U.S. patent application number 16/441472 was filed with the patent office on 2019-10-17 for method of detecting cleaved snap25 in tissue samples.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to Ron S. Broide, Brian Cai, Ester Fernandez-Salas, Joseph Francis, Catherine Rheaume.
Application Number | 20190317095 16/441472 |
Document ID | / |
Family ID | 55016834 |
Filed Date | 2019-10-17 |
United States Patent
Application |
20190317095 |
Kind Code |
A1 |
Broide; Ron S. ; et
al. |
October 17, 2019 |
METHOD OF DETECTING CLEAVED SNAP25 IN TISSUE SAMPLES
Abstract
Methods and compositions for detecting BoNT/A enzymatic activity
in tissues or a tissue sample are described herein. The invention
encompasses antibodies that bind preferentially to BoNT/A cleaved
SNAP25 and is able to preferentially detect BoNT/A cleaved SNAP25,
as compared to intact (non-cleaved) SNAP25, in a tissue sample.
Inventors: |
Broide; Ron S.; (San marcos,
CA) ; Cai; Brian; (Irvine, CA) ;
Fernandez-Salas; Ester; (Ann Arbor, MI) ; Francis;
Joseph; (Laguna Niguel, CA) ; Rheaume; Catherine;
(Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
55016834 |
Appl. No.: |
16/441472 |
Filed: |
June 14, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14792795 |
Jul 7, 2015 |
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16441472 |
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62021379 |
Jul 7, 2014 |
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62158900 |
May 8, 2015 |
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62163829 |
May 19, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
G01N 33/573 20130101; C07K 2317/72 20130101; G01N 2333/952
20130101; C07K 2317/567 20130101; C07K 2317/34 20130101; C07K 16/18
20130101 |
International
Class: |
G01N 33/573 20060101
G01N033/573; C07K 16/18 20060101 C07K016/18 |
Claims
1. A method of diagnosing if a tissue has been exposed to BoNT/A
enzymatic activity comprising contacting a tissue sample suspected
of having been exposed to BoNT/A enzymatic activity with an
anti-SNAP25 antibody wherein the antibody binds preferentially to
BoNT/A cleaved SNAP25; and detecting whether the anti-SNAP25
antibody bound to the tissue sample, wherein the presence of the
anti-SNAP25 antibody binding to the tissue sample indicates that
the tissue sample has been exposed to BoNT/A activity.
2. The method of claim 1, wherein the antibody does not bind to
full length SNAP25.
3. The method of claim 1, wherein the antibody is able to
preferentially bind to BoNT/A cleaved SNAP25 in a tissue.
4. The method of claim 1, wherein the tissue is a biopsy
sample.
5. The method of claim 1, wherein the antibody comprises a light
chain sequence of SEQID NO:1 or SEQ ID NO:2 and is a recombinant
murine antibody.
6. The method of claim 1, wherein the antibody comprises a heavy
chain sequence of SEQID NO:3 or SEQID NO:4 and is a recombinant
murine antibody.
7. The method of claim 1, wherein the antibody comprises a light
chain sequence of SEQID NO: 5 or SEQID NO: 6 and is a recombinant
human antibody.
8. The method of claim 1, wherein the antibody comprises a heavy
chain sequence of SEQID NO:7 or SEQID NO:8 and is a recombinant
human antibody.
9. The method of claim 1, wherein the antibody binds to the same
epitope of an antibody with a heavy chain and/or light chain
comprising one or more sequences of SEQID NOs:1-8.
10. A kit for diagnosing if a tissue has been exposed to BoNT/A
enzymatic activity comprising an anti-SNAP25 antibody, wherein the
antibody binds preferentially to BoNT/A cleaved SNAP25.
11. The kit of claim 10, wherein the antibody does not bind to full
length SNAP25.
12. The kit of claim 10, wherein the antibody is able to
preferentially bind to BoNT/A cleaved SNAP25 in a tissue.
13. The kit of claim 10, wherein the tissue is a biopsy sample.
14. The kit of claim 10, wherein the antibody comprises a light
chain sequence of SEQID NO:1 or SEQ ID NO:2 and is a recombinant
murine antibody.
15. The kit of claim 10, wherein the antibody comprises a heavy
chain sequence of SEQID NO:3 or SEQID NO:4 and is a recombinant
murine antibody.
16. The kit of claim 10, wherein the antibody comprises a light
chain sequence of SEQID NO: 5 or SEQID NO: 6 and is a recombinant
human antibody.
17. The kit of claim 10, wherein the antibody comprises a heavy
chain sequence of SEQID NO:7 or SEQID NO:8 and is a recombinant
human antibody.
18. The kit of claim 10, wherein the antibody binds to the same
epitope of an antibody with a heavy chain and/or light chain
comprising one or more sequences of SEQID NOs:1-8.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/792,795 filed Jul. 7, 2015 which claims the
benefit of U.S. Provisional Application Ser. No. 62/021,379 filed
Jul. 7, 2014; 62/158,900 filed May 8, 2015; and 62/163,829 filed
May 19, 2015, all incorporated entirely by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present disclosure relates to methods and compositions
for detecting cleaved SNAP25 in tissue samples, such as botulinum
neurotoxin cleavage of SNAP25. The invention further provides for
antibodies that bind to cleaved SNAP25, including the SNAP25
cleavage product by botulinum neurotoxin serotype A.
BACKGROUND OF THE INVENTION
[0003] The therapeutic utility of botulinum neurotoxin type A
(BoNT/A) has grown considerably over the past several decades and
Allergan's product, onabotulinumtoxinA, is now approved globally
for 11 major therapeutic and cosmetic indications, including
treatment for various neuromuscular disorders (Ramirez-Castaneda,
J. and Jankovic, J., (2014). "Long-term efficacy, safety, and side
effect profile of botulinum toxin in dystonia: a 20-year
follow-up." Toxicon 90, 344-348.; Yablon, S. A., Brin, M. F.,
VanDenburgh, A. M., Zhou, J., Garabedian-Ruffalo, S. M.,
Abu-Shakra, S., and Beddingfield, F. C., III (2011). "Dose response
with onabotulinumtoxinA for post-stroke spasticity: a pooled data
analysis."Mov Disord. 26, 209-215.), smooth muscle and autonomic
dysfunctions (Ellsworth, P. and Travis, M., (2014). "Onabotulinum
toxin A: a therapeutic option for refractory neurogenic detrusor
overactivity and idiopathic overactive bladder." Urol. Nurs. 34,
165-171.; Grunfeld, A., Murray, C. A., and Solish, N., (2009).
"Botulinum toxin for hyperhidrosis: a review." Am. J. Clin.
Dermatol. 10, 87-102.) and for nociceptive pain syndromes (Aoki, K.
R. and Francis, J., (2011). "Updates on the antinociceptive
mechanism hypothesis of botulinum toxin A." Parkinsonism. Relat
Disord. 17 Suppl 1, S28-S33.; Burstein, R., Zhang, X., Levy, D.,
Aoki, K. R., and Brin, M. F., (2014). "Selective inhibition of
meningeal nociceptors by botulinum neurotoxin type A: therapeutic
implications for migraine and other pains." Cephalalgia 34,
853-869.).
[0004] While the general mechanism of action (MoA) for BoNT/A at
the presynaptic nerve terminal is well established (Montal, M.,
(2010). "Botulinum neurotoxin: a marvel of protein design." Annu.
Rev. Biochem. 79, 591-617.), there are still many unanswered
questions regarding the intracellular trafficking patterns and
general `life-cycle` of the toxin. Resolving these questions partly
depends on the ability to precisely detect the toxin's location,
distribution, and movement within a cell. Direct detection of
BoNT/A using antibodies is difficult due to its high potency and
therefore, extremely low concentration within neurons. An
alternative approach for detecting the presence of BoNT/A has been
to track its enzymatic activity via immuno-staining for the cleaved
SNAP25 product (SNAP25.sub.197). Both commercial and proprietary
antibodies have been used to trace the expression of full-length
SNAP25 (SNAP25.sub.206) or BoNT/A-cleaved SNAP25 (SNAP25.sub.197).
However, the terminal epitope of SNAP25.sub.197 that is generated
following BoNT/A cleavage is difficult to specifically target with
an antibody without also recognizing the intact SNAP25 protein
(Mort, J. S. and Buttle, D. J., (1999). "The use of cleavage site
specific antibodies to delineate protein processing and breakdown
pathways." Mol. Pathol. 52, 11-18.). Consequently, immuno-staining
results have been misleading, with some antibodies being
assay-dependent while other antibodies are tissue-specific. There
is therefore a need for a very selective antibody against
BoNT/A-cleaved SNAP25 that can identify SNAP25.sub.197 in any
tissue-type and in multiple assays following exposure to
BoNT/A.
[0005] The present disclosure addresses these issues by providing
methods and compositions for detecting botulinum toxin cleaved
SNAP25, including BoNT/A cleaved SNAP25 and any other
BoNT/A-related compounds that cleave SNAP25 at position `197`, in
various different assays, such as but not limited to
immunohistochemistry using highly specific recombinant monoclonal
antibodies (rMAb) against SNAP25.sub.197. These antibodies can be
used to detect SNAP25.sub.197 in a variety of tissues from
different species, such as but not limited to human. These
antibodies can also be used as tools to diagnose activity and
efficacy in tissues from humans that have been treated with
neurotoxin, such as but not limited to onabotulinumtoxinA.
SUMMARY OF THE INVENTION
[0006] The invention encompasses an anti-SNAP25 antibody wherein
the antibody binds preferentially to a BoNT/A cleaved SNAP25. In
some embodiments, the anti-SNAP25 antibody binds preferentially to
a SNAP25 that has been cleaved by a recombinant botulinum toxin
with enzymatic (light chain) activity of botulinum toxin serotype
A. In other embodiments, the anti-SNAP25 antibody does not bind to
full length or uncleaved SNAP25.
[0007] In other embodiments, the anti-SNAP25 antibody is able to
detect BoNT/A cleaved SNAP25 (or SNAP25 cleaved by a recombinant
botulinum toxin with enzymatic (light chain) activity of botulinum
toxin serotype A) in a tissue. In some embodiments, the tissue is a
biopsy sample. In other embodiments, the tissue is a skin
punch.
