Anti-amyloid precursor protein immunoglobulins inhibit amyloid-b production by steric hindrance Rhian S. Thomas 1 , J. Eryl Liddell 2 and Emma J. Kidd 1 1 Welsh School of Pharmacy, Cardiff University, UK 2 Monoclonal Antibody Unit, School of Biosciences, Cardiff University, UK Introduction Alzheimer’s disease (AD) is characterized pathologi- cally by an over-accumulation in the brain of intracel- lular neurofibrillary tangles, amyloid-b (Ab)-containing extracellular senile plaques and neuronal loss [1]. The ‘amyloid hypothesis’ suggests that Ab accumulation in the brain is an initiating event in AD [2], although it does not explain all aspects of AD pathology [3]. Despite this, it is still the dominant theory used to explain the disease and many therapeutic strategies have therefore concentrated on attempting to modify Ab accumulation in the brain [4]. Ab is a 38–43-mer peptide that is cleaved from amyloid precursor protein (APP) [5]. APP can be processed by one of two proteolytic pathways. The Keywords Alzheimer’s disease; amyloid precursor protein; amyloid-b; monoclonal antibodies; b-secretase cleavage site Correspondence E. J. Kidd, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK Fax: +44 29 20874149 Tel: +44 29 20875803 E-mail: KiddEJ@cf.ac.uk Website: http://www.cardiff.ac.uk/phrmy/ contactsandpeople/fulltimeacademicstaff/ kidd-emmanew-overview_new.html (Received 15 July 2010, revised 30 September 2010, accepted 27 October 2010) doi:10.1111/j.1742-4658.2010.07942.x The cleavage of amyloid precursor protein (APP) by b- and c-secretases results in the production of amyloid-b (Ab) in Alzheimer’s disease. We raised two monoclonal antibodies, 2B3 and 2B12, that recognize the b-secretase cleavage site on APP but not Ab. We hypothesized that these antibodies would reduce Ab levels via steric hindrance of b-secretase. Both antibodies decreased extracellular Ab levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPPa from the non- amyloidogenic a-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhib- ited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site, whereas 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Ab levels. After 60 min incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where b-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPPa by b-sec- retase in a cell-free assay where the effects of antibody internalization and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, generated following b-secretase cleavage (bCTF), were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site IgGs can generically reduce Ab levels via inhibition of b-secretase by steric hindrance and may provide a novel alternative therapy for Alzhei- mer’s disease. Abbreviations Ab, amyloid-b; AD, Alzheimer’s disease; APP, amyloid precursor protein; BACE1, beta-site APP cleaving enzyme; bCTF, b-cleaved C-terminal APP fragments; CI, confidence interval; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with 0.05% Tween 20. FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS 167 non-amyloidogenic route involves cleavage of APP by a-secretase [6] within the Ab region to release sAPPa [7]. In the amyloidogenic pathway, b-secretase, identified as beta-site APP cleaving enzyme (BACE1) [8–10], first cleaves APP to liberate sAPPb and C99 [11]. The c-sec- retase complex [12–15] then cleaves C99 to produce Ab and a C-terminal intracellular fragment [11]. Although there is still some debate regarding the co-localization of the enzymes and substrates involved, it is generally con- sidered that, after synthesis, a proportion of APP is transported to the cell membrane and is then internal- ized for processing in the endosomal–lysosomal system, where it may be further processed to Ab [16,17]. Current therapy in the UK is limited to symptom- atic treatment with acetyl-cholinesterase inhibitors for moderate AD only [18]. Recently, there has been much attention given to the development of novel immuno- therapeutic approaches in AD. Ab vaccination, both passive and active, has been used successfully in trans- genic mice to reduce Ab plaque deposition [19] and improve cognition [20–24]. This led to a phase IIA clinical trial involving vaccination with aggregated Ab42, AN1792, but this was halted when several patients developed meningoencephalitis [25]. Alterna- tive immunotherapeutic approaches are therefore required. Here we present data relating to two monoclonal antibodies, 2B12 and 2B3. Unlike the previous approaches, our antibodies do not bind to Ab, but bind to APP in the vicinity of the b-secretase cleavage site. We previously demonstrated that 2B12 reduced levels of extracellular Ab40 from cell lines endoge- nously expressing native APP in a time- and concen- tration-dependent manner. The mode of action was, however, unclear [26]. Here we present data character- izing 2B3 and comparing the two antibodies. We dem- onstrate that 2B3 also reduces Ab levels from a native cell line and is more potent than 2B12. This effect on Ab levels was specific to the cleavage site antibodies. We suggest that these antibodies bind to the extracellu- lar region of APP when it is transported to the cell membrane and become internalized with the protein into the endosomal ⁄ lysosomal system where they inhi- bit BACE1 cleavage via steric hindrance. This will drastically reduce levels of Ab produced, representing an alternative therapeutic strategy to treat AD. Results 2B3 and 2B12 have different epitopes on APP Both 2B3, an IgG1 isotype, and 2B12, an IgG2b iso- type, detected full-length APP from MOG-G-UVW cell lysates in a western blot (Fig. 1). Both antibod- ies detected bands of 