U.S. patent application number 10/162889 was filed with the patent office on 2003-04-24 for agents and compositions and methods utilizing same useful in diagnosing and/or treating or preventing plaque forming.
This patent application is currently assigned to Ramot University Authority for Applied Research & Industrial Development. Invention is credited to Frenkel, Dan, Hanan, Eilat, Solomon, Beka.
Application Number | 20030077252 10/162889 |
Document ID | / |
Family ID | 27387248 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077252 |
Kind Code |
A1 |
Solomon, Beka ; et
al. |
April 24, 2003 |
Agents and compositions and methods utilizing same useful in
diagnosing and/or treating or preventing plaque forming
Abstract
A method of immunizing against plaque forming diseases using
display technology is provided. The method utilize novel agents, or
pharmaceutical compositions for vaccination against plaque forming
diseases which rely upon presentation of an antigen or epitope on a
display vehicle. The method further includes agents, or
pharmaceutical compositions for vaccination against plaque forming
diseases, which rely upon presentation of an antibody, or an active
portion thereof, on a display vehicle. Whether antigens or
antibodies are employed, disaggregation of plaques results from the
immunization.
Inventors: |
Solomon, Beka; (Herzlia
Pituach, IL) ; Hanan, Eilat; (Tel Aviv, IL) ;
Frenkel, Dan; (Rehovot, IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
ATTORNEYS AT LAW
PATENT AND TRADEMARK CAUSES
624 NINTH STREET, N.W., SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Ramot University Authority for
Applied Research & Industrial Development
Tel Aviv
IL
|
Family ID: |
27387248 |
Appl. No.: |
10/162889 |
Filed: |
June 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10162889 |
Jun 6, 2002 |
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09629971 |
Jul 31, 2000 |
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09629971 |
Jul 31, 2000 |
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09473653 |
Dec 29, 1999 |
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60152417 |
Sep 3, 1999 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456; 514/44R |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 16/18 20130101; A61P 25/28 20180101; C07K 2317/622 20130101;
A61K 38/1709 20130101; C07K 2319/00 20130101; A61K 49/0004
20130101; A61K 2039/6075 20130101; C07K 2317/74 20130101; C07K
14/4711 20130101; A61P 19/08 20180101; A61P 35/00 20180101; A61K
39/0007 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/93.2 ;
514/44; 435/456; 435/235.1 |
International
Class: |
A61K 048/00; C12N
007/01; C12N 015/86 |
Claims
What is claimed is:
1. A method for inhibiting aggregation of .beta.-amyloid in a
subject or disaggregating aggregated .beta.-amyloid in a subject
comprising administering to a subject in need thereof an effective
amount of a filamentous bacteriophage which displays an antibody or
epitope binding fragment thereof which binds to said .beta.-amyloid
so as to inhibit aggregation of said .beta.-amyloid in said subject
and/or to cause disaggregation of said .beta.-amyloid aggregate in
said subject.
2. The method of claim 1, wherein said bacteriophage displays an
antibody or epitope binding fragment which binds to SEQ ID NO:
1.
3. The method of claim 2, wherein said bacteriophage displays an
antibody or epitope binding fragment which binds to SEQ ID NO: 1 in
a peptide comprising SEQ ID NO: 1.
4. The method of claim 3, wherein said peptide is selected from the
group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 21, and SEQ ID NO: 22.
5. The method of claim 1, wherein said antibody or epitope binding
fragment thereof is displayed via coat glycoprotein VIII on said
bacteriophage.
6. A method for inhibiting aggregation of a prion protein in a
subject or disaggregating aggregated prion protein in a subject
comprising administering to a subject in need thereof an effective
amount of a filamentous bacteriophage which displays an antibody or
epitope binding fragment thereof which binds to said prion protein
so as to inhibit aggregation of said prion protein in said subject
and/or to cause disaggregation of said prion protein aggregate in
said subject.
7. The method of claim 6, wherein said prion protein is scrapie
isoform (PrP.sup.sc).
8. The method of claim 7, wherein said antibody or fragment binds
to SEQ ID NO: 26.
9. The method of claim 8, wherein said antibody or fragment binds
to SEQ ID NO: 26 in a peptide comprising SEQ ID NO: 26.
10. The method of claim 9, wherein said peptide is SEQ ID NO:
25.
11. The method of claim 6, wherein said antibody or epitope binding
fragment thereof is displayed via coat glycoprotein VIII on said
bacteriophage.
12. the method of claim 6, wherein said antibody is selected from
the group consisting of mAb 3-11 and mAb 2-40.
13. An antibody or epitope binding fragment thereof which binds to
a prion protein so as to inhibit aggregation of said prion protein
and/or to cause disaggregation of said prion protein aggregate.
14. The antibody or epitope binding fragment thereof of claim 13,
wherein said prion protein is scrapie isoform (PrP.sup.sc).
15. The antibody or epitope binding fragment thereof of claim 14,
wherein said antibody or epitope binding fragment binds to SEQ ID
NO: 26.
16. The antibody or epitope binding fragment thereof of claim 15,
wherein said antibody or epitope binding fragment binds to SEQ ID
NO: 26 in a peptide comprising SEQ ID NO: 26.
17. The antibody or epitope binding fragment thereof of claim 16,
wherein said peptide is SEQ ID NO: 25.
18. The antibody of claim 13, which is selected from the group
consisting of mAb 3-11 and mAb 2-40.
19. A pharmaceutical composition in unit dosage form, comprising a
pharmaceutically acceptable carrier and, as active ingredient, a
filamentous bacteriophage displaying an antibody or epitope binding
fragment thereof which binds to .beta.-amyloid so as to inhibit
aggregation of said .beta.-amyloid in a subject and/or to cause
disaggregation of any .beta.-amyloid aggregate in the subject.
20. The pharmaceutical composition of claim 19, wherein said
bacteriophage displays an antibody or epitope binding fragment
which binds to SEQ ID NO: 1.
21. The pharmaceutical composition of claim 20, wherein said
antibody or epitope binding fragment is displayed via coat
glycoprotein VII on said bacteriophage.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 09/629,971, filed Jul. 31, 2000, which is a
continuation-in-part of U.S. application Ser. No. 09/473,653, filed
Dec. 29, 1999, which is a continuation-in-part of U.S. provisional
application 60/152,417, filed Sep. 3, 1999, the entire contents of
each being incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to agents and compositions and
to methods of utilizing same for treating plaque-forming diseases.
More particularly, the methods according to the present invention
involve the use of (i) plaque derived antigens cloned and displayed
on the surface of a display vehicle for in vivo elicitation of
antibodies capable of preventing plaque formation and of
disaggregating existing plaques; and (ii) antibodies raised against
plaque derived antigens, at least an immunologic portion of which
is cloned and displayed on a display vehicle, which immunologic
portion is capable of preventing plaque formation and of
disaggregating existing plaques. The present invention firer
relates to a method of targeting a display vehicle to the brain of
an animal, including man and to a method for detecting the presence
of plaque forming prions.
[0003] Plaques forming diseases are characterized by the presence
of amyloid plaques deposits in the brain as well as neuronal
degeneration. Amyloid deposits are formed by peptide aggregated to
an insoluble mass. The nature of the peptide varies in different
diseases but in most cases, the aggregate has a beta-pleated sheet
structure and stains with Congo Red dye. In addition to Alzheimer's
disease (AD), early onset Alzheimer's disease, late onset
Alzheimer's disease, presymptomatic Alzheimer's disease, other
diseases characterized by amyloid deposits are, for example, SAA
amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and
prion diseases. The most common prion diseases in animals are
scrapie of sheep and goats and bovine spongiform encephalopathy
(BSE) of cattle (Wilesmith and Wells (1991) Curr Top Microbiol
Immunol 172: 21-38). Four prion diseases have been identified in
humans: (i) kuru, (ii) Creutzfeldt-Jakob Disease (CJD), (iii)
Gerstmann-Streussler-Sheinker Disease (GSS), and (iv) fatal
familial insomnia (FFI) (Gajdusek (1977) Science 197: 943-960;
Medori, Tritschler et al. (1992) N Engl J Med 326: 444-449).
Etiology of Prion Diseases
[0004] s Prion diseases involve conversion of the normal cellular
prion protein (PrP.sup.C) into the corresponding scrapie isoform
(PrP.sup.Sc). Spectroscopic measurements demonstrate that the
conversion of PrP.sup.c into the scrapie isoform (PrP.sup.Sc)
involves a major conformational transition, implying that prion
diseases, like other amyloidogenic diseases, are disorders of
protein conformation. The transition from PrP.sup.C to PrP.sup.Sc
is accompanied by a decrease in -helical secondary structure (from
42% to 30%) and a remarkable increase in .beta.-sheet content (from
3% to 43%) (Caughey et. al. 1991, Pan et. al. 1993). This
rearrangement is associated with abnormal physiochemical
properties, including insolubility in non-denaturing detergents and
partial resistance to proteolysis. Previous studies have shown that
a synthetic peptide homologous with residues 106-126 of human PrP
(PrP106-126) exhibits some of the pathogenic and physicochemical
properties of PrP.sup.Sc (Selvaggini et. al. 1993, Tagliavini et.
al. 1993, Forloni, et. al. 1993). The peptide shows a remarkable
conformational polymorphism, acquiring different secondary
structures in various environments (De Gioia et al. 1994). It tends
to adopt a .beta.-sheet conformation in buffered solutions, and
aggregates into amyloid fibrils that are partly resistant to
digestion with protease. Recently, the x-ray crystallographic
studies of a complex of antibody 3F4 and its peptide epitope (PrP
104-113) provided a structural view of this flexible region that is
thought to be a component of the conformational rearrangement
essential to the development of prion disease (Kanyo et al. 1999).