[0008] In some embodiments, the anti-SNAP25 antibody comprises a
light chain sequence of SEQID NO:1 or SEQID NO: 2 and is a
recombinant murine antibody. In other embodiments, the anti-SNAP25
antibody comprises a heavy chain sequence of SEQID NO:3 or SEQID
NO:4 and is a recombinant murine antibody. In other embodiments,
the anti-SNAP25 antibody comprises a light chain sequence of SEQID
NO:5 or SEQID NO: 6 and is a recombinant human antibody. In other
embodiments, the anti-SNAP25 antibody comprises a heavy chain of
SEQID NO:7 or SEQID NO:8 and is a recombinant human antibody. In
other embodiments, the anti-SNAP25 antibody is an antibody that
binds to the same epitope of an antibody with a heavy chain and/or
light chain comprising one or more sequences of SEQID NOs:1-8.
[0009] The invention also encompasses a method of diagnosing if a
tissue has been exposed to BoNT/A enzymatic activity comprising
contacting a tissue sample suspected of having been exposed to
BoNT/A enzymatic activity with an anti-SNAP25 antibody wherein the
antibody preferentially binds to a BoNT/A cleaved SNAP25; and
detecting whether the anti-SNAP25 antibody bound to the tissue
sample, wherein the presence of the anti-SNAP25 antibody binding to
the tissue sample indicates that the tissue sample has been exposed
to BoNT/A enzymatic activity. In some embodiments, the BoNT/A
enzymatic activity is from a native BoNT/A. In other embodiments,
the BoNT/A activity is from a recombinant botulinum toxin with
enzymatic (light chain) activity of botulinum toxin serotype A. In
some embodiments, the anti-SNAP25 antibody used in the method
comprises a light chain sequence of SEQID NO:1 or SEQID NO: 2 and
is a recombinant murine antibody. In other embodiments, the
anti-SNAP25 antibody used in the method comprises a heavy chain
sequence of SEQID NO:3 or SEQID NO:4 and is a recombinant murine
antibody. In other embodiments, the anti-SNAP25 antibody used in
the method comprises a light chain sequence of SEQID NO:5 or SEQID
NO: 6 and is a recombinant human antibody. In other embodiments,
the anti-SNAP25 antibody used in the method comprises a heavy chain
of SEQID NO:7 or SEQID NO:8 and is a recombinant human antibody. In
other embodiments, the anti-SNAP25 antibody used in the method is
an antibody that binds to the same epitope of an antibody with a
heavy chain and/or light chain comprising one or more sequences of
SEQID NOs:1-8.
[0010] In another aspect, the invention encompasses a kit for
diagnosing if a tissue has been exposed to BoNT/A enzymatic
activity comprising an anti-SNAP25 antibody, wherein the antibody
binds preferentially to BoNT/A cleaved SNAP25. In some embodiments,
the BoNT/A enzymatic activity is from a native BoNT/A. In other
embodiments, the BoNT/A activity is from a recombinant botulinum
toxin with enzymatic (light chain) activity of botulinum toxin
serotype A. In some embodiments, the anti-SNAP25 antibody used in
the kit comprises a light chain sequence of SEQID NO:1 or SEQID NO:
2 and is a recombinant murine antibody. In other embodiments, the
anti-SNAP25 antibody used in the kit comprises a heavy chain
sequence of SEQID NO:3 or SEQID NO:4 and is a recombinant murine
antibody. In other embodiments, the anti-SNAP25 antibody used in
the kit comprises a light chain sequence of SEQID NO:5 or SEQID NO:
6 and is a recombinant human antibody. In other embodiments, the
anti-SNAP25 antibody used in the kit comprises a heavy chain of
SEQID NO:7 or SEQID NO:8 and is a recombinant human antibody. In
other embodiments, the anti-SNAP25 antibody used in the kit is an
antibody that binds to the same epitope of an antibody with a heavy
chain and/or light chain comprising one or more sequences of SEQID
NOs:1-8.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows a diagrammatic representation of the putative
epitope for the humanized and murine recombinant monoclonal
antibodies against SNAP25.sub.197. The diagram shows the 12-residue
peptide, minus the N-terminal cysteine residue (used for
conjugating to Keyhole Limpet Hemocyanin) that was used to generate
the original monoclonal antibody.
[0012] FIG. 2 (A-F) shows Western Blot analysis comparing the
specificity of different antibodies for detecting full-length
SNAP25.sub.206 and cleaved SNAP25.sub.197 in rat embryonic cortical
cell lysates treated with (L.sub.3) or without (L.sub.2) BoNT/A at
3 nM concentration. (A) Blot probed with a commercially available
anti-SNAP25 mAb (SMI-81R) that recognizes both the full-length
(206) and cleaved (197) forms of SNAP25. In lane 2, only
SNAP25.sub.206 is detected, whereas in lane 3, both SNAP25.sub.206
(arrow) and SNAP25.sub.197 (arrowhead) are detected. (B) Blot
probed with a commercially available anti-SNAP25 mAb (MC-6050) that
reportedly recognizes both SNAP25.sub.206 and SNAP25.sub.197. Only
SNAP25.sub.197 appears as a single band in lane 3 (arrowhead). (C)
Blot probed with a commercially available anti-SNAP25 mAb (MC-6053)
that is reportedly specific for SNAP25.sub.197. This antibody
recognizes a thin, faint SNAP25.sub.197 band in lane 3. (D) Blot
probed with Ab632 anti-SNAP25.sub.197 rMAb. In lane 2, no band is
detected, whereas in lane 3, a single band for SNAP25.sub.197 is
detected (arrowhead). (E) Blot probed with Ab635
anti-SNAP25.sub.197 rMAb. In lane 2, no band is detected, whereas
in lane 3, a single band for SNAP25.sub.197 is detected
(arrowhead). (F) Blot probed with RGT-1092 anti-SNAP25.sub.197 pAb.
This antibody primarily recognizes SNAP25.sub.197 in lane 3
(arrowhead), although two faint bands are visible just above and
below the SNAP25.sub.197 band. Lane 1, protein ladder; Lane 2,
untreated cortical cell lysate; Lane 3, BoNT/A-treated (3 nM)
cortical cell lysate.
[0013] FIG. 3 (A-F) shows Western Blot analysis comparing the
specificity of different antibodies for detecting full-length
SNAP25.sub.206 and cleaved SNAP25.sub.197 in SiMa cell lysates
treated with (L.sub.3) or without (L.sub.2) BoNT/A at 3 nM
concentration. (A) Blot probed with a commercially available
anti-SNAP25 mAb (SMI-81R) that recognizes both the full-length
(206) and cleaved (197) forms of SNAP25. In lane 2, only
SNAP25.sub.206 is detected, whereas in lane 3, both SNAP25.sub.206
(arrow) and SNAP25.sub.197 (arrowhead) are detected. (B) Blot
probed with a commercially available anti-SNAP25 mAb (MC-6050) that
reportedly recognizes both SNAP25.sub.206 and SNAP25.sub.197. Only
SNAP25.sub.197 appears as a single band in lane 3 (arrowhead). (C)
Blot probed with a commercially available anti-SNAP25 mAb (MC-6053)
that is reportedly specific for SNAP25.sub.197. The antibody does
not appear to recognize any bands. (D) Blot probed with Ab632
anti-SNAP25.sub.197 rMAb. In lane 2, no band is detected, whereas
in lane 3, a single band for SNAP25.sub.197 is detected
(arrowhead). (E) Blot probed with Ab635 anti-SNAP25.sub.197 rMAb.
In lane 2, no band is detected, whereas in lane 3, a single band
for SNAP25.sub.197 is detected (arrowhead). (F) Blot probed with
RGT-1092 anti-SNAP25.sub.197 pAb. This antibody primarily
recognizes SNAP25.sub.197 in lane 3 (arrowhead), although two faint
bands are visible just above and below the SNAP25.sub.197 band.
Lane 1, protein ladder; Lane 2, untreated SiMa cell lysate; Lane 3,
BoNT/A-treated (0.01 nM) SiMa cell lysate.
[0014] FIG. 4(A, B) shows control Western Blot analysis for the
cortical cell studies in FIG. 2 and SiMa cell studies in FIG. 3
probed with anti-GAPDH mAb demonstrating equal loading of samples
in lanes 2 and 3. Lane 1, protein ladder; Lane 2, untreated cell
lysate; Lane 3, BoNT/A-treated (3 nM) cortical cell lysate and
(0.01 nM) SiMa cell lysate. FIG. 4(C, D) shows Western Blot
analysis demonstrating the epitope specificity of Ab632-rMAb using
SiMa cell lysates treated with BoNT/A (L.sub.3), BoNT/C (L.sub.4),
BoNT/E (L.sub.5) or with no toxin (L.sub.2). (C) Blot probed with a
commercially available anti-SNAP25 mAb (SMI-81R) that recognizes
both the full-length (206) and cleaved (197 for BoNT/A, 198 for
BoNT/C and 180 for BoNT/E) forms of SNAP25. In lane 2, only
SNAP25.sub.206 is detected, whereas in lane 3, both SNAP25.sub.206
(arrow) and SNAP25.sub.197 are detected. In lane 4, both
SNAP25.sub.206 (arrow) and SNAP25.sub.198 are detected and in lane
5, both SNAP25.sub.206 (arrow) and SNAP25.sub.180 are detected; (D)
Blot probed with Ab632 anti-SNAP25.sub.197 rMAb. In lane 2, 4 and
5, no band is detected, whereas in lane 3, a single band for
SNAP25.sub.197 is detected. Lane 1, protein ladder; Lane 2,
untreated SiMa cell lysate; Lane 3, BoNT/A-treated SiMa cell
lysate; Lane 4, BoNT/C-treated SiMa cell lysate; Lane 5,
BoNT/E-treated SiMa cell lysate.
[0015] FIG. 5 (A-J) shows immunohistochemical analysis comparing
the specificity of antibodies against SNAP25 in sections of rat
bladder following treatment with either onabotulinumtoxinA (10
U/kg) or vehicle. (A-E) Confocal images of bladder detrusor muscle
(DM) injected with onabotulinumtoxinA and probed with (A)
commercial mAb (SMI-81R) against full-length (206) and cleaved
(197) SNAP25, (B) a second commercial mAb (MC-6050) against
SNAP25.sub.197 & SNAP25.sub.206, (C) commercial mAb (MC-6053)
against SNAP25.sub.197, (D) Ab632-rMAb against SNAP25.sub.197 and
(E) RGT-1092 pAb against SNAP25.sub.197. (F-J) Confocal images of
control rat bladder injected with vehicle and probed with the same
five antibodies. Arrows (F and J) point to IR-signal within nerve
fibers from vehicle treated rat bladder. DM, detrusor muscle; Scale
bar=50 .mu.m.
