An internalizing antibody targeting of cell surface GRP94 effectively suppresses tumor angiogenesis of colorectal cancer

https://doi.org/10.1016/j.biopha.2022.113051Get rights and content
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Highlights

  • Novel fully human monoclonal antibodies specific to GRP94 are developed using phage display technology.

  • An internalizing antibody has potent antiangiogenic activity in vitro and in vivo.

  • The antibody reduces HCT116 tumor growth without severe toxicity in vivo.

  • Endothelial cell surface GRP94 is a novel potential antiangiogenic target in CRC.

  • This antibody can be applied pharmaceutically to treat GRP94-mediated CRC.

Colorectal cancer (CRC) is one of the life-threatening malignancies worldwide. Thus, novel potential therapeutic targets and therapeutics for the treatment of CRC need to be identified to improve the clinical outcomes of patients with CRC. In this study, we found that glucose-regulated protein 94 (GRP94) is overexpressed in CRC tissues, and its high expression is correlated with increased microvessel density. Next, through phage display technology and consecutive in vitro functional isolations, we generated a novel human monoclonal antibody that specifically targets cell surface GRP94 and shows superior internalizing activity comparable to trastuzumab. We found that this antibody specifically inhibits endothelial cell tube formation and simultaneously promotes the downregulation of GRP94 expression on the endothelial cell surface. Finally, we demonstrated that this antibody effectively suppresses tumor growth and angiogenesis of HCT116 human CRC cells without causing severe toxicity in vivo. Collectively, these findings suggest that cell surface GRP94 is a novel potential anti-angiogenic target in CRC and that antibody targeting of GRP94 on the endothelial cell surface is an effective strategy to suppress CRC tumor angiogenesis.

Abbreviations

CRC
colorectal cancer
GRP94
glucose-regulated protein 94
MVD
microvessel density
mAb
monoclonal antibody
5-FU
5-fluorouracil
FOLFIRI
leucovorin, 5-FU and irinotecan
FOLFOX
Leucovorin, 5-FU, and oxaliplatin
FDA
Food and Drug Administration
EGF
epidermal growth factor
VEGF
vascular endothelial growth factor
ER
endoplasmic reticulum
TMA
tissue microarray
IHC
immunohistochemistry
HUVECs
human umbilical vein endothelial cells
FBS
fetal bovine serum
rhGRP94
recombinant human GRP94
PCR
polymerase chain reaction
scFv
single-chain variable fragment
ELISA
enzyme-linked immunosorbent assay
CDR
complementarity-determining region
IgG
immunoglobulin G
BSA
bovine serum albumin
PBS
phosphate-buffered saline
PBS-T
PBS containing 0.05% (v/v) Tween 20
HRP
horseradish peroxide
TMB
3,3′,5,5′-tetramethylbenzidine
SPR
surface plasmon resonance
PFA
paraformaldehyde
hTNFα
human tumor necrosis factor α
ICAM-1
intercellular cell adhesion molecule-1
VCAM-1
vascular cell adhesion molecule-1
TBS-T
Tris buffered saline with 0.1% (v/v) Tween 20
ERK
extracellular signal-regulated kinase
OCT
optimal cutting temperature
TUNEL
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
DAPI
4′,6-diamidino-2-phenylindole, dihydrochloride
GOT
glutamic oxaloacetic transaminase
GPT
glutamic pyruvic transaminase
TBIL
total bilirubin
CRE
creatinine
BUN
blood urea nitrogen
ANOVA
analysis of variance
SEM
standard error of the mean

Keywords

Cell surface GRP94
Tumor angiogenesis
Fully human antibody
Internalization
Downregulation

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These authors contributed equally to this work.