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Fusion protein comprising IL-2 protein and CD80 protein, and use thereof

A fusion protein, IL-2 technology, applied in the field of fusion proteins, can solve problems such as side effects

Pending Publication Date: 2020-11-20
GI INNOVATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological, and systemic side effects have been reported in patients receiving IL-2 immunotherapy

Method used

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  • Fusion protein comprising IL-2 protein and CD80 protein, and use thereof
  • Fusion protein comprising IL-2 protein and CD80 protein, and use thereof
  • Fusion protein comprising IL-2 protein and CD80 protein, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0189] Preparation Example 1. Preparation of hCD80-Fc-IL-2 variant (2M): GI101

[0190] To generate fusion proteins comprising human CD80 fragment, Fc domain and IL-2 variants, polynucleotides were synthesized by ThermoFisher Scientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide comprises a nucleotide sequence (SEQ ID NO:8) encoding a sequence comprising a signal peptide (SEQ ID NO:1), a CD80 fragment (SEQ ID NO:2), an Ig hinge (SEQ ID NO:3 ), an Fc domain (SEQ ID NO:4), a linker (SEQ ID NO:5) and a fusion of an IL-2 variant (2M) with two amino acid substitutions (R38A, F42A) (SEQ ID NO:6) proteins, starting from the N-terminus. Insert the polynucleotide into the pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein as shown in SEQ ID NO:9. After introducing the vector, at 37°C, 125RPM and 8% CO 2 concentration for 7 days. Then, the culture is harvested and the fusion protein...

preparation Embodiment 2

[0193] Preparation Example 2. Preparation of mCD80-Fc-IL-2 variant (2M): mGI101

[0194] To generate fusion proteins comprising mouse CD80, Fc domain and IL-2 variants, polynucleotides were synthesized by ThermoFisher Scientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide comprises a nucleotide sequence (SEQ ID NO:14) encoding a sequence comprising a signal peptide (SEQ ID NO:1), mCD80 (SEQ ID NO:13), an Ig hinge (SEQ ID NO:3) , an Fc domain (SEQ ID NO:4), a linker (SEQ ID NO:5) and a fusion protein of an IL-2 variant with two amino acid substitutions (2M) (R38A, F42A) (SEQ ID NO:6) , starting from the N-terminus in turn. Insert the polynucleotide into the pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein as shown in SEQ ID NO:15. After introducing the vector, at 37°C, 125RPM and 8% CO 2 concentration for 7 days. Then, the culture is harvested, and the fusion protein is pu...

preparation Embodiment 3

[0196] Preparation Example 3. Preparation of hCD80-Fc: GI101C1

[0197]To generate a fusion protein comprising the human CD80 fragment and the Fc domain, polynucleotides were synthesized by ThermoFisher Scientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide comprises a nucleotide sequence (SEQ ID NO: 16) encoding a sequence comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3 ) and Fc domain (SEQ ID NO: 4) fusion protein. Insert the polynucleotide into the pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein as shown in SEQ ID NO:17. After introducing the vector, at 37 °C, 125 RPM and 8% CO 2 concentration for 7 days. Then, the culture is harvested, and the fusion protein is purified therefrom. The purified fusion protein was named "GI101C1".

[0198] Purification and collection of the fusion protein were performed in the same...

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Abstract

The present invention provides a fusion protein comprising an IL-2 protein and a CD80 protein. A fusion protein comprising a CD80 fragment, an immunoglobulin Fc, and an IL-2 variant, in one embodiment, can activate immune cells, such as natural killer cells, and, at the same time, can control the immune cell regulatory activity of regulatory T cells. Therefore, a pharmaceutical composition comprising the fusion protein as an active ingredient can increase the immune activity in vivo and can be effectively used for not only cancer but also infectious diseases, and thus is highly industrially applicable.

Description

technical field [0001] The invention relates to a fusion protein comprising IL-2 protein and CD80 protein and its application. Specifically, the present invention relates to a novel fusion protein, which has cancer therapeutic effect and immune enhancing effect. Background technique [0002] Interleukin 2 (IL-2), also known as T-cell growth factor (TCGF), is a globular glycoprotein that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 has a protein size of 15.5 kDa to 16 kDa and consists of 133 amino acids. IL-2 mediates various immune effects by binding to the IL-2 receptor, which consists of three distinct subunits. [0003] Moreover, IL-2 is mainly synthesized by activated T cells, especially by CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells, and induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C07K14/705A61P31/12A61P35/00A61K38/00
CPCC07K14/705A61P31/12A61K38/00C07K14/55A61P35/00C12N15/62C07K2319/00C07K2319/31C07K14/70532
Inventor 张明浩
Owner GI INNOVATION INC