[0016] FIG. 6 (A-J) shows immunohistochemical analysis comparing
the specificity of antibodies against SNAP25 in sections of rat
glabrous skin following treatment with either onabotulinumtoxinA
(30 U/kg) or vehicle. (A-E) Confocal images of rat skin injected
with onabotulinumtoxinA and probed with (A) commercial mAb
(SMI-81R) against full-length (206) and cleaved (197) SNAP25, (B) a
second commercial mAb (MC-6050) against SNAP25.sub.197 &
SNAP25.sub.206, (C) commercial mAb (MC-6053) against
SNAP25.sub.197, (D) Ab632-rMAb against SNAP25.sub.197 and (E)
RGT-1092 pAb against SNAP25.sub.197. (F-J) Confocal images of
control rat skin injected with vehicle and probed with the same
five antibodies. Arrows (F, G, H and J) point to IR-signal within
nerve fibers from vehicle treated rat skin. Asterisks (B, C, G and
H) point to non-specific IR-signal within the lumen of blood
vessels. BV, blood vessel; CT, connective tissue; Scale bar=50
.mu.m.
[0017] FIG. 7 (A-H) shows immunohistochemical analysis comparing
the specificity of antibodies against SNAP25 in skeletal muscle
underlying rat glabrous skin following treatment with either
onabotulinumtoxinA (30 U/kg) or vehicle. (A-D) Confocal images
showing motor nerve terminals (MNT, arrows) within the underlying
muscle of the rat paw injected with onabotulinumtoxinA and probed
with (A) commercial mAb (SMI-81R) against full-length (206) and
cleaved (197) SNAP25; (B) a second commercial mAb (MC-6050) against
SNAP25.sub.197 & SNAP25.sub.206; (C) a commercial mAb (MC-6053)
against SNAP25.sub.197 and (D) Ab632-rMAb against SNAP25.sub.197;
(E-H) Confocal images from control rat paw injected with vehicle
and probed with the same four antibodies. SNAP25-IR signal is in
green (shown as gray in black and white drawings) and DIC
illumination was used to delineate the underlying muscle fibers
(MF). Arrow (E) points to IR-signal within a MNT from vehicle
treated rat paw; asterisks (B,C,F and G) point to non-specific
IR-signal within the muscle. Scale bar=50 .mu.m.
[0018] FIG. 8 (A-T) shows immunohistochemical analysis comparing
the specificity of various SNAP25.sub.197-specific antibodies in
sections of rat glabrous skin (FIGS. 7A-7J) and rat bladder (FIGS.
7K-7T) following treatment with either BOTOX.RTM. or vehicle. (A-J)
Confocal images of rat skin injected with BOTOX.RTM. and probed
with (A) an older generated lot (May 2, 2011) of the humanized
Ab632-rMAb, (B) a more recent lot of humanized Ab632-rMAb, (C) a
recent lot of the murine Ab635-rMAb, (D) the Ab507 mAb (clone 2E2A6
used for the Cell-Based Assay in 18383-CIP), and (E) the original
`non-recombinant` 3C1A5 mAb purified from ascites. (F-J) Confocal
images of control rat skin injected with vehicle and probed with
the same five antibodies. Arrows (A, B, C) point to specific
immunoreactive-signal within nerve fibers surrounding blood
vessels. (K-T) Confocal images of bladder smooth muscle (SM)
injected with BOTOX.RTM. and probed with (K) an older generated lot
(May 2, 2011) of the humanized Ab632-rMAb, (L) a more recent lot of
humanized Ab632-rMAb, (M) a recent lot of the murine Ab635-rMAb,
(N) the Ab507 mAb (clone 2E2A6 used for the Cell-Based Assay in
18383-CIP), and (O) the original `non-recombinant` 3C1A5 mAb
purified from ascites. (P-T) Confocal images of control rat bladder
injected with vehicle and probed with the same five antibodies.
Arrows (K, L, M and O) point to specific immunoreactive-signal in
nerve fibers within the bladder smooth muscle. BV, blood vessel;
CT, connective tissue; SM, detrusor muscle. Scale bar=50 .mu.m.
[0019] FIG. 9 (A-H) shows immunohistochemical comparison of the
commercially-available (MC-6053) mAb against SNAP25.sub.197 vs.
Ab635-rMAb in sections of human back skin following treatment with
either onabotulinumtoxinA (10U) or vehicle. Confocal images of
blood vessels in onabotulinumtoxinA (A, B) and vehicle-treated (E,
F) human skin probed with either (A, E) the MC-6053 mAb or (B, F)
Ab635-rMAb. Sweat glands in onabotulinumtoxinA (C, D) and
vehicle-treated (G, H) human skin probed with either (C, G) the
MC-6053 mAb or (D, H) Ab635-rMAb. Arrows point to IR-signal within
nerve fibers from vehicle treated human skin. BV, blood vessel; CT,
connective tissue; SG, sweat gland; Scale bar=50 .mu.m.
[0020] FIG. 10 (A-F) shows confocal images of BoNT/A (3 nM)- or
vehicle-treated dorsal root ganglia (DRG) cultures probed with
antibodies to different forms of SNAP25. (A-C) DRG cultures exposed
to BoNT/A for 3 hr and later stained with (A) commercial (SMI-81R)
mAb against full-length (206) and cleaved (197) SNAP25, (B)
commercial (MC-6053) mAb against SNAP25.sub.197 and (C) Ab632-rMAb
against SNAP25.sub.197. (D-F) Control DRG cultures exposed to
vehicle and probed with the same three antibodies. Arrowhead in E
points to a neuronal soma exhibiting background labeling.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. As used herein,
the following terms have the meanings ascribed to them unless
specified otherwise.
[0022] "BoNT/A" refers to botulinum toxin serotype A produced by
Clostridium botulinum.
[0023] "Onabotulinumtoxin A" refers to the trade name of
BOTOX.RTM., which is an FDA-approved formulation of the 900 kDa
botulinum neurotoxin serotype A complex.
[0024] An epitope that "specifically binds" or "preferentially
binds" (used interchangeably herein) to an antibody or a
polypeptide is a term that is well understood in the art, and
methods to determine such specific or preferential binding are also
well known in the art. A molecule is said to exhibit "specific
binding" or "preferential binding" if it reacts or associates more
frequently, more rapidly, with greater duration and with greater
affinity with a particular cell or substance than it does with
alternative cells or substances. An antibody "specific binding" or
"preferential binding" to a target if it binds with greater
affinity, avidity, more readily, more exclusively and/or with
greater duration than it binds to other substances. It is
understood by reading this definition that, for an example, an
antibody (or moiety or epitope) that specifically or preferentially
binds to a first target may or may not specifically or
preferentially bind to a second target. As such, "specific binding"
or "preferential binding" does not necessarily require (although it
can include) exclusive binding. Generally, but not necessarily,
reference to binding herein means preferential binding.
[0025] "SNAP25.sub.197" as used herein refers to the 197 amino acid
fragment of synaptosomal-associated protein, 25 kDa (SNAP25) that
is produced when full-length SNAP25 protein is cleaved by botulinum
toxin serotype A or a recombinant botulinum toxin with enzymatic
(light chain) activity of botulinum toxin serotype A.
[0026] "SNAP25.sub.206" as used herein refers to the full length
SNAP25 protein containing 206 amino acids.
[0027] It is to be understood that this invention is not limited to
particularly exemplified antibodies, formulations, or methods of
using such antibodies, which as such, may vary. It is also to be
understood that the technical terminology used herein is for the
purpose of describing particular embodiments of the invention only,
and is not intended to be limiting.
II. Antibodies that Bind to SNAP25.sub.197
[0028] BoNT/A molecular targets, including synaptic vesicle
glycoprotein 2C (SV2C), fibroblast growth factor receptor 3 (FGFR3)
and SNAP25.sub.206, are broadly expressed and co-localized in
autonomic and sensory nerve fibers throughout the body, including
rat and primate urinary bladder and glabrous skin. Consequently,
the nerve endings are likely equally susceptible to the inhibitory
effects of BoNT/A. Although antibodies exist against various
molecular targets of BoNT/A, there has not been an optimal antibody
identified that is useful for detecting cleaved (i.e., BoNT/A
activity) SNAP25 (e.g., SNAP25.sub.197 fragment) in both Western
blotting technique and also in tissue samples (e.g., using
immunofluorescence).
[0029] Aspects of the present disclosure comprise, in part, an
anti-SNAP25 antibody that is specific for the BoNT/A SNAP25
cleavage product that can be used in ELISA assays, Western blot
applications, in cell culture assays and in tissue samples to
detect BoNT/A activity. Another aspect of the present invention is
an anti-SNAP25 antibody with an epitope to the carboxy terminus of
the SNAP25.sub.197 fragment that does not substantially bind to
full length SNAP25 (SNAP25.sub.206).
[0030] Methods for making antibodies (monoclonal or polyclonal) are
known in the art. One method which may be employed is the method of
Kohler and Milstein, Nature 256:495-497 (1975) or a modification
thereof. Typically, monoclonal antibodies are developed in
non-human species such as mice. In general, a mouse or rat is used
for immunization but other animals may also be used. Also, once an
antibody is identified to have the binding characteristics that are
desired, the antigen-binding site including their complementarity
determining regions (CDRs) can be fused to the constant domains or
supporting framework region of antibodies of other species
including human. In some instances, producing these recombinant
monoclonal antibodies can minimize unwanted immunological response
in patients or host animals that these antibodies are injected
into. In other instances, producing these recombinant monoclonal
antibodies can expand the utility of a specific antibody with
certain binding characteristics for diagnostic or use for detecting
BoNT/A activity in tissues of different species of animals. In some
instances, recombinant monoclonal antibodies may have an advantage
of minimal "off target" signal because of the selectively of not
only the CDR, but also due to the recombinant IgG backbone, which
can be very different than the endogenous IgG of the tissue of
interest.
[0031] Anti-SNAP25.sub.197 antibodies were generated and described
in US Patent Publication No. US2012/0225436A1, hereby incorporated
by reference. The CDRs of one of these monoclonal antibodies,
selected for its superior performance in immunohistochemical (IHC)
assays, were sequenced and recombinantly engineered into
immunoglobulin backbones from either human (IgG1) or murine (IgG2A)
origin. These antibodies were further characterized along with
commercially available antibodies for their specificity to cleaved
SNAP25 (SNAP25.sub.197) in both Western blot assays and for use in
tissue samples (either rat tissue or human tissue) using
immunofluorescence. Table 1 below includes a list of anti-SNAP25
that were used in this comparison.
