Generation and characterization of a human nanobody against VEGFR-2.
OBJECTIVE
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.


CONCLUSIONS
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.
VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Lin
L
Ma
VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|State Key Laboratory of Natural Medicines, School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China.
Kai
K
Gu
VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Cheng-Hai
CH
Zhang
VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Xue-Tao
XT
Chen
VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yi
Y
Jiang
Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, 333 Bostwick Ave, NE, Grand Rapids, MI 49503, USA.
Karsten
K
Melcher
State Key Laboratory of Natural Medicines, School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China.
Juan
J
Zhang
State Key Laboratory of Natural Medicines, School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China.
Min
M
Wang
VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, 333 Bostwick Ave, NE, Grand Rapids, MI 49503, USA.
H Eric
HE
Xu
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D000915:Antibody Affinity; D049109:Cell Proliferation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000276:immunology
D020533:Angiogenesis Inhibitors; D019151:Peptide Library; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D040301:Vascular Endothelial Growth Factor Receptor-2