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

A bifunctional antibody, PD-L1 technology, applied in the field of tumor immunology, can solve problems such as complex process, evaluation model, and difficulty in the development of bifunctional antibodies

Active Publication Date: 2019-06-28
HUABO BIOPHARM
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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-PD-L1/VEGF bifunctional antibody and application thereof
  • Anti-PD-L1/VEGF bifunctional antibody and application thereof
  • Anti-PD-L1/VEGF bifunctional antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0261] Example 1 Construction of bifunctional antibody 900387 and 900388 expression vector and protein expression and purification

[0262] The anti-PD-L1 antibody of the present invention is a mouse monoclonal antibody obtained by immunizing mice with human PD-L1-His protein (heavy chain variable region and light chain variable region sequences are as SEQ ID NO.: 1 and SEQ ID NO.: 2), and then humanized the humanized monoclonal antibody (900339). The heavy chain sequence is shown in SEQ ID NO.: 8 and the light chain sequence is shown in SEQ ID NO.: 9. The artificially synthesized VEGFR1D2 (sequence shown in SEQ ID NO.: 10) is connected to the 5'end or 3'end of the heavy chain expression vector by linking Linker (GGSGGSGGSGGSGGS), and is co-transfected with the light chain vector (1:1) In CHO-S cells, 37 degrees, 5% CO 2 After culturing at 130 rpm / min for 7 days, the supernatant was collected by centrifugation. The supernatant was centrifuged at 4000 rpm for 10 min, and filtere...

Embodiment 2

[0270] Example 2 Affinity detection of bispecific antibodies

[0271] In this example, the SPR method was used to determine the antibody-antigen binding kinetics and affinity.

[0272] Materials and instruments:

[0273] Recombinant human PD-L1, Sino Biological, 10084-H08H

[0274] Recombinant human VEGF165, Sino Biological, 11066-HNAH

[0275] Amino coupling kit, GE, BR-1000-50

[0276] HBS-EP(10X), GE, BR-1006-69

[0277] Human Antibody Captrue Kit, GE, BR-1008-39

[0278] S series sensor chip (Series S Sensor Chip CM5), GE, BR-1005-30

[0279] BIACORE, GE, Biacore 8K

[0280] Method: Prepare Anti-Human Capture-CM5 chip according to the amino coupling method of human antibody capture kit. Place the chip at room temperature to equilibrate for 20-30 minutes and load it into the Biacore 8K instrument; dilute the transient protein to the experimental working concentration with equilibration buffer; dilute the antigen to 50 nM with equilibration buffer, and then 3 times dilution 7 concentratio...

Embodiment 3

[0286] Example 3 Non-specific adsorption effect of bispecific antibodies and non-target molecules

[0287] In this example, the SPR method was used to determine the non-specific adsorption effect of antibodies and non-target molecules.

[0288] Materials and instruments:

[0289] Egg white lysozyme, Sigma, L3790

[0290] Soybean pancreatic inhibitor 1-S type, Sigma, T-2327

[0291] Amino coupling kit, GE, BR-1000-50

[0292] HBS-EP(10X), GE, BR-1006-69

[0293] Rabbit polyclonal antibody against lysozyme, ABcam, Ab391

[0294] Anti-trypsin inhibitor antibody, LifeSpan Biosciences, LS-C76609

[0295] 0.85% phosphoric acid solution, ProteOn, 176-2260

[0296] 50mM hydroxide, ProteOn, 176-2230

[0297] S series sensor chip CM5, GE, BR-1005-30

[0298] BIACORE, GE, Biacore 8K

[0299] Method: Place Series S Sensor Chip CM5 at room temperature to equilibrate for 20-30 minutes, and load it into Biacore 8K instrument. The amino coupling kit was used to fix egg white lysozyme and soybean pancreatic in...

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PUM

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Abstract

The invention provides an anti-PD-L1 / VEGF bispecific antibody and application thereof, and particularly provides a bifunctional antibody. The bifunctional antibody comprises an anti-PD-L1 antibody orelement (a) and an anti-VEGF antibody or element (b) connected with the anti-PD-L1 antibody or element. The bifunctional antibody can be simultaneously bound with VEGF and PD-L1, thereby exerting a therapeutic effect on VEGF and PD-L1-positive tumor cells (especially malignant tumor cells).

Description

Technical field [0001] The present invention belongs to the field of tumor immunology, and specifically relates to anti-PD-L1 / VEGF bifunctional antibodies and uses thereof. Background technique [0002] Tumors can be divided into two categories: benign tumors and malignant tumors according to the cell characteristics of new organisms and the degree of harm to the body. Among them, malignant tumor diseases are the major diseases that endanger human health in today's society, and their lethality ranks second. , Common tumors include liver cancer, lung cancer, stomach cancer, breast cancer, bladder cancer, etc. According to reports, there were approximately 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018 (17 million and 9.5 million respectively after excluding non-melanoma skin cancer), of which 18.1 million new cancer cases, Asia accounted for nearly half Of the 9.6 million cancer deaths, Asia accounts for nearly 70%. [0003] Due to individual differe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/85C12N5/10G01N33/574A61K39/395A61K47/68A61P35/00
CPCA61P35/00C12N5/10C12N15/85G01N33/574C07K19/00C12N15/63C07K16/2827C07K2317/31C07K16/22A61K2039/505C07K2317/92C07K2317/73C07K2317/24C07K14/705C07K2319/33C12N15/62C07K2317/565A61K47/6879
Inventor 朱向阳张凤雪蔡明清张雷陈时俞玲
Owner HUABO BIOPHARM
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