103 kDa (± 2.5 kDa) and 56 kDa (± 0.6 kDa). However, 2B3 recognized the 56 kDa fragment, possibly a thrombin cleavage frag- ment [27], more strongly than it did the 103 kDa fragment of APP, whereas 2B12 recognized the 103 kDa fragment more strongly than it did the 56 kDa fragment. Neither antibody detected Ab40 in an ELISA or western blot [26]; data not shown. The antibodies differentially recognized sAPPa and sAPPb in an ELISA (Fig. 1). There was no signifi- cant difference in the recognition by 2B12 of sAPPa or sAPP b. Similarly, there were no significant differ- ences in the amount of 2B3 bound to sAPPa when compared with the amount of 2B12 bound to either sAPPa or sAPPb. However, significantly less 2B3 bound to sAPPb than sAPPa (P < 0.01). The amount of 2B3 bound to sAPPb was also signifi- cantly less than the binding of 2B12 to either sAPPa (P < 0.001) or sAPPb (P < 0.001). 2B3 2B12 103 kDa A B 56 kDa 0 2 4 6 8 10 12 14 16 Absorbance (% of standard antibody) 2B3 2B12 sAPPα sAPPβ ** *** *** Fig. 1. (A) Representative western blot of MOG-G-UVW lysate (50 lg) detected with either 2B3 or 2B12 at 2 lgÆmL )1 on a 10% gel. Both antibodies detected full-length APP at 103 kDa (± 2.5 kDa) and a smaller protein at 56 kDa (± 0.6 kDa), probably a thrombin cleavage fragment of APP. 2B3 preferentially recognized the 56 kDa fragment, n = 4–5. (B) Differential 2B3 and 2B12 recognition of sAPPa and sAPPb as determined by indirect ELISA. Data are expressed as a mean (± standard error of the mean) percentage of the standard antibody (6E10) at 0.1 lgÆmL )1 . 2B12 recognized sAPPa and sAPPb equally well. However, significantly more 2B3 bound to sAPPa than to sAPPb. The amount of 2B3 bound to sAPPb was also significantly less than the amount of 2B12 bound to either sAPPa or sAPPb.**P < 0.01, ***P < 0.001 after one-way ANOVA and Bonferroni post-hoc tests, n =4. Antibodies inhibit amyloid-b production R. S. Thomas et al. 168 FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS 2B3 recognizes full-length APP and a peptide spanning the cleavage site on APP more strongly than 2B12 To determine the relative affinities of 2B3 and 2B12 for APP, we first compared their binding to a peptide, Kb, which contains the b-secretase cleavage site on APP (Fig. 2). The antibodies differentially bound to the peptide, as evidenced by their differential Hill slopes, 0.90 for 2B3 and 0.63 for 2B12 (P < 0.001). The antibody concentration at which half-maximal binding was reached was significantly lower for 2B3, 1.279 lgÆmL )1 [95% confidence interval (CI) 1.153– 1.418], than for 2B12, 2.963 lgÆmL )1 (95% CI 1.696– 5.177) (P < 0.001) and the maxima reached for 2B3, 103.7% (95% CI 100.7–106.7) was significantly greater than that for 2B12, 73.44% (95% CI 62.34–84.54) (P < 0.001). Furthermore, significantly more 2B3 than 2B12 was detected bound to Kb at all antibody con- centrations greater than 0.1 lgÆmL )1 (P < 0.05). We next compared their binding efficiency to full- length APP in a competition ELISA using MOG-G- UVW cell lysate as a source of APP. Both 2B3 and 2B12 interfered with the binding of a commercial detection antibody for APP in a concentration-depen- dent manner (Fig. 3). At 5, 10 and 20 lgÆmL )1 , 2B12 significantly reduced the binding of the commercial antibody to 90.5% (P < 0.01), 89.13% (P < 0.001) and 82.9% (P < 0.01) of control levels, respectively. This was similar to previously reported levels [26]. At 5, 10 and 20 lgÆmL )1 , 2B3 also significantly reduced the binding of the second commercial antibody to 87.4% (P < 0.01), 82.33% (P < 0.001) and 72.94% (P < 0.001) of control levels. Even at the highest con- centration, 20 lgÆmL )1 , a control IgG did not signifi- cantly reduce the binding of the second detection antibody when compared with control levels. The three antibodies differentially inhibited the binding of the APP detection antibody at 1 lgÆmL )1 (P < 0.05), 5 lgÆmL )1 (P < 0.001), 10 lgÆmL )1 (P < 0.001) and 20 lgÆmL )1 (P < 0.001). 2B3 and 2B12 produced a significantly greater inhibition in the binding of the APP detection antibody than did the control IgG at all concentrations tested (P < 0.05). At 10 lgÆmL )1 (P < 0.01) and 20 lgÆmL )1 (P < 0.001), 2B3 also sig- nificantly inhibited binding of the detection antibody more than 2B12. The majority of both 2B3 and 2B12 remained bound to their antigen, Kb, after incubation at different pH values To determine the effects of pH on the antibodies, 2B3 and 2B12 were bound to the Kb peptide at pH 7.4 and then incubated for 0 or 60 min at pH 4.0 or 7.4. There 0 20 40 60 80 100 120 0.00001 0.0001 0.001 0.01 0.1 1 10 100 Antibody concentration (µg·mL –1 ) Absorbance (% of standard antibody) 2B3 2B12 * * * * * * * * * Fig. 2. Half-maximal binding of 2B3 and 2B12 to a peptide, Kb, spanning the b-secretase cleavage site, as determined by ELISA. Data are expressed as a mean (± standard error of the mean) per- centage of the standard antibody (6E10) at 0.05 lgÆmL )1 . The con- centration at which half-maximal binding was reached was significantly lower for 2B3 than for 2B12 (P < 0.001). *Significant differences between 2B3 and 2B12 with two-tailed Student’s t-tests, P < 0.05, n = 3–4. 70 75 80 85 90 95 100 105 0 5 10 15 20 25 Antibody concentration (µ g ·mL –1 ) APP (% of control) 2B3 2B12 Control IgG a aa a b b *** *** ** b ** b b c ** b *** c Fig. 3. Binding of 2B3, 2B12 or a control IgG to APP as determined by a competition sandwich ELISA for APP. Data are expressed as mean (± standard error of the mean) percentage of the media con- trol. One-way ANOVA indicated that the antibodies differentially inhibited the binding of a commercial anti-APP IgG at each antibody concentration. Antibody data points followed by different letters (either a, b or c) differ significantly from each other in their ability to inhibit the commercial antibody at that particular antibody concen- tration after one-way ANOVA and Bonferroni (P < 0.05). 