Publication Date: 2022-03-01
BIOTHEUS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although bifunctional antibodies are the direction of antibody drug development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and differences in quality control Therefore, the research and development of bifunctional antibodies has always been difficult

Method used

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  • Anti-VEGF/PD-L1 bispecific antibody and application thereof
  • Anti-VEGF/PD-L1 bispecific antibody and application thereof
  • Anti-VEGF/PD-L1 bispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0227] Example 1: Cloning and expression of anti-VEGF / PD-L1 bispecific antibody

[0228] 1.1 Antibody structure design

[0229] In this example, six anti-VEGF / PD-L1 bispecific antibodies were constructed, namely:

[0230] Ava-2GS-NSD / Ava-4GS-NSD: composed of 2 polypeptide chains, the schematic diagram of its structure is as follows figure 1 As shown in A, the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which includes the VH amino acid sequence derived from the anti-VEGF antibody Bevacizumab (Patent No.: WO1998045332) (SEQ ID NO:3), the C-terminus of the VH amino acid sequence is directly connected to the CH amino acid sequence derived from human IgG1 (introducing LALA mutation to reduce the Fc function, SEQ ID NO:4), and the anti-PD-L1 Nanobody C - The N-terminal of Ye-18-5 (patent application number: 2019108631090) (SEQ ID NO: 5) passes through 11 amino acid residues (G 4 S) 2 G (SEQ ID NO:6) (Ava-2GS-NSD) or 21 amino acid residues ...

Embodiment 2

[0236] Example 2: Determination of antibody affinity for bispecific antibodies

[0237] ForteBio affinity determination was performed according to the existing method (Estep, P et al., Solution-based measurement of high-throughput antibody-antigen affinity and epitope fractionation, MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline for 30 min in the assay buffer, then measured online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded onto the AHC sensor online. Then put the sensor into 100nM PD-L1 or VEGF antigen for 5 minutes, and then transfer the sensor to PBS for 5 minutes to dissociate. Analysis of kinetics was performed using a 1:1 binding model.

[0238] Table 1. Binding affinity of candidate molecules and PD-L1

[0239] Numbering KD(M) Kon(1 / Ms) Koff(1 / s) Ava-2GS-NSD 8.48E-09 3.19E+05 2.70E-03 Luc-2GS-NSD 9.52E-09 2.81E+05 2.67E-03 Ava-4GS-NSD 7.12E-09 3.54E+05...

Embodiment 3

[0243] Example 3: Binding of purified bispecific antibodies to PD-L1

[0244] CHO cells overexpressing human PD-L1 (CHO-hPD-L1 cells) were generated by transfecting pCHO1.0 vector (purchased from Invitrogen) cloned into MCS of human PD-L1 cDNA (purchased from Sino Biological). Adjust the cell density of the expanded CHO-hPD-L1 cells to 2×10 6 Cells / ml, 100 μl / well was added to a 96-well flow plate, and centrifuged for later use. Dilute the purified PD-L1 antibody with PBS, start at 400nM and start 3-fold dilution for a total of 12 points. Add 100 μl / well of the above-mentioned diluted sample into the above-mentioned 96-well flow plate with cells, incubate at 4°C for 30 minutes, and wash with PBS twice. Goat F(ab')2 anti-human IgG-Fc (PE) (purchased from Abcam) diluted 100 times with PBS was added to 100 μl / well, incubated at 4°C for 30 min, and washed twice with PBS. Add 100 μl / well of PBS to resuspend the cells, detect on CytoFlex (Bechman) flow cytometer and calculate the...

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Abstract

The invention relates to an application of an anti-PD-L1 / VEGF (platelet death-L1 / vascular endothelial growth factor) bispecific antibody. Specifically, the invention provides an anti-PD-L1 / VEGF bispecific antibody, and the antibody has a function of blocking the combination of PD-L1 and a receptor PD-1 and a function of blocking the combination of VEGF and a receptor thereof. The bispecific antibody can play a better role in killing tumors by activating an immune system and blocking tumor angiogenesis at the same time. Moreover, the invention provides an amino acid sequence of the bispecific antibody, a corresponding expression vector, a host cell capable of expressing the bispecific antibody, and a production method of the bispecific antibody.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to an anti-VEGF / PD-L1 bispecific antibody and its application. Background technique [0002] Programmed death 1 ligand 1 (programmed death 1 ligand 1, PD-L1), also known as CD274, is a member of the B7 family and a ligand of PD-1. PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and an intracellular region consisting of 30 amino acids. Unlike other B7 family molecules, PD-L1 negatively regulates the immune response. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages and dendritic cells, etc. In addition to lymphocytes, PD-L1 is also expressed in other tissues such as thymus, heart, placenta, etc. Endothelial cells, and various non-lymphoid systems such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K47/68A61K51/10A61K39/395A61P35/00
CPCC07K16/22C07K16/2827A61K47/6845A61K47/6849A61K47/6879A61K51/1027A61K51/1021A61P35/00C07K2317/31C07K2317/569C07K2317/56C07K2317/52C07K2317/76C07K2317/92C07K2317/94C07K2317/73A61K2039/505C07K2317/24C07K2317/524C07K2317/71G01N2333/475G01N2333/70532C07K16/468C07K2317/565
Inventor 缪小牛陈乘袁志军黄威峰曾竣玮孙左宇
Owner BIOTHEUS INC
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