The identification of classes of sequences that participate in
folding-unfolding and/or solubilization-aggregation processes may
open new direction for the treatment of plaque forming disease,
based on the prevention of aggregation and/or the induction of
dissaggregation (Silen and Agard, 1989, Frenkel et al. 1998,
Horiuchi and Caughey, 1999).
Alzheimer's Disease--Clinical Overview
[0005] Alzheimer's disease (AD) is a progressive disease resulting
in senile dementia. Broadly speaking, the disease falls into two
categories: late onset, which occurs in old age (typically above 65
years) and early onset, which develops well before the senile
period, e.g., between 35 and 60 years. In both types of the
disease, the pathology is similar, but the abnormalities tend to be
more severe and widespread in cases beginning at an earlier age.
The disease is characterized by two types of lesions in the brain,
senile plaques and neurofibrillary tangles. Senile plaques are
areas of disorganized neutrophils up to 150 mm across with
extracellular amyloid deposits at the center, visible by
microscopic analysis of sections of brain tissue. Neurofibrillary
tangles are intracellular deposits of tau protein consisting of two
filaments twisted about each other in pairs.
Senile Plaques and Other Amyloid Plaques
[0006] The principal constituent of the senile plaques is a peptide
termed A.beta. or beta-amyloid peptide (.beta.AP). The amyloid beta
peptide is an internal fragment of 39-43 amino acids of a precursor
protein termed amyloid precursor protein (APP). Several mutations
within the APP protein have been correlated with the presence of
Alzheimer's disease (See, e.g., Goate et al., Nature 349,704, 1991,
valine.sup.717 to isoleucine; Chartier Harlan et al. 15 Nature 353,
844, 1991, valine.sup.717 to glycine; Murrell et al., Science 254,
97, 1991, valine.sup.717 to phenylalanine; Mullan et al., Nature
Genet. 1, 345, 1992, a double mutation changing lysine.sup.595
-methionine.sup.596 to asparagine.sup.595-leucine.sup.596).
[0007] Such mutations are thought to cause Alzheimer's disease by
increased or altered processing of APP to beta-amyloid,
particularly processing of APP to increased amounts of the long
form of beta-amyloid (i.e., A.beta.1-42 and A.beta.1-43). Mutations
in other genes, such as the presenilin genes, PS1 and PS2, are
thought indirectly to affect processing of APP to generate
increased amounts of long form beta-amyloid (see Hardy, TINS 20,
154, 1997). These observations indicate that beta-amyloid, and
particularly its long form, is a causative element in Alzheimer's
disease.
[0008] Other peptides or proteins with evidence of self aggregation
are also known, such as, but not limited to, amylin (Young AA. et
al., 1994, FEBS Lett, 343(3);237-41); bombesin, caerulein,
cholecystokinin octapeptide, eledoisin, gastrin-related
pentapeptide, gastrin tetrapeptide, somatostatin (reduced),
substance P; and peptide, luteinizing hormone releasing hormone,
somatostatin N-Tyr (Banks and Kastin, Prog Brain Res.,
91:139-4,1992).
[0009] Binding of high affinity monoclonal antibodies (mAbs) to
such regions may alter the molecular dynamics of the whole protein
chain or assembly. By appropriate selection, mAbs have been found
to recognize incompletely folded epitopes and to induce native
conformation in partially or wrongly folded protein (Frauenfelder
et al. 1979, Blond and Goldberg 1987, Karplus and Petsko 1990,
Carlson and Yarmush 1992, Solomon and Schwartz 1995).
Treatment
[0010] U.S. Pat. No. 5,688,561 to Solomon teaches methods of
identifying monoclonal antibodies effective in disaggregating
protein aggregates and preventing aggregation of such proteins.
Specifically, U.S. Pat. No. 5,688,561 demonstrates
anti-beta-amyloid monoclonal antibodies effective in disaggregating
beta-amyloid plaques and preventing beta-amyloid plaque formation
in vitro. U.S. Pat. No. 5,688,561 stipulates the in vivo use of
such antibodies to prevent plaque formation by aggregation of
beta-amyloid or to disaggregate beta-amyloid plaques which have
already formed. These teachings do not, however, identify an
epitope to be employed to generate such antibodies. In addition,
these teachings do not provide means with which to enable the
penetration of such antibodies into the brain through the blood
brain barrier (BBB). Furthermore, this patent fails to teach the
use of phage display technology as a delivery method for antigens
or antibodies. Yet furthermore, no experimental results
demonstrating the in vivo effectiveness of such antibodies are
demonstrated by U.S. Pat. No. 5,688,561.
[0011] EP 526511 by McMichael teaches administration of homeopathic
dosages (less than or equal to 10.sup.-2 mg/day) of beta-amyloid to
patients with pre-established AD. In a typical human with about 5
liters of plasma, even the upper limit of this dosage would be
expected to generate a concentration of no more than 2 pg/ml. The
normal concentration of beta-amyloid in human plasma is typically
in the range of 50-200 pg/ml (Seubert et al., Nature 359, 325-327
1992). Because this proposed dosage would barely alter the level of
endogenous circulating beta-amyloid and because EP 526511 does not
recommend the use of an adjuvant, it seems implausible that any
therapeutic benefit would result therefrom.
[0012] PCT/US98/25386 by Schenk and a Nature paper by Schenk et al.
(Nature, 400:173-177, 1999) teach administration of beta-amyloid
immunogens to a patient in order to generate antibodies to prevent
formation of plaques or dissolve existing plaques. According to
Schenk, 50 to 100 mg of antigen are required, 1 to 10 mg if an
adjuvant is employed. These teachings also stipulate that a similar
effect may be achieved by direct administration of antibodies
against beta-amyloid, in both cases disregarding the blood brain
barrier which, under normal circumstances, prevents the penetration
of antibodies into the brain.
[0013] It is also important to note-that these teachings are
typically restricted to the use of ". . . any of the naturally
occurring forms of beta-amyloid peptide, and particularly the human
forms (i.e., A.beta.39, A.beta.40, A.beta.41, A.beta.42 or
A.beta.43)" or ". . . longer polypeptides that include, for
example, a beta-amyloid peptide, active fragment or analog together
with other amino acids", or "multimers of monomeric immunogenic
agents".
[0014] These teachings ignore, however, earlier data teaching that
the first 28 amino acids of beta-amyloid are sufficient to elicit
antibodies which both disaggregate and inhibit aggregation of
beta-amyloid plaques in vitro (Hanan and Solomon, Amyloid: Int. J.
Exp. Clin. Invest. 3:130-133, 1996; Solomon et al., Proc. Natl.
Acad. Sci. U.S.A. 93:452-455, 1996; Solomon et al., Proc. Natl.
Acad. Sci. U.S.A. 94:4109-4112, 1997).
[0015] Schenk and Schenk et al. both fail to teach the use of the
N-terminal epitope of beta-amyloid plaques which is known to be a
sequential epitope composed of only four amino acid residues (EFRH,
SEQ ID NO: 1) located at positions 3-6 of the beta-amyloid peptide
(Frenkel D., J. Neuroimmunol., 88:85-90,1998). Antibodies against
this epitope have subsequently been shown to disaggregate
beta-amyloid fibrils, restore beta-amyloid plaques solubilization
and prevent neurotoxic effects on PC 12 cells (Solomon, B. et al.,
Proc. Natl. Acad. Sci. USA. 94:4109-4112, 1997; and Solomon, B., et
al., Proc. Natl. Acad. Sci. USA., 93:452-455,1996).
[0016] This epitope has been independently confirmed as the epitope
bound by anti-aggregating antibodies using random combinatorial
hexapeptide phage display (Frenkel and Solomon, J. of Neuroimmunol.
88:85-90, 1998).
[0017] The EFRH (SEQ ID NO: 1) epitope is available for antibody
binding when beta-amyloid peptide is either in solution or in
aggregates. Blocking of this epitope by a monoclonal antibody
prevents self-aggregation and enables resolubilization of already
formed aggregates.
[0018] These findings suggest that the teachings of Schenk and
colleagues are inefficient at best. Since, as has already been
mentioned hereinabove, the normal concentration of beta-amyloid in
human serum is 50-200 pg/ml, immunization with that peptide could
be expected to produce either low antibody titers or high toxicity
if strong adjuvants are used and as such it is not applicable for
therapy. Indeed, in order to achieve significant serum titers of
antibody against beta-amyloid a series of 11 monthly injections was
required (Schenk et al., Nature, 400:173-177, 1999). The degree to
which these serum titers will persist over time is not yet known,
and this point is especially crucial with respect to early onset
Alzheimer's disease.
[0019] Schenk and colleagues further teach that an immunogenic
peptide such as beta-amyloid may be displayed upon the surface of a
virus or bacteria. However, they fail to teach use of an antigen so
displayed to effect immunization. No mention is made of defining an
epitope in this context and no experimental data is provided
either. In addition, delivery of antibody displayed on a display
vehicle is not taught by Schenk or Schenk et al. altogether.
[0020] Collectively, the prior art fails to teach means with which
an effective titer of anti-aggregation antibodies can be generated
in vivo in a short time and/or be introduced into the brains of
patients suffering a plaque-forming diseases. In addition, the
persistence of titers generated via prior art teachings has not
been established.
[0021] There is thus a widely recognized need for, and it would be
highly advantageous to have, effective means of disaggregating
amyloid plaques in vivo which would have lasting effect, high
efficiency, rapid onset, no adverse effect on the treated subject
and which is readily amenable to large scale production.
SUMMARY OF THE INVENTION
[0022] According to one aspect of the present invention there is
provided a method of treating a plaque forming disease comprising
the steps of (a) displaying a polypeptide on a display vehicle, the
polypeptide representing at least one epitope of an aggregating
protein associated with plaque formation in the plaque forming
disease, the at least one epitope being capable of eliciting
antibodies capable of disaggregating the aggregating protein and/or
of preventing aggregation of the aggregating protein; and (b)
introducing the display vehicle into a body of a recipient so as to
elicit the antibodies capable of disaggregating the aggregating
protein and/or of preventing aggregation of the aggregating
protein.