2B3 and 2B12 also significantly reduced the binding of the commercial anti- body in comparison with media controls at 100%. **P < 0.01, ***P < 0.001, significantly different from media controls using two- tailed Student’s t-tests. The control IgG did not significantly inter- fere with the binding of the detection antibody, n =4. R. S. Thomas et al. Antibodies inhibit amyloid-b production FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS 169 were significant differences between the persistence in binding of the antibodies to Kb under the various con- ditions tested (P < 0.001) (Fig. 4). There was also a significant interaction between the antibody type (2B3 or 2B12) and pH (P < 0.05). The complex formed between 2B12 and Kb was not significantly affected by pH or incubation time. The complex formed between 2B3 and Kb was also not significantly affected by the incubation period, but there was a significant reduction in 2B3 binding at pH 4.0 after 60 min (P < 0.05). Incubation of Kb with phosphate-buffered saline with 0.05% Tween 20 (PBST) alone at pH 4.0 for 1 h, prior to incubation with antibody at pH 7.4, did not affect the binding of either 2B3 or 2B12 (data not shown), indicating that the above results are not due to degra- dation of the antigen. Importantly, even at pH 4.0, there was still significantly more 2B3 bound to Kb than 2B12. 2B3 is more effective at reducing extracellular Ab40 and Ab42 levels in cell culture media than 2B12 The antibodies tested differentially inhibited levels of extracellular Ab40 (P < 0.001) (Fig. 5A). 2B12 signifi- cantly reduced levels of Ab40 in MOG-G-UVW cell media to 65.3% of media control levels (P < 0.05). This was similar to levels previously reported [26]. 2B3 significantly reduced levels of extracellular Ab40 to 36.8% of media control levels (P < 0.001). Neither the control IgG nor the anti-N-terminal APP IgG had any significant effect on Ab40 levels. Both 2B3 and 2B12 significantly reduced Ab40 more than the anti- N-terminal APP IgG (P < 0.001, P < 0.05, respec- tively) and 2B3 significantly reduced Ab40 levels more than 2B12 (P < 0.05). The antibodies also differentially inhibited levels of Ab42 (P < 0.05). 2B12 significantly reduced Ab42 lev- els to 54.8% of the media control (P < 0.01). 2B3 was again more effective and reduced these levels to 21.9% of media control levels (P < 0.01) (Fig. 5B). Ab42 levels 0 10 20 30 40 50 60 70 80 90 100 0 min 60 min 0 min 60 min Absorbance (% of standard antibody) 2B3 2B12 *** *** * * pH 7.4 pH 4.0 Fig. 4. Effect of pH and incubation time on persistence of 2B3 and 2B12 binding to Kb as determined by ELISA. Data are expressed as mean (± standard error of the mean) percentage absorbance of the standard antibody, 6E10 (0.05 lgÆmL )1 ). Antibodies were first allowed to form a complex with Kb at pH 7.4 and the complex was then incubated with buffers of pH 4.0 or 7.4 for 0 and 60 min. The complex formed between Kb and 2B3 was not affected by incuba- tion time, but was significantly affected by the pH of the buffer. The persistence in binding of 2B12 to Kb was not significantly affected by either incubation time or pH. At all values tested, signif- icantly more 2B3 remained bound to the Kb peptide than 2B12. *P < 0.05, ***P < 0.001, 2B3 significantly different to 2B12. Data were analysed for statistical significance with Generalized Linear Model univariate analysis and Bonferroni post-hoc tests, n = 3–4. 0 20 40 60 80 100 120 140 A B 2B3 2B12 IgG N Aβ40 (% of control) *** ** a b 0 20 40 60 80 100 120 140 2B3 2B12 N Aβ42 (% of control) ** ** c Fig. 5. Levels of extracellular Ab40 (A) and Ab42 (B) from MOG- G-UVW culture media after incubation with 2B3, 2B12, an irrelevant mouse IgG (Ab40 only) or an anti-N-terminal APP IgG (N), all at 10 lgÆmL )1 for 48 h. Data are expressed as mean (± standard error of the mean) percentage of media control Ab levels as detected in a sandwich ELISA and corrected for total cell protein concentration. Both 2B3 and 2B12 significantly reduced both forms of Ab from media controls. Neither of the control antibodies had any significant effect on Ab levels. *P < 0.05, **P < 0.01, ***P < 0.001, signifi- cantly different from media controls (100%) with two-tailed Student’s t-tests. a P < 0.05, significantly different from all other groups; b P < 0.05 significantly different from 2B3 and N; c P < 0.05 significantly different from N after ANOVA and Tukey’s Honestly Significant Difference, n = 3–6 (A) and n = 4 (B). Antibodies inhibit amyloid-b production R. S. Thomas et al. 170 FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS remained at 100.1% of control levels after incubation with the anti-N-terminal APP IgG. Again, 2B3 signifi- cantly reduced Ab42 levels more than the anti-N-ter- minal IgG (P < 0.05). Anti-b-secretase cleavage site IgGs do not alter levels of APP, sAPPa, or affect cell viability, but reduce b-cleaved C-terminal APP fragment (bCTF) levels Neither 2B12, 2B3 nor the irrelevant IgG had any effect on levels of intracellular APP as measured in an ELISA (Fig. 6A). Furthermore, they did not have any signifi- cant effect on the number of viable cells, as measured using an MTS assay with [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra- zolium, inner salt (Fig. 6B). The anti-N-terminal APP IgG appeared to either increase the number of cells or alter the