A Class of Small Proteins and Their Applications

A protein and G protein technology, applied in the field of small proteins, can solve the problems of poor structural stability of polypeptide inhibitors, unfavorable for the design of small molecule inhibitors, no deep binding pockets, etc., to achieve weak IgG binding ability, no T cells. Depletion side effects, good tissue penetration effect

Active Publication Date: 2021-02-26
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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  • A Class of Small Proteins and Their Applications
  • A Class of Small Proteins and Their Applications
  • A Class of Small Proteins and Their Applications

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 invention discloses a class of small proteins and applications thereof. The backbone protein of the small protein is a mutant of the IgG-binding domain of Streptococcal protein G, and the small protein includes an alpha-helix and four beta-sheets, the alpha-helix running through a region surrounded by four beta-sheets , and at the same time, key amino acid sites capable of binding to PD-1 protein are also distributed on the β-sheet surface formed by the four β-sheets. The small protein of the present invention has a high structural similarity with PD‑L1 protein, can inhibit PD‑1 / PD‑L1 binding, and is a potential PD‑1 / PD‑L1 inhibitor, which has weak IgG binding ability and can be used in vivo The circulation residence time is long, and has high structural stability, can be used to inhibit the interaction between PD‑1 / PD‑L1, is simple to prepare, can be used for large-scale production and application, and facilitates the 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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/315A61K38/16A61P35/00
CPCA61K38/00A61P35/00C07K14/315
Inventor 费浩杨修竹
Owner SHANGHAI UNIV
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