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Multifunctional fusion protein and application thereof

A fusion protein and multi-functional technology, applied in the biological field, can solve problems such as difficult expression, structural design problems, and cytotoxicity

Pending Publication Date: 2022-03-01
SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this structural antibody can activate effector T cells and NK cells and direct their activity to target against disease cells, resulting in disease-specific cytotoxicity
However, since IL-15 and IL-15Rα are easily degraded by enzymes in vivo and difficult to express in vitro, the stability of this antibody is not satisfactory
[0009] Therefore, target design combinations to achieve good multi-mechanism synergy is a preferred strategy, but multi-target design also brings structural design problems

Method used

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  • Multifunctional fusion protein and application thereof
  • Multifunctional fusion protein and application thereof
  • Multifunctional fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] The acquisition and optimization of embodiment 1 nucleotide sequence

[0115] The light chain and heavy chain amino acid sequence information of the multifunctional fusion protein CCI (or BCI) is selected from the public or self-developed Claudin18.2 (or B7H3) target monoclonal antibody sequence information, and the variable region and constant region of the sequence are obtained by analysis information. The variant sequence of the natural IL-15 and IL-15Rα complex was inserted into the amino acid sequence of one heavy chain, and the scFv sequence of the CD3 antibody was inserted at the corresponding position of the other heavy chain. According to needs, adjust the Fc of the amino acid sequence of the multifunctional fusion protein to other IgG types, such as IgG4, etc., and further design the required form of amino acid mutation in each heavy chain, thereby obtaining the amino acid sequence of the target multifunctional fusion protein, for:

[0116] Multifunctional f...

Embodiment 2

[0125] Embodiment 2 Gene synthesis and the construction of expression vector

[0126] 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 multifunctional fusion protein CCI and BCI antibody expression light chain and heavy chain nucleotide sequence, the vector and the target fragment were double-digested with HindIII and XhoI, after recovery, enzyme-linked by DNA ligase and transformed Escherichia coli competent cells DH5α, selected positive clones and carried out plasmid extraction and enzyme digestion verification, obtained the full-length first heavy chain, second heavy chain, first light chain and second light chain containing the fusion protein CCI Recombinant plasmids, respectively CCI-1 (the firs...

Embodiment 3

[0127] Example 3 plasmid extraction

[0128] 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 multifunctional fusion protein, and particularly provides a multifunctional fusion protein which is targeted to TAA and CD3 and has a biological effect of an IL-15 / IL-15R alpha compound. The multifunctional fusion protein can solve the problems of drug resistance and relapse of a single-target antibody drug, reduce the onset dose, more effectively kill tumor cells, prolong the half-life period of IL-15 / IL-15Ralpha serum, improve the tumor targeting property and reduce the toxic and side effects of the IL-15 / IL-15Ralpha serum. By utilizing the combination of the CD3 antibody and the tumor targeting antibody, the tumor can be directed, the T cells can be recruited to approach the tumor cells, and the effect of mediating the T cells to kill the tumor cells is achieved. Through structural design of formation of immune synapses, relative distance between antibody functional sites and antigens and the like, the structure of the fusion protein is more stable. The IL-15 / IL-15R alpha compound promotes the lasting recruitment and activation effects on T cells and NK cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a multifunctional fusion protein, in particular to a multifunctional fusion protein targeting TAA and CD3 and having the biological effect of IL-15 / IL-15Rα complex. Background technique [0002] The occurrence and development of tumors are accompanied by the invasion of the immune system, and immunocompromised individuals often have a higher incidence of cancer and a poorer prognosis. Cytokines (cytokine, CK) are immunomodulatory molecules, which can activate or inhibit the immune system according to their properties, dosage concentration and activity site. Therefore, this method of immunostimulatory cytokine therapy can achieve the effect of improving the immune function of cancer patients. [0003] Cytokines, as immunotherapy, have certain benefits, but their clinical use has the disadvantage of poor single-drug targeting. Only high-concentration administration can achi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/395A61K38/20A61K38/17A61P35/00
CPCC07K14/5443C07K14/7155C07K16/28C07K16/30C07K16/2809C12N15/85A61P35/00C07K2319/30C07K2319/31C07K2317/622C07K2317/51C12N2800/107C12N2800/22A61K38/00
Inventor 周冲吴崇兵王艺臻姜晓玲殷刘松
Owner SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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