levels of metabolic activity above control levels (P < 0.01). Neither 2B12 nor the control IgG had any significant effect on the levels of sAPPa (Fig. 7A). However, incubation with 2B3 significantly reduced bCTF levels to 64.6% (P < 0.05) of control values in MOG-G-UVW cells (Fig. 7B). Anti-b-secretase cleavage site IgGs reduce BACE1 cleavage via steric hindrance in a cell-free assay Neither 2B3, 2B12, the control IgG nor the anti-N-ter- minal APP IgG had any detectable effects on total lev- els of BACE1 in MOG-G-UVW cells, as investigated by western blotting (data not shown). However, differ- ences were observed in BACE1 activity after in vitro incubation of sAPPa with BACE1 and the antibodies. The antibodies tested differentially inhibited the cleav- age of sAPPa by BACE1, as detected by western blot- ting (P < 0.001) (Fig. 8A,B). There was a decrease in 0 20 40 60 80 100 120 A B 2B3 2B12 IgG APP (% of control) 0 20 40 60 80 100 120 140 160 180 200 2B3 2B12 IgG N Absorbance (% of control) ** Fig. 6. Levels of intracellular APP as determined by ELISA (A) and cell viability as determined by MTS assay (B) in MOG-G-UVW cells after incubation with 2B3, 2B12, an anti-N-terminal APP IgG (N, MTS only) or a control irrelevant mouse IgG, all at 10 lgÆ mL )1 for 48 h. Data are expressed as mean (± standard error of the mean) percentage of control (media only). APP levels were corrected for total cell protein concentration. None of the antibodies tested had any significant effect on levels of APP or cell viability, apart from the anti-N-terminal APP IgG, which appeared to stimulate growth. **P < 0.01, significantly different from media control using two- tailed Student’s t-tests, n =3. 0 20 40 60 80 100 120 140 160 180 200 A B 2B12 IgG sAPPα (% of control) 0 20 40 60 80 100 120 2B3 2B12 βCTF (% of control) * Fig. 7. Levels of extracellular sAPPa (A) and intracellular bCTF (B) as determined by ELISA from MOG-G-UVW cells after incubation with 2B12, 2B3 or a control irrelevant mouse IgG, all at 10 lgÆmL )1 for 48 h. Data are expressed as mean (± standard error of the mean) percentage of control (media only). All levels were corrected for total cell protein concentration. Neither 2B12 nor the IgG anti- body significantly affected levels of sAPPa. However, 2B3 signifi- cantly reduced levels of bCTF from control levels. *P < 0.05, significantly different from media controls using two-tailed Student’s t-tests, n =3. R. S. Thomas et al. Antibodies inhibit amyloid-b production FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS 171 sAPPa levels from control levels (sAPPa alone) after incubation with BACE1 (media control) of )41.91 A units. Neither the control IgG nor the anti-N-terminal APP IgG had any significant effect on the activity of BACE1 and decreases in sAPPa on the addition of BACE1 were similar to those observed in the presence of control media alone (–)6.9 and )44.4 A units, respectively). The addition of both 2B3 and 2B12 sig- nificantly inhibited the action of BACE1 from control conditions. 2B12 significantly reduced this decrease in sAPPa levels to only )17.29 A units (P < 0.05). 2B3 virtually abolished the reduction in sAPPa caused by BACE1 and the decrease in sAPPa levels was only )0.05 A units (P < 0.01). Levels of sAPPa were very similar to the levels observed when sAPPa was incu- bated with 2B3 alone. Discussion Both 2B3 and 2B12 were raised to the same immuno- gen, yet they clearly have different epitopes, as evi- denced by their binding profile to APP in western blotting and by their differential binding to sAPPa and sAPPb. 2B3 binds significantly less to sAPPb than it does to sAPPa (25% less), whereas 2B12 recog- nizes sAPPb and sAPPa equally well. The evidence suggests that 2B12 binds upstream of the b-secretase cleavage site (towards the N-terminal of APP), as it recognizes both sAPPa and sAPPb. Its epitope, there- fore, is in a region common to both peptides. Although 2B3 also recognizes both of these APP frag- ments, more 2B3 bound to sAPPa than to sAPPb. However, 2B3 does not recognize the Ab peptide. This suggests that 2B3 binds across the b-secretase cleavage site, and only slightly into the Ab region (Fig. 9). Results from a BLAST (http://www.ncbi.nlm.nih. gov) search on the Ka peptide used to raise both anti- bodies indicated that they should be specific to APP as this sequence of amino acids is highly conserved in APP and not found in other mammalian sequences (data not shown). In addition, we have previously demonstrated that there was no cross-reaction between 2B12 and a range of peptides tested by ELISA or wes- tern blotting [26]. 2B3 binds more effectively to APP than 2B12, as demonstrated by a competition assay, in which 2B3 interfered with the binding of a second detection anti- body for APP more efficiently than 2B12. Although these results could be due to the differential epitopes or isotypes of 2B3 and 2B12, meaning that 2B3 might interfere more effectively with the binding of the APP detection antibody, they do suggest that 2B3 has a higher relative affinity for APP than 2B12. This is sup- ported by the results of the affinity rankings of the two antibodies for the Kb peptide, a 15-mer frag- ment spanning the b-secretase cleavage site on APP. Significantly more 2B3 than 2B12 bound to Kb at all sAPPα and BACE1 incubated with :- –60 –50 –40 –30 –20 –10 0 10 2B3 2B12 IgG N Media control Change in sAPPα from relevant control (OD units) ** * 12 A B 34 5678 910 Fig. 8. (A) Representative western blot of sAPPa incubated with or without BACE1 and 2B12, 2B3, control IgG, anti-N terminal APP IgG (N) (all at 1 lgÆmL )1 ) or media control. Lane 