Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration.
Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 +/- 77, 20 +/- 3, 288 +/- 86 and 322 +/- 82 ng/ml (mean +/- standard deviation; n > or = 10), respectively. Anti-NRP1(B), a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1(B) antibody. In rats and monkeys receiving single injections of anti-NRP1(B) at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1(B), respectively. In addition to the soluble NRP1 isoforms, for the first time, a approximately 120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1(B) treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1(B) induced membrane NRP1 shedding.
Lu
Yanmei
Y
Department of Assay and Automation Technology, Genentech Inc., South San Francisco, CA 94080, USA. yanmei@gene.com
Xiang
Hong
H
Liu
Peter
P
Tong
Raymond R
RR
Watts
Ryan J
RJ
Koch
Alexander W
AW
Sandoval
Wendy N
WN
Damico
Lisa A
LA
Wong
Wai Lee
WL
Meng
Y Gloria
YG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000494:pharmacology*; D015153:Blotting, Western; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D051379:Mice; D039942:Neuropilin-1 / Q000097:blood*; D051381:Rats
D000911:Antibodies, Monoclonal; D039942:Neuropilin-1