[0023] According to another aspect of the present invention there
is provided an agent for treating a plaque forming disease
comprising a display vehicle displaying a polypeptide representing
at least one epitope of an aggregating protein associated with
plaque formation in the plaque forming disease, the at least one
epitope being capable of eliciting antibodies capable of
disaggregating the aggregating protein and/or of preventing
aggregation of the aggregating protein.
[0024] According to yet another aspect of the present invention
there is provided a pharmaceutical composition for treating a
plaque forming disease comprising an effective amount of a display
vehicle displaying a polypeptide, the polypeptide representing at
least one epitope of an aggregating protein associated with plaque
formation in the plaque forming disease, the at least one epitope
being capable of eliciting an effective amount of antibodies
capable of disaggregating the aggregating protein and/or of
preventing aggregation of the aggregating protein, the
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier.
[0025] According to still another aspect of the present invention
there is provided a method of preparing a display vehicle for
treating a plaque forming disease, the method comprising the step
of genetically modifying a genome of a display vehicle by inserting
therein a polynucleotide sequence encoding a polypeptide
representing at least one epitope of an aggregating protein
associated with plaque formation in the plaque forming disease, the
at least one epitope being capable of eliciting antibodies capable
of disaggregating the aggregating protein and/or of preventing
aggregation of the aggregating protein, such that when the display
vehicle propagates the polypeptide is displayed by the display
vehicle.
[0026] According to an additional aspect of the present invention
there is provided a method of treating a plaque forming disease
comprising the steps of (a) displaying a polypeptide representing
at least an immunological portion of an antibody being for binding
at least one epitope of an aggregating protein associated with
plaque formation in the plaque forming disease, the binding capable
of disaggregating the aggregating protein and/or of preventing
aggregation of the aggregating protein; and (b) introducing the
display vehicle into a body of a recipient so as to disaggregate
the aggregating protein and/or prevent its aggregation.
[0027] According to still an additional aspect of the present
invention there is provided a method of introducing a display
vehicle lacking an engineered targeting moiety into a brain of a
recipient, the method comprising the step of administering the
display vehicle intranasally to the recipient.
[0028] According to further features in preferred embodiments of
the invention described below, the step of introducing the display
vehicle into the body of the recipient so as to disaggregate the
aggregating protein is effected through an olfactory system of the
recipient.
[0029] According to yet another additional aspect of the present
invention there is provided an agent for treating a plaque forming
disease comprising a display vehicle displaying a polypeptide
representing at least an immunological portion of an antibody which
can bind at least one epitope of an aggregating protein associated
with plaque formation in the plaque forming disease, the
immunological portion of the antibody being capable of
disaggregating said aggregating protein and/or of preventing
aggregation of the aggregating protein.
[0030] According to yet an additional aspect of the present
invention there is provided a pharmaceutical composition for
treating a plaque forming disease comprising an effective amount of
a display vehicle displaying a polypeptide representing at least an
immunological portion of an antibody which can bind at least one
epitope of an aggregating protein associated with plaque formation
in the plaque forming disease, the immunological portion of the
antibody being capable of disaggregating the aggregating protein
and/or of preventing aggregation of the aggregating protein, the
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier.
[0031] According to still an additional aspect of the present
invention there is provided a method of preparing a display vehicle
for treating a plaque forming disease comprising the step of
genetically modifying a genome of a display vehicle by inserting
therein a polynucleotide sequence encoding at least an
immunological portion of an antibody capable of binding at least
one epitope of an aggregating protein associated with plaque
formation in the plaque forming disease, the immunological portion
of the antibody being capable of disaggregating the aggregating
protein and/or of preventing aggregation of the aggregating
protein.
[0032] According to a further aspect of the present invention there
is provided a polypeptide comprising at least an immunological
portion of an antibody being capable disaggregating a prion protein
aggregate and/or of preventing aggregation of said prion
protein.
[0033] According to further features in preferred embodiments of
the invention described below, the polypeptide is capable of
binding at least one epitope formed by an amino acid sequence set
forth in SEQ ID NO: 25.
[0034] According to yet a further aspect of the present invention
there is provided a method of detecting a presence or an absence of
a prion protein in a biological sample, the method comprising the
steps of: (a) incubating an anti-prion antibody or an immunological
portion thereof with the biological sample; (b) determining a
presence or an absence of antigen complexes formed with the
anti-prion antibody or the immunological portion thereof, to
thereby determine the presence or the absence of the prion protein
in the biological sample.
[0035] According to still further features in the described
preferred embodiments the plaque forming disease is selected from
the group consisting of early onset, Alzheimer's disease, late
onset Alzheimer's disease, presymptomatic Alzheimer's disease, SAA
amyloidosis, hereditary Icelandic syndrome, senility and multiple
myeloma.
[0036] According to still further features in the described
preferred embodiments the plaque forming disease is selected from
the group consisting of scrapie, bovine spongiform encephalopathy
(BSE), kuru, Creutzfeldt-Jakob Disease (CJD),
Gerstmann-Streussler-Sheinker Disease (GSS) and fatal familial
insomnia (FFI).
[0037] According to still further features in the described
preferred embodiments the aggregating protein is selected from the
group consisting of beta-amyloid, serum amyloid A, cystantin C, IgG
kappa light chain and prion protein.
[0038] According to still further features in the described
preferred embodiments the display vehicle is selected from the
group consisting of a virus, a bacteria and a polypeptide
carrier.
[0039] According to still further features in the described
preferred embodiments the virus is selected from the group
consisting of a double stranded DNA virus, a single stranded DNA
virus, a positive strand RNA virus and a negative strand RNA
virus.
[0040] According to still further features in the described
preferred embodiments the display vehicle is a bacteriophage.
[0041] According to still further features in the described
preferred embodiments the display vehicle is a filamentous
bacteriophage.
[0042] According to still further features in the described
preferred embodiments the bacteriophage display vehicle is capable
of propagating within bacterial flora of the host.
[0043] According to still further features in the described
preferred embodiments the bacteriophage display vehicle is capable
of propagating within E. coli.
[0044] According to still further features in the described
preferred embodiments the bacteriophage display vehicle is fd.
[0045] According to further features in the described preferred
embodiments of the invention, the display vehicle is incapable of
propagation in vivo.
[0046] According to still further features in the described
preferred embodiments a triple dose of 10.sup.10 units of the
chosen display vehicle induces an antibody titer of at least
1:50,000 within 30 days of administration, as measured by
ELISA.
[0047] According to still further features in the described
preferred embodiments the at least one epitope of said prion
protein is formed by an amino acid sequence set forth in SEQ ID NO:
25.
[0048] According to still further features in the described
preferred embodiments the immunological portion of an antibody
serves for binding at least one epitope of an aggregating protein
associated with plaque formation in a plaque forming disease, said
immunological portion of said antibody being capable of
disaggregating said aggregating protein and/or of preventing
aggregation of said aggregating protein.
[0049] According to still further features in the described
preferred embodiments the prion protein is the aggregating protein
associated with plaque formation
[0050] According to still further features in the described
preferred embodiments the biological sample is derived from tissues
and/or body fluids of a human, a primate, a monkey, a pig, a
bovine, a sheep, a deer, an elk, a cat, a dog and a chicken.
[0051] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
methods, agents, and pharmaceutical compositions for preventing or
reversing the progression of a plaque forming disease. The present
invention further includes methods for preparing agents and
pharmaceutical compositions useful for preventing or treating
plaque forming diseases and to a method of detecting the presence
of a pathogenic prion protein in a biological sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show details of the invention in more
detail than is necessary for a fundamental understanding of the
invention, the description taken with the drawings making apparent
to those skilled in the art how the several forms of the invention
may be embodied in practice.
[0053] In the drawings:
[0054] FIG. 1a is a schematic depiction of an IgM antibody.
[0055] FIG. 1b is a photograph of an ethidium bromide stained 1.5%
agarose gel showing cDNA fragments of the heavy and the light
chains of IgM508. Lane 1: Kb Wadder); Lanes 2 and 3 V.sub.H and
V.sub.L fragments, respectively, as indicated by arrows.
[0056] FIG. 1c is a photograph of an ethidium bromide stained 1.5%
agarose gel showing scFv DNA fragment derived from antibody IgM
508. is Lane 1: Kb (Ladder); Lane 2: scFv 508 DNA (750 bp).
[0057] FIG. 1d is a schematic depiction of filamentous phage
displaying an scFv.
[0058] FIG. 1e is a schematic depiction of a soluble scFv.
[0059] FIG. 2 is a physical map of plasmid pCC-508F which is used
for the production of scFv-508-CBD fusion protein (also referred to
herein as 508(Fv)-CBD) under control of lac promoter. Amp res--a
gene encoding .beta.-lactamase; V.sub.L and V.sub.L--sequences
coding for the variable domains of the heavy and light chains of
scFv-508, respectively; Lin--a gene coding for a
(Gly.sub.4Ser).sub.3 (SEQ ID NO: 2) linker present between the
variable domains V.sub.H and V.sub.L. Restriction sites and
positions thereof are also shown.
[0060] FIG. 3 is a physical map of plasmid pfFEKCA-508 which is
used according to the present invention for cytoplasmic expression
of the scFv-508-CBD fusion protein under the control of a
T.sub.7-promoter. Amp res--a gene encoding .beta.-lactamase;
V.sub.L and V.sub.L--sequences coding for the variable domains of
the heavy and light chains of scFv-508, respectively; Lin--a gene
coding for a (Gly.sub.4Ser).sub.3 (SEQ ID NO: 2) linker present
between the variable domains V.sub.H and V.sub.L. T7-promoter and
T7 term-T7 promoter and T7 terminator sequences, respectively.
