Construction, expression, purification, and characterization of a dual-targeting PD-1/VEGF-A fusion protein (P-V)
Niliang Qian1, Liucun Gao2, Lihou Dong2
1Anhui Medical University, Hefei 230032, China; Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Abstract:
Targeting programmed death-1 (PD-1) is regarded as a novel and promising means for the treatment of many types of solid tumor. In the tumor microenvironment (TME), VEGF expression is dramatically up-regulated, and compounds that neutralize VEGF or block the interaction of VEGF with its receptors exhibit potent antitumor activity, and blocking PD-1 might promote T cell infiltration into TME and significantly enhance local immune activation. Thus, we fused domain II and domain III of kinase-insert domain receptor (KDR), the receptor of VEGF-A, to the Fc side of an anti-PD-1 monoclonal antibody with a (Gly4Ser)3 linker to generate a dual targeting fusion protein. The recombinant plasmid was successfully constructed and the fusion protein was expressed in 293E cells. Protein purification was performed in a single step by using protein A affinity chromatography. The molecular weight of the fusion protein was approximately 220kDa, and the yield was approximately 2.97g/L. Specific binding of recombinant protein to PD-1 and VEGF was detected by enzyme-linked immunosorbent assay (ELISA) analysis. Half maximal effective concentration (EC50) values were 0.561nM for PD-1 and 0.682nM for VEGF-A; accordingly, half maximal inhibitory concentration (IC50) values were 0.914nM and 0.583nM, respectively. Proliferation inhibition assays indicated that the fusion protein could inhibit the growth of human umbilical vein endothelial cells effectively. Taken together, the results indicate that this novel fusion protein can simultaneously target PD-1 and VEGF and may be beneficial for combining anti-angiogenesis with immunotherapeutic approaches for the treatment of patients with cancer.
Insights
A novel fusion protein simultaneously targets programmed death-1 (PD-1) and vascular endothelial growth factor (VEGF) to enhance cancer immunotherapy and anti-angiogenesis for solid tumors.
Area of Science:
- Oncology
- Immunotherapy
- Biotechnology
Background:
- Targeting programmed death-1 (PD-1) is a promising cancer therapy.
- Vascular Endothelial Growth Factor (VEGF) is upregulated in the tumor microenvironment (TME) and contributes to tumor growth.
- Blocking PD-1 enhances T cell infiltration and immune activation within the TME.
Purpose of the Study:
- To develop a novel dual-targeting fusion protein combining anti-PD-1 and anti-VEGF functionalities.
- To evaluate the efficacy of this fusion protein in vitro for cancer treatment.
Main Methods:
- A fusion protein was engineered by linking VEGF receptor domains (KDR) to an anti-PD-1 antibody.
- The fusion protein was expressed in 293E cells and purified using protein A affinity chromatography.
- Binding affinity, inhibitory concentration (IC50), and anti-proliferative effects on endothelial cells were assessed using ELISA and proliferation assays.
Main Results:
- The fusion protein was successfully expressed with a molecular weight of ~220kDa and a yield of ~2.97g/L.
- Specific binding to both PD-1 and VEGF-A was confirmed, with low nanomolar EC50 and IC50 values.
- The fusion protein effectively inhibited the proliferation of human umbilical vein endothelial cells.
Conclusions:
- The novel fusion protein demonstrates simultaneous targeting of PD-1 and VEGF-A.
- This dual-targeting approach holds potential for combining anti-angiogenesis and immunotherapy in cancer treatment.
