Generation and characterization of a human nanobody against VEGFR-2
Abstract
Aim: Nanobody is an antibody fragment consisting of a single monomeric variable antibody domain, which can be used for a variety of biotechnological and therapeutic purposes. The aim of this work was to isolate and characterize a human signal domain antibody against VEGFR-2 domain3 (VEGFR D3) from a phage display library.
Methods: To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning. For nanobody expression and purification, four VEGFR2 D3-blocking clones were subcloned into a pETduet-biotin-MBP expression vector. The recombinant proteins carried an MBP tag to facilitate purification by affinity chromatography. Recombinant NTV(1–4) was obtained after an additional gel filtration chromatography step. The interactions between VEGFR2 D3 and NTV(1–4) were assessed with luminescence-based AlphaScreen assay and SPR assay. Anti-angiogenesis effects were examined in human umbilical vein endothelial cells (HUVECs).
Results: In the AlphaScreen assay, NTV1 (100 and 200 nmol/L) elicited the highest binding signal with VEGFR2 D3; NTV2 showed moderate interactions with VEGFR2 D3; NTV3 and NTV4 exhibited little or no interaction with VEGFR2 D3. In the SPR assay, NTV1 displayed a high affinity for VEGFR2 D3 with an equilibrium dissociation constant (KD) of 49±1.8 nmol/L. NTV1 (1–1000 nmol/L) dose-dependently inhibited the proliferation of HUVECs and the endothelial tube formation by the HUVECs.
Conclusion: The nanobody NTV1 is a potential therapeutic candidate for blocking VEGFR2. This study provides a novel and promising strategy for development of VEGFR2-targeted nanobody-based cancer therapeutics.
Keywords:
nanobody; VEGFR2; anticancer agent; anti-angiogenesis; phage display
Methods: To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning. For nanobody expression and purification, four VEGFR2 D3-blocking clones were subcloned into a pETduet-biotin-MBP expression vector. The recombinant proteins carried an MBP tag to facilitate purification by affinity chromatography. Recombinant NTV(1–4) was obtained after an additional gel filtration chromatography step. The interactions between VEGFR2 D3 and NTV(1–4) were assessed with luminescence-based AlphaScreen assay and SPR assay. Anti-angiogenesis effects were examined in human umbilical vein endothelial cells (HUVECs).
Results: In the AlphaScreen assay, NTV1 (100 and 200 nmol/L) elicited the highest binding signal with VEGFR2 D3; NTV2 showed moderate interactions with VEGFR2 D3; NTV3 and NTV4 exhibited little or no interaction with VEGFR2 D3. In the SPR assay, NTV1 displayed a high affinity for VEGFR2 D3 with an equilibrium dissociation constant (KD) of 49±1.8 nmol/L. NTV1 (1–1000 nmol/L) dose-dependently inhibited the proliferation of HUVECs and the endothelial tube formation by the HUVECs.
Conclusion: The nanobody NTV1 is a potential therapeutic candidate for blocking VEGFR2. This study provides a novel and promising strategy for development of VEGFR2-targeted nanobody-based cancer therapeutics.