Multi-functional antibody as well as preparation method and uses thereof

A multi-functional, antibody technology, applied in the direction of antibodies, antibody mimics/scaffolds, applications, etc., can solve problems such as short half-life, achieve the effects of improved purity, production process and druggability, and easy quality control

Active Publication Date: 2021-02-26
SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the toxicity of cytokines such as IL-15 under high-dose administration and short-term repeated administration due to short half-life still exist, which has become a major constraint for combination strategies

Method used

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  • Multi-functional antibody as well as preparation method and uses thereof
  • Multi-functional antibody as well as preparation method and uses thereof
  • Multi-functional antibody as well as preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The acquisition and optimization of embodiment 1 nucleotide sequence

[0063] The light chain and heavy chain amino acid sequence information of antibodies 6, 16, and 17 were selected from public or self-developed PD-1 target monoclonal antibody sequence information, and the variable region and constant region information of the sequence was obtained by analysis. The natural IL-15 sequence or IL-15 variant sequence is inserted into the amino acid sequence of one heavy chain, and the IL-15 receptor sequence, preferably IL-15RαSushi sequence, is inserted at the corresponding position of the other heavy chain. According to needs, adjust the Fc of the amino acid sequence of the antibody 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, which are respectively:

[0064] Antibody 6 - the first heavy chain is SEQ ID NO: 1, the second heavy ch...

Embodiment 2

[0072] Embodiment 2 Gene synthesis and the construction of expression vector

[0073] The pcDNA3.1-G418 vector is used as a special vector for expressing the light chain and heavy chain of the multifunctional antibody. The pcDNA3.1-G418 vector contains the promoter CMVPromoter used for the heavy chain, the eukaryotic screening marker G418 tag and the prokaryotic screening tag Ampicilline. Gene synthesis obtained antibodies 6, 16, and 17 to express the nucleotide sequences of the light chain and heavy chain. HindIII and XhoI were used to double-enzyme digest the vector and the target fragment, and after recovery, enzyme-linked by DNA ligase and transformed Escherichia coli competent cells DH5α, positive clones were selected and subjected to 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 antibody 6 , respectively pcDNA3.1-G418-6-1,...

Embodiment 3

[0074] Example 3 plasmid extraction

[0075] According to the method described in "Molecular Cloning Experiment Guide" (2002, Science Press), the recombinant plasmids containing the above-mentioned genes of interest were transformed into Escherichia coli competent cells DH5α, and the transformed bacteria were coated on a medium containing 100 μg / ml ampicillin. Culture on LB plates, select plasmid clones and culture them in liquid LB medium, shake the bacteria at 260rpm for 14 hours, extract plasmids from the endotoxin-free plasmid extraction kit, dissolve them in sterile water, and measure the concentration with a nucleic acid protein quantifier.

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Abstract

The invention provides a multi-functional antibody which is obtained through a genetic engineering technology, and synchronously has the biological effects of an IL-15 / IL-15R(alpha) compound as targeted PD1. The invention further discloses a nucleic acid molecule for encoding the antibody, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, as well as a preparation method and medical application of the multi-functional antibody. The multi-functional antibody can effectively solve the problems of drug resistance and disease recurrenceof single-target antibody drugs, as well as more effectively kill tumor cells and prolong survival times of in-situ tumor model animals at a reduced effective dose. Compared with an IL-15 or IL-15 / IL-15 receptor compound, the multi-functional antibody is capable of prolonging serum half-life and improving tumor targeting property with toxic and side effects reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multifunctional antibody targeting PD-1 and having a biological effect of IL-15 / IL-15Rα complex, a nucleic acid molecule encoding the antibody, and a recombinant vector comprising the nucleic acid molecule , the recombinant cell containing the recombinant vector, the preparation method of the multifunctional antibody and its medical application. Background technique [0002] Programmed cell death protein 1 (programmed cell death protein 1, PD-1) and its receptors PD-L1, PD-L2 are important regulators of T cell activity. The combination of PD-1 on the surface of T cells and PD-L1 / 2 on the surface of other cells results in the inhibition of T cells, which plays an important role in the process of avoiding autoimmune diseases and generating immune tolerance. Tumor cells use the PD-1 / PD-L1 checkpoint, a self-protecting regulatory mechanism of the immune system, to express ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/85C12N5/10A61K39/395A61P35/00
CPCC07K16/2818C07K14/5443C07K14/7155A61P35/00C07K2319/30C07K2317/92C07K2317/94A61K2039/505
Inventor 姜晓玲姜东成吴崇兵
Owner SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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