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Fusion protein of PD-1 extracellular domain mutant with high affinity as well as pharmaceutical composition and application of fusion protein

A technology of PD-1 and fusion protein, which is applied in the field of tumor therapy and molecular immunology, can solve the problems of not having ADCC, CDC, etc., and achieve the effect of high efficacy

Active Publication Date: 2022-02-11
江苏东抗生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since related PD-L1 / L2 and PD-1 are also expressed on immune cells, considering that if PD-1 / PD-L1 antibodies have too strong ADCC, CDC, ADCP and other activities, they will kill their own immune cells, so PD-1 / PD-L1 antibodies are mostly designed as blocking antibodies, without ADCC, CDC, etc.

Method used

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  • Fusion protein of PD-1 extracellular domain mutant with high affinity as well as pharmaceutical composition and application of fusion protein
  • Fusion protein of PD-1 extracellular domain mutant with high affinity as well as pharmaceutical composition and application of fusion protein
  • Fusion protein of PD-1 extracellular domain mutant with high affinity as well as pharmaceutical composition and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Expression vector construction of PD-1 variant-Fc fusion protein

[0049] The amino acid sequences and mutation sites of the wild-type PD-1 prepared by the present invention and its four high-affinity PD-1 extramembrane mutants are as follows: figure 1 As shown, the amino acid sequence of PD-1 is SEQ ID NO.1; the amino acid sequence of variant M2C5 is SEQ ID NO.2; the amino acid sequence of variant M3H6 is SEQ ID NO.3; the amino acid sequence of variant M4B3 is SEQ ID NO. NO.4; the amino acid sequence of the variant M5G8 is SEQ ID NO.5.

[0050] PD-1 and its variants are linked to IgG1 Fc (amino acid sequence: SEQ ID NO.8), IgG4 Fc (amino acid sequence: SEQ ID NO.9) or 6×His ( Amino acid sequence: SEQ ID NO.20) etc. are connected to form a fusion protein, and the schematic diagram of its protein structure is as follows figure 2 shown.

[0051] In order to secrete and express PD-1 and its variants in mammalian cells, a signal peptide (amino acid sequence: ...

Embodiment 2

[0055] Example 2: Expression and purification of PD-1 and its variant fusion proteins

[0056] Recombinant PD-1 and its variant fusion proteins were expressed by transient transfection of 293E cells (purchased by Invitrogen). 293E cells in the logarithmic growth phase were treated with 4×10 5 / mL density inoculated in a shaker flask at 37°C, 5% CO 2 Transfection was carried out after 24 hours of incubation on a shaking table at 125r / min.

[0057] For every 100 mL of 293E cells, take 5 mL of OPTI-MEM and add 200 μg of plasmid, shake and mix and incubate at room temperature for 5 minutes to obtain a plasmid solution; take another 5 mL of OPTI-MEM and add 600 μg of PEI, shake and mix for 5 minutes at room temperature to obtain a PEI solution ; Mix the plasmid and PEI solution, vortex and incubate at room temperature for 20 minutes, add the reaction mixture dropwise to the cells, and place at 37°C, 5% CO 2 Cultivate on a shaker at 125r / min, fed feed on the 4th and 6th day, and ...

Embodiment 3

[0058] Example 3: Analysis of the binding activity of PD-1 and its variant fusion proteins to PD-L1

[0059] Dilute the PD-L1 / His antigen (purchased from Sino Biological, Cat. No. 10084-H08H) in the coating solution to 1 μg / mL, add 100 μL to each well of the enzyme-linked plate, and place it in a humid box at 4°C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute the fusion protein of PD-1 and its mutants with 1×PBS to 15 μg / mL, and after 3-fold serial dilution, add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, and wash the enzyme-linked plate 3 times, add goat anti-human Fc-HRP secondary antibody to react at room temperature for 45min, wash the enzyme-linked plate 5 times and add 100μL TMB substrate for color development, react for 3min and wash with 100μL 2N H 2 SO 4 The reaction was terminated, and the enzyme-linked immunosorb...

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Abstract

The invention discloses a PD-1 (programmed death-1) extracellular domain mutant with high affinity. The PD-1 extracellular domain mutant is composed of an amino acid sequence selected from SEQ ID NO.3. The invention also discloses a preparation method of the PD-1 extracellular domain mutant. The invention further provides a fusion protein of the PD-1 extracellular domain mutant with high affinity and application of the fusion protein. According to the invention, the PD-1 extracellular domain mutant with high affinity or the fusion protein of the PD-1 extracellular domain mutant is creatively synthesized, and compared with wild type PD-1, the fusion protein has higher affinity for combining with human PD-L1 or PD-L2 and higher efficacy. The fusion protein disclosed by the invention provides a wider way for immunotherapy of tumors.

Description

technical field [0001] The invention relates to the fields of tumor therapy and molecular immunology, and relates to a high-affinity fusion protein of a mutant of the extramembrane region of PD-1 and its pharmaceutical composition and application. Background technique [0002] Tumor immunotherapy is to stimulate and enhance the immune function of the body to achieve the purpose of controlling and killing tumor cells. Tumor immunotherapy is one of the current hot spots in the field of tumor treatment, and clinical trials have also achieved remarkable therapeutic effects. PD-1 and its ligand PD-L1 / L2 mediate the inhibitory "exhaustion" of T cells and the induction of immune tolerance. The highly expressed PD-L1 / L2 on the surface of tumor cells interacts with PD-1 to cause tumor The immune escape of cells, therefore inhibiting the PD1-PD-L1 / L2 signaling pathway and reactivating the suppressed immune system has become a hot spot in immunotherapy recently. [0003] Programmed de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K19/00G01N33/68C12N15/85C12N5/10A61K38/17A61P35/00A61P35/02
CPCA61P35/00A61P35/02C07K14/70521A61K38/00C07K2319/21C07K2319/30G01N2333/70521G01N33/68G01N33/57492G01N2333/70532Y02A50/30
Inventor 魏化伟黄亚杰
Owner 江苏东抗生物医药科技有限公司
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