1, sAPPa alone; lane 2, sAPPa and BACE1 (media control); lane 3, sAPPa and 2B12; lane 4, sAPPa, BACE1 and 2B12; lane 5, sAPPa and 2B3; lane 6, sAPPa, BACE1 and 2B3; lane 7, sAPPa and IgG; lane 8, sAPPa, BACE1 and IgG; lane 9, sAPPa and N; lane 10, sAPPa, BACE1 and N. Only 2B12 and 2B3 inhibited the action of BACE1 in this system. (B) Quantification of western blots showing the mean (± standard error of the mean) change in sAPPa from relevant con- trol levels (i.e. sAPPa and relevant antibody alone) after sAPPa incubation with BACE1 and either 2B3, 2B12, control IgG, anti-N terminal APP IgG (N) or media alone. *P < 0.05, significantly differ- ent from media control and N; **P < 0.01 significantly different from media control, IgG and N, after one-way ANOVA and Bonfer- roni post-hoc tests, n = 3–6. α α -secretase cleavage site 2B3 A β COOHNH 2 2B12 β -secretase cleavage site sAPP β sAPP α APP Fig. 9. Hypothesized epitopes of 2B3 and 2B12 on APP. 2B3 binds across the b-secretase cleavage site, whereas 2B12 binds upstream of this. Neither antibody recognizes Ab when cleaved from APP. Antibodies inhibit amyloid-b production R. S. Thomas et al. 172 FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS concentrations greater than 0.1 lgÆmL )1 and the con- centration for half-maximal binding was significantly lower for 2B3. All these data suggest that 2B3 has a higher relative affinity for APP than 2B12. We hypothesize that 2B3 and 2B12 will bind to the APP ectodomain after APP has trafficked to the cell membrane and will be endocytosed into the cell with the protein. Indeed, Tampellini et al. [28] demonstrated that antibodies to the N- and mid-region of Ab bound first to the ectodomain of APP and were then internalized. This immunocomplex would be formed at ‘normal phys- iological pH’, presumably around pH 7.4. Once inter- nalized, however, the complex may enter organelles, where it would be subjected to much lower pH values, potentially as low as pH 4.5 [29,30]. Low pH is well known to affect antibody binding [31]. Therefore, we tested the persistence of the immunocomplex, after it had formed at pH 7.4, at two different pH values. The persistence of the immunocomplex formed when 2B12 bound to Kb was not significantly affected by pH or incubation time. Similarly, the Kb ⁄ 2B3 immunocom- plex was not affected by incubation time, but a decrease in pH did significantly reduce its persistence. Neverthe- less, significantly more 2B3 than 2B12 remained bound to the Kb peptide at all pH values and time points tested, and 2B3 also retained nearly 70% of its original binding capacity. We therefore suggest that both anti- bodies would retain a large proportion of their biologi- cal activity, even under the low pH conditions found in the endosomal ⁄ lysosomal system [29,30]. Furthermore, the lack of an effect of time suggests that the immuno- complexes formed will persist for a biologically relevant period of time. Having demonstrated that 2B3 bound more effi- ciently to APP than 2B12, we then investigated whether it reduced the production of Ab40 in a similar manner to 2B12. Both 2B3 and 2B12 significantly reduced extracellular levels of Ab40. However, neither the anti-N-terminal APP IgG nor the control mouse IgG had any effect on Ab40. This suggests that it is not sufficient to have an antibody that binds to APP in order to reduce Ab, but that the antibodies must bind in the vicinity of the b-secretase cleavage site to accomplish this effect. We also investigated whether either antibody affected the more aggregatory species of Ab,Ab42, as there have been suggestions that the majority of this peptide is cleaved from APP within the trans-golgi network or the endoplasmic reticulum, prior to the trafficking of APP to the cell membrane [32]. If this were indeed the case, then our anti-cleav- age site IgGs might be ineffective against this species of Ab. Again, both 2B3 and 2B12 significantly reduced Ab42 from control levels, but the N-terminal antibody had no effect. This would suggest that at least a por- tion of this peptide is produced elsewhere in the cell rather than in the secretory pathway, and after APP translocation to the cell membrane. It is interesting to note that, in both cases, 2B3 reduced levels of Ab40 and Ab42 more than 2B12, although this was only sig- nificant in the case of Ab40. This could be because of the higher relative affinity of 2B3 for APP over 2B12. Alternatively, it could be a function of its epitope, as by binding closer to the cleavage site than 2B12, it may block the access of BACE1 to APP via steric hin- drance more effectively than 2B12. Clearly, informa- tion regarding the mode of action of the antibodies is important. There are three predominant theories that have been used to explain how Ab-specific antibodies may bring about the clearance of Ab [33]: disruption of Ab aggre- gates or neutralization of Ab oligomers, Fc-receptor- mediated phagocytosis of Ab by microglia and the peripheral sink hypothesis, in which the sequestration of circulating Ab causes an efflux of Ab from the brain to the plasma [34,35]. 