TABLE-US-00001 TABLE 1 List of anti-SNAP25 antibodies Anti- IgG-
SNAP25.sub.197 body Specificity Vendor Species/Type isotype antigen
SMI-81R SNAP25.sub.206/197 Covance, Murine/mAb IgG1 Uncleaved
Princeton, SNAP25 NJ MC-6050 SNAP25.sub.206/197 R&D Abs,
Murine/mAb n/a 15-mer, LV, NV C.sub.OOH-term MC-6053 SNAP25.sub.197
R&D Abs, Murine/mAb n/a 15-mer, LV, NV C.sub.OOH-term Ab507
SNAP25.sub.197 Allergan Murine/mAb n/a 12-mer, C.sub.OOH-term Ab632
SNAP25.sub.197 Allergan rHuman/rMAb IgG1 12-mer, C.sub.OOH-term
Ab635 SNAP25.sub.197 Allergan rMurine/rMAb IgG2A 12-mer,
C.sub.OOH-term RGT- SNAP25.sub.197 Allergan Rabbit/pAb IgG 7-mer,
1092 C.sub.OOH-term n/a = not available; mAb = mouse monoclonal
antibody; rMAb = recombinant monoclonal antibody; pAb = rabbit
polyclonal antibody
III. Characterization of Anti-SNAP25.sub.197 Antibodies
[0032] Several methods can be used to characterize anti-SNAP25
antibodies. One method is to identify the epitope to which it
binds. Epitope mapping is commercially available from various
sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH
Lelystad, The Netherlands). Epitope mapping can be used to
determine the sequence to which an anti-SNAP25 antibody binds. The
epitope can be a linear epitope, i.e., contained in a single
stretch of amino acids, or a conformational epitope formed by a
three-dimensional interaction of amino acids that may not
necessarily be contained in a single stretch. Peptides of varying
lengths (e.g., at least 4-6 amino acids long) can be isolated or
synthesized (e.g., recombinantly) and used for binding assays with
anti-SNAP25 antibodies. The epitope to which anti-SNAP25 antibody
binds can be determined in a systematic screening by using
overlapping peptides derived from the extracellular sequence and
determining binding by an anti-SNAP25 antibody.
[0033] Another method that can be used to characterize an
anti-SNAP25 antibody is to use competition assays with other
antibodies that bind to the same antigen or even the same epitope
on the same antigen. Competition assays are well known to those
skilled in the art.
[0034] Another method of characterizing anti-SNAP25 antibodies is
by the antigen to which it binds. Anti-SNAP25 antibodies can be
used in Western blots. Specifically, in some embodiments,
anti-SNAP25 antibodies were used in Western blot assays to
determine its specificity to BoNT/A cleaved SNAP25
(SNAP25.sub.197). Anti-SNAP25 antibodies that preferentially bind
to SNAP25.sub.197 and not full-length (i.e., uncleaved) SNAP25 in
Western blot assays are preferred embodiments of the present
disclosure. Characterization of anti-SNAP25 antibodies in Western
blot assays are detailed in the Examples below.
IV. Methods of Diagnosing BoNT/A Activity in Tissue Samples
[0035] Antibodies that binds to BoNT/A cleaved SNAP25
(SNAP25.sub.197) may be used to identify the presence or absence of
BoNT/A activity in a variety of tissues. Such tissues may include
skin, including but not limited to, glabrous or hairy, muscle,
including but not limited to skeletal muscle and smooth muscle,
bladder, glandular tissues, including but not limited to prostate,
lacrimal gland, endocrine glands, and exocrine glands, blood
vessels, spinal cord and brain, including nerve fibers within any
and all of these tissues. Such tissues may be procured as part of a
biopsy or skin punch.
[0036] An antibody that is suitable for use to detect BoNT/A
activity in tissues would need to (1) be specific to binding to
SNAP25.sub.197 and not to full-length or uncleaved SNAP 25
(SNAP25.sub.206); and (2) be able to detect SNAP25.sub.197
preferentially (and not full-length or uncleaved SNAP25) in tissue
samples. Another characteristic that would be desirable is that the
antibody would preferentially bind to BoNT/A cleaved SNAP25
(SNAP25.sub.197) in tissue samples without substantially binding to
non-specific antigens (i.e., low or no background, non-specific
binding).
[0037] Determining the absence or presence of BoNT/A, or
BoNT/A-like compound activity in a particular tissue sample may be
important for a variety of clinical and non-clinical diagnostic
purposes including, but not limited to: 1) understanding the
mechanism of action for BoNT/A in a particular tissue or clinical
indication; 2) assessment of BoNT/A activity and/or spread beyond
the site of injection; 3) assessment of suspected immunity to
BoNT/A; 4) assessment and understanding of local diffusion of
BoNT/A (e.g., from an injected muscle to a neighboring muscle or
tissue); 5) assessment of potential exposure to BoNT/A in the
setting of human botulism performed by biopsy; and 6) clinical
pharmacological studies.
[0038] In one embodiment, the use can involve the formation of a
complex between SNAP25.sub.197 and an antibody that specifically
binds to SNAP25.sub.197. In one embodiment of the diagnostic
methods of this invention, the anti-SNAP25.sub.197 antibody can
bear a detectable label. Examples of labels may be used include a
radioactive agent, a fluorophore, chemical label, a biological
agent such as but not limited to, biotin/streptavidin detection, or
an enzymatic substrate label. In other embodiments, a secondary
antibody of another species that can bear a detectable label can be
used to detect the anti-SNAP25.sub.197 antibody. The use of a
secondary antibody may in some cases, boost the signal of the
primary anti-SNAP25.sub.197 antibody, thereby detecting low/lower
levels of BoNT/A activity in tissue samples.
V. Compositions of this Invention
[0039] This invention also encompasses compositions comprising an
anti-SNAP25.sub.197 antibody that can preferentially detect BoNT/A
cleaved SNAP25 without detecting uncleaved SNAP25 (SNAP25.sub.206)
in ELISA, Western blot assays and in tissue samples. In some
embodiments, the anti-SNAP25.sub.197 antibody is a recombinant
monoclonal antibody where the CDR has been fused with the
supporting framework of an antibody of a different species. In
other embodiments, the anti-SNAP25.sub.197 antibody is a
recombinant monoclonal antibody where the CDR has been fused with
the supporting framework of an antibody of the same species. In
some embodiments, the anti-SNAP25.sub.197 antibody is a recombinant
monoclonal antibody where the CDR has been fused with the
supporting framework of a human antibody. In some embodiments, the
anti-SNAP25.sub.197 antibody is a recombinant monoclonal antibody
where the CDR has been fused with the supporting framework of a
mouse antibody.
[0040] It was found that one antibody, 3C1A5, that has been
previously preliminarily described in US2012/0225436 was especially
useful in the present invention because of its inherent ability to
detect SNAP25.sub.197 in BoNT/A-treated tissues. It was known that
3C1A5 preferentially bound to SNAP25.sub.197 in an immuno-based
(e.g., ELISA) assay and/or in a cell-based assay. FIG. 1 shows a
depiction of the putative epitope binding site of the 3C1A5
antibody on SNAP25.sub.197. As described in detail in the Examples,
this antibody (and its recombinant human and murine versions) also
preferentially detected (or bind to) BoNT/A cleaved SNAP25
(SNAP25.sub.197) in Western blot assays. Surprisingly, this
antibody (and its recombinant human and murine versions) also was
able to preferentially detect (or bind to) BoNT/A cleaved SNAP25
(SNAP25.sub.197) in rat and human tissue samples. Although there
exists reports of anti-cleaved SNAP25 antibodies being able to
preferentially detect (or bind to) SNAP25.sub.197 in Western blot
assays and in tissue sample, as shown in detail in the Examples,
3C1A5 (or its recombinant human and murine versions) was the only
antibody that consistently detected SNAP25.sub.197 in all assays
and on different tissues and did not exhibit non-specific binding
to other epitopes.
[0041] In some cases, the antibody of the present invention is
3C1A5. In other cases, the antibody of the present invention is a
recombinant antibody comprising the antigen binding site of 3C1A5,
but has been fused with the framework regions of another antibody
from the same or different species.
[0042] In one embodiment, the light chain/heavy chain of the
anti-SNAP25.sub.197 antibody comprises one or more of the sequences
listed below in Table 2.
TABLE-US-00002 TABLE 2 Ab635 and 632 Antibody Sequence Murine-3C1A5
(Ab635) Sequence-(pOptiVec/pcDNA3) Light Chain Murine 3C1A5 (Ab635)
SEQ ID NO: 1 DVVMTQTPLTLSVTIGQPASISCKSSQSLLNTNGKTYLTWLIQRPGQSPQ
RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQSSHFP
FTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDIN
VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE
ATHKTSTSPIVKSFNRNEC Light Chain Variable Domain Murine 3C1A5
(Ab635) SEQ ID NO: 2
DVVMTQTPLTLSVTIGQPASISCKSSQSLLNTNGKTYLTWLIQRPGQSPQ
RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQSSHFP FTFGSGTKLEIK
Heavy Chain Murine 3C1A5 (Ab635) SEQ ID NO: 3
QVKLQESGAELVKPGASVKISCKASGYTFTDHSIHWVKQKPGQGLEWIGY
LFPGNGNFEYNEKFKGKATLTADKSSSTVYMYLNSLTSEDSAVYFCKRMG
YWGQGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT
VTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPA
SSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTC
VVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQ
DWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKD
KVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLN
VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Heavy Chain Variable Domain
Murine 3C1A5 (Ab635) SEQ ID NO: 4
QVKLQESGAELVKPGASVKISCKASGYTFTDHSIHWVKQKPGQGLEWIGY
LFPGNGNFEYNEKFKGKATLTADKSSSTVYMYLNSLTSEDSAVYFCKRMG YWGQGTTVTVSS
Human-3C1A5 (Ab632) Sequence-(pOptiVec/pcDNA3) Light Chain Human
3C1A5 (Ab632) SEQ ID NO: 5
DVVMTQTPLTLSVTIGQPASISCKSSQSLLNTNGKTYLTWLIQRPGQSPQ
RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQSSHFP
FTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC Light Chain Variable Domain Human 3C1A5 (Ab632)
SEQ ID NO: 6 DVVMTQTPLTLSVTIGQPASISCKSSQSLLNTNGKTYLTWLIQRPGQSPQ
RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQSSHFP FTFGSGTKLEIK
Heavy Chain Human 3C1A5 (Ab632) SEQ ID NO: 7
QVKLQESGAELVKPGASVKISCKASGYTFTDHSIHWVKQKPGQGLEWIGY
LFPGNGNFEYNEKFKGKATLTADKSSSTVYMYLNSLTSEDSAVYFCKRMG
YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy Chain Variable
Domain Human 3C1A5 (Ab632) SEQ ID NO: 8
QVKLQESGAELVKPGASVKISCKASGYTFTDHSIHWVKQKPGQGLEWIGY
LFPGNGNFEYNEKFKGKATLTADKSSSTVYMYLNSLTSEDSAVYFCKRMG YWGQGTTVTVSS
[0043] In some cases, the antibodies of the invention comprises a
light chain sequence of SEQID NO:1 or SE ID NO:2 and is a
recombinant murine antibody. In other cases, the antibodies of the
invention comprises a heavy chain sequence of SEQID NO:3 or SEQID
NO:4 and is a recombinant murine antibody. In other cases, the
antibodies of the invention comprises a light chain sequence of
SEQID NO: 5 or SEQID NO: 6 and is a recombinant human antibody. In
other cases, the antibodies of the invention comprises a heavy
chain sequence of SEQID NO:7 or SEQID NO:8 and is a recombinant
human antibody.
