Interleukin 2 mutant

A mutant and interleukin technology, applied in the field of protein engineering, can solve problems such as reduced affinity and reduced biological activity of IL-2 mutants

Pending Publication Date: 2021-11-19
SHANGHAI GP BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The IL-2 quadruple mutant (CN103492411A) mutates the amino acids at the three positions of F44A, Y45A and L72G in IL-2, reduces the affinity of the IL-2 protein to the high-affinity IL-2 receptor and retains the mutation The affinity of the somatic IL-2 protein for the medium-affinity IL-2 receptor, but at the same time the biological activity of the resulting IL-2 mutant is also reduced

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1. Synthesis of Mutant Interleukin-2 (IL-2) Proteins

[0119] 1. Gene synthesis

[0120] 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 the example, adding an HSA tag to the end of the gene fragment facilitates purification, and this tag is also a common means to prolong 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.

[0121] Number of mutations mutation site Example mutant names protein tag 3 3, 42 and 44 IL-2gm17 (T3A, F42N, F44T) HSA

[0122] 2. Plasmid construction

[0123] The synthesized gene was subcloned into the pTT5 plasmid using molecular biology reagents according to the manufacturer's instructions.

[...

Embodiment 2

[0126] Example 2. Preparation of receptor protein

[0127] In order to study the binding ability of IL-2 mutant molecules to IL-2Rα receptor and IL-2Rβγ heterodimerization receptor, human IL-2Rα receptor and IL-2Rβγ heterodimerization receptor proteins were prepared.

[0128] The human IL-2Rα receptor was designed by connecting the coding sequence of the extracellular domain of IL-2Rα to the coding sequence of 6×His Tag (SEQ ID NO: 3), and cloning it into the eukaryotic expression vector. 293E cells cultured in Freestyle medium were used for transient transfection expression of IL-2Rα receptor. 24 hours before transfection, inoculate 0.5×10 6 Cells / ml 150ml of 293E cells, 37°C 5% CO 2 Cultivate on a shaker at 120rpm in an incubator. During transfection, add 150 μl of 293fectin to 2.85 ml of OptiMEM, mix well, and incubate at room temperature for 2 minutes; meanwhile, dilute 150 μg of the plasmid used to express IL-2Rα receptor to 3 ml with OptiMEM. Mix the above-mentioned ...

Embodiment 3

[0130] Example 3. Using biacore to detect the affinity experiment of binding receptors

[0131] To study the affinity of IL-2 mutants to the receptor relative to wild type, IL-2 mutant molecules IL-2gm17-HSA and wild-type Affinity of type IL-2-HSA to human IL-2Rα subunit: Human IL-2Rα subunit was immobilized on a CM5 chip (190RU). IL-2gml7-HSA and IL-2-HSA were used as analytes in HBS-EP buffer at 25°C. For IL-2Rα, the analyte concentration was 200 nM down to 1.526 nM (1 :2 dilution) at a flow rate of 30 μl / min (180 sec association time, 300 sec dissociation time). For IL-2Rα, regeneration was performed with 20 mM NaOH, 30 ul / min for 10 seconds. For IL-2Rα, 1:1 binding was used, RI≠0, Rmax=global fit data.

[0132] The result is as figure 2 As shown, the Rmax value of IL-2gm17-HSA was 0, and the Rmax value of IL-2-HSA was 140. IL-2gm17-HSA abolishes the affinity for IL-2Rα relative to IL-2-HSA.

[0133] The affinity of IL-2 mutant molecule IL-2gm17-HSA and wild-type IL-...

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Abstract

The invention provides an interleukin 2 mutant, a fusion protein, an antibody or a conjugate containing the mutant, and a pharmaceutical composition containing the mutant, the fusion protein, the antibody or the conjugate. Compared with wild-type interleukin 2, the mutant eliminates binding with IL-2R[alpha], reduces binding with an IL-2R[beta][gamma] dimer, but keeps stimulating proliferation of tumor immune cells, including but not limited to proliferation of T cells and NK cells. According to the IL-2 mutant, the affinity to a high-affinity IL-2 receptor is eliminated, and meanwhile, the affinity to a medium-affinity IL-2 receptor is reduced. The interleukin 2 mutant provided by the invention is more suitable for the conjugate of the fusion protein and the antibody. The interleukin 2 mutant provided by the invention has extremely few mutation sites relative to wild type interleukin 2, so that the potential immunogenicity is extremely low. Meanwhile, various side effects caused by immunotherapy by using natural IL-2 are avoided.

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 have eliminated the IL Binding of -2Rα reduces binding to IL-2Rβγ dimers, but retains stimulation of proliferation of tumor immune cells, including but not limited to T cells and NK cells. The interleukin 2 mutant of the present invention is more suitable for fusion protein and antibody conjugates. It can be used for immunotherapy, but it does not have various side effects caused by the use of natural IL-2 for immunotherapy. Background technique [0002] Interleukin-2 (IL-2, Interleukin-2) is a type I cytokine produced by activated CD4+ and CD8+ T cells, also known as T cell growth factor. Activated dendritic cells (DCs), natural killer (NK) cells and NKT cells can also produce IL-2. IL-2 is a multifunctional cytokine that plays a very important role in the hom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C07K19/00A61K38/20A61P35/00
CPCC07K14/55A61P35/00C07K2319/31C07K2319/30A61K38/00
Inventor 胡辉
Owner SHANGHAI GP BIOTECH CO LTD
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