CD47 and PD-L1 targeting bifunctional fusion protein

A fusion protein, PD-L1 technology, applied in the field of biomedicine, can solve the problems of insufficient efficacy and safety of antibody drugs, achieve good anti-tumor efficacy and blood safety, improve tumor antigen presentation, and improve tumor cell targeting. sexual effect

Active Publication Date: 2017-12-12
TAIZHOU MABTECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The present invention provides a bifunctional fusion protein targeting CD47 and PD-L1, which can not only bind to PD-L1 to block the PD-1 / PD-L1 signaling pathway, but also bind to CD47 to block the CD47 / SIRPα signal Pathway to solve the problem of insufficient efficacy and safety of existing immune checkpoint antibody drugs

Method used

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  • CD47 and PD-L1 targeting bifunctional fusion protein
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  • CD47 and PD-L1 targeting bifunctional fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Construction and expression of a bifunctional fusion protein targeting CD47 and PD-L1

[0044] According to SEQ ID NO: 4 (encoding the fusion peptide segment formed by connecting the SIRPα mutant and the Fc segment of the antibody, with the Hole structure formed by mutations of Y349C, T366S, L368A, and Y407V), SEQ ID NO: 5 (encoding anti-PD- L1 antibody light chain), SEQ IDNO: 6 (encoding anti-PD-L1 antibody heavy chain, with Knob structure formed by mutations of T366W and S354C), synthesize the corresponding DNA fragments, and insert them into pcDNA3.1 vector respectively , Constructed into expression vectors pcDNA3.1-SIRPα-m-Fc, pcDNA3.1-antiPDL1-L and pcDNA3.1-antiPDL1-H, co-transfected into CHO-dhfr-expressing host cells, and inhibited with dihydrofolate reductase High-expressing clones were screened under pressure with methotrexate (MTX), and the high-expressing clones obtained were cultured in DMEM / F-12K medium (Thermofisher) under the conditions of 37℃ an...

Embodiment 2

[0045] Example 2. Purification of a bifunctional fusion protein targeting CD47 and PD-L1

[0046] The protein content in the cell culture fluid collected in Example 1 was determined by OD280 ultraviolet absorption method to be 1.5 mg / ml, and the pH value was determined to be 7.0.

[0047] Use a depth filtration system (Pall Company) to clarify and filter the cell culture solution. First use a depth filter with a pore size of 0.6-9μm to remove cells, cell debris and insoluble substances, and then use a depth filter with a pore size of 0.1μm or less to remove fine particles. Collect the filtrate.

[0048] The collected filtrate was purified by affinity chromatography, using GE's rProteinA Sepharose 4 FastFlow purification medium and Avant 150 protein purification system, and the sample was loaded at a dose of 30mg protein / ml medium, and the binding buffer was pH7.0 20mM phosphate buffer, Elution Buffer is 25mM citrate buffer with pH 3.5, flow rate 5ml / min, collect the eluate containin...

Embodiment 3

[0049] Example 3. SDS-PAGE electrophoresis identification of the bifunctional fusion protein targeting CD47 and PD-L1

[0050] Under reducing (dithiothreitol DTT) and non-reducing conditions, the purified fusion protein was identified by SDS-PAGE electrophoresis, using a polyacrylamide gel with a separation gel concentration of 7.5%, and a sample amount of 50μg protein per well , See the results of electrophoresis image 3 (Non-reduced SDS-PAGE), Figure 4 (Reduced SDS-PAGE), lanes 1 and 2 are two duplicate samples of the bifunctional fusion protein targeting CD47 and PD-L1. The electrophoresis results show that the molecular weight of the target protein is about 110kD and the purity is greater than 98%.

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Abstract

The invention relates to a CD47 and PD-L1 targeting bifunctional fusion protein, belongs to the field of biomedicine, and solves the problems that anti-PD-1 / PD-L1 treatment is poor in a low-immunogenicity tumor treatment effect and anti-CD47 treatment is poor in a targeting ability. The fusion protein is composed of a CD47 binding part and a PD-L1 binding part which are linked by a disulfide bond, can block the binding of CD47 and SIRPalpha, can block the binding of PD-L1 and PD-1, can achieve the effects of activating macrophages to phagocytize tumor cells and promoting antigen presentation in natural immunity, can achieve an effect of promoting the activation of tumor specific T cells in acquired immunity, and has lower blood toxicity; and as compared with the single use of anti-PD-L1 or anti-CD47 for treatment, the fusion protein has better antitumor curative effects and blood safety.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a recombinant fusion protein targeting CD47 molecules and PD-L1 molecules. Background technique [0002] The "immune escape" of cancer cells is considered to be the main mechanism of tumorigenesis, development and drug resistance. Tumors generally protect themselves from being eliminated by the immune system by directly or indirectly suppressing T cell signals. Tumor immune checkpoint therapy (immune chenkpoint therapy) is a type of treatment that modulates the activity of T cells through a series of pathways such as co-suppression or costimulation signals to improve anti-tumor immune responses. [0003] Immune checkpoints use costimulatory and co-suppressive molecules in the immune response process to "turn on" or "turn off" the immune response. They play an important role in regulating the amplitude and duration of T cell responses and maintaining self-tolerance. Prevent damage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/395A61K39/44A61P35/00A61P37/02
CPCA61K2039/507C07K14/4703C07K16/2818C07K19/00C07K2319/00C07K2319/03
Inventor 郭亚军寇庚钱卫珠郭怀祖徐进
Owner TAIZHOU MABTECH PHARM CO LTD
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