Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration

Abstract

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.

Figure 1

Circulating NRP1 concentration versus time profiles. (A) Circulating NRP1 increases in athymic nude mice receiving a single intravenous bolus dose of 10 mg/kg anti-NRP1^B. Serum samples were collected post-dose at the following time points: 15 minutes; 1, 4, 8 and 16 hours; and 2, 4, 7, 10, 14, 21 and 28 days. Error bars are standard deviations of four animals per time point. (B) Circulating NRP1 increases dose-dependently in Sprague-Dawley rats receiving a single intravenous bolus dose of 2, 10 or 50 mg/kg anti-NRP1^B. Serum samples were collected pre-dose and at the following post-dose time points: 15 minutes; 2, 4, 8, 12 and 24 hours; and 2, 3, 5, 7, 10, 14, 21 and 28 days. Concentrations of circulating NRP1 in prebleed were 21 ± 4, 20 ± 9 and 22 ± 5 ng/ml for dose groups of 2, 10 and 50 mg/kg, respectively. N = 3 animals per time point. (C) Circulating NRP1 increases dose-dependently in cynomolgus monkeys receiving a single intravenous dose of 0, 0.5, 3, 15 or 50 mg/kg anti-NRP1^B. Serum samples were collected at pre-dose and the following post-dose time points: 10 and 45 minutes; 2, 4, 7, 12 and 24 hours; and 2, 3, 5, 7, 10, 13, 16, 20, 24, 28, 35, 42, 49 and 56 days. Concentrations of circulating NRP1 in prebleed were 271 ± 13, 304 ± 118, 268 ± 125 and 258 ± 70 ng/ml for dose groups of 0.5, 3, 15 and 50 mg/kg, respectively. N = 4 animals per time point. (D) Circulating NRP1 levels in rats receiving multiple doses of 10 mg/kg anti-NRP1^B. Sprague-Dawley rats received five intravenous doses of vehicle or 10 mg/kg anti-NRP1^B on days 0, 3, 6, 9 and 12. Serum samples were collected at the following time points for both groups: day 0, 15 minutes post-dose, day 1, day 3 pre-dose, day 9 pre-dose, day 12 pre-dose and 15 minutes post-dose, day 13 and day 15; and day 29 for the anti-NRP1^B dose group. N = 3 animals per time point. Symbols represent mean ± standard deviation for (A and D) and mean + standard deviation for (B and C) for clarity.

Figure 2

Western blot analysis of circulating NRP1. (A) Circulating NRP1 proteins in sera from untreated or anti-NRP1^B treated rodents. Serum (100 µl) from 10 mg/kg anti-NRP1^B human IgG1 treated mouse (day 2) (lane 2) or rat (mixture of day 3 and day 5) (lane 4) and equal volume of untreated mouse (lane 1) or rat (lane 3) serum with the addition of a matching amount (∼2 µg) of anti-NRP1^B antibody were incubated with goat anti-rat NRP1 ECD antibody-conjugated beads. The immunoprecipitated (IP) proteins from both mouse and rat sera were separated on the same gel, and the membrane was immunoblotted (IB) with the goat anti-rat NRP1 ECD antibody. (B) Circulating NRP1 proteins in untreated and anti- NRP1^B treated cynomolgus monkey sera. Serum (100 µl) from 50 mg/kg anti-NRP1^B human IgG1 treated monkey (day 35) (lane 2) and an equal volume of serum from the same monkey before treatment (lane 1) with the addition of a matching amount (∼5 µg) of anti-NRP1^B antibody were incubated with rabbit anti-human sNRP1 antibody-conjugated beads. The immunoprecipitated proteins were immunoblotted with the rabbit anti-human sNRP1 antibody. These results are representative of sera from four individual animals. (C) Circulating NRP1 proteins in pooled sera from untreated humans. The amount of 2 µg anti-NRP1^B mouse IgG2a (lane 2) or isotype control antibody (lane 1) was added to 200 µl blank human serum pool. The antibodies were immunoprecipitated with goat anti-mouse IgG Fc antibody-conjugated beads, and the associated proteins were immunoblotted with rabbit anti-human sNRP1 antibody.

Figure 3

Tandem mass spectrometry analysis confirms the presence of the MAM domain in the 120 kDa circulating NRP1 protein. Affinity purified circulating NRP1 proteins from human serum were separated by SDS-PAGE. The 120 kDa gel band was digested with trypsin and analyzed. The most abundant protein hit was human NRP1. Total protein coverage was 31% and three peptides from the MAM domain (amino acids 650–811) were identified. The representative peptide shown above covers residues 680–702 (mass/charge = 845.71, charge = 3) where matched peaks are underlined. b-ions originating from the N-terminus and y-ions originating from the C-terminus of the peptide are labeled as per the standard nomenclature convention of Andersson and Porath.