Mutant protein for proliferating immune cells

A mutant and protein technology, applied in the field of protein engineering, can solve the problems of easy generation of multimers, unfavorable production and quality control, and mutual binding between different molecules.

Pending Publication Date: 2020-11-17
SHANGHAI GP BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fusion protein constructed eliminates IL-2’s high-affinity IL-2Rα binding effect on the cell surface through self-binding, but the fusion protein may have self-combination or mutual binding between different molecules, which is easy to produce multimers, which is not conducive to production and quality control; the fusion protein has a binding and dissociation balance in vivo, which cannot really completely eliminate the binding effect on IL-2Rα with high affinity on the cell surface, and may still bind to IL-2Rα on the cell surface

Method used

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  • Mutant protein for proliferating immune cells
  • Mutant protein for proliferating immune cells
  • Mutant protein for proliferating immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1. Synthesis of Mutant Interleukin-2 (IL-2) Proteins

[0139] 1. Gene synthesis

[0140] The nucleotide sequence encoding the mutant amino acid sequence of the interleukin-2 (IL-2) protein is obtained by an automatic gene synthesis method. In some embodiments, adding HIS tags to the ends of gene fragments facilitates purification; in some embodiments, adding IgG1-Fc to the ends of gene fragments facilitates purification, and Fc tags are also a common means of extending the half-life of protein drugs. The gene fragment is flanked by a single restriction enzyme cleavage site. All gene synthesis sequences were designed with a 5' DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells.

[0141]

[0142] 2. Plasmid construction

[0143] The synthesized gene was subcloned into the pcDNA3.4 plasmid using molecular biology reagents according to the manufacturer's instructions.

[0144] 3. Expression of mutant interleukin-...

Embodiment 2

[0147] Example 2.Expression of CD25 protein

[0148] gene synthesis

[0149] The nucleotide sequence encoding the amino acid sequence of CD25 protein (SEQ ID NO: 23) is obtained by automatic gene synthesis. SEQ ID NO: 24 (GGGSGGGSGGGSGGGS) is the amino acid sequence of the linker. In some embodiments, the gene fragment is co-expressed with IgG1-Fc through a linker, which is convenient for purification. The gene fragment is flanked by a single restriction enzyme cleavage site. All gene synthesis sequences were designed with a 5' DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells. An exemplary leader peptide sequence is given in SEQ ID NO:25. The synthesized gene was subcloned into the pcDNA3.4 plasmid using molecular biology reagents according to the manufacturer's instructions.

[0150] Use Expi293F cells (Thermo Fisher Scientific) to carry out the transfection of the plasmid, and the cells are cultivated at 37°C in a shaker ...

Embodiment 3

[0152] Example 3. Using ELISA, Fortebio or biacore to detect the binding affinity experiment of CD25

[0153] The present inventors detected the binding ability of IL-2 mutants to CD25 by enzyme-linked immunosorbent assay.

[0154] CD25 (derived from Example 2) was coated onto a 96-well high-adsorption microplate (3590, Costar), washed and blocked. Dilute the sample to be tested to an appropriate concentration and add it to the well. TMB color development, microplate reader (M5, Molerlder Devies), the wavelength is 450 / 650nm, read the signal value of each well. rhIL-2 is recombinant human interleukin-2 for injection (Quanqi).

[0155] Table 1. Summary of binding activity of rhIL-2 to IL-2 mutants and CD25

[0156]

[0157] Note: The binding activity of each concentration point of IL-2gm was compared with rhIL-2 (binding activity 100%).

[0158] The results are shown in figure 1 . It can be seen from the figure that at the experimental concentration, it can be clearly ...

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Abstract

The invention provides IL-2 mutant protein, fusion protein or a conjugate comprising the IL-2 mutant protein, and a pharmaceutical composition comprising the IL-2 mutant protein, and the fusion protein or conjugate. Compared with the wild type IL-2 protein, the glycosylation pattern of the IL-2 mutant protein in the invention is altered, so that the affinity of the mutant IL-2 protein to a high-affinity IL-2 receptor is eliminated or reduced, and the affinity of the mutant IL-2 protein to a medium-affinity IL-2 receptor is substantially retained. The IL-2 mutant protein in the invention has extremely low immunogenicity, so that the IL-2 mutant protein can be used for immunotherapy, but does not have various side effects generated by immunotherapy by utilizing natural IL-2.

Description

technical field [0001] The present invention relates to the field of protein engineering. In particular, the present invention relates to novel interleukin-2 (IL-2) mutants and methods for their preparation, which are compared to wild-type IL-2 protoprotein and its binding partner IL The binding ability of -2 receptor α subunit is reduced, but it retains the binding ability to IL-2 receptor β subunit and IL-2 receptor γ subunit, as well as the corresponding biological activity, which can better stimulate T Proliferation of effector cells and NK cells. Background technique [0002] Interleukin-2 (IL-2, Interleukin-2) is a kind of cell growth factor in the immune system, which can regulate the cell activity of white blood cells in the immune system, promote the proliferation of Th0 and CTL, and also participate in antibody response, hematopoiesis and tumor surveillance. IL-2 mediates its action by binding to the IL-2 receptor (IL-2R). IL-2R is composed of α, β and γ chains, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C12N15/26C12N15/62C12N15/85C12N5/10A61K38/20A61P37/04
CPCC07K14/55C12N15/85A61P37/04C07K2319/00A61K38/00A61P37/00A61P35/00C12N5/0646C12N5/0638C12N2501/2302C07K2319/21C07K2319/30A61K38/2013C12N15/62C12N2510/00C12N2800/107
Inventor 胡辉张莹
Owner SHANGHAI GP BIOTECH CO LTD
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