2B3 and 2B12 are unlikely to exert their effects by any of these mechanisms, as nei- ther binds to Ab. We previously demonstrated that 2B12 was not toxic to cells in culture [26] and neither the cleavage site antibodies nor the control IgG anti- body affected cell viability levels here. The cleavage site antibodies are therefore not reducing levels of Ab by initiating cell death. In contrast, the anti-N-terminal APP IgG led to increased absorbance levels in the MTS assay. However, we did not explore this finding any further because we had already demonstrated that this commercially available antibody did not affect Ab levels and therefore was likely to behave in a very dif- ferent manner to the cleavage site antibodies. Further- more, 2B12 did not alter detectable cleavage by the non-amyloidogenic pathway in MOG-G-UVW cells, as sAPPa levels remained unchanged. It has been demonstrated that anti-N-terminal Ab Igs, which can reduce Ab pathology in vivo, can reduce intracellular levels of Ab in vitro only when internalized into cells [28], yet none of the theories described above fully explain how intracellular Ab lev- els may be reduced. We have demonstrated that 2B3 and 2B12 can reduce levels of extracellular A b.Itis therefore probable that our antibodies are also reduc- ing levels of intracellular Ab. This is particularly likely as neither of the antibodies bind to the Ab peptide itself and cannot therefore be increasing its extracellu- lar degradation, or interfering with the assay. We were, however, unable to measure intracellular Ab40 or Ab42 because of the low levels detectable in our native cell lines. R. S. Thomas et al. Antibodies inhibit amyloid-b production FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS 173 Internalized anti-Ab Igs or anti-cleavage site IgGs may inhibit the action of b-secretase. However, Tam- pellini et al. [28] saw no evidence of this with anti-Ab Igs. Tampellini et al. [28] observed that Ab and APP ectodomain antibodies induced increased APP internal- ization from the cell surface, which actually led to enhanced cleavage by b-secretase and subsequently to enhanced clearance of the antibody-bound bCTF frag- ments in the lysosomal system. 2B3 and 2B12 did not alter intracellular APP levels. Therefore, it seems unli- kely that their mode of action is via increased degrada- tion of APP. Unlike Tampellini et al. [28], we observed a significant decrease in bCTF levels after incubation with 2B3. This would imply that the cleavage site anti- bodies do not induce increased internalization of APP leading to enhanced cleavage by b-secretase, but that they are reducing Ab by a different mechanism. It sug- gests that they are inhibiting the cleavage of APP by b-secretase. The lack of a significant effect after incu- bation with 2B12 may be a result of the smaller effect that this antibody has on Ab levels. We hypothesized that 2B3 and 2B12 were blocking the action of b-secretase by steric hindrance. We there- fore devised a simple cell-free system to investigate this hypothesis that avoided complications from other cellular components and overcame the low levels of APP fragments in the native cell lines. Both 2B3 and 2B12 drastically reduced or nearly abolished BACE1 cleavage of sAPPa. This clearly demonstrates that anti-cleavage site IgGs are capable of inhibiting BACE1 in vitro. The presence of a large protein (IgG) did not nonspecifically block the action of BACE1. Crucially, our results demonstrate that antibody epi- topes are vitally important to this inhibition, as the anti-N-terminal APP IgG, which binds some distance from the cleavage site, had no such effect on BACE1. In this simple in vitro system, any effects of APP inter- nalization or enzyme ⁄ substrate co-localization are eliminated. In conjunction with the observed decrease in bCTF levels, results from the cell-free assay system suggest that the mode of action of our cleavage site antibodies is probably via steric hindrance. Similar effects on Ab levels were obtained by Arbel et al. [36], who produced monoclonal antibodies using a peptide containing part of the Swedish mutation at the b-secretase cleavage site. They also demonstrated a reduction in both extracellular and intracellular Ab levels, but in cell lines over-expressing APP. These antibodies have also been shown to improve cognition in the Tg2576 Swedish mutation mouse model of AD pathology [37] and to reduce Ab levels in the V717I London mutation mouse model [38]. We believe that our use of model cell lines that do not over-express APP is very important, as the majority of cases of AD occur in people with much lower levels of APP than those associated with transfected cells. As far as we are aware, we have demonstrated for the first time that the most likely mode of action for such antibodies is via steric hindrance. Immunotherapy for AD remains an exciting pros- pect, despite the failure of the AN1792 clinical trial [39]. Passive immunization with b-secretase cleavage site antibodies might alleviate some of the problems associated with this trial, such as the T-lymphocyte meningoencephalitis and cerebral micro-haemorrhages [40,41]. These antibodies would not bind to existing Ab and would not therefore stimulate the T-lympho- cyte response or lead to the excessive complement activation that some believe would be a problem with Ab antibodies [42]. In conjunction with other immuno- therapeutic strategies to reduce plaque load, such anti- bodies may have a considerable impact on the development of disease-modifying treatments for AD. Materials and Methods Materials and cell culture All chemicals and reagents were purchased from Sigma- Aldrich (Poole, UK) or Fisher Scientific (Leicester, UK) and all reactions were performed at room temperature unless otherwise specified. Astrocytoma cells, MOG-G-UVW (ECACC, Porton Down, UK), were cultured in a 1 : 1 mix of Ham’s F10 and Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Perbio Science UK Ltd, Cramlington, UK) and 2 mml-glutamine. Antibody production and isotyping Full details of the immunization protocol and hybridoma development are detailed elsewhere [26]. Antibodies (2B12 and 2B3) were raised to a 15-mer