Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

doi:10.1007/s13238-011-1076-4

Prot Cell

2011, Vol. 2

Issue (7) : 573-584 https://doi.org/10.1007/s13238-011-1076-4 PMID: 21822802

RESEARCH ARTICLE

Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

Guoxin Wu¹(

), Sethu Sankaranarayanan¹, Donna L. Montgomery², Adam J. Simon¹, Zhiqiang An², Mary J. Savage¹

1. Department of Neurology, Merck Research Laboratory, West Point, PA 19486, USA; 2. Department of Biologics Research, Merck Research Laboratory, West Point, PA 19486, USA

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Abstract

We have previously described a novel artificial NFEV β-secretase (BACE1) cleavage site, which when introduced into the amyloid-β precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC₅₀ of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC₅₀ for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.

Keywords scFv antibody BACE1 amyloid-β precursor protein (APP) immunoassay

Corresponding Author(s): Wu Guoxin,Email:Guoxin_wu@merck.com

Issue Date: 01 July 2011

Cite this article:

Guoxin Wu,Sethu Sankaranarayanan,Donna L. Montgomery, et al. Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product[J]. Prot Cell, 2011, 2(7): 573-584.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1076-4
https://academic.hep.com.cn/pac/EN/Y2011/V2/I7/573

Tab.1 Design of biotin labeled peptides

Fig.1
Eight scFv clones (EV1–EV8) were identified that bind specifically to the EV peptide but not to the NFEV peptide.

Fig.2
Amino acid differences in complementary determining regions (CDR) 1, 2 and 3 of both heavy and light chains are bold and underlined. CDR portions in both heavy and light chains among the 8 clones are aligned and underlined. The amino acid sequence in CDR region shows that clone EV2 is the same as EV6 and EV3 is identical to EV7.

Fig.3
The human APP_WT BACE cleavage site is indicated by KMDA, while the Swedish mutation is NLDA and the artificial BACE substrate is the NFEV sequence. The secretase cleavage sites are indicated by arrows. α, alpha-secretase; β, beta-secretase; γ, gamma-secretase. Cleavage of the APP_NFEV sequence by β-secretase followed by γ-secretase leads to the production of Aβ_EV40 and Aβ_EV42 peptides.

Fig.4
The identified EV-specific phage clones were counter-screened using an array of peptides (Table 1) to assess their specificity for binding to the N-terminal EV peptide. All 5 identified EV reactive clones (EV1, EV2, EV3, EV5 and EV8) were highly specific to the N-terminal EV peptide.

Fig.5
(A) EV3-IgG1 shows robust signal with the EV N-terminal peptide and greater than 6000-fold signal to noise ratio without detectable cross reactivity to other N or C-terminal modified peptides. (B) The EV rabbit polyclonal antibody showed significant reactivity with other counter-screening peptides in addition to the EV N-terminal peptide. (C) Sensitivity for the detection of synthetic EV N-terminal peptide by EV3-IgG-AP. The lower limit of reliable quantitation of the EV peptide is ~0.3 nmol/L.

Fig.6
(A) BACE1 concentration response in an BACE1 enzymatic reaction using NFEV substrate, followed by detection of the EV N-terminal peptide with EV3-IgG1-AP. A linear dose-dependent increase in EV peptide signal is observed as a function of BACE1 concentration ( = 0.99, <0.001, = 3). (B) Time course of production of EV N-terminal peptides following cleavage of NFEV substrate by 0.3 nmol/L BACE1 in an enzymatic reaction. A linear increase in EV N-terminal peptide signal was observed over time ( = 0.912, <0.001, = 3). (C) A specific BACE1 inhibitor MRK-3 was tested at a range of concentrations in the BACE1 enzymatic reaction with NFEV peptide substrate. A dose-dependent inhibition of EV N-terminal peptide production was observed with MRK-3 with an IC of 2.4 nmol/L.

Tab.2 IC for BACE1 inhibitor MRK-3 using NFEV substrate ( = 5)

Fig.7
(A) Production of EV_Aβ peptides from SH-SY5Y cells stably expressing APP_NFEV was tested at a range of concentrations of MRK-3. EV_Aβ peptides were measured using the detecting antibody EV3-IgG1-AP in a sandwich ELISA following capture with 4G8. A dose-dependent inhibition of EV-Aβ production was observed with an EC of ~236 nmol/L. (B) Measurement of EV_Aβ peptides using an EV rabbit polyclonal detecting antibody yielded comparable EC of ~254 nmol/L in the same experiment as in (A). (C) MRK-3 treatment led to a dose-dependent increase in sAPPα (EC ~332 nmol/L) from the same experiment as in (A), indicating no cellular toxicity. (D) Assay specificity in EV_Aβ detection with EV3-IgG1-AP antibody. Immunodepletion of Aβ peptides with Aβ-selective antibodies 6E10 or 4G8, but not mouse IgG control antibody led to a complete loss of signal in the EV3-IgG1-AP detection system.

Fig.8
Brain Aβ gene-dosage dependence, with homozygote (Homo) displaying 2 fold higher levels than heterozygote (Heter) litter mates. Wild type animals show no detectable signal. (A) Aβ_EV40 and (B) Aβ_EV42 levels. ( = 3, Mean±SEM).

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