[0044] Still in other embodiments the antibodies of the invention
can be an antibody that binds to the same epitope of an antibody
with a heavy chain and/or light chain comprising one or more
sequences of SEQID NOs:1-8. In some cases, the antibodies of the
invention can be an antibody that will compete for binding to the
same epitope of an antibody with a heavy chain and/or light chain
comprising one or more sequences of SEQID NOs:1-8.
[0045] The following Examples are provided to illustrate, but not
to limit the invention.
IV. EXAMPLES
Example 1. Western Blot Comparison
[0046] SNAP25 antibodies (listed in Table 1) were first compared by
Western blot analysis in their ability to recognize the full-length
(206) or BoNT/A-cleaved (197) forms of SNAP25 from rat embryonic
cortical cell lysates and in SiMa cell lysates treated with and
without BoNT/A. (See FIGS. 2 and 3).
[0047] Rat cortical neurons were harvested from embryonic pups
(E18) and digested in a papain dissociation system (Worthington
Biochemical Corp., Lakewood, N.J.) at 37.degree. C. for 15 minutes
to obtain individual cells. Cortical cells were then transferred to
Neurobasal medium (Life Technologies, Carlsbad, Calif.) containing
B-27 supplements, 0.5 mM L-glutamine and penicillin/streptomycin.
Rat dorsal root ganglia (DRG) harvested from neonatal pups (P7-P14)
were pooled and digested in papain-containing HBSS (final
concentration of 20 units of papain per ml in 1 mM L-cysteine) at
37.degree. C. for 15 minutes. Ganglia were washed and subsequently
digested in Ca2+/Mg2+-free HBSS containing Type 1 collagenase (1.7
mg/ml, Sigma, St Louis, Mo.) and incubated at 37.degree. C. for an
additional 15 minutes. The ganglia were then washed in Neurobasal-A
media (Life Technologies, Carlsbad, Calif.) containing B-27
supplements, 0.5 mM L-glutamine, penicillin/streptomycin and 20
ng/ml 2.5S nerve growth factor (NGF) and gently triturated through
Pasteur pipettes. Cortical and DRG cells were homogenously
dispersed, plated onto poly-D-lysine/laminin-coated 12-mm
coverslips (BD Biosciences, San Jose, Calif.) placed in 100-mm
culture dishes and grown for 6 to 7-DIV prior to treatment. All
animal protocols and procedures were approved by the Allergan
Institutional Animal Care and Use Committee and performed in
accordance with NIH guidelines.
[0048] On select days, cultures were treated with or without 3 nM
BoNT/A (150 kDa; Metabiologics, Madison, Wis.) for 3 hr at
37.degree. C. Following treatment, cells were rinsed with and then
incubated in fresh culture medium overnight. Cortical cells were
then washed with PBS, lysed in freshly prepared Lysis Buffer (20 mM
Tris pH 7.5, 0.15 M sodium chloride, 1 nM EDTA, 1 mM EGTA, 10%
Triton X-100 and one tablet of EDTA-free protease inhibitors) for
20 min on ice and then centrifuged at 4000 rpm for 20 min to
eliminate debris prior to Western blot (WB) analysis. DRG cells
were washed, fixed with 4% paraformaldehyde for 10-15 min and
processed for immunocytochemistry according to the protocol
below.
[0049] SiMa cells (DSMZ, Germany) were cultured in BD Biosciences
brand Collagen IV flasks (VWR, Radnor, Pa.) with vented caps
(Marini, P., MacLeod, R. A., Treuner, C., Bruchelt, G., Bohm, W.,
Wolburg, H., Schweizer, P., and Girgert, R., 1999. SiMa, a new
neuroblastoma cell line combining poor prognostic cytogenetic
markers with high adrenergic differentiation. Cancer Genet.
Cytogenet. 112, 161-164.). Growth media consisted of RPMI 1640, 0.1
mM Non-Essential Amino-Acids, 10 mM HEPES, 1 mM Sodium Pyruvate,
100 U/mL Penicillin, 100 .mu.g/mL Streptomycin, and 10% Fetal
Bovine Serum. Cells were treated with or without BoNT/A (0.01 nM)
for 24 hrs at 37.degree. C. Following treatment, SiMa cells were
washed with PBS, lysed in freshly prepared Lysis Buffer for 20 min
on ice and then centrifuged at 4000 rpm for 20 min to eliminate
debris prior to WB analysis.
[0050] For WB assays, we employed rat embryonic cortical neurons as
well as a human neuroblastoma cell line (SiMa), which is known for
its sensitivity to BoNT/A-mediated SNAP25 cleavage
(Fernandez-Salas, E., Wang, J., Molina, Y., Nelson, J. B., Jacky,
B. P., and Aoki, K. R., 2012. Botulinum neurotoxin serotype A
specific cell-based potency assay to replace the mouse bioassay.
PLoS. One. 7, e49516.). Total cell lysates from these cultures were
separated by electrophoresis (Biorad TGX Any Kd gel) and the gel
was transferred onto a PVDF membrane. Blots were blocked in buffer
(5% dry milk in 1.times.TBS-0.1% Tween-20) for 1 hr at room
temperature and then incubated overnight at 4.degree. C. with
primary antibodies in blocking buffer. Following washes, blots were
incubated with HRP-conjugated secondary antibodies (Bio Rad,
Hercules, Calif.) and developed by ECL Plus (GE Healthcare,
Pittsburgh, Pa.). A separate control blot was probed for
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to show equal
loading of cell lysate samples. Western blots were scanned using a
variable mode GE Typhoon 9410 imager and analyzed with ImageQuant
TL v. 2005 software (GE Healthcare, Pittsburgh, Pa.).
[0051] First a commercially available and widely-used monoclonal
antibody (SMI-81R) directed against all forms of the SNAP25 protein
recognized both SNAP25.sub.206 and SNAP25.sub.197 (FIG. 2A). In
contrast, a second commercially available monoclonal antibody
(MC-6050) described as recognizing both forms of SNAP25 was
surprisingly specific for SNAP25.sub.197 in lysates from
toxin-treated cells (FIG. 2B). Furthermore, another antibody from
the same company (MC-6053), described as recognizing only
BoNT/A-cleaved SNAP25 revealed a thin, faint band exclusively in
the toxin-treated lane (FIG. 2C).
[0052] The human (Ab632) and murine (Ab635) rMAbs directed against
BoNT/A-cleaved SNAP25 were very specific for SNAP25.sub.197; only a
single band was detected in toxin-treated lysates, while no bands
were detected in the untreated, control lanes (FIG. 2D, E).
Similarly, our in-house rabbit pAb (RGT-1092) against
SNAP25.sub.197 only detected a band in the BoNT/A-treated sample,
while no bands were detected in the control lane (FIG. 2F). It was
noted however, that the pAb recognized two additional faint bands,
one just above the SNAP25.sub.197 band and another at .about.20 kDa
present only in toxin-treated samples, which could not be readily
explained. Nevertheless, the upper band is not likely to be intact
SNAP25, as no band was observed in the untreated lane. Similar WB
results were obtained using lysates from BoNT/A-treated and
untreated SiMa cell cultures (FIG. 3), except that no band was
observed for MC-6053 in either control or toxin-treated lanes (FIG.
3C). Control blots probed for GAPDH showed equal loading of all
samples for both cortical and SiMa cell lysate experiments (FIG.
4A, B).
[0053] A separate WB analysis was performed to examine the epitope
specificity of our rMAbs using BoNT/C- and BoNT/E-treated SiMa cell
lysates compared to the BoNT/A-treated lysate. It is well
established that BoNT/C cleaves SNAP25 at amino acid (aa) residue
198, while BoNT/E cleaves SNAP25 at aa residue 180 [11]. While the
SMI-81R mAb recognized both full length and BoNT-cleaved forms of
SNAP25 (FIG. 4C), our human rMAb only detected a single band in the
BoNT/A-treated lysate sample, as expected (FIG. 4D).
[0054] FIG. 2 shows WB analysis comparing the specificity of
antibodies against SNAP25 using rat embryonic cortical neuronal
cell lysates treated with (L3) or without (L2) BoNT/A. (A) Blot
probed with a commercially available anti-SNAP25 mAb (SMI-81R) that
recognizes both the full-length (206) and cleaved (197) forms of
SNAP25. In lane 2, only SNAP25206 is detected, whereas in lane 3,
both SNAP25206 (arrow) and SNAP25197 (arrowhead) are detected. (B)
Blot probed with a commercially available anti-SNAP25 mAb (MC-6050)
that reportedly recognizes both SNAP25206 and SNAP25197. Only
SNAP25197 appears as a single band in lane 3 (arrowhead). (C) Blot
probed with a commercially available anti-SNAP25 mAb (MC-6053) that
is reportedly specific for SNAP25197. This antibody recognizes a
thin, faint SNAP25197 band in lane 3. (D) Blot probed with Ab632
anti-SNAP25197 rMAb. In lane 2, no band is detected, whereas in
lane 3, a single band for SNAP25197 is detected (arrowhead). (E)
Blot probed with Ab635 anti-SNAP25197 rMAb. In lane 2, no band is
detected, whereas in lane 3, a single band for SNAP25197 is
detected (arrowhead). (F) Blot probed with RGT-1092 anti-SNAP25197
pAb. This antibody primarily recognizes SNAP25197 in lane 3
(arrowhead), although two faint bands are visible just above and
below the SNAP25197 band. Lane 1, protein ladder; Lane 2, untreated
cortical cell lysate; Lane 3, BoNT/A-treated (3 nM) cortical cell
lysate.
