Anti-amyloid precursor protein immunoglobulins inhibit amyloid-β production by steric hindrance

Anti-amyloid precursor protein immunoglobulins inhibitamyloid-b production by steric hindranceRhian S. Thomas1, J. Eryl Liddell2 and Emma J. Kidd1

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)-containingextracellular 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: [email protected]

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-secretasesresults 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. Bothantibodies 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 Aband 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 sAPPaor sAPPb. 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 kDaA

B

56 kDa

0

2

4

6

8

10

12

14

16

Abs

orba

nce

(% o

f sta

ndar

d an

tibod

y) 2B32B12

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 reducedthe 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 100Antibody concentration (µg·mL–1)

Abs

orba

nce

(% o

f sta

ndar

d an

tibod

y) 2B32B12 *

**

*

**

*

*

*

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 25Antibody concentration (µg·mL–1)

AP

P (

% o

f con

trol

)

2B32B12Control IgG

aa a

ab

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


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

Abs

orba

nce

(% o

f sta

ndar

d an

tibod

y)2B32B12*** ***

**

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

140A

B

2B3 2B12 IgG N

Aβ4

0 (%

of c

ontr

ol)

***

**

a

b

0

20

40

60

80

100

120

140

2B3 2B12 N

Aβ4

2 (%

of c

ontr

ol)

**

**

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. aP < 0.05, significantly different from all other

groups; bP < 0.05 significantly different from 2B3 and N; cP < 0.05

significantly different from N after ANOVA and Tukey’s Honestly

Significant Difference, n = 3–6 (A) and n = 4 (B).



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

120A

B

2B3 2B12 IgG

AP

P (

% o

f con

trol

)

0

20

40

60

80

100

120

140

160

180

200

2B3 2B12 IgG N

Abs

orba

nce

(% o

f con

trol

)

**

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 lgÆmL)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

200A

B

2B12 IgG

sAP

Pα

(%

of c

ontr

ol)

0

20

40

60

80

100

120

2B3 2B12

βCT

F (

% o

f con

trol

)

*

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.



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-secretasecleavage 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 NMediacontrol

Cha

nge

in s

AP

Pα

from

rel

evan

tco

ntro

l (O

D u

nits

)

**

*

1 2

A

B

3 4 5 6 7 8 9 10

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.

αα-secretasecleavage site

2B3

Aβ COOHNH2

2B12β-secretase

cleavage sitesAPPβsAPPαAPP

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.



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 Ab40and 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 Abby 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 Ablevels 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 AbIgs, 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 Ab. It is

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 Ab40or Ab42 because of the low levels detectable in our

native cell lines.



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-AbIgs. 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 Ablevels, 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 mm l-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].



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

Na2CO3, 35 mm NaHCO3, 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 Na2HPO4, pH

5.0), incubated for 20 min. The reaction was stopped with

2.5 m H2SO4 and the absorbance determined at 492 nm.

The diluent used on day 2 was phosphate-buffered saline

(PBS; 137 mm NaCl, 1.5 mm KH2PO4, 8 mm Na2HPO4,

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 lgÆmL)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 lgÆmL)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 H3PO4.

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, asthe detection antibody. For analysis of Ab42, media was

collected after antibody treatment and tested in a sandwich



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�

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 C2H3O2Na (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� 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� 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, sAPPaalone, 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.

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Anti-amyloid precursor protein immunoglobulins inhibit amyloid-β production by steric hindrance

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