Restriction sites and positions thereof are also shown.
[0061] FIG. 4 shows an analysis of .beta.AP binding by antibody
508(Fv)-CBD in an ELISA assay. The analyzed antibodies were added
to .beta.AP coated wells. Bound antibodies were detected with HRP
conjugated secondary antibodies. The parental 508 IgM antibody was
used as a positive control. The unrelated anti-.beta.-galactosidase
antibody Gal6(Fv)-CBD was used as a negative control.
[0062] FIG. 5 shows PCR analysis of phage DNA inserts. DNA isolated
from pCC-508(Fv), lane 2, and pCC-Gal6(Fv), Lane 3, were PCR
amplified and separated on a 1.5% agarose gel. Ethidium bromide
staining and UV illumination were used to visualize the bands. Lane
1 contains a DNA size marker. The arrow marks the position of an
intact scFv migrating at about 750 bp.
[0063] FIG. 6 demonstrates expression and purification of
508(Fv)-CBD. 5-10 .mu.g protein were loaded in each lane of a 14%
SDS polyacrylamide gel. Proteins were visualized by Coomassie
brilliant blue staining. The arrow marks the position of the
scFv-CBD fusion protein. Lane 1--total cell extract from
non-induced BL21(DE3) cells carrying 508((Fv)-CBD expression
vector. Lane 2--total cell extract from BL21(DE3) cells carrying
508((Fv)-CBD expression vector induced for 3 hours with IPTG. Lane
3--washed, solubilized and reduced inclusion bodies that were used
in refolding. Lane 4--protein that did not bind to cellulose during
cellulose-assisted refolding. Lane 5--protein washed away from
cellulose with TBS. Lane 6--protein washed away from crystalline
cellulose with distilled water. Lane 7--soluble 508(Fv)-CBD
recovered from cellulose by high-pH elution and neutralization.
[0064] FIG. 7 demonstrates the stability of 508(v)-CBD. Purified
508(Fv)-CBD protein was stored at 4.degree. C. for one day (dark
squares) or one week (dark circles), and then analyzed for .beta.AP
binding in an ELISA assay, as described in the legend to FIG. 4.
The unrelated antibody Gal6(Fv)-CBD served as a negative control
(open squares).
[0065] FIG. 8 demonstrates quantitation of 508(Fv) mutants
affinity-enrichment by PCR and DNA restriction analysis. The DNA of
19 508(Fv)-mutant micro library clones before (FIG. 8a) and of 11
clones picked up after one cycle of affinity selection (FIG. 8b)
were analyzed. The DNA was digested with PvuI and separated on a
1.5% agarose gel. A non-mutated scFv-CBD appears as an intact 1250
bp fragment (upper arrow). A mutated clone is indicated by the
appearance of both 700 bp (middle arrow) and 550 bp (lower arrow)
fragments. A DNA size marker is shown in lane 1.
[0066] FIG. 9 shows an analysis of .beta.AP binding (FIG. 9a) and
stability (FIG. 9b) of mutated 508(Fv) derivatives in an ELISA
assay. The analyzed antibodies were added to .beta.AP coated wells.
Bound antibodies stored at 4.degree. C. for one day or for one week
were detected as described in the legend to FIG. 7. 508(Fv) wild
type (open squares), C96F (dark squares), C96Y (dark circles), C96S
(dark triangles). The unrelated anti-.beta.-galactosidase antibody
Gal6(Fv)-CBD was used as a negative control (open squares).
[0067] FIG. 10 shows an analysis of the specific inhibition of
.beta.AP binding by antibody 508F(Fv) in a competitive ELISA assay.
The antibody was pre-incubated with varying concentrations of the
competing peptides: .beta.AP (acids 1-16 of SEQ ID NO: 3) (dark
squares) or the unrelated peptide WVLD (SEQ ID NO: 4) (open
squares), before being added to .beta.AP coated wells. Bound
antibodies were detected as described in the legend to FIG. 7.
[0068] FIGS. 11a and 11b show nucleotide (SEQ ID NO: 5) and deuced
amino acid (SEQ ID NO: 6) sequences of scFv 508F heavy chain (FIG.
11a); and the linker and the variable region of the light chain
(FIG. 11b). The amino acid sequence is presented by a three-letter
code; CDRs and the linker are underlined.
[0069] FIG. 12 demonstrates the prevention of .beta.AP mediated
toxic effect on PC12 cells by 508F(Fv). Cells were incubated with
fibrillar .beta.A alone, or with fibrillar .beta.A that had been
incubated with antibodies at different molar ratio of
antibody/.beta.AP, as indicated. An
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay was used to estimate cell survival.
[0070] FIG. 13 demonstrates the disaggregation of fibrillar .beta.A
by 509F(Fv). The fibrillar state of pre-formed .beta.A fibrils were
measured with or without incubation with antibodies at different
molar ratio of antibody/.beta.AP, as indicated. The fluorescence of
thioflavin-T (ThT) reagent in a ThT assay which is proportional to
fibril .beta.A was used to assess the fibril morphology.
[0071] FIGS. 14a-d demonstrate the detection of filamentous phage
(f88-EFRH) in brain sections via immunofluorescence one day
following a single dose applied intranasally. Appearance of
filamentous phage in mouse olfactory bulb and hippocampus sections
using fluorescent rabbit anti-phage antibody (FIGS. 14a and 14c,
respectively) as is compare to an untreated mouse brain (FIGS. 14b
and 14d, respectively). The sections were observed using a
fluorescence microscope at a final magnification of .times.10.
[0072] FIGS. 15a-d demonstrate the disappearance of filamentous
phage (f88-EFRH) from mouse brain 28 days following a single
intranasal administration. Disappearance of filamentous phage from
mouse olfactory bulb and hippocampus is demonstrated in sections of
these organs using fluorescent rabbit anti-phage antibody (FIGS.
15a and 15c, respectively), as is compared to an untreated mouse
brain (FIGS. 15b and 15d, respectively). The sections were observed
using a fluorescence microscope at a final magnification of
.times.10.
[0073] FIGS. 16a-d show histology of mouse brain sections after
phage f88-EFRH clearance. Brain sections of olfactory organ (FIG.
16a) and hipocampous (FIG. 16c) after 28 days following phage
f88-EFRH administration were stained with hematoxylin and eosin,
and compared to sections of an untreated brain (FIGS. 16b and 16d,
respectively). The stained sections were examined and photographed
at a final magnification is of .times.40.
[0074] FIGS. 17a-d show fluorescence detection of biotin of phage
pCC-508F coupled to biotinylated .beta.AP (acids 1-16 of SEQ ID NO:
3) in mouse brain sections following a single intranasal
administration. Appearance of .beta.AP (acids 1-16, SEQ ID NO: 3)
coupled to filamentous phage displaying scFv508F in mice olfactory
bulb and hippocampus sections using streptavidin coupled to PE
(FIGS. 17a and 17c, respectively) as is compare to an untreated
mouse brain (FIGS. 17b and 17d, respectively). The sections were
observed using a fluorescence microscope at a final magnification
of .times.20.
[0075] FIGS. 18a-d show histology of mouse brain after phage
pCC-508F coupled to biotinylated .beta.AP (acids 1-16 of SEQ ID NO:
3) administration. Olfactory organ (FIG. 18a) and hippocampus (FIG.
18b) sections one day following phage administration were stained
with hematoxylin and eosin, and were compared to untreated mouse
brain sections (FIGS. 18c and 18d, respectively). The stained
sections were examined and photographed at a final magnification of
.times.40.
[0076] FIG. 19 is a diagram of immunization schedule with
filamentous phage displaying the EFRH (SEQ ID NO: 1) epitope of
.beta.-amyloid peptide.
[0077] FIGS. 20a and 20b show immunization with f3 filamentous
phage displaying EFRH (SEQ ID NO: 1) epitope of .beta.-amyloid
peptide as a fusion of phage glycoprotein III (gpIII). Serum IgG
titer of different bleeds from mice immunized with the EFRH-phage
according to the schedule of FIG. 19 against wild type filamentous
phage coat proteins (FIG. 20a) and the N-terminal epitope (acids
1-16, SEQ ID NO: 3) of .beta.-amyloid (FIG. 20b).
[0078] FIG. 21 demonstrates long lasting immunization with f3
filamentous phage. Serum IgG titer of different bleeds from mice
immunized with EFRH-phage against wild type filamentous phage coat
proteins and the N-terminal (acids 1-16, SEQ ID NO: 3) of
.beta.-amyloid.
[0079] FIG. 22 show binding of anti-aggregating .beta.AP monoclonal
antibody (mAb 10D5) to peptide-presenting phage selected from an
f88 phage library. Unrelated mAb 5.5 raised against acetylcholine
receptor was used as a negative control. Antibodies were added to
phage-coated wells and ELISA was used to detect binding.
[0080] FIG. 23 show binding of anti-aggregating .beta.AP mAb (10D5)
to a YYEFRH (SEQ ID NO: 7)-phage and VHEPHEFRHVALNPV (SEQ ID NO:
8)-phage. Antibody in concentration of 1 .mu.g/ml was added to
phage-coated wells and binding was analyzed by ELISA. Filamentous
phage without insert was used as a control.
[0081] FIGS. 24a-b show immunization with f88 filamentous phage
displaying EFRH (SEQ ID NO: 1) epitope of .beta.-amyloid peptide as
a fusion of phage glycoprotein VIII (gpVIII). Serum IgG titer of
different bleeds from mice immunized with EFRH-phage against wild
type filamentous phage coat proteins (FIG. 24a) and the N-terminal
epitope (acids 1-16, SEQ ID NO: 3) of .beta.-amyloid peptide (FIG.
24b).