[0055] FIG. 3 shows WB analysis comparing the specificity of
antibodies against SNAP25 using SiMa cell lysates treated with (L3)
or without (L2) BoNT/A. (A) Blot probed with a commercially
available anti-SNAP25 mAb (SMI-81R) that recognizes both the
full-length (206) and cleaved (197) forms of SNAP25. In lane 2,
only SNAP25206 is detected, whereas in lane 3, both SNAP25206
(arrow) and SNAP25197 (arrowhead) are detected. (B) Blot probed
with a commercially available anti-SNAP25 mAb (MC-6050) that
reportedly recognizes both SNAP25206 and SNAP25197. Only SNAP25197
appears as a single band in lane 3 (arrowhead). (C) Blot probed
with a commercially available anti-SNAP25 mAb (MC-6053) that is
reportedly specific for SNAP25197. The antibody does not appear to
recognize any bands. (D) Blot probed with Ab632 anti-SNAP25197
rMAb. In lane 2, no band is detected, whereas in lane 3, a single
band for SNAP25197 is detected (arrowhead). (E) Blot probed with
Ab635 anti-SNAP25197 rMAb. In lane 2, no band is detected, whereas
in lane 3, a single band for SNAP25197 is detected (arrowhead). (F)
Blot probed with RGT-1092 anti-SNAP25197 pAb. This antibody
primarily recognizes SNAP25197 in lane 3 (arrowhead), although two
faint bands are visible just above and below the SNAP25197 band.
Lane 1, protein ladder; Lane 2, untreated SiMa cell lysate; Lane 3,
BoNT/A-treated (0.01 nM) SiMa cell lysate.
[0056] FIG. 4(A, B) shows control blots for the cortical cell
studies in FIG. 2 and SiMa cell studies in FIG. 3 probed with
anti-GAPDH mAb showing equal loading of samples in lanes 2 and 3.
Lane 1, protein ladder; Lane 2, untreated cell lysate; Lane 3,
BoNT/A-treated (3 nM) cortical cell lysate and (0.01 nM) SiMa cell
lysate. FIG. 4(C, D) shows Western Blot analysis demonstrating the
epitope specificity of Ab632-rMAb using SiMa cell lysates treated
with BoNT/A (L.sub.3), BoNT/C (L.sub.4), BoNT/E (L.sub.5) or with
no toxin (L.sub.2). (C) Blot probed with a commercially available
anti-SNAP25 mAb (SMI-81R) that recognizes both the full-length
(206) and cleaved (197 for BoNT/A, 198 for BoNT/C and 180 for
BoNT/E) forms of SNAP25. In lane 2, only SNAP25.sub.206 is
detected, whereas in lane 3, both SNAP25.sub.206 (arrow) and
SNAP25.sub.197 are detected. In lane 4, both SNAP25.sub.206 (arrow)
and SNAP25.sub.198 are detected and in lane 5, both SNAP25.sub.206
(arrow) and SNAP25.sub.180 are detected; (D) Blot probed with Ab632
anti-SNAP25.sub.197 rMAb. In lane 2, 4 and 5, no band is detected,
whereas in lane 3, a single band for SNAP25.sub.197 is detected.
Lane 1, protein ladder; Lane 2, untreated SiMa cell lysate; Lane 3,
BoNT/A-treated SiMa cell lysate; Lane 4, BoNT/C-treated SiMa cell
lysate; Lane 5, BoNT/E-treated SiMa cell lysate.
Example 2. Immunohistochemical Comparison--Rat Tissue
[0057] The specificity of the antibodies was then tested using
immunohistochemistry (IHC) in rat bladder and glabrous skin that
had been treated with either onabotulinumtoxinA or saline and then
harvested 2-days post-injection. Throughout these IHC studies,
adjacent sections processed without primary antibodies showed only
background staining (data not shown).
[0058] Male Sprague-Dawley rats (Charles River Laboratories,
Wilmington, Mass.) were used for this study. Rats were pair-housed
in the RD3 vivarium with free access to food and water on a 12 hour
light-dark cycle. All procedures were approved by the Allergan
Institutional Animal Care and Use Committee and adhered to NIH
guidelines.
[0059] Working solutions of onabotulinumtoxinA (BOTOX.RTM.,
Allergan, Inc., Irvine, Calif.) and BoNT/A (150 kDa; Metabiologics,
Madison, Wis.) were prepared in either 0.9% saline or 0.5% BSA/0.9%
saline, respectively. For bladder injections, rats were first
anesthetized and prepped for surgery. A lower midline abdominal
incision was made, exposing the urinary bladder, seminal vesicles,
and prostate gland. The urinary bladder wall was then injected at
four equidistant sites along the midline circumference with 10
.mu.l of onabotulinumtoxinA (2.5 U/kg) yielding a final toxin load
of 10 U/kg. Control animals received 10 .mu.l injections of vehicle
(0.9% saline) into comparable target sites. For glabrous skin
injections, onabotulinumtoxinA (30 U/kg) or vehicle was
administered as a single intradermal (ID) injection (25 .mu.l) into
the center of the right hindlimb paw between the footpads.
[0060] Rats were sacrificed at 2-days post-injection and bladders
or the central portion of the planter surface of the hindpaws were
harvested and fixed overnight at 4.degree. C. in Zamboni's fixative
(American MasterTech, Lodi, Calif.). Tissues were washed and
cryoprotected in 30% sucrose/PBS solution overnight at 4.degree. C.
Bladder and skin samples were hemisected along the midline,
embedded in O.C.T. (Tissue-Tek) and stored frozen at -80.degree. C.
until sectioning. Tissue blocks were cryostat-sectioned (14
.mu.m-thick), slide-mounted and slides were kept at -20.degree. C.
until use.
[0061] Slide-mounted tissue sections and cell culture coverslips
were first blocked for non-specific signal in blocking buffer
(1.times.PBS+0.1% Triton X-100+10% Normal Donkey Serum) and then
incubated with primary antibodies at the desired concentrations in
blocking buffer overnight at 4.degree. C. Following several washes,
sections and coverslips were incubated with secondary antibodies
(Jackson ImmunoResearch, West Grove, Pa.) diluted in blocking
buffer for 2 hr at 4.degree. C. and then washed again. Coverslips
with cultures were inverted and mounted onto microscope slides
using Fluoromount-G (EM Sciences, Hatfield, Pa.) containing 1.5
.mu.g/ml DAPI. Slide-mounted sections were coversliped using the
same mounting media. Adjacent sections processed without primary
antibodies served as negative controls to show background signal.
Alternate sections were stained with hematoxylin & eosin for
better anatomical identification.
[0062] Images were captured and analyzed using either a Zeiss
LSM-710 confocal microscope with ZEN software (Carl Zeiss,
Thornwood, N.Y.) or an Olympus FV1000 confocal microscope (Olympus,
Center Valley, Pa.). Imaris.RTM. (Bitplane, South Windsor, Conn.)
software was utilized for qualitative analysis of nerve fibers.
Nerve fiber-types were identified on the basis of their morphology
and neurochemistry.
[0063] In the rat bladder, the SMI-81R antibody directed against
SNAP25 showed IR-signal in nerve fibers throughout the detrusor
muscle. The SNAP25-IR pattern was identical in both
onabotulinumtoxinA and saline-treated bladders, as expected (FIG.
5A, F). The MC-6050 monoclonal antibody, which is reported to
recognize both intact and BoNT/A-cleaved forms of SNAP25,
demonstrated IR-signal in nerve fibers from the detrusor muscle of
toxin-treated, but not saline-treated bladder (FIG. 5B, G),
comparable to the results from the Western Blot analysis. Likewise,
the MC-6053 monoclonal antibody demonstrated IR-signal only in
toxin-treated, but not saline-treated bladder (FIG. 5C, H). In
contrast with the specificity detected in the WB analysis, RGT-1092
pAb generated against SNAP25.sub.197 demonstrated IR-signal in both
toxin-treated and saline-treated bladders (FIG. 5E, J). Most
importantly, Ab632-rMAb showed clear IR-signal in nerve fibers from
the detrusor muscle of toxin-treated bladders (FIG. 5D), while no
signal was detected in saline-treated control bladders (FIG. 51).
Moreover, in separate studies, Ab635-rMAb showed similar
specificity as Ab632-rMAb to SNAP25.sub.197 in rat bladder (FIG.
8M, R)).
[0064] In the rat glabrous skin, the SMI-81R antibody exhibited
IR-signal in nerve fibers surrounding blood vessels (among other
skin regions). As expected for this antibody, the SNAP25-IR pattern
was identical in both onabotulinumtoxinA and saline-treated skin
(FIG. 6A, F). The MC-6050 monoclonal antibody demonstrated
IR-signal primarily in nerve fibers from toxin-treated skin.
However, slight IR-signal was also evident in nerve fibers from
saline-treated skin (FIG. 6B, G). The MC-6050 antibody also showed
strong IR-signal in the lumen of blood vessels (FIG. 6B, G). But as
this particular IR-signal was never demonstrated by other SNAP25
antibodies, it was determined to be non-specific. Similarly, the
MC-6053 monoclonal antibody showed IR-specific signal in nerve
fibers surrounding blood vessels from both toxin and saline-treated
skin, as well as non-specific signal in the lumen of blood vessels
(FIG. 6C, H). Once again, RGT-1092 pAb generated against
SNAP25.sub.197 demonstrated IR-signal in both toxin-treated and
saline-treated rat skin (FIG. 6E, J), suggesting that despite its
specificity in WB analysis, this antibody is not amenable for IHC.
In clear contrast, Ab632-rMAb exhibited IR-signal in nerve fibers
only in toxin-treated, but not in saline-treated skin (FIG. 6D, I)
supporting its superb specificity.
