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Multifunctional antibody composition

A composition and multi-functional technology, applied in the direction of antibodies, drug combinations, anti-tumor drugs, etc., can solve the problems of light and heavy chain mismatch, complex structure, etc., and achieve good pharmacodynamics and high purity

Pending Publication Date: 2022-07-08
SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the disclosed antibody containing IL-15 / IL-15Ra structure, due to its complex structure, there is still the problem of mismatching light and heavy chains, and there is no relevant research on its related high stability preparations

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Acquisition and Optimization of Nucleotide Sequences

[0065] The amino acid sequence information of the light chain and heavy chain of the multifunctional antibody is selected from the public or self-developed PD-1 target monoclonal antibody sequence information, and the variable region and constant region information of the sequence are obtained by analysis. The native IL-15 sequence or the IL-15 variant sequence is inserted into the amino acid sequence of one heavy chain, and the IL-15 receptor sequence, preferably the IL-15RαSushi sequence, is inserted in the corresponding position of the other heavy chain. According to needs, adjust the Fc of the antibody amino acid sequence to other IgG types, such as IgG1, etc., and further design the desired form of amino acid mutation in each heavy chain, thereby obtaining the amino acid sequence of the target antibody, respectively:

[0066] The first heavy chain is SEQ ID NO:1, the second heavy chain is SEQ ID NO:3,...

Embodiment 2

[0070] Example 2 Gene synthesis and construction of expression vector

[0071] The pcDNA3.1-G418 vector was used as a dedicated vector for expressing the light and heavy chains of the multifunctional antibody. The pcDNA3.1-G418 vector contains the promoter CMVPromoter used for the heavy chain, the eukaryotic selection marker G418 tag and the prokaryotic selection tag Ampicilline. The nucleotide sequence of the light chain and heavy chain of the antibody of the multifunctional antibody obtained by gene synthesis was double digested with HindIII and XhoI, and the DNA ligase was used for enzymatic ligation after recovery, and transformed into E. coli competent Cell DH5α, select positive clones and carry out plasmid extraction and enzyme digestion verification to obtain recombinant plasmids containing the full-length first heavy chain, second heavy chain, first light chain and second light chain of the multifunctional antibody, respectively pcDNA3.1-G418-16-1, pcDNA3.1-G418-16-2 ...

Embodiment 3

[0072] Example 3 Plasmid extraction

[0073]According to the method described in "Molecular Cloning Experiment Guide" (Science Press, 2002), the recombinant plasmids containing the above target genes were transformed into E. coli competent cells DH5α, and the transformed bacteria were spread on 100 μg / ml ampicillin-containing Cultivate on LB plate, select plasmid clones and culture in liquid LB medium, shake bacteria at 260 rpm for 14 hours, extract plasmids with endotoxin-free plasmid extraction kit, dissolve with sterile water and measure the concentration with nucleic acid protein quantifier.

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Abstract

The present invention relates to a stable pharmaceutical composition of a multifunctional antibody and a use thereof. Specifically, the invention relates to a pharmaceutical composition which comprises a multifunctional antibody which targets PD-1 and has the biological effect of an IL-15 / IL-15R alpha compound, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a surfactant, sugar and amino acid. The pharmaceutical composition disclosed by the invention has relatively high stability and PD-1 binding force.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a pharmaceutical composition of a multifunctional antibody targeting PD-1 and having the biological effect of IL-15 / IL-15Rα complex. Background technique [0002] PD-1 (programmed death protein-1) is an inhibitory costimulatory molecule encoded by the PDCD1 gene. It is a 50-55kDa type I transmembrane glycoprotein composed of 288 amino acid residues. The transmembrane structure region and the intracellular tail structure are composed of three parts. It can be expressed on the surface of activated T cells, B cells, NK cells and other monocytes and dendritic cells (DCs), suggesting that PD-1 plays a more critical role in immune regulation. As a negative costimulatory molecule expressed on activated T cells, PD-1 binds to PD-L1 expressed by tumor cells, which can inhibit the proliferation of T cells and promote the apoptosis of activated T cells; while PD-1 antibody can block the The c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61K9/19A61K47/02A61K47/26A61P35/00
CPCC07K16/2818A61K9/0019A61K9/19A61K47/02A61K47/26A61P35/00A61K2039/505C07K2317/92
Inventor 姜晓玲吴崇兵朱彩林杜雾晨高超李琼
Owner SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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