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Monoclonal Antibodies and Their Applications

A monoclonal antibody, mouse anti-human technology, applied in the direction of antibodies, applications, anti-tumor drugs, etc., can solve the problems of immune infeasibility, achieve the effect of promoting proliferation, good curative effect and safety, and strengthening immune activation

Active Publication Date: 2019-03-19
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When murine MAb production became mature, researchers expected to produce human MAb through conventional hybridoma technology, but because human hybridoma cell lines cannot stably produce high-yield antibodies, and for many antigens, human In vivo immunity is not feasible

Method used

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  • Monoclonal Antibodies and Their Applications
  • Monoclonal Antibodies and Their Applications
  • Monoclonal Antibodies and Their Applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Embodiment 1: the preparation of hybridoma and carrier

[0145] 1. Antigen and Lymphocyte Preparation and Immunization

[0146] 1.1 Antigen preparation:

[0147] 1.1.1 Preparation of 293F cells expressing human PDL1 protein

[0148] Fusion of human PDL1 amino acid sequence and TEV-mlgG2aFc amino acid sequence, the amino acid sequence design is shown in SEQ ID No.13:

[0149] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERKLENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0150] The amino acid sequence corresponding to the above-designed fusion protein (PDL1-TEV-mIgG2aFc) was artificially optimized for codons,...

Embodiment 2

[0184] Embodiment 2: the preparation of positive control antibody (PCAB)

[0185] 1. PCAb antibody sequence is as follows:

[0186] PCABH, as shown in SEQ ID No.14:

[0187] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDYFYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0188] PCABL, as shown in SEQ ID No.15:

[0189] EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESGNSQESVTEQDSKDSTYSLSSTLNTLSKADGYEKHKVYACEVTHS

[0190] 2. Artificially optimize the codons of the amino acid sequence corresponding to the above antibody se...

Embodiment 4

[0216] Example 4: Preparation and Identification of Monoclonal Antibody

[0217] 1. Preparation of antibodies by in vitro culture method

[0218] Resuscitate the DMEM medium containing 15% fetal bovine serum and 1% penicillin and streptomycin and use vials to culture H017-9H10.4.7, H017-6F11.3.2, H017-9C10.2.8, H017-2F12.2.2, H017-9F7 .5.4.10, H017-6H3.5.1.3.5, H017-13H10.2.6, H017-1B8.4.3.1, H018-17B5.5.1, after the cells are transferred to the medium bottle, replace with 10% fetal bovine serum, 1% Penicillin and streptomycin are cultured in DMEM medium, and the middle bottle is replenished to 20ml, and then the roller bottle is cultured to 200ml.

[0219] 2. Antibody Purification

[0220] Take out the cell supernatant cultured in the roller bottle for about 7 days, vacuum filter through a 0.22um mixed cellulose microporous membrane, store at 4°C, and perform protein A affinity chromatography for antibody purification. Loading: after equilibrating the purification column w...

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Abstract

The invention belongs to the technical field of antibody medicines, and in particular relates to a mouse anti-human PD-L1 monoclonal antibody and an application thereof. The antibody derives from mouse hybridoma cells and is subjected to PD-L1 affinity screening. The mouse-derived antibody has excellent affinity and specificity to monkey PD-L1 or / and human PD-L1, and can block the combination of human PD-L1 and human PD-1 in dose dependence, effectively promote T leukomonocyte proliferation and stimulate the secretion of lymphadenoma cell IL-2 and IFN gamma. Meantime, the anti-PD-L1 can promote the proliferation of tumor antigen-specific T cells by blocking PD-1 / PD-L1 signals, so as to exert the action of killing tumor cells. The treatment performed by blocking the PD-1 and PD-L1 signal channels is leading immunotherapy due to good treatment effect and safety, thereby being a hot target in the lung cancer treatment field in the last two years.

Description

technical field [0001] The present invention relates to the field of antibody medicine, in particular to monoclonal antibody and its application. Background technique [0002] PD-L1: [0003] Programmed death receptor-1 (programmed death-1 PD-1) protein was originally obtained in apoptotic T cell hybridoma by subtractive hybridization method, and was named programmed death because it is related to apoptosis -1 receptor. Two PD-1 ligands have been found so far, namely PD-L1 (also known as B7-H1, which belongs to the B7 superfamily) and PD-L2 (also known as B7-DC). The human PD-L1 gene is located on chromosome 9P24, and its open reading frame encodes a type I transmembrane protein containing 290 amino acids. The charged intracellular region consists of. The extracellular segment of human PD-L1 has high homology with B7-1 and B7-2, and the peptide contains 4 obvious cysteine ​​residues, which are convenient for the heavy chain and light chain of the extracellular segment f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P37/02A61P35/00A61P31/12
CPCA61K2039/505C07K16/2818C07K2317/565C07K2317/92
Inventor 冯晓金磊王英武尉继征陈宇珩王成忠张士权
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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