peptide spanning the b-secretase cleavage site on APP, EEISEVKMDAEFRHD, termed Ka. Both antibodies were concentrated from culture medium using Amicon Centriplus YM-100 filters (Millipore, Watford, UK) with a nominal molecular mass cut-off of 100 kDa and the isotype determined using the Isostrip mouse monoclonal antibody isotyping kit (Serotec, Oxford, UK). Western blotting was performed using standard meth- ods. Briefly, samples were resolved on 10% polyacrylamide gels, transferred on to 0.2 lm nitrocellulose membranes (Amersham Biosciences, Little Chalfont, UK), incubated with the relevant antibody and detected as previously described [26]. Antibodies inhibit amyloid-b production R. S. Thomas et al. 174 FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS Determination of antibody epitopes The epitopes of 2B3 and 2B12 on APP were investigated by western blotting, as above, and by comparing the relative binding profiles of the antibodies with cleavage products of APP in an indirect ELISA. Recombinant sAPPa and sAPPb (Sigma-Aldrich) were adsorbed to a 96-well microtitre plate (Greiner Bio-One, Stonehouse, UK) at 5 lgÆmL )1 in carbonate ⁄ bicarbonate buffer (15 mm Na 2 CO 3 ,35mm NaHCO 3 , pH 9.8) overnight at 4 °C. Plates were blocked with 1% nonfat milk powder for 1 h, then 2B3 or 2B12 was subsequently incubated at 1 lgÆmL )1 for 2 h. Antibodies were detected with a secondary anti- mouse IgG conjugated to horseradish peroxidase, 1 : 2500 (Pierce, Rockford, IL, USA) for 1 h and visualized with the enzyme substrate, o-phenylenediamine in a 0.1 m citrate phosphate buffer (24 mm citric acid, 51 mm Na 2 HPO 4 ,pH 5.0), incubated for 20 min. The reaction was stopped with 2.5 m H 2 SO 4 and the absorbance determined at 492 nm. The diluent used on day 2 was phosphate-buffered saline (PBS; 137 mm NaCl, 1.5 mm KH 2 PO 4 ,8mm Na 2 HPO 4 , 2.5 mm KCl, pH 7.4) with 0.05% Tween 20 (PBST). All results were expressed as a proportion of the standard anti- body (6E10, Cambridge BioScience Ltd, Cambridge, UK) at 0.1 lgÆmL )1 bound to sAPPa, to correct for any inter- plate variation. Quantification of APP APP was quantified using the APP DuoSet (R&D Systems, Abingdon, UK) following the manufacturer’s guidelines [26]. Briefly, the capture antibody was used at 4 lgÆmL )1 in PBS overnight. Plates were blocked with 1% bovine serum albumin and 5% sucrose in PBS and samples were quanti- fied using a six-point standard curve. The biotinylated detection antibody was used at 300 ngÆmL )1 and detected using streptavidin–horseradish peroxidase and o-phenylene- diamine. Affinity ranking of 2B3 and 2B12 for an APP fragment Affinity ranking of the two antibodies was accomplished by comparing their binding properties to a peptide, Kb, which spans the b-secretase cleavage site on APP, in an indirect ELISA. This peptide represents a 15-mer sequence (SEV- KMDAEFRHDSGY), slightly further into the Ab region of APP than Ka, the immunizing peptide. ELISA methods followed those detailed above with the following exceptions. Kb was adsorbed to a 96-well microtitre plate at a concen- tration of 10 l g ÆmL )1 in carbonate ⁄ bicarbonate buffer overnight at 4 °C. 2B3 or 2B12 was incubated for 2 h at concentrations ranging from 0.00001 to 30 lgÆmL )1 and detected as above. All results were expressed as a propor- tion of the standard antibody (6E10) at 0.05 lgÆmL )1 . Binding of 2B3 and 2B12 to full-length APP A competition assay, in conjunction with the sandwich ELISA for APP (R&D Systems) described above, was used to determine relative binding of 2B3 and 2B12 to APP from cell lysates. MOG-G-UVW cells were lysed and concen- trated through a filter with a nominal cut-off of 100 kDa (Millipore) to provide predominantly full-length APP at a concentration of 30 ngÆmL )1 , as described previously [26]. After formation of the APP ⁄ capture antibody complex on the 96-well plate and prior to incubation with the detection antibody, the test antibodies, 2B3, 2B12 or control IgG (Pierce), were incubated at concentrations ranging from 1 to 20 lgÆ mL )1 for 1 h. Binding of these antibodies was then inferred by a decrease in binding of the detection antibody compared with the PBST control alone. Persistence of 2B3 and 2B12 binding at different pH values The binding persistence of 2B3 and 2B12 to Kb was investi- gated at two different pH values using an indirect ELISA. The methods followed those detailed above with the follow- ing modifications. Kb was adsorbed to a 96-well plate and blocked as above. 2B3 and 2B12 (5 lgÆmL )1 ) were incubated with Kb for 1 h in PBST (pH 7.4) and the antibody solution was aspirated. The immunocomplex was then incubated for a further 1 h in PBST at either pH 7.4 or 4.0 for 0 or 60 min. To ensure that the antigen was not degraded or dis- sociated from the plate by the pH treatment, Kb was also incubated with PBST alone at pH 4.0 for 1 h prior to incu- bation with 2B3 or 2B12 at pH 7.4. Binding of both antibod- ies was detected as above and all results were expressed as a proportion of a standard antibody (6E10) at 0.05 lgÆmL )1 . PBST was adjusted to the correct pH with H 3 PO 4 . Effects of 2B3 and 2B12 on levels of Ab40, Ab42, sAPPa and bCTF All experiments were performed in 24-well cluster plates, in triplicate, with a starting density of 25 000 MOG-G-UVW cells per well. Cells were allowed to attach overnight and were then incubated with control media, 2B3, 2B12, an anti-N-terminal APP IgG (22C11, Millipore) or an irrele- vant control mouse IgG (Sigma-Aldrich) (all at 10 lgÆmL )1 ) for 48 h at 37 °C. This was repeated on a min- imum of three different passage numbers, where each n = 1 passage. For