[0082] FIG. 25 shows inhibition of serum of an immunized mice in
binding to .beta.AP by synthetic peptides derived from the
N-terminal of .beta.-amyloid peptide. The assay was done with
1:3000 dilution of serum after a third immunization with f88-EFRH
reacted with the various peptides in various concentrations per
well, as indicated. The peptide WVLD (SEQ ID NO: 4) was used as a
negative control.
[0083] FIG. 26 demonstrates prevention of .beta.AP mediated toxic
effect on PC12 cells by serum antibodies raised against
f88-EFRH-phage. Cells were incubated with fibrillar .beta.A alone,
or with fibrillar .beta.A that has been incubated with serum from
the third bleeding at different concentration. The negative control
was serum from a non-immunized mouse. The MTT assay was used to
estimate cell survival.
[0084] FIG. 27 demonstrates interference with fibrillar
.beta.-amyloid formation by serum antibodies raised against the
f88-EFRH-phage. Estimation of the fluorescence of ThT which
correlates with the amount of fibrillar .beta.-amyloid formed after
incubation for a week at 37.degree. C. in the presence of serum
samples diluted as indicated. The negative control was serum from a
non-immunized mouse. The positive control was without serum. Fibril
formation was measured by the ThT assay.
[0085] FIG. 28 illustrates the amino acids sequence corresponding
to the human prion protein 106-126 (SEQ ID NO: 25) and to the mouse
homologue.
[0086] FIG. 29 demonstrates the neurotoxicity effect of the PrP
peptide as measured by MTT assay. PC12 cells were seeded in 96 well
plates in a DMEM medium supplemented with 2 mM insulin, 2 .mu.M
L-glutamine and 100 units penicillin/streptomycin. Cell viability
was assessed by the MTT assay following incubation with PrP
106-126, (at different concentrations). PrP 106-126 was either
preincubated for 4 days at 37.degree. C. and then added to the
cells for 3 days (grey bars), or was preincubated for 4 days at
37.degree. C. and then added to the cells for 5 days (white bars)
or was preincubated for 7 days at 37.degree. C. and was then added
to the cells for 5 days (black bars).
[0087] FIG. 30 illustrates the extent of aggregation of the PrP
peptide, using ThT binding assay. PrP 106-126 (0-0.8 mg/ml) was
incubated for 7 days at 37.degree. C. and emmision at 482 nm was
measured to determine the extent of aggregation.
[0088] FIG. 31 demonstrates the protective effect of mAbs 3-11,
2-40 on PrP peptide neurotoxicity. PC12 cells were seeded in a 96
wells plate in a DMEM medium supplemented with 2 mM insulin 2 mM
L-glutamine and 100 units penicillin/streptomycin and were
incubated for three days. The following treatments were conducted:
(1) Positive Control, untreated cells; (2) 100 .mu.M PrP 106-126
that was preincubated for 7 days at 37.degree. C.; (3,4,5) an
aggregated peptide that was preincubated for 1 hour before exposure
to the cells together with the mAbs 3-11 (treatment 3), 2-40
(treatment 4) and 3F4 (treatment 5). Cell viability was assessed
using the MTT assay.
[0089] FIG. 32 illustrates the modulation of PrP conformation by
the mAbs. PrP 106-126 (0.3 mg/ml) was incubated for 7 days at
37.degree. C. (1) and with mAbs 2-40, 3-11 and 3F4 (treatments 2, 3
and 4, respectively). The antibodies were incubated with the sample
for 24 hours either prior to the PrP incubation (grey bars) or
following a one week PrP incubation (white bars). Fibril formation
was assessed by the ThT binding assay.
[0090] FIG. 33 shows the concentration dependent protective effect
of mAb 3-11 against PrP fibrillar aggregate formation. PrP 106-126
(0.3 mg/ml) was incubated for 7 days at 37.degree. C. with diluted
mAb 3-11 (1:1, 1:10, 1:50, corresponding to treatments 1, 2, and 3,
respectively). The antibody was incubated with the sample for 24
hours either prior to (grey bars), or following (white bars), the
one week incubation of PrP. Amyloid fibril formation was assessed
using ThT binding assay
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0091] The present invention is of methods, pharmaceutical agents
and compositions, which can be used for treating plaque-forming
diseases, including, but not limited to, Alzheimer's disease and
prion generated plaque forming diseases. Specifically the present
invention can be used to (i) induce active immunity to plaque
derived antigens in a recipient by immunizing with at least one
epitope of an aggregating protein associated with plaque formation
in a plaque forming disease on a display vehicle, so that
antibodies elicited in response to immunization are capable of
preventing plaque formation and/or of disaggregating existing
plaques; and (ii) induce passive immunity by administering at least
an immunological portion of an antibody which can bind to at least
one epitope of an aggregating protein associated with plaque
formation in a plaque forming disease, raised against plaque
derived antigens, cloned and displayed on a display vehicle,
capable of preventing plaque formation and of disaggregating
existing plaques. This passive immunity may be of exceptionally
long duration if the display vehicle employed is capable of
replicating within the recipient. The present invention further
relates to a method of targeting a display vehicle to the brain of
an animal, including man, so that plaques present in the brain,
such as beta amyloid plaques in brains of Alzheimer's disease
patients, may be disaggregated. Finally, the present invention also
related to a method of detecting aggregate forming prion proteins
in a biological sample.
[0092] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0093] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description. The invention is capable of other embodiments or of
being practiced or carried out in various ways. Also, it is to be
understood that the phraseology and terminology employed herein is
for the purpose of description and should not be regarded as
limiting.
[0094] While reducing one aspect of the present invention to
practice, as is further exemplified in Examples 1-15 of the
Examples section that follows, antigens derived from beta amyloid
peptide were displayed on the surface of a filamentous phage which
was used for immunization of experimental animals. All of the
peptides employed contained the EFRH epitope (SEQ ID NO: 1,
residues 3-6, SEQ ID NO: 3) of beta amyloid peptide (SEQ ID NO: 3).
The epitope was presented as a fusion protein of fd phage coat
glycoprotein III or VIII. Doses ranging from 10.sup.10 to 10.sup.12
phages per injection were employed on 8 week old female BALB/c
mice. A typical immunization schedule included three injections at
14-day intervals, administered either intraperitoneally or
intranasally.
[0095] During and after the immunization process, the antibody
serum titer of subject mice was tested for the production of
A.beta. specific antibodies by enzyme linked immunosorbent assay
(ELISA) as detailed in methods and materials hereinbelow. Serum
titers were subsequently shown to persist for 11 months in response
to a protocol including only 3 immunizations. While all tested
epitopes containing EFRH produced a titer, displaying the epitope
on the surface of a display vehicle produced far highest and
unexpected titers. These high titers are believed to be a result of
the great number of copies presented to the immune system using
this method, and this idea is supported by results of binding
assays using controlled amounts of sera.
[0096] The anti-aggregating properties of the obtained polyclonal
antibody raised against EFRH epitopes with respect to beta-amyloid
fibril formation was measured by the ThT binding assay. Serum, at
dilution of 1:10 and 1:100, disrupted formation of fibril structure
of .beta.-amyloid with extensive deterioration of fibril
morphology, as indicated by a substantial decrease in ThT
fluorescence. The unrelated serum used as control (serum from
un-immunized mouse) did not significantly inhibit fibril
formation.
[0097] The effect of the same serum on disruption of already formed
.beta.A fibril (the toxic form of .beta.AP) was also determined.
Serum of EFRH immunized mice incubated with pre-formed .beta.A
fibrils disrupted the fibril structure. The unrelated control
antibody had no significant effect on fibril morphology. Together,
these results confirm the ability of EFRH epitope presented by
suitable display vehicles to evoke production of anti-aggregation
antibodies, which can inhibit or reverse the process of fibril
formation.
[0098] Diluted serum produced according to this embodiment of the
present invention prevented the neurotoxicity of beta amyloid
peptide. This result implies potential clinical utility in
preventing brain deterioration of patients suffering from amyloid
plaque diseases.
[0099] While reducing another aspect of the present invention to
practice, and as is further exemplified in Examples 15-21 of the
Examples section which follows, it was uncovered that site-directed
antibodies (designated mAbs 3-11 and 2-40), which were generated
against a prion derived peptide, are useful in preventing or
disaggregating prion generated plaques.
[0100] Binding of the prion derived peptide (PrP 106-126) to these
mAbs led to a significant protective effect against aggregation as
was measured by the ThT and MTT assays. The mAbs generated by the
preset invention also significantly decrease the peptide fibrillar
aggregation and reverse the aggregated form to a disaggregated
conformation as assayed by the ThT binding assay.
[0101] The binding of mAbs 3-11 and 2-40 to the PrP peptide either
in solution or to the aggregate suggests that this epitope is
involved in aggregation process and may act as a regulatory site
controlling both the solubilization and disaggregation process of
PrP peptide and perhaps the whole PrP protein.
[0102] Thus, according to one aspect of the present invention there
is provided a method of treating a plaque forming disease. The
method according to this aspect of the present invention is
effected by displaying a polypeptide on a display vehicle, the
polypeptide representing at least one epitope of an aggregating
protein associated with plaque formation in the plaque forming
disease, the at least one epitope being capable of eliciting
antibodies capable of disaggregating the aggregating protein and/or
of preventing aggregation of the aggregating protein, and
introducing the display vehicle into a body of a recipient, so as
to elicit the antibodies capable of disaggregating the aggregating
protein and/or of preventing aggregation of the aggregating
protein.
[0103] According to a preferred embodiment of the present invention
the display vehicle is selected such that less than 30 days
following an introduction of a triple dose of 10.sup.10 units
thereof to the recipient, a titer of the antibodies in the
recipient is above 1:50,000, as is determined by ELISA.
