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Anti-PD-L1 monoclonal antibody, and application thereof in preparation of anticancer drugs

A monoclonal antibody, PD-L1 technology, applied in the biological field, can solve the problem of no PD-L1 antibody drug, and achieve the effect of good affinity

Active Publication Date: 2020-04-17
冠科生物技术(中山)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nivolumab and Pembrolizumab have also been launched in my country. At the same time, two PD-1 antibodies, Toripalimab (trade name: Tuoyi) and sintilimab (trade name: Tyvyt), developed in China, were also released on the same day. Time has been approved for marketing in China, but no PD-L1 antibody drug is currently on the market in China

Method used

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  • Anti-PD-L1 monoclonal antibody, and application thereof in preparation of anticancer drugs
  • Anti-PD-L1 monoclonal antibody, and application thereof in preparation of anticancer drugs
  • Anti-PD-L1 monoclonal antibody, and application thereof in preparation of anticancer drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of anti-PD-L1 monoclonal antibody of the present invention

[0031] Technical route of the present invention is:

[0032] After immunizing mice with PD-L1 protein, isolate the mouse spleen, extract total RNA, reverse transcribe into cDNA, and amplify antibody light and heavy chain genes by PCR, and then construct a single-chain antibody phage library , Obtain the target antibody through screening.

[0033] 1. Experimental materials

[0034] 1. Strain E.coli TG1; E.coli DH5α, used for the expression of Fab antibody. Laboratory preservation.

[0035] 2. Plasmid vector pComb3XTT. Laboratory preservation.

[0036] 3. Reagents Trizol total RNA extraction kit, M-MLV reverse transcriptase, and RNase inhibitors were purchased from Invitrogen.

[0037] Tag DNA polymerase, recombinant ligase and endonuclease were purchased from NEB Company. DNA gel recovery kit was purchased from Shanghai Sangong Company.

[0038] 2. Experimental steps

[0039] 1. I...

Embodiment 2

[0074] Example 2 Detection of anti-PD-L1 monoclonal antibody

[0075] 1. Antibody Western blot detection

[0076] (1) Preparation of polyacrylamide gel: configure 12% separation gel liquid pure water to solidify after 15 minutes of sealing, then add concentrated gel and insert the corresponding comb, and it can solidify and use after 15 minutes. It is ready to use now to ensure the experimental effect .

[0077] (2) Treat the sample: Dilute an appropriate amount of PD-L1 to 40 μL, add 5x deformation non-reducing buffer at a ratio of 4:1, boil in a metal bath at 100°C for 5 minutes, and centrifuge at 12,000 rpm for 5 minutes.

[0078] (3) Electrophoresis: Add 20 μL of sample to each well, add 5 μL of Rainbow Marker as a control, and perform electrophoresis at a constant current of 50 mA until the indicator reaches the bottom of the gel plate.

[0079] (4) Membrane transfer: Cut off the glue at the size of the target protein according to the appropriate area according to the r...

Embodiment 3

[0095] Example 3 Blocking effect of anti-human PD-L1 monoclonal antibody on PD-1 / PD-L1 interaction

[0096] CHO cells expressing human PD-L1 were suspended in FACS buffer. Add the anti-PD-L1 antibody (0.5 ug / ml) prepared by the present invention to the cell suspension and incubate at 4°C for 30 minutes. Unbound antibody was washed away and FITC-labeled PD-1 recombinant protein (1 ug / ml) was added to the tube and incubated at 4°C for 30 minutes.

[0097] Flow cytometric analysis was performed using a FACScan flow cytometer. According to the measurement of the mean fluorescence intensity (MFI) of the staining, the results showed that the anti-PD-L1 monoclonal antibody blocked the binding of PD-1 to the CHO cells expressing PD-L1, Such as Figure 4 shown. It is proved that the anti-PD-L1 monoclonal antibody prepared by the present invention can significantly inhibit the interaction between PD-1 and PD-L1 on the cell surface.

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Abstract

The invention discloses an anti-PD-L1 monoclonal antibody. A mouse is immunized with a PD-L1 protein, the spleen of the mouse is separated after successful immunization is determined, total RNA is extracted, reverse transcription is carried out to obtain cDNA, PCR amplification is performed to obtain antibody light-chain and heavy-chain genes, a single-chain antibody phage library is constructed,and screening is performed to obtain the target antibody. The anti-PD-L1 monoclonal antibody screened from the phage antibody library has good affinity to PD-L1, and can inhibit the combination of PD-L1 and PD-1. The anti-PD-L1 monoclonal antibody is used as an immune checkpoint inhibitor after being further humanized, and can be used for treating cancers and infectious diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-PD-L1 monoclonal antibody, and the application of the antibody in the preparation of anticancer drugs. Background technique [0002] Cancer is known as one of the five terminal illnesses in the world. The exploration of cancer treatment has never stopped all over the world. However, cancer treatment is still a worldwide problem, which has caused huge economic pressure on society and patients (including their families) . The basic unit of cancer lesions is cancer cells. The endless proliferation of cancer cells consumes a large amount of nutrients in the patient's body. At the same time, cancer cells can also release a variety of toxins, causing a series of symptoms in the human body. If the detection and treatment are not timely, It can also be transferred to grow and reproduce throughout the body. Traditional means of treating cancer include radiotherapy, chemotherapy, sur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2827C07K2317/565C07K2317/92
Inventor 张守涛郭亚楠万骐鸣
Owner 冠科生物技术(中山)有限公司
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