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I Ntrabody‐based Strategies For Inhibition Of Vascular Endothelial Growth Factor Receptor‐2: Effects On Apoptosis, Cell Growth, And Angiogenesis
Yurong Yang Wheeler, T. Kute, M. Willingham, S. Chen, D. Sane
Published 2003 · Chemistry, Medicine
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VEGF, an endothelial‐specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR. We hypothesized that an intrabody could bind newly synthesized KDR and block receptor transport to the cell surface, thereby inhibiting important VEGF effects. We expressed a single chain antibody (p3S5) to KDR with or without the endoplasmic reticulum (ER) retention signal (KDEL), using either a plasmid (p3S5‐HAK) or a tet‐off adenoviral system (Ad‐HAK). Plasmid‐mediated expression of the tethered intrabody significantly reduced KDR expression (from 82.5±12.5% to 27.9±13.6% of cells; P<0.01) and thymidine incorporation in successfully transfected cells. Ad‐HAK infection resulted in intrabody expression in >90% of human umbilical vein endothelial cells (HUVECs), producing marked (80%) apoptosis at 48 h postinfection. The intrabody was essential for these effects, as confirmed by inhibiting its expression with doxycycline or by expressing irrelevant genes (lacZ, GFP). Cell death was dependent on KDR, because Ad‐HAK infection of cell lines with minimal or no KDR had little effect on cell viability. Infected HUVECs were unable to form tubes on Engelbreth Holm‐Swarm (EHS) tumor gel matrix. These results demonstrate the potential for development of an intrabody‐based strategy to block angiogenesis and prevent tumor growth.
This paper references
Degradation from the endoplasmic reticulum: Disposing of newly synthesized proteins
J. Lippincott-Schwartz (1988)
Intrabody and immunotoxin-producing cell approaches for cancer gene therapy
Technology evaluation: IMC-1C11, ImClone Systems.
S. Hunt (2001)
Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo
K. J. Kim (1993)
Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis.
K. Plate (1993)
Signaling by Receptor Tyrosine Kinases
W. Fantl (1993)
Vascular endothelial growth factor-toxin conjugate specifically inhibits KDR/flk-1-positive endothelial cell proliferation in vitro and angiogenesis in vivo.
S. Ramakrishnan (1996)
Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.
J. H. Richardson (1995)
Characterization of Vascular Endothelial Cell Growth Factor Interactions with the Kinase Insert Domain-containing Receptor Tyrosine Kinase
S. Cunningham (1999)
Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor.
G. Conn (1990)
Pharmacokinetics and interspecies scaling of a novel VEGF receptor inhibitor, SU5416
J. Sukbuntherng (2001)
A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial.
R. Alvarez (2000)
Vascular endothelial growth factor is a potential tumor angiogenesis factor in human glioma in vivo
K. H. Plate (1992)
Significance of vascular endothelial growth factor/vascular permeability factor for solid tumor growth, and its inhibition by the antibody.
S. Kondo (1993)
Vascular Endothelial Growth Factor Induces Expression of the Antiapoptotic Proteins Bcl-2 and A1 in Vascular Endothelial Cells*
H. Gerber (1998)
Tumor suppressor loci on mouse chromosomes 9 and 16 are lost at distinct stages of tumorigenesis in a transgenic model of islet cell carcinoma.
S. Parangi (1995)
Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody.
W. Marasco (1993)
Peptide-binding specificity of the molecular chaperone BiP
G. Flynn (1991)
KDEL receptor expression is not coordinatedly up-regulated with ER stress-induced reticuloplasmin expression in HeLa cells.
D. H. Llewellyn (1997)
Prolonged tumor dormancy by prevention of neovascularization in the vitreous.
S. Brem (1976)
Vascular Endothelial Growth Factor Regulates Endothelial Cell Survival through the Phosphatidylinositol 3′-Kinase/Akt Signal Transduction Pathway
H. Gerber (1998)
Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis.
R. Shaheen (1999)
Intracellular Stability of Anti-caspase-3 Intrabodies Determines Efficacy in Retargeting the Antigen*
A. Rajpal (2001)
Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression.
J. Nör (1999)
Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.
N. Ferrara (1989)
An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells.
G. Siemeister (1998)
Antigen‐independent selection of stable intracellular single‐chain antibodies
A. Auf der Maur (2001)
Targeted tumor killing via an intracellular antibody against erbB-2.
J. Deshane (1995)
Akt Mediates Cytoprotection of Endothelial Cells by Vascular Endothelial Growth Factor in an Anchorage-dependent Manner*
Y. Fujio (1999)
Molecular cloning of the beta‐subunit of human prolyl 4‐hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.
T. Pihlajaniemi (1987)
Vascular permeability factor gene expression in normal and neoplastic human ovaries.
T. Olson (1994)
Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy
L. Witte (2004)
Halting angiogenesis suppresses carcinoma cell invasion
M. Skobe (1997)
Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.
L. Benjamin (1997)
The splice variants of vascular endothelial growth factor (VEGF) and their receptors.
C. J. Robinson (2001)
Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms.
R. Berkman (1993)
Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant
B. Millauer (1994)
E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein
J. S. Orlando (2002)
Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells.
J. Tran (1999)
Intrabodies: turning the humoral immune system outside in for intracellular immunization
W. Marasco (1997)
Tumor interactions with the vasculature: angiogenesis and tumor metastasis.
C. Blood (1990)
Dormancy of micrometastases: Balanced proliferation and apoptosis in the presence of angiogenesis suppression
L. Holmgren (1995)
ERD2 proteins mediate ER retention of the HNEL signal of LRP's receptor-associated protein (RAP).
