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Anti-PD-L1/VEGF fusion protein

A PD-L1 and fusion protein technology, applied in the field of fusion proteins, can solve the problems of slow tumor growth and inability to achieve metastasis, and achieve the effect of excellent blocking ability

Pending Publication Date: 2021-12-07
ZEDA BIOPHARMACEUTICALS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If there is no angiogenesis within the tumor, the primary tumor grows slowly and metastasis cannot be achieved

Method used

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  • Anti-PD-L1/VEGF fusion protein
  • Anti-PD-L1/VEGF fusion protein
  • Anti-PD-L1/VEGF fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Construction of anti-PD-L1 / VEGF bifunctional fusion protein

[0057] In the present invention, the anti-PD-L1 monoclonal antibody is connected in series with the D2 domain of VEGFR1 to construct an anti-PD-L1 / VEGF bifunctional fusion protein, and the structural diagram is as follows figure 1 shown.

[0058] fusion protein M8-D2

[0059] The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 1) and the C-terminus of the heavy chain (SEQ ID NO: 2) of the anti-PD-L1 monoclonal antibody M8 are linked by a peptide linker L (SEQ ID NO: 3) , to obtain the heavy chain of the fusion protein (SEQ ID NO: 4), and the sequence of the light chain of the fusion protein is SEQ ID NO: 5.

[0060] Fusion protein M8-D2-M2

[0061] The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 6) and the C-terminus of the heavy chain (SEQ ID NO: 2) of the anti-PD-L1 monoclonal antibody M8 are connected by a peptide linker L (SEQ ID NO: 3) , to obtain the heavy chain of the fusion prot...

Embodiment 2

[0063] Example 2. Expression and purification of anti-PD-L1 / VEGF bifunctional fusion protein

[0064] The nucleic acid sequence of the heavy chain of M8-D2 is SEQ ID NO: 8, and the nucleic acid sequence of the light chain is SEQ ID NO: 9. The nucleic acid sequence of the heavy chain of M8-D2-M2 is SEQ ID NO: 10, and the nucleic acid sequence of the light chain is SEQ ID NO: 9. The DNA fragments of the heavy and light chains of the anti-PD-L1 / VEGF bifunctional fusion protein were subcloned into pcDNA3.4 vectors (purchased from thermofisher, A14697), and the recombinant plasmids were extracted and co-transfected into CHO cells and / or 293F cells. After the cells were cultured for 7 days, the culture solution was filtered by high-speed centrifugation and vacuum filtration with a microporous membrane, and then loaded onto the HiTrap MabSelect SuRe column, and the protein was eluted with 100mM citric acid, pH3.5 eluent in one step, and the target sample was recovered and dialyzed C...

Embodiment 3

[0065] Example 3. Determination of the affinity of the anti-PD-L1 / VEGF bifunctional fusion protein to the antigen by enzyme-linked immunosorbent assay (ELISA) 3.1 ELISA detection of the affinity of the anti-PD-L1 / VEGF bifunctional fusion protein to PD-L1

[0066]Self-made recombinant PD-L1-ECD-Fc protein (refer to WO2018 / 137576A1 for the preparation method), coated with 100ng / well, overnight at 4°C. Wash the plate 3 times with PBST, add 200 μl / well blocking solution, place at 37°C for 1 hour, wash the plate once with PBST and set aside. Dilute the antibody with diluent to 100 μg / ml, and dilute it 4 times to form 12 concentration gradients (the highest concentration is 100000 ng / ml, the lowest concentration is 0.02 ng / ml), and add to the blocked ELISA plate in turn, 100 μl / well, 37 °C for 1 hour. Wash the plate three times with PBST, add HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat. #ab87422), and place at 37°C for 30 minutes. After washing the plate wi...

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Abstract

The invention relates to the technical field of fusion proteins, and particularly relates to an anti-PD-L1 / VEGF fusion protein. The fusion protein comprises an anti-PD-L1 antibody, a peptide linker L and a D2 structural domain of VEGFR 1. The N terminal of the D2 structural domain of VEGFR1 is connected to the C terminal of an anti-PD-L1 antibody heavy chain through the peptide linker L. The fusion protein has the potential of treating diseases related to PD-L1 and VEGF activity.

Description

technical field [0001] The present invention relates to the technical field of fusion proteins, more specifically, to an anti-PD-L1 / VEGF fusion protein. Background technique [0002] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids, which is one of the main known immune checkpoints (Immune Checkpoint) (Blank et al, 2005, Cancer Immunotherapy , 54:307-314). PD-1 is expressed in activated T lymphocytes, and it binds to ligands PD-L1 (programmed cell death receptor-ligand 1, programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- Ligand 2, programmed cell death-Ligand 2) binding can inhibit the activity of T lymphocytes and related cellular immune responses in vivo. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B and T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/395A61K38/17A61P35/00A61P35/02
CPCC07K16/2827C07K14/71C12N9/12C12Y207/10001C12N15/85A61P35/00A61P35/02C07K2317/565C07K2319/00C12N2800/107A61K38/00A61K38/17A61K39/395C07K19/00C12N15/62A61K38/18C12N15/63
Inventor 黄浩旻邓岚李理朱祯平
Owner ZEDA BIOPHARMACEUTICALS INC
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