[0104] According to another aspect of the present there is provided
an agent for treating a plaque forming disease. The agent according
to this aspect of the present invention comprising a display
vehicle displaying a polypeptide, the polypeptide representing at
least one epitope of an aggregating protein associated with plaque
formation in the plaque forming disease, the at least one epitope
being capable of eliciting antibodies capable of disaggregating the
aggregating protein and/or of preventing aggregation of the
aggregating protein.
[0105] According to still another aspect of the present invention
there is provided a pharmaceutical composition for treating a
plaque forming disease. The composition according to this aspect of
the present invention comprising an effective amount of a display
vehicle displaying a polypeptide, the polypeptide representing at
least one epitope of an aggregating protein associated with plaque
formation in the plaque forming disease, the at least one epitope
being capable of eliciting an effective amount of antibodies
capable of disaggregating the aggregating protein and/or of
preventing aggregation of the aggregating protein, and a
pharmaceutically acceptable carrier.
[0106] According to yet another aspect of the present invention
there is provided a method of preparing a display vehicle for
treating a plaque forming disease. The method according to this
aspect of the present invention is effected by genetically
modifying a genome of a display vehicle by inserting therein a
polynucleotide sequence encoding a polypeptide representing at
least one epitope of an aggregating protein associated with plaque
formation in the plaque forming disease, the at least one epitope
being capable of eliciting antibodies capable of disaggregating the
aggregating protein and/or of preventing aggregation of the
aggregating protein, such that when the display vehicle propagates
the polypeptide is displayed by the display vehicle.
[0107] Use of beta amyloid peptide antigens in conjunction with
adjuvants to effect immunization has previously been difficult due
to a combination of high toxicity and low titers which result.
Using prior art methods as a starting point, immunization of a
mouse with a 16 amino acids peptide of beta-amyloid conjugated to
KLH (SEQ ID NO: 9) was carried out. This immunization produced a
low but measurable antibody titer against beta-amyloid.
[0108] While reducing one aspect of the present invention to
practice, splenectomy of the immunized mouse facilitated
preparation of IgM hybridoma 508 expressing scFvAb with specificity
to beta-amyloid. RNA was subsequently extracted from this hybridoma
and was employed for antibody cloning. IgM 508 hybridoma showed
specific activity to A.beta. n preventing its toxic affect on PC12
cells (Anavi, S. 1998, M.Sc. thesis from the department of
Molecular Microbiology and Biotechnology of the Tel-Aviv
University, Israel). V.sub.H and V.sub.L sequences of IgM 508 were
cloned separately and linked using a commercially available vector
to form a single chain antibody with anti-beta amyloid specificity.
This single chain antibody was subsequently expressed as a fusion
protein in a phage display library and clones with anti beta
amyloid activity were selected for propagation in E. coli.
[0109] Further reduction to practice was demonstrated by
determining the apparent binding constants of the purified antibody
presenting phage to amyloid beta were measured by ELISA test, and
half-maximal binding was obtained at an antibody concentration of
340 ng/ml, corresponding to 8.times.10.sup.-6 M. This result
anticipate that the prepared single chain antibody will be
effective under in vivo conditions. This phage was also able to
disrupt already formed fibril structures confirming that the
purified single chain antibody is biologically active, as suggested
by the binding constant determination.
[0110] While reducing another aspect of the present invention to
practice, it was uncovered that monoclonal antibodies raised
against a peptide sequence of a prion protein were effective in
disaggregating, or preventing the formation, of prion plaques.
[0111] A model for assessing PrP 106-126 toxicity was established
by the present inventors and utilized to test the effectiveness of
two immunoglobulin clones [designated mAb 3-11 (IgM) and mAb 2-40
(IgG1)] for neuroprotective and disaggregative capabilities.
[0112] As is further detailed in Examples 15-21, both mAb 3-11 and
mAb 2-40 significantly reduced the dose dependent toxic effects of
PrP 106-126 on PC-12 cells. Co-incubation of mAb 3-11 with PrP
106-126 prevented fibrillar aggregation, while administration of
mAb 3-11 to already formed aggregates, resulted in disaggregation
of 50% of the amyloid fibrils (Example 21).
[0113] Thus, according to an additional aspect of the present
invention there is provided a method of treating a plaque forming
disease. The method according to this aspect of the present
invention is effected by displaying a polypeptide representing at
least an immunological portion of an antibody being for binding at
least one epitope of an aggregating protein associated with plaque
formation in the plaque forming disease, the binding capable of
disaggregating the aggregating protein and/or of preventing
aggregation of the aggregating protein, and introducing the display
vehicle into a body of a recipient so as to disaggregate the
aggregating protein and/or prevent its aggregation.
[0114] According to a preferred embodiment of the present
invention, and as is further described hereinbelow and exemplified
hereinunder in the Examples section, introducing the display
vehicle into the body of the recipient so as to disaggregate the
aggregating protein and/or prevent the aggregation of the
aggregating protein is effected through an olfactory system of the
recipient.
[0115] According to yet an additional aspect of the present
invention there is provided an agent for treating a plaque forming
disease. The agent according to this aspect of the present
invention comprising a display vehicle displaying a polypeptide
representing at least an immunological portion of an antibody which
can bind at least one epitope of an aggregating protein associated
with plaque formation in the plaque forming disease, the
immunological portion of the antibody being capable of
disaggregating said aggregating protein and/or of preventing
aggregation of the aggregating protein.
[0116] According to still an additional aspect of the present
invention there is provided a pharmaceutical composition for
treating a plaque forming disease. The composition according to
this aspect of the present invention comprising an effective amount
of a display vehicle displaying a polypeptide representing at least
an immunological portion of an antibody which can bind at least one
epitope of an aggregating protein associated with plaque formation
in the plaque forming disease, the immunological portion of the
antibody being capable of disaggregating the aggregating protein,
and a pharmaceutically acceptable carrier.
[0117] According to a further aspect of the present invention there
is provided a method of preparing a display vehicle for treating a
plaque forming disease. the method according to this aspect of the
present invention is effected by genetically modifying a genome of
a display vehicle by inserting therein a polynucleotide sequence
encoding at least an immunological portion of an antibody capable
of binding at least one epitope of an aggregating protein
associated with plaque formation in the plaque forming disease, the
immunological portion of the antibody being capable of
disaggregating the aggregating protein.
[0118] For purposes of this specification and the accompanying
claims the terms "patient", "subject" and "recipient" are used
interchangeably. They include humans and other mammals which are
the object of either prophylactic, experimental, or therapeutic
treatment.
[0119] For purposes of this specification and the accompanying
claims, the terms "beta amyloid peptide" is synonymous with
".beta.-amyloid peptide", ".beta.AP", ".beta.A", and "A.beta.". All
of these terms refer to a plaque forming peptide derived from
amyloid precursor protein.
[0120] As used herein, "PrP protein", "PrP", "prion" and "prion
protein" refer to polypeptides which are capable, under appropriate
conditions, of inducing the formation of aggregates responsible for
plaque forming diseases. For example, normal cellular prion protein
(PrP.sup.C) is converted under such conditions into the
corresponding scrapie isoform (PrP.sup.Sc) which is responsible for
plaque forming diseases such as, but not limited to, bovine
spongiform encephalopathy (BSE), or mad cow disease, feline
spongiform encephalopathy of cats, kuru, Creutzfeldt-Jakob Disease
(CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal
familial insomnia (FFI).
[0121] As used herein in the specification and in the claims the
term "disaggregating" refers to solubilization of aggregated
proteins typically held together by non covalent bonds.
[0122] For purposes of this specification and the accompanying
claims the terms "comprising" refers to inclusion of one or more
recited element but does not exclude other elements not
specifically recited. For example, a composition that comprises
A.beta. peptide encompasses both an isolated A.beta. peptide and
A.beta. peptide as a component of a larger polypeptide sequence.
Similarly, an immunological portion of an antibody may be included
as a part of a larger of the antibody, say the entire antibody.
[0123] As used herein in the specification and in the claims
section that follows, the term "treating" includes substantially
inhibiting, slowing or reversing the progression of a disease,
substantially ameliorating clinical symptoms of a disease or
substantially preventing the appearance of clinical symptoms of a
disease.
[0124] As used herein in the specification and in the claims
section that follows, the term "plaque forming disease" refers to
diseases characterized by formation of plaques by an aggregating
protein (plaque forming peptide), such as, but not limited to,
beta-amyloid, serum amyloid A, cystantin C, IgG kappa light chain
or prion protein, in diseases such as, but not limited to, early
onset Alzheimer's disease, late onset Alzheimer's disease,
presymptomatic Alzheimer's disease, SAA amyloidosis, hereditary
Icelandic syndrome, senility, multiple myeloma, and to prion
diseases that are known to affect humans, such as for example,
kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Sche-
inker disease (GSS), and fatal familial insomnia (FFI) and animals,
such as, for example, scrapie and BSE.
[0125] Because amyloid plaques associated with the diseases
described hereinabove are located within the brain, any proposed
treatment modality must demonstrate an ability to cross the blood
brain barrier (BBB) as well as an ability to dissolve amyloid
plaques. Normally, the average size of molecules capable of
penetrating the BBB is approximately 2 kDa. Monoclonal antibodies
are typically in the range 135-900 kDa. Therefore, future
therapeutic use of antibodies in treating amyloid plaque diseases
must is rely on either reduction of their size concurrent with
retention of activity, or on development of novel delivery
strategies.
[0126] Small synthetic peptides consisting of antigen epitopes,
such as the EFRH (SEQ ID NO: 1) epitope of A.beta. or the PrP
106-126 peptide (SEQ ID NO: 25) described herein, are in general
poor antigens and need to be coupled to a larger carrier. Even
after coupling they may induce only a low affinity immune response.
For example, injection of A.beta.-KLH or A.beta.-fibril leads to
very slow immune response (Anavi, S., 1998, M. Sc. thesis from the
Department of Molecular Microbiology and Biotechnology of the
Tel-Aviv University) and many efforts have been made to circumvent
low affinity response, with limited success.
