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

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

References

[1] An, Z., Forrest, G., Moore, R., Cukan, M., Hayto, P., Huang, L., Vitelli, S., Zhao, J., Lu, P., Hua, J., Gibson, C.R., Harvey, B.R., Montgomery, D., Zaller, D., Wang, F., Strohl, W. (2009). IgG2m4, an engineered antibody isotype with reduced Fc function. mAbs 1 , 572–579 .
[2] Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D.R., Price, D.L., and Wong, P.C. (2001). BACE1 is the major β-secretase for generation of Abeta peptides by neurons.Nat Neurosci 4, 233–234 .11224536
[3] Copeland, R.A. (2005). Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists.Methods Biochem Anal 46, 1–265 .16350889
[4] Harrison, S.M., Harper, A.J., Hawkins, J., Duddy, G., Grau, E., Pugh, P.L., Winter, P.H., Shilliam, C.S., Hughes, Z.A., Dawson, L.A., (2003). BACE1 (beta-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes.Mol Cell Neurosci 24, 646–655 .14664815
[5] McCampbell, A., Wessner, K., Marlatt, M.W., Wolffe, C., Toolan, D., Podtelezhnikov, A., Yeh, S., Zhang, R., Szczerba, P., Tanis, K.Q., (2011). Induction of Alzheimer’s-like changes in brain of mice expressing mutant APP fed excess methionine.J Neurochem 116, 82–92 .21054384
[6] Miller, M.D., Geleziunas, R., Bianchi, E., Lennard, S., Hrin, R., Zhang, H., Lu, M., An, Z., Ingallinella, P., Finotto, M., (2005). A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.Proc Natl Acad Sci U S A 102, 14759–14764 .16203977
[7] Montgomery, D.L., Wang, Y., Hrin, R., Luftig, M., Su, B., Miller, M.D., Wang, F., Haytko, P., Huang, L., Vitelli, S., Condra, J., Liu, X., Hampton, R., Carfi, A., Pessi, A., Bianchi, E., Joyce, J., Lloyd, C., Geleziunas, R., Bramhill, D., King, V.M., Finnefrock, A.C., Strohl, W., An, Z. (2009). Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41. mAbs 1 , 462–474 .
[8] Palys, T.J., Schmid, K.E., Scherer, J.M., and Schoepp, R.J. (2006). Conversion of a mouse Fab into a whole humanized IgG antibody for detecting botulinum toxin.Hum Antibodies 15, 125–132 .17522434
[9] Pietrak, B.L., Crouthamel, M.C., Tugusheva, K., Lineberger, J.E., Xu, M., DiMuzio, J.M., Steele, T., Espeseth, A.S., Stachel, S.J., Coburn, C.A., (2005). Biochemical and cell-based assays for characterization of BACE-1 inhibitors.Anal Biochem 342, 144–151 .15958191
[10] Rüfenacht, P., Güntert, A., Bohrmann, B., Ducret, A., and D?beli, H. (2005). Quantification of the A beta peptide in Alzheimer’s plaques by laser dissection microscopy combined with mass spectrometry.J Mass Spectrom 40, 193–201 .15706631
[11] Sankaranarayanan, S., Holahan, M.A., Colussi, D., Crouthamel, M.C., Devanarayan, V., Ellis, J., Espeseth, A., Gates, A.T., Graham, S.L., Gregro, A.R., (2009). First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.J Pharmacol Exp Ther 328, 131–140 .18854490
[12] Sankaranarayanan, S., Price, E.A., Wu, G., Crouthamel, M.C., Shi, X.P., Tugusheva, K., Tyler, K.X., Kahana, J., Ellis, J., Jin, L., (2008). In vivo beta-secretase 1 inhibition leads to brain Abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin-1.J Pharmacol Exp Ther 324, 957–969 .18156464
[13] Shi, X., Tugusheva, K., Bruce, J.E., Luca, A., Chen, D.E., Hu, B., Wu, G.X., Price, E., Register, R.B., Lineberger, J., (2005). Novel mutation introduced at the β-site of amyloid precursor protein enhances β-secretase cleavage in vitro and in cells.J Alzheimers Dis 7, 139–148 15851852
[14] Shi, X.P., Chen, E., Yin, K.C., Na, S., Garsky, V.M., Lai, M.T., Li, Y.M., Platchek, M., Register, R.B., Sardana, M.K., (2001). The pro domain of β-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.J Biol Chem 276, 10366–10373 .11266439
[15] Simon, A.J., Chen, L., Price, E., Xu, M., Lucka, A., Tang, M., Chen, E., Espeseth, A.S., Sardana, M., Shi, X.P., (2004). A genetically engineered mouse model with an enhanced beta-secretase substrate exhibits increased amyloid generation.Neurobiol Aging 25, S242.
[16] Sinha, S., and Lieberburg, I. (1999). Cellular mechanisms of β-amyloid production and secretion.Proc Natl Acad Sci U S A 96, 11049–11053 .10500121
[17] Stachel, S.J., Coburn, C.A., Steele, T.G., Jones, K.G., Loutzenhiser, E.F., Gregro, A.R., Rajapakse, H.A., Lai, M.T., Crouthamel, M.C., Xu, M., (2004). Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1).J Med Chem 47, 6447–6450 .15588077
[18] Tomasselli, A.G., Qahwash, I., Emmons, T.L., Lu, Y., Leone, J.W., Lull, J.M., Fok, K.F., Bannow, C.A., Smith, C.W., Bienkowski, M.J., (2003). Employing a superior BACE1 cleavage sequence to probe cellular APP processing.J Neurochem 84, 1006–1017 .12603825
[19] Turner, R.T. 3rd, Koelsch, G., Hong, L., Castanheira, P., Ermolieff, J., Ghosh, A.K., and Tang, J. (2001). Subsite specificity of memapsin 2 (β-secretase): implications for inhibitor design.Biochemistry 40, 10001–10006 .11513577
[20] Vassar, R., and Citron, M. (2000). Abeta-generating enzymes: recent advances in β- and γ-secretase research.Neuron 27, 419–422 .11055423
[21] Vaughan, T.J., Williams, A.J., Pritchard, K., Osbourn, J.K., Pope, A.R., Earnshaw, J.C., McCafferty, J., Hodits, R.A., Wilton, J., and Johnson, K.S. (1996). Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.Nat Biotechnol 14, 309–314 .9630891
[22] Willem, M., Garratt, A.N., Novak, B., Citron, M., Kaufmann, S., Rittger, A., DeStrooper, B., Saftig, P., Birchmeier, C., and Haass, C. (2006). Control of peripheral nerve myelination by the beta-secretase BACE1.Science 314, 664–666 .16990514
[23] Wu, G., Sankaranarayanan, S., Hsieh, S.H., Simon, A.J., and Savage, M.J. (2011). Decrease in brain soluble amyloid precursor protein β (sAPPβ) in Alzheimer’s disease cortex.J Neurosci Res 89, 822–832 .21433051
[24] Wu, G., Sankaranarayanan, S., Tugusheva, K., Kahana, J., Seabrook, G., Shi, X.P., King, E., Devanarayan, V., Cook, J.J., and Simon, A.J. (2008). Decrease in age-adjusted cerebrospinal fluid β-secretase activity in Alzheimer’s subjects.Clin Biochem 41, 986–996 .18489907
[25] Yang, L.B., Lindholm, K., Yan, R., Citron, M., Xia, W., Yang, X.L., Beach, T., Sue, L., Wong, P., Price, D., (2003). Elevated β-secretase expression and enzymatic activity detected in sporadic Alzheimer disease.Nat Med 9, 3–4 .12514700