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Small proteins and application thereof

A protein and G protein technology, applied in the field of small proteins, can solve the problems of poor structural stability of polypeptide inhibitors, not conducive to the design of small molecule inhibitors, and no deep binding pockets, etc., to achieve weak IgG binding ability and no T cells Depleted side effects, good tissue penetration effects

Active Publication Date: 2019-10-08
SHANGHAI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

combination of the two Hydrophobic and relatively flat, without deep binding pockets, such a binding surface is not conducive to the design of small molecule inhibitors
However, the structural stability of polypeptide inhibitors is poor, the affinity is not high enough, and the short circulation time in the body also limits its application.

Method used

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  • Small proteins and application thereof
  • Small proteins and application thereof
  • Small proteins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Comparison of the preferred small protein sequence and the wild-type GB1 backbone sequence

[0069] Table 1 lists the 11 mutant small proteins and skeleton protein GB1 involved in this example. Each of the mutant small proteins can be obtained through simple prokaryotic expression, and the prokaryotic expression vector of the small protein is obtained by molecular cloning, and then expressed by prokaryotic, and the pure mutant small protein can be obtained by affinity purification. Wherein, the prokaryotic expression process of the small protein is as follows: the prokaryotic expression plasmid of each mutant small protein can be obtained by molecular cloning. After the plasmid sequence is correct, it is chemically transformed into BL21(DE3) competent E. coli cells, and the cells are cultured overnight in LB medium containing antibiotics consistent with the plasmid resistance gene (such as LB medium containing kanamycin for pET28a) , to obtain overnight bacte...

Embodiment 2

[0073] Example 2 Comparison of conformation similarity between small protein backbone and PD-L1 protein

[0074] Using the pair fitting function of the PyMOL software Wiazrd menu, select the C of 12 key amino acids on the binding surface of PD-L1 and PD-1 α Position C corresponding to the β sheet of the small protein backbone α For comparison, according to the root mean square deviation (root-mean-square deviation, RMSD, the unit is ) to evaluate the similarity between the small protein surface and the PD-L1 structure, the smaller the value, the closer the spatial structure, when , the representative structure is completely overlapped. The RMSD comparison values ​​of selected small protein backbones and PD-L1 were all within Left and right, the RMSD value of the GB1 skeleton and PD-L1 selected in the present invention is only Such as Figure 5 As shown in Table 2, Table 2 lists the RMSD values ​​obtained from the comparison of the conformational similarity between t...

Embodiment 3

[0078] 1) Surface Plasmon Resonance (SPR) detection of mutant small protein 8 inhibiting PD-1 / PD-L1 binding

[0079] SPR detection is to detect the change of the chip's refractive index and reflect the change of the weight of the binding substance on the chip surface. When the analyte flows through the chip surface and binds to the coupled ligand on the chip, the chip's refractive index changes, and the instrument records the response of the change. Value (Response Unit, RU). The RU value reflects the change in the weight of the conjugate on the chip surface, reflecting the binding or dissociation of the analyte and the ligand. Such as Figure 6As shown, the PD-1 protein was coupled on the chip, and different concentrations of GB1 mutant GBM8 were mixed in the analyte PD-L1. With the increase of the concentration of GB1 mutant GBM8, the combination of PD-L1 and PD-1 decreased, It reflects the inhibitory effect of GB1 mutant GBM8 on the binding of PD-1 / PD-L1.

[0080] 2) Sur...

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Abstract

The present invention discloses small proteins and an application thereof. The skeleton protein of the small protein is a mutant of an IgG binding domain of streptococcus G protein, and the small protein includes an alpha helix and four beta folding, The alpha helix penetrates through a region formed by the inclusion of four beta folding, while a key amino acid site capable of binding to the PD-1protein is also distributed on a beta folding surface formed by the four beta folding. The small protein of the present invention has a high structural similarity to the PD-L1 protein and inhibits PD-1 / PD-L1 binding, is a potential PD-1 / PD-L1 inhibitor, has weak IgG binding ability, long circulation time in vivo, and high structural stability, which can be used to inhibit the interaction between PD-1 / PD-L1. The preparation method is simple and can be used for large-scale production and application for immunotherapy of cancer.

Description

technical field [0001] The present invention relates to a class of small proteins, in particular to a class of small proteins based on the skeleton of GB1 (Immunoglobulin G-binding protein G, referred to as GB1 for short), and such small proteins At the same time, it has the ability to bind IgG, and its application as a PD-1 / PD-L1 inhibitor belongs to the technical field of PD-1 / PD-L1 inhibitors. Background technique [0002] The immune system plays a vital role in the fight against cancer. Tumor cells express tumor-specific antigens on the cell surface due to changes in genetic material. Immune cells can kill tumor cells through the recognition of these specific antigens. However, tumor cells will adopt immune escape mechanisms to allow them to survive anti-tumor immunity. Therefore, blocking the escape of tumor cells is beneficial to the killing of tumor cells. [0003] PD-1 (programmed cell death 1, CD279) programmed cell death protein is a type I membrane protein and...

Claims

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Application Information

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IPC IPC(8): C07K14/315A61K38/16A61P35/00
CPCA61K38/00A61P35/00C07K14/315
Inventor 费浩杨修竹
Owner SHANGHAI UNIV
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