[0127] Since the pathological effects of plaque forming
polypeptides are maintained only in the central nervous system
(CNS), the capability of highly specific Abs in preventing such
plaques in vivo is dependent on the permeability of the blood brain
barrier (BBB). For example, in the progressive stage of AD,
evidence shows alteration in the permeability of the BBB, which may
lead to direct delivery of such antibody from the periphery to the
CNS to disaggregate already formed plaques and minimize further
toxic effects (Schenk et al., Nature 1999, 100:173-177). Preferred
embodiments of the present invention include direct delivery of
monoclonal Abs presented on display vehicles to the brain across
the blood brain barrier.
[0128] An increasing body of evidence shows that olfactory deficits
and degenerative changes in the central olfactory pathways are
affected early in the clinical course of AD. Moreover, the anatomic
patterns involved in AD suggest that the olfactory pathway may be
the initial stage in the development of AD.
[0129] Olfactory receptor neurons are bipolar cells that reside in
the epithelial lining of the nasal cavity. Their axons traverse the
cribriform plate and project to the first synapse of the olfactory
pathway in the olfactory pathway in the olfactory bulb of the
brain. This configuration makes them a highway by which viruses or
other transported substances may gain access to the CNS across the
BBB.
[0130] In the early stages of AD, the BBB may limit the entry of
antibody circulating in the periphery to the CNS. In contrast
A.beta. anti-aggregating antibodies displayed on a phage surface
have the potential not only be delivered directly to the CNS by
intranasal administration but also to prevent olfactory permanent
damage by .beta.A in the patients. As previously shown, intranasal
administration (Mathison et al., J. Drug Target, 1998) 5(6),
415-441; Chou et al., Biopharm Drug Dispos. 1997) 18(4):335-46; et
al., Gene Therapy 1995) 2:418-423) enables the direct entry of
viruses and macromolecules into the CSF or CNS.
[0131] Use of olfactory receptor neurons as a point of delivery for
an adenovirus vector to the brain is reported in the literature.
This method reportedly causes expression of a reporter gene in the
brain for 12 days without apparent toxicity (Draghia et al., Gene
Therapy 2:418-423, 1995).
[0132] Thus, according to a preferred embodiment of the present
invention, a vehicle displaying an immunological portion of an
antibody capable of disaggregating, or preventing the formation of,
a polypeptide aggregate associated with a plaque forming disease is
delivered via this route to the brain.
[0133] As A.beta. is produced continuously by cells in peripheral
tissues which cross the blood brain barrier (BBB) leading to
localized toxic effects in specific neuronal populations,
intranasal administration of such a vehicle may also prevent the
progression of the disease by minimizing the amount of peripheral
A.beta. available to form plaques.
[0134] The use of display vehicles such as filamentous phages as a
drug delivery system to the CNS opens new horizons for therapeutic
approaches for Alzheimer's disease, as well as for other
neurodegenerative diseases involving toxic extracellular
aggregation of human peptides such as for example, prion generated
diseases.
[0135] The display vehicle according to the present invention can
be of any type including viral (e.g., bacteriophage, such as
filamentous bacteriophage, fd, for example), bacterial and prion
display vehicles. Thus, for example, the display vehicle can be a
double stranded DNA virus, a single stranded DNA virus, an RNA
virus (positive or negative strand), a bacteria and a polypeptide
carrier. According to a preferred embodiment of the present
invention the display vehicle is capable of propagation in the
recipient. Thus, for example, a bacteriophage display vehicle can
be propagated in bacterial flora, such as Escherichia coli residing
in the recipient's body. Alternatively, the display vehicle is an
in vivo non-propagateable particle.
[0136] The phage or virus vehicle has promise as a targetable in
vivo therapy approach. Although concerns about the potential
infection of the natural intestinal flora (Delmastro et al.,
Vaccine 1997, 15: 1276-1285, Willis et al., Gene. 1993, 128:79-83;
Poul et al., J. Mol. Biol. 1999, 288, 203-211) have been expressed,
UV inactivation of phage showed (Delmastro et al., Vaccine 1997,
15: 1276-1285) that they are as immunogenic as their infective
counterparts. Use of inactivated phage may preclude incorporation
of phage encoded transgenes into the nucleus for subsequent
expression in host cells (Larocca et al., 1998, Hum. Gene Ther.
9:2393-2399), an important practical consideration. Therefore,
according to alternate preferred embodiments, the display vehicles
employed in the present invention may be either replicating or
non-replicating.
[0137] Phage or virus display involves the expression of cDNA
clones as fusion proteins with phage or virus coat proteins. If the
cDNAs selected for expression encode antigens, the phage or virus
may then be employed as an antigen presenting vehicle, which can
optionally replicate within a recipient.
[0138] As described above, according to preferred embodiments of
the present invention, antigens displayed by a phage or virus may
be used directly for vaccination, without antigen purification. In
this case, the bulk of the coat proteins serve to stimulate a
general immune response because they are "non-self" with respect to
the vaccinated subject. The antigen-coat protein fusion elicits a
specific antibody against epitopes in the displayed cDNA gene
product.
[0139] Antibody phage or virus display is accomplished, for
example, by is fusing the coding sequence of the antibody variable
regions to a phage or virus coat protein. To this end, the variable
(V) regions (V.sub.H and V.sub.L) mRNA isolated from
antibody-producing cells is reverse-transcribed into cDNA, and
heavy and light chains assembled randomly to encode single chain Fv
(scFv). These cassettes are cloned directly into a suitable vector
such as a phagemid vector for expression and display on the phage
or virus surface. This linkage between antibody genotype and
phenotype allows the enrichment of antigen specific phage or virus
antibodies, using immobilized or labeled antigen. Phage or virus
that display a relevant antibody will be retained on a surface
coated with antigen, while non-adherent phages or viruses will be
washed away. Bound phages or viruses can be recovered from the
surface, re-infected into suitable host cells and re-grown for
further enrichment and, eventually for binding analysis.
[0140] The success of antibody phage or virus display hinges on the
combination of this display and enrichment method. Phage or virus
antibody genes can be sequenced, mutated and screened to improve
antigen binding.
[0141] It is possible to rearrange the genes which code for the
various regions of an antibody molecule such that its specificity
and affinity for an antigen are altered. The antibody can be
maintained on the surface of the phage or virus for further
manipulation or be released as soluble scFv (.about.25 kDa)
fragment.
[0142] Since its invention at the beginning of the 1990's, antibody
phage display has revolutionized the generation of monoclonal
antibodies and their engineering. This is because phage display
allows antibodies to be made completely in vitro, bypassing the
immune system and the immunization procedure, and allowing in vitro
tailoring of the affinity and specificity of the antibody. It is
therefore anticipated that the most efficient new vaccine
development strategies will employ this technology.
[0143] Additional features can be added to the vector to ensure its
safety and/or enhance its therapeutic efficacy. Such features
include, for example, markers that can be used to negatively select
against cells infected with the recombinant virus such as
antibiotic sensitivity. Negative selection is therefore a means by
which infection can be controlled because it provides inducible
suicide through the addition of antibiotic. Such protection ensures
that if, for example, mutations arise that produce altered forms of
the viral vector or recombinant sequence, cellular transformation
will not occur. Features that limit expression to particular cell
types can also be included. Such features include, for example,
promoter and regulatory elements that are specific for the desired
cell type.
[0144] Viruses are very specialized infectious agents that have
evolved, in many cases, to elude host defense mechanisms.
Typically, viruses infect and propagate in specific cell types. The
targeting specificity of viral vectors utilizes its natural
specificity to specifically target predetermined cell types and
thereby introduce a recombinant gene into the infected cell.
[0145] As used herein in the specification and in the claims
section that follows, the term polypeptide refers to a stretch of
amino acids covalently linked there amongst via peptide bonds.
Different polypeptides have different functionalities according to
the present invention. While according to one aspect a polypeptide
is derived from an immunogen designed to induce an active immune
response in a recipient, according to another aspect of the
invention, a polypeptide is derived from an antibody which results
following the elicitation of an active immune response, in, for
example, an animal, and which can serve to induce a passive immune
response in the recipient. In both cases, however, the polypeptide
is encoded by a polynucleotide according to any possible codon
usage.
[0146] As used herein the phrase "immune response" or its
equivalent "immunological response" refers to the development of a
beneficial humoral (antibody mediated) and/or a cellular (mediated
by antigen-specific T cells or their secretion products) response
directed against an aggregating protein (plaque forming peptide) in
a recipient patient. Such a response can be an active response
induced by administration of immunogen or a passive response
induced by administration of antibody or primed T-cells. A cellular
immune response is elicited by the presentation of polypeptide
epitopes in association with Class I or Class II MHC molecules, to
activate antigen-specific CD4.sup.+ T helper cells and/or CD8.sup.+
cytotoxic T cells. The response may also involve activation of
monocytes, macrophages, NK, cells, basophils, dendritic cells,
astrocytes, microglia cells, eosinophils or other components of
innate immunity.
[0147] As used herein "active immunity" refers to any immunity
conferred upon a subject by administration of an antigen.
[0148] As used herein "passive immunity" refers to any immunity
conferred upon a subject without administration of an antigen.
"Passive immunity" therefore includes, but is not limited to,
administration of a replicating display vehicle which includes an
immunological portion of an antibody presented on its surface to a
recipient. Although replication of such a vehicle is active, the
immune response is passive from the standpoint of the
recipient.