analysis of Ab40, media was subjected to immunoprecipitation and ELISA as described previously [26]. Briefly, the ELISA employed the N-terminal Ab anti- body 6E10 (5 lgÆmL )1 ) as the capture antibody and affin- ity-purified BAM401AP (0.45 lgÆmL )1 Autogen Bioclear, Calne, UK), specific to the C-terminus of human Ab40, as the detection antibody. For analysis of Ab42, media was collected after antibody treatment and tested in a sandwich R. S. Thomas et al. Antibodies inhibit amyloid-b production FEBS Journal 278 (2011) 167–178 ª 2010 The Authors Journal compilation ª 2010 FEBS 175 ELISA (Biosource, Invitrogen, Paisley, UK). To determine the effect of the antibodies on sAPPa, MOG-G-UVW cells were incubated as before and media was tested in a sand- wich ELISA (IBL, Hamburg, Germany). All cells were lysed and intracellular APP and bCTF (IBL) levels were detected by ELISA, as described above. All data were nor- malized to total cell protein concentration as determined by bicinchoninic acid protein assay (Pierce). Effect of 2B3 and 2B12 on MOG-G-UVW cell viability Viability studies were performed on MOG-G-UVW cells after incubation with 2B3, 2B12, control IgG (Sigma- Aldrich), anti-N-terminal APP IgG (22C11, Millipore) or media control, all at 10 lgÆmL )1 , using the CellTiter 96 Ò MTS Aqueous One Solution Cell Proliferation Assay (Pro- mega, Southampton, UK) in 96-well cluster plates. MOG- G-UVW cells were first allowed to adhere overnight after plating at a concentration of 2000 per well, and were then incubated with treatments for 48 h. Viability was assessed following the manufacturer’s guidelines. Effect of 2B3 and 2B12 on BACE1 activity in a cell-free assay The effect of 2B3 and 2B12 on BACE1 levels was investi- gated, after antibody treatment as detailed above, and analy- sed by western blotting. 12.5 g total protein was run on the polyacrylamide gel and detected with anti-BACE1 IgG (0.27 lgÆmL )1 , Santa Cruz Biotechnology, Santa Cruz, USA). The effect of the antibodies on BACE1 activity was also investigated in a cell-free assay. Recombinant human sAPPa (4 lgÆmL )1 , R&D Systems), containing the b-secre- tase cleavage site, was incubated in the presence or absence of BACE1 (82.5 lgÆmL )1 , R&D Systems) for 1 h at 37 °C, with the addition of one of the following treatments, 2B3, 2B12, control IgG (Pierce), anti-N-terminal APP IgG or media control. Prior to the addition of BACE1, the antibod- ies and sAPPa were allowed to form a complex for 3 min. The total reaction volume was 20 lL; all reactions were per- formed in 50 mm C 2 H 3 O 2 Na (pH 4.5) and all antibodies were used at 1 lgÆmL )1 . The reaction was stopped by the addition of 3· Laemmli sample buffer [43]. A volume equiv- alent to 26.67 ng starting sAPPa was analysed in a western blot and detected with the anti-N-terminal APP IgG (33.3 ngÆmL )1 ). Statistical analyses Data generated in ELISA assays were quantified by com- paring data with standard curves included on each plate, using graphpad prism Ò 4. The results were first normalized to total protein concentration, where relevant, and expressed as a percentage of media control values. MTS and ELISA results were then analysed using a Student’s t- test at the two-tailed significance level to determine if con- centrations were significantly different to media controls (100%). Where relevant, ELISA data were subsequently analysed with one-way ANOVA. To compare the relative affinities of 2B3 and 2B12 for the Kb peptide, log antibody concentration was plotted against the percentage absorbance (of standard antibody) and a sigmoidal dose–response curve fitted to the data using graphpad prism Ò 4. Curve parameters were com- pared using the F-test and differences between 2B3 and 2B12 at each concentration were compared using a Stu- dent’s t-test. The persistence in binding of 2B3 and 2B12 to Kb at different pH values was investigated using General- ized Linear Model univariate analysis, with absorbance as the dependent variable and pH, antibody and time as fac- tors. Both antibodies were subsequently investigated inde- pendently with ANOVA and Bonferroni. Western blots for the inhibitory effects of 2B3 and 2B12 on BACE1 were quantified using nih imager. All bands were first normalized to, and expressed as a percentage of, sAPPa alone, to allow comparisons between blots. The normalized control density of sAPPa and antibody was subtracted from the relevant experimental condition of sAPPa, antibody and BACE1 to determine the change in sAPPa after incubation with BACE1. The resulting change in sAPPa was analysed using one-way ANOVA and Bonferroni. Where necessary, data were transformed to fulfil the assumptions of normality and homoskedasticity and, there- fore, to allow the use of parametric testing. Acknowledgements This work was funded by grant number 79 from the Alzheimer’s Society, UK. We would like to thank Katrin Hack, Pavlina Doubkova, Lynne Murphy and Shahista Jaffer for their technical assistance with this project. References 1 Selkoe DJ (2001) Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev 81, 741–766. 2 Hardy J & Allsop D (1991) Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. 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Eryl Liddell 2 and Emma. is cleaved from amyloid precursor protein (APP) [5]. APP can be processed by one of two proteolytic pathways. The Keywords Alzheimer’s disease; amyloid precursor protein; amyloid-b; monoclonal. via inhibition of b-secretase by steric hindrance and may provide a novel alternative therapy for Alzhei- mer’s disease. Abbreviations Ab, amyloid-b; AD, Alzheimer’s disease; APP, amyloid precursor