G. Bu (1997)
Vascular endothelial growth factor and platelet-derived growth factor are potential angiogenic and metastatic factors in human breast cancer.
K. Anan (1996)
Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.
S. Cheng (1996)
Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer.
L. F. Brown (1995)
Bioactivity of anti-angiogenic ribozymes targeting Flt-1 and KDR mRNA
T. J. Parry (1999)
Signalling by receptor tyrosine kinases.
W. Fantl (1993)
Vascular endothelial growth factor as a marker of tumor endothelium.
R. Brekken (1998)
Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy
Ji-Dai Chen (1997)
Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion.
P. Steinberger (2000)
Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells.
H. Zhang (1995)
Tumor suppressor loci in mouse chromosome 9 and chromosome 16 are lost at distinct stages of tumorigenesis in a transgenic mouse model of islet cell carcinoma
S. Parangi (1995)
Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway: Requirement for Flk1/KDR activation
H. P. Gerber (1998)
A C-terminal signal prevents secretion of luminal ER proteins
S. Munro (1987)
Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2.
D. Lu (1999)
Mutational analysis of the human KDEL receptor: distinct structural requirements for Golgi retention, ligand binding and retrograde transport.
F. Townsley (1993)
Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.
L. Benjamin (1999)
Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo
K. Plate (1992)
An antagonistic vascular endothelial growth factor (VEGF) –stimulated receptor autophosphorylation and proliferation of human endothelial cells
G. Siemeister (1998)
Amino acid and cDNA sequence of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor
G. Conn (1990)
Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer.
M. Gordon (2001)
Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer.
J. P. Crew (1997)
The role of VEGF in normal and neoplastic hematopoiesis
H. Gerber (2002)
Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence.
M. Saleh (1996)
Vascular permeability factor (VPF, VEGF) in tumor biology
D. Senger (2004)
Intrakines--evidence for a trans-cellular mechanism of action.
B. Engel (2000)
Quality control of ER synthesized proteins: an exposed thiol group as a three‐way switch mediating assembly, retention and degradation.
A. Fra (1993)
Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor.
R. Jain (1998)
Sequence of a second human KDEL receptor.
M. Lewis (1992)
Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth.
K. Peters (1993)
Regulation of protein export from the endoplasmic reticulum.
J. Rose (1988)
Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity
T. Alon (1995)
ER Quality Control: The Cytoplasmic Connection
R. Kopito (1997)
A human homologue of the yeast HDEL receptor
M. Lewis (1990)
Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.
R. Kendall (1993)
Technology evaluation: IMC-1C11
S. Hunt (2001)
This paper is referenced by
Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation
K. Paz (2005)
Intrabody targeting vascular endothelial growth factor receptor-2 mediates downregulation of surface localization
E. Alirahimi (2017)
Vascular Endothelial Growth Factor Receptor Antibodies for Anti-Angiogenic Therapy
P. Bohlen (2008)
Flt-1 intraceptor induces the unfolded protein response, apoptotic factors, and regression of murine injury-induced corneal neovascularization.
N. Singh (2006)
Rationale and development of multispecific antibody drugs
Jijie Gu (2010)
ER Intrabodies: Potent Molecules for Specific Knockdown of Proteins Passingthe ER
Thomas Boldicke (2015)
Chemically modified siRNA directed against the KDR gene inhibits the proliferation of breast cancer cells.
Yin-lin Ge (2009)
Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9.
Nutjeera Intasai (2008)
Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER
T. Böldicke (2007)
Intracellular antibodies (intrabodies) and their therapeutic potential.
A. S. Lo (2008)
Intrabody applications in neurological disorders: progress and future prospects.
T. Miller (2005)
Affinity protein based inhibition of cancer related signaling pathways
Erik Vernet (2009)
Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display.
David W. Colby (2004)
Isolation, engineering, and characterization of intracellular antibodies specific for the huntingtin protein
David W. Colby (2005)
Organelle-targeted delivery of biological macromolecules using the protein transduction domain: potential applications for Peptide aptamer delivery into the nucleus.
T. Yoshikawa (2008)
Nanotools for megaproblems: probing protein misfolding diseases using nanomedicine modus operandi.
V. Uversky (2006)
CHAPTER 11 – Gene Therapy for CNS Diseases Using Intrabodies
Todd W. Miller (2006)
Intrabodies: production and promise.
Martin R Stocks (2004)
An anti-major histocompatibility complex class I intrabody protects endothelial cells from an attack by immune mediators.
C. Doebis (2006)
Antibody Structure and Function: The Basis for Engineering Therapeutics
M. Chiu (2019)
Recent Advances with ER Targeted Intrabodies.
Andrea L. J. Marschall (2016)
Targeting Pyk2 for therapeutic intervention
C. Lipinski (2010)
Rôle de la protéine tyrosine phosphatase DEP-1 dans la régulation du programmeangiogénique induit par le VEGF
C. Chabot (2010)
Protein i: interference at protein level by intrabodies.
J. Manikandan (2007)
Morphological Alterations of Microvasculature and Neoangio-genesis in the Pressure Ulcers Repair in Paraplegics
R. Scelsi (2007)
Intrabody and intrakine strategies for molecular therapy.
Yurong Yang Wheeler (2003)
ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi
Oliver Backhaus (2016)
Novel highly efficient intrabody mediates complete inhibition of cell surface expression of the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR).
T. Böldicke (2005)
Intraovarian actions of anti-angiogenic agents disrupt periovulatory events during the menstrual cycle in monkeys.
F. Xu (2005)
The Pyk2 FERM domain as a target to inhibit glioma migration
J. Loftus (2009)
Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology
Congcong Zhang (2020)
Specific in vivo knockdown of protein function by intrabodies
Andrea L. J. Marschall (2015)See more