[0149] For purposes of this specification and the accompanying
claims the terms "epitope" and "antigenic determinant" are used
interchangeably to refer to a site on an antigen to which B and/or
T cells respond. B-cell epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
and more usually, at least 5 or 8-10 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed. (1996). Antibodies that recognize the same epitope
can be identified in a simple immunoassay showing the ability of
one antibody to block the binding of another antibody to a target
antigen. T-cells recognize continuous epitopes of about nine amino
acids for CD8 cells or about 13-15 amino acids for CD4 cells. T
cells that recognize the epitope can be identified by in vitro
assays that measure antigen-dependent proliferation, as determined
by .sup.3H-thymidine incorporation by primed T cells in response to
an epitope (Burke et al., J. Inf. Dis. 170, 1110-19, 1994), by
antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et
al., J. Immunol. 156, 3901-3910) or by cytokine secretion.
[0150] The presence of a cell-mediated immunological response can
be determined by proliferation assays (CD4.sup.+ T cells) or CTL
(cytotoxic T lymphocyte) assays. The relative contributions of
humoral and cellular responses to the protective or therapeutic
effect of an immunogen can be distinguished by separately isolating
IgG and T-cells from an immunized syngeneic animal and measuring
protective or therapeutic effect in a second subject.
[0151] As used herein and in the claims, the terms "antibody" or
"immunoglobulin" are used interchangeably and refer to any of
several classes of structurally related proteins that function as
part of the immune response of an animal or recipient, which
proteins include IgG, IgD, IgE, IgA, IgM and related proteins.
[0152] Under normal physiological conditions antibodies are found
in plasma and other body fluids and in the membrane of certain
cells and are produced by lymphocytes of the type denoted B cells
or their functional equivalent. Antibodies of the IgG class are
made up of four polypeptide chains linked together by disulfide
bonds. The four chains of intact IgG molecules are two identical
heavy chains referred to as H-chains and two identical light chains
referred to as L-chains.
[0153] In order to produce polyclonal antibodies, a host, such as a
rabbit or goat, is immunized with the antigen or antigen fragment,
generally with an adjuvant and, if necessary, coupled to a carrier.
Antibodies to the antigen are subsequently collected from the sera
of the host. The polyclonal antibody can be affinity purified
against the antigen rendering it monospecific. Previous experience
has shown that standard production of polyclonal antibodies is not
the method of choice for preparation of disaggregating antibodies
for plaque forming peptides due to problems of poor titer and
toxicity.
[0154] In order to produce monoclonal antibodies hyperimmunization
of an appropriate donor, generally a mouse, with the antigen is
undertaken. Isolation of splenic antibody producing cells is then
carried out. These cells are fused to a cell characterized by
immortality, such as a myeloma cell, to provide a fused cell hybrid
(hybridoma) which can be maintained in culture and which secretes
the required monoclonal antibody. The cells are then be cultured,
in bulk, and the monoclonal antibodies harvested from the culture
media for use. By definition, monoclonal antibodies are specific to
a single epitope. Monoclonal antibodies often have lower affinity
constants than polyclonal antibodies raised against similar
antigens for this reason.
[0155] Monoclonal antibodies may also be produced ex-vivo by use of
primary cultures of splenic cells or cell lines derived from spleen
(Anavi, S., 1998. Locking the N-terminal of the Alzheimer
.beta.-amyloid peptide prevents the neurotoxicity in cell cultures,
M.Sc. thesis. In order to produce recombinant antibody (see
generally Huston et al, 1991; Johnson and Bird, 1991; Mernaugh and
Mernaugh, 1995), messenger RNAs from antibody producing
B-lymphocytes of animals, or hybridoma are reverse-transcribed to
obtain complementary DNAs (cDNAs). Antibody cDNA, which can be full
length or partial length, is amplified and cloned into a phage or a
plasmid. The cDNA can be a partial length of heavy and light chain
cDNA, separated or connected by a linker. The antibody, or antibody
fragment, is expressed using a suitable expression system to obtain
recombinant antibody. Antibody cDNA can also be obtained by
screening pertinent expression libraries.
[0156] The antibody can be bound to a solid support substrate or
conjugated with a detectable moiety or be both bound and conjugated
as is well known in the art. For a general discussion of
conjugation of fluorescent or enzymatic moieties see Johnstone
& Thorpe, Immunochemistry in Practice, Blackwell Scientific
Publications, Oxford, 1982. The binding of antibodies to a solid
support substrate is also well known in the art. See for a general
discussion Harlow & Lane Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Publications, New York, 1988 and
Borrebaeck, Antibody Engineering--A Practical Guide, W. H. Freeman
and Co., 1992.
[0157] As used herein and in the claims, the phrase "an
immunological portion of an antibody" include an F(ab').sub.2
fragment of an antibody, an Fab fragment of an antibody, an Fv
fragment of an antibody, a heavy chain of an antibody, a light
chain of an antibody, an unassociated mixture of a heavy chain and
a light chain of an antibody, a heterodimer consisting of a heavy
chain and a light chain of an antibody, a catalytic domain of a
heavy chain of an antibody, a catalytic domain of a light chain of
an antibody, a variable fragment of a light chain of an antibody, a
variable fragment of a heavy chain of an antibody, and a single
chain variant of an antibody, which is also known as scFv. In
addition, the term includes chimeric immunoglobulins which are the
expression products of fused genes derived from different species,
one of the species can be a human, in which case a chimeric
immunoglobulin is said to be humanized. Typically, an immunological
portion of an antibody competes with the intact antibody from which
it was derived for specific binding to an antigen.
[0158] Optionally, an antibody or preferably an immunological
portion of an antibody, can be chemically conjugated to, or
expressed as, a fusion protein with other proteins. For purposes of
this specification and the accompanying claims, all such fused
proteins are included in the definition of antibodies or an
immunological portion of an antibody.
[0159] As used herein the terms "immunogenic agent" or "immunogen"
or "antigen" are used interchangeably to describe a molecule
capable of inducing an immunological response against itself on
administration to a recipient, either alone, in conjunction with an
adjuvant, or presented on a display vehicle.
[0160] As used herein the term "adjuvant" refers to a compound
that, when administered in conjunction with an antigen, augments
the immune response to the antigen, but when administered alone
does not generate an immune response to the antigen. Adjuvants can
augment an immune response by several mechanisms including
lymphocyte recruitment, stimulation of B and/or T cells, and
stimulation of macrophages.
[0161] A pharmaceutical preparation according to the present
invention includes, as an active ingredient, a display vehicle
displaying at least one epitope of an aggregating protein
associated with plaque formation in a plaque forming disease, the
at least one epitope being capable of eliciting antibodies capable
of disaggregating the aggregating protein. Alternatively, a
pharmaceutical composition according to the present invention
includes, as an active ingredient, a display vehicle displaying at
least an immunological portion of an antibody being for binding at
least one epitope of an aggregating protein associated with plaque
formation in said plaque forming disease, said immunological
portion of said antibody being capable of disaggregating said
aggregating protein.
[0162] The preparation according to the present invention can be
administered to an organism per se, or in a pharmaceutical
composition where it is mixed with suitable carriers or
excipients.
[0163] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0164] Herein the term "active ingredient" refers to the
preparation accountable for the biological effect.
[0165] Hereinafter, the phrases. "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0166] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0167] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0168] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, inrtaperitoneal, intranasal, or
intraocular injections.
[0169] Alternately, one may administer a preparation in a local
rather than systemic manner, for example, via injection of the
preparation directly into the brain of a patient.
[0170] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0171] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0172] For injection, the active ingredients of the invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0173] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for oral ingestion by a patient. Pharmacological
preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries if desired,
to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable
polymers such as polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0174] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0175] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0176] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0177] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroetha- ne or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0178] The preparations described herein may be formulated for
parenteral administration, e.g., by bolus injection or continues
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0179] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0180] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0181] The preparation of the present invention may also be
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0182] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients effective to prevent,
alleviate or ameliorate symptoms of disease or prolong the survival
of the subject being treated.
[0183] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0184] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
circulating antibody concentration or titer. Such information can
be used to more accurately determine useful doses in humans.
[0185] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p.1).
[0186] Dosage amount and interval may be adjusted individually to
provide plasma or brain levels of antibodies which are sufficient
to prevent aggregation or disaggregate existing aggregates (minimal
effective concentration, MEC). The MEC will vary for each
preparation, but can be estimated from in vitro data. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. Binding assays can be
used to determine plasma concentrations.
[0187] Dosage intervals can also be determined using the MEC value.
Preparations, should be administered using a regimen, which
maintains plasma levels above the MEC for 10-90% of the time,
preferable between 30-90% and most preferably 50-90%.
[0188] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0189] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0190] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition, as if further detailed
above.
[0191] The present invention also relates to a method of detecting
both the pathogenic and non-pathogenic form of a prion protein in a
biological sample.
[0192] Thus according to another aspect of the present invention
there is provided a method of detecting a presence or an absence of
a prion protein in a biological sample, the method comprising the
steps of: (a) incubating an anti-prion antibody or an
immunoilogical portion thereof with the biological sample; and (b)
determining a presence or an absence of antibody-antigen complexes,
thereby determining the presence or the absence of the prion
protein in the biological sample.
[0193] It will be appreciated that such complexes can be detected
via any one of several methods known in the art, which methods can
employ biochemical and/or optical detection schemes.
[0194] Thus, this aspect of the present invention provides a method
of assaying or screening biological samples, such as body tissue or
fluid suspected of including a prion protein either in a native
non-disease conformation or a disease related conformation.
[0195] The detection method according to this aspect of the present
invention, can also be utilized for rapid and cost effective
screening of products such as pharmaceuticals (derived from natural
sources), foods, cosmetics or any materials which might contain
prions.
[0196] It will be appreciated that such a detection method can also
be utilized in an assay for uncovering potential anti-prion drugs
usefull in prevention or disaggregation of prion aggregates.
[0197] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0198] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0199] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., as New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
[0200] Reference is made to the following materials and methods,
which were employed in experiments described in the following
examples.
Materials