[0065] Our samples of rat glabrous skin often contain underlying
skeletal muscle, providing an excellent opportunity to validate our
rMAbs on their ability to recognize BoNT/A-cleaved SNAP25 within
motor nerve terminals (MNT). Similar results for antibody
specificity were observed in MNTs as in other skin nerve
fiber-types (FIG. 7). While the SMI-81R mAb recognized both full
length and BoNT/A-cleaved forms of SNAP25 in MNTs and axons, the
commercial mAbs, MC-6050 and MC-6053 demonstrated IR-signal
primarily in nerve fibers from toxin-treated skin. However,
non-specific IR-signal was also observed in saline-treated tissue
for these commercial mAbs (FIG. 7F,G). In contrast, Ab632-rMAb
exhibited IR-signal in MNTs and axons only in toxin-treated, but
not in saline-treated skin (FIG. 7D,H).
[0066] To further exemplify the Ab632-rMAb's superior specificity
for SNAP25.sub.197 in tissues, we compared the immuno-reactive
signal of Ab632 and Ab635 to an initial batch lot of the human rMAb
(May 2, 2011), the original, native 3C1A5 mAb purified from ascites
and the 2E2A6 (Ab507) mAb used for Allergan's cell-based potency
assay for BOTOX.RTM. (Fernandez-Salas, E., Wang, J., Molina, Y.,
Nelson, J. B., Jacky, B. P., and Aoki, K. R., 2012. Botulinum
neurotoxin serotype A specific cell-based potency assay to replace
the mouse bioassay. PLoS. One. 7, e49516.). The IR signal for these
antibodies was compared in rat glabrous skin and bladder tissues
following treatment with onabotulinumtoxin A.
[0067] In rat glabrous skin, both Ab632 and Ab635 showed strong
IR-signal for SNAP25.sub.197 in nerve fibers surrounding blood
vessels (FIG. 8B, C) and other areas (data not shown) following
BOTOX.RTM. treatment, but this IR-signal was absent in nerve fibers
from vehicle-treated controls (FIG. 8G, H). Similarly, an older and
less refined batch lot of the human rMAb showed good IR-signal in
skin nerve fibers following toxin treatment (FIG. 8A), but not in
saline-treated controls (FIG. 8F). In contrast, no specific
IR-signal for SNAP25.sub.197 was detected in toxin-treated skin
nerve fibers using either the native 3C1A5 mAb or the Ab507 mAb
(FIG. 8D, E).
[0068] Comparable results were demonstrated in the rat bladder
following BOTOX.RTM. treatment. All three rMAbs (Ab632, Ab635 and
the older human batch lot) showed specific IR-signal for
SNAP25.sub.197 in nerve fibers throughout the detrusor muscle of
the bladder (FIG. 8K, L, M). Specific IR-signal was also observed
in rat bladder nerve fibers with the native 3C1A5 mAb (FIG. 8O).
However, as in the rat glabrous skin, no specific SNAP25.sub.197
IR-signal was detected in toxin-treated bladders using the Ab507
mAb (FIG. 8N), which is used in Allergan's cell-based potency assay
for BOTOX.RTM.. No IR-signal was detected in vehicle-treated
control rat bladders with any of the antibodies utilized (FIG.
8P-T).
[0069] FIG. 5 shows immunohistochemical analysis comparing the
specificity of antibodies against SNAP25 in sections of rat bladder
following treatment with either onabotulinumtoxinA (10 U/kg) or
vehicle. (A-E) Confocal images of bladder detrusor muscle (DM)
injected with onabotulinumtoxinA and probed with (A) commercial mAb
(SMI-81R) against full-length (206) and cleaved (197) SNAP25, (B) a
second commercial mAb (MC-6050) against SNAP25.sub.197 &
SNAP25.sub.206, (C) commercial mAb (MC-6053) against
SNAP25.sub.197, (D) Ab632-rMAb against SNAP25.sub.197 and (E)
RGT-1092 pAb against SNAP25.sub.197. (F-J) Confocal images of
control rat bladder injected with vehicle and probed with the same
five antibodies. Arrows (F and J) point to IR-signal within nerve
fibers from vehicle treated rat bladder. DM, detrusor muscle; Scale
bar=50 .mu.m.
[0070] FIG. 6 shows immunohistochemical analysis comparing the
specificity of antibodies against SNAP25 in sections of rat
glabrous skin following treatment with either onabotulinumtoxinA
(30 U/kg) or vehicle. (A-E) Confocal images of rat skin injected
with onabotulinumtoxinA and probed with (A) commercial mAb
(SMI-81R) against full-length (206) and cleaved (197) SNAP25, (B) a
second commercial mAb (MC-6050) against SNAP25.sub.197 &
SNAP25.sub.206, (C) commercial mAb (MC-6053) against
SNAP25.sub.197, (D) Ab632-rMAb against SNAP25.sub.197 and (E)
RGT-1092 pAb against SNAP25.sub.197. (F-J) Confocal images of
control rat skin injected with vehicle and probed with the same
five antibodies. Arrows (F, G, H and J) point to IR-signal within
nerve fibers from vehicle treated rat skin. Asterisks (B, C, G and
H) point to non-specific IR-signal within the lumen of blood
vessels. BV, blood vessel; CT, connective tissue; Scale bar=50
.mu.m.
[0071] FIG. 7. shows immunohistochemical analysis comparing the
specificity of antibodies against SNAP25 in skeletal muscle
underlying rat glabrous skin following treatment with either
onabotulinumtoxinA (30 U/kg) or vehicle. (A-D) Confocal images
showing motor nerve terminals (MNT, arrows) within the underlying
muscle of the rat paw injected with onabotulinumtoxinA and probed
with (A) commercial mAb (SMI-81R) against full-length (206) and
cleaved (197) SNAP25; (B) a second commercial mAb (MC-6050) against
SNAP25.sub.197 & SNAP25.sub.206; (C) a commercial mAb (MC-6053)
against SNAP25.sub.197 and (D) Ab632-rMAb against SNAP25.sub.197;
(E-H) Confocal images from control rat paw injected with vehicle
and probed with the same four antibodies. SNAP25-IR signal is in
green (shown as gray in black and white drawings) and DIC
illumination was used to delineate the underlying muscle fibers
(MF). Arrow (E) points to IR-signal within a MNT from vehicle
treated rat paw; asterisks (B,C,F and G) point to non-specific
IR-signal within the muscle. Scale bar=50 .mu.m.
[0072] FIG. 8 shows immunohistochemical analysis comparing the
specificity of SNAP25.sub.197-specific antibodies in sections of
rat glabrous skin (A-J) and rat bladder (K-T) following treatment
with BOTOX.RTM. or vehicle. (A-E) Confocal images of rat skin
following BOTOX.RTM. injection show IR-signal for SNAP25.sub.197 in
nerve fibers surrounding blood vessels with all the rMAbs (old and
new batches, arrow). The 2E2A6 clone (Ab507) and the 3C1A5 ascites
antibodies both failed to detect any SNAP25.sub.197 signal in this
tissue. (F-J) In vehicle-treated controls, no SNAP25-IR is detected
with any of the antibodies. (K-O) SNAP25.sub.197-IR signal is
detected in the nerve fibers coursing through the detrusor muscle
in rat bladder (arrows) following BOTOX.RTM. treatment with all the
antibodies except the 2E2A6 clone. (P-T) In vehicle-treated
controls, no SNAP25.sub.197-IR is detected with any of the
antibodies. BV, blood vessel; CT, connective tissue; SM, smooth
muscle.
Example 3. Immunohistochemical Comparison--Human Tissue
[0073] Among the commercially available antibodies against SNAP25,
the MC-6053 monoclonal antibody targeting SNAP25.sub.197 is most
similar to our murine Ab635-rMAb with regard to the type and
species of antibody (Table 1). We therefore performed a
head-to-head comparison of the SNAP25-IR expression patterns
between our Ab635-rMAb and the MC-6053 mAb in biopsy samples of
onabotulinumtoxinA and saline-treated human back skin. The
presumption was that since this was a probe of human tissue using a
mouse antibody, the non-specific IR-signal (regardless of the
source) would be minimal.
[0074] Human skin biopsy samples were obtained through a Phase 1
Allergan clinical study. The study was conducted in accordance with
the guidelines and regulations for Good Clinical Practice and all
relevant local and country privacy guidelines. The study protocol,
informed consent, and all appropriate study-related documents were
approved by the Institutional Review Board/Ethics Committee.
[0075] Adult human back skin was injected ID with either 10 U of
onabotulinumtoxinA or vehicle. Punch biopsy samples from back skin
were harvested 14-days post-treatment and fixed overnight in the
same fixative. Tissues were washed and cryoprotected in 30%
sucrose/PBS solution overnight at 4.degree. C. Skin samples were
hemisected along the midline, embedded in O.C.T. (Tissue-Tek) and
stored frozen at -80.degree. C. until sectioning. Tissue blocks
were cryostat-sectioned (14 .mu.m-thick), slide-mounted and slides
were kept at -20.degree. C. until use. IHC and data analysis was
performed as outlined above.
[0076] In the human back skin, the IR-signal for both antibodies
was observed in nerve fibers surrounding blood vessels and sweat
glands within the skin (FIG. 9). IR-signal for the MC-6053 mAb was
observed in nerve fibers from both onabotulinumtoxinA and
saline-treated back skin. In sharp contrast, IR-signal for our
murine Ab635-rMAb was only observed in nerve fibers from
onabotulinumtoxinA-treated, but not saline-treated human back skin
(FIG. 9) demonstrating its superior specificity and more
importantly, its utility as a clinical diagnostics tool.
[0077] FIG. 9 shows immunohistochemical comparison of the
commercially-available (MC-6053) mAb against SNAP25.sub.197 vs.
Ab635-rMAb in sections of human back skin following treatment with
either onabotulinumtoxinA (10U) or vehicle. Confocal images of
blood vessels in onabotulinumtoxinA (A, B) and vehicle-treated (E,
F) human skin probed with either (A, E) the MC-6053 mAb or (B, F)
Ab635-rMAb. Sweat glands in onabotulinumtoxinA (C, D) and
vehicle-treated (G, H) human skin probed with either (C, G) the
MC-6053 mAb or (D, H) Ab635-rMAb. Arrows point to IR-signal within
nerve fibers from vehicle treated human skin. BV, blood vessel; CT,
connective tissue; SG, sweat gland; Scale bar=50 .mu.m.
[0078] Given the difficulty in detecting BoNT/A location and
movement within cells, the proprietary recombinant humanized
.alpha.-SNAP25.sub.197 and proprietary recombinant murine
.alpha.-SNAP25.sub.197 can be used to cross detect SNAP25.sub.197
in the other species. While other .alpha.-SNAP25 antibodies are
capable of detection, using recombinant murine
.alpha.-SNAP25.sub.197 to detect SNAP25.sub.197 in human tissue or
using recombinant humanized .alpha.-SNAP25.sub.197 to detect
SNAP25.sub.197 in murine allows for in-depth analysis of BoNT/A
mechanism of action not possible with other available
antibodies.
Example 4. Immunocytochemical Comparison
[0079] Some antibodies may work better for one assay/indication
over another. Therefore, in order to complete our analysis, we
compared the IR-signal from several of the antibodies in DRG cell
cultures that were treated with either BoNT/A (3 nM) or saline.
[0080] DRG cell cultures were prepared and treated as outlined
above. Immunocytochemistry and data analysis was performed as
detailed above.
[0081] As in the tissues, the the SMI-81R antibody showed strong
IR-signal in both BoNT/A and saline-treated cultures (FIG. 10A, D).
Both the MC-6053 commercially available mAb and our human
Ab632-rMAb demonstrated specific SNAP25.sub.197-IR signal in
neuronal cells from BoNT/A-treated cultures (FIG. 10B, C). No
signal was detected in saline-treated cultures (FIG. 10E, F).
However, the MC-6053 mAb exhibited a faint background signal over
the neuronal soma in the saline-treated cultures (FIG. 10E).
[0082] FIG. 10 shows confocal images of BoNT/A (3 nM)- or
vehicle-treated dorsal root ganglia (DRG) cultures probed with
antibodies to different forms of SNAP25. (A-C) DRG cultures exposed
to BoNT/A for 3 hr and later stained with (A) commercial (SMI-81R)
mAb against full-length (206) and cleaved (197) SNAP25, (B)
commercial (MC-6053) mAb against SNAP25.sub.197 and (C) Ab632-rMAb
against SNAP25.sub.197. (D-F) Control DRG cultures exposed to
vehicle and probed with the same three antibodies. Arrowhead in E
points to a neuronal soma exhibiting background labeling.
[0083] The presence of active BoNT/A in cells expressing SNAP25 can
often be determined by using a selective antibody against the
cleaved substrate (SNAP25.sub.197). In the present study, we
introduce several rMAbs that were developed in-house against
SNAP25.sub.197 and compared their immuno-reactive signal against
that of commercial antibodies using a variety of different methods
(Table 3). Both our human and murine rMAbs consistently detected
SNAP25.sub.197 in all assays and on different tissues, and as
expected, did not detect full-length SNAP25 (SNAP25.sub.206). This
was not the case with other purportedly SNAP25.sub.197-selective
antibodies, which displayed variable assay-dependent specificity.
These results confirm that the BoNT/A-cleaved SNAP25 epitope is
difficult to target specifically with an antibody without also
recognizing the intact SNAP25 protein, which could lead to
potential misinterpretation of results if the proper controls are
not in place. Therefore, any given SNAP25.sub.197 antibody should
be tested under multiple conditions and tissue types to ensure its
fidelity in detecting the presence of BoNT/A-cleaved SNAP25.
[0084] Site-specific antibodies are increasingly being used for
both in vitro and in vivo analysis. These antibodies can detect
sites of phosphorylation or sites of enzymatic cleavage and are
invaluable tools for our understanding of the maturation, activity
and degradation of proteins (Mort, J. S. and Buttle, D. J., 1999.
The use of cleavage site specific antibodies to delineate protein
processing and breakdown pathways. Mol. Pathol. 52, 11-18.; Mort,
J. S., Flannery, C. R., Makkerh, J., Krupa, J. C., and Lee, E. R.,
2003. Use of anti-neoepitope antibodies for the analysis of
degradative events in cartilage and the molecular basis for
neoepitope specificity. Biochem. Soc. Symp. 107-114.; Nagata, K.,
Izawa, I., and Inagaki, M., 2001. A decade of site- and
phosphorylation state-specific antibodies: recent advances in
studies of spatiotemporal protein phosphorylation. Genes Cells 6,
653-664.). Within the field of botulinum neurotoxins, cleavage
site-specific antibodies can help detect the activity of minute
quantities of BoNT light-chain that may otherwise be very difficult
to perceive. To that end, the use of polyclonal antibodies may be
of limited value because a mixed immunoglobulin population could be
produced, not all of which would have the required specificity for
the cleavage epitope (Mort, J. S. and Buttle, D. J., 1999. The use
of cleavage site specific antibodies to delineate protein
processing and breakdown pathways. Mol. Pathol. 52, 11-18.).
Furthermore, the peptide antigen should be relatively short in
order to reduce the possibility of generating antibodies that bind
to a part of the sequence remote from the target epitope.
[0085] The anti-SNAP25.sub.197 mAb presented in the current study
was initially screened and selected for its superior performance in
IHC assays. The rMAbs (Ab632 and Ab635) that were subsequently
generated from this antibody demonstrated superb specificity to
BoNT/A-cleaved SNAP25 in several different assays, including IHC.
Furthermore, these rMAbs showed superior SNAP25.sub.197 specificity
compared to other antibodies tested (Table 3). Accordingly, our
rMAbs represent effective new tools for the detection of BoNT/A
activity within cells and in clinical samples, and will be utilized
in future studies to characterize the efficacy of BoNT/A in tissues
of interest. The specific sequences for these antibodies are
presented in Table 2 above.
[0086] Overall, the antibodies tested in Western blot assays,
immunohistochemistry on tissues and immunocytochemistry on cells
showed various specificity to BoNT/A cleaved SNAP25
(SNAP25.sub.197) and uncleaved SNAP25 (SNAP25.sub.206). A summary
of the results are shown in Table 3 below.
TABLE-US-00003 TABLE 3 Summarized Results of Antibody Specificity
to full length SNAP25 (206) or BoNT/A-cleaved SNAP25 (197). Rat
Human Rat DRG Antibody Specificity Western Blot bladder Rat skin
skin culture SMI-81R SNAP25.sub.206/197 206 + 197 206 + 197 206 +
n/t 206 + 197 197 MC-6050 SNAP25.sub.206/197 197 197 206 + n/t n/t
197 + b MC-6053 SNAP25.sub.197 197 197 206 + 206 + 197 197 + b 197
+ b Ab632 SNAP25.sub.197 197 197 197 n/t 197 Ab635 SNAP25.sub.197
197 197 n/t 197 n/t RGT- SNAP25.sub.197 197 206 + 197 206 + n/t n/t
1092 197 n/t = not tested; b = background
[0087] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application. All publications, patents and
patent applications cited herein are hereby incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent or patent application were
specifically and individually indicated to be so incorporated by
reference.
Sequence CWU 1
1
91219PRTMus sp. 1Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser
Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln
Ser Leu Leu Asn Thr 20 25 30Asn Gly Lys Thr Tyr Leu Thr Trp Leu Ile
Gln Arg Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser
Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95Ser His Phe Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Ala Asp
Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125Gln
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135
140Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
Arg145 150 155 160Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu 180 185 190Arg His Asn Ser Tyr Thr Cys Glu
Ala Thr His Lys Thr Ser Thr Ser 195 200 205Pro Ile Val Lys Ser Phe
Asn Arg Asn Glu Cys 210 2152112PRTMus sp. 2Asp Val Val Met Thr Gln
Thr Pro Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr 20 25 30Asn Gly Lys Thr
Tyr Leu Thr Trp Leu Ile Gln Arg Pro Gly Gln Ser 35 40 45Pro Gln Arg
Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser
85 90 95Ser His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
Lys 100 105 1103436PRTMus sp. 3Gln Val Lys Leu Gln Glu Ser Gly Ala
Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp His 20 25 30Ser Ile His Trp Val Lys Gln
Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Leu Phe Pro Gly
Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr
Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr65 70 75 80Met Tyr Leu
Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Lys Arg
Met Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105
110Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
115 120 125Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys
Gly Tyr 130 135 140Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly
Ser Leu Ser Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu
Glu Ser Asp Leu Tyr Thr Leu 165 170 175Ser Ser Ser Val Thr Val Pro
Ser Ser Pro Arg Pro Ser Glu Thr Val 180 185 190Thr Cys Asn Val Ala
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 195 200 205Ile Val Pro
Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 210 215 220Glu
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu225 230
235 240Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile
Ser 245 250 255Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
Asp Val Glu 260 265 270Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu
Gln Phe Asn Ser Thr 275 280 285Phe Arg Ser Val Ser Glu Leu Pro Ile
Met His Gln Asp Trp Leu Asn 290 295 300Gly Lys Glu Phe Lys Cys Arg
Val Asn Ser Ala Ala Phe Pro Ala Pro305 310 315 320Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 325 330 335Val Tyr
Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 340 345
350Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
355 360 365Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn
Thr Gln 370 375 380Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr
Ser Lys Leu Asn385 390 395 400Val Gln Lys Ser Asn Trp Glu Ala Gly
Asn Thr Phe Thr Cys Ser Val 405 410 415Leu His Glu Gly Leu His Asn
His His Thr Glu Lys Ser Leu Ser His 420 425 430Ser Pro Gly Lys
4354112PRTMus sp. 4Gln Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val
Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp His 20 25 30Ser Ile His Trp Val Lys Gln Lys Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Leu Phe Pro Gly Asn Gly Asn
Phe Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Val Tyr65 70 75 80Met Tyr Leu Asn Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Lys Arg Met Gly Tyr
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 1105219PRTHomo
sapiens 5Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr
Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu
Leu Asn Thr 20 25 30Asn Gly Lys Thr Tyr Leu Thr Trp Leu Ile Gln Arg
Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu
Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu
Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95Ser His Phe Pro Phe Thr Phe
Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150
155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 2156112PRTHomo sapiens 6Asp Val Val Met Thr Gln Thr Pro
Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys
Lys Ser Ser Gln Ser Leu Leu Asn Thr 20 25 30Asn Gly Lys Thr Tyr Leu
Thr Trp Leu Ile Gln Arg Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile
Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95Ser
His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105
1107442PRTHomo sapiens 7Gln Val Lys Leu Gln Glu Ser Gly Ala Glu Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp His 20 25 30Ser Ile His Trp Val Lys Gln Lys Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Leu Phe Pro Gly Asn Gly
Asn Phe Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Ser Thr Val Tyr65 70 75 80Met Tyr Leu Asn Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Lys Arg Met Gly
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 110Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120
125Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
130 135 140Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235
240Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 340 345 350Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360
365Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 4408112PRTHomo sapiens 8Gln Val Lys Leu Gln Glu Ser
Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30Ser Ile His Trp Val
Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Leu Phe
Pro Gly Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr65 70 75 80Met
Tyr Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95Lys Arg Met Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
100 105 110912PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 9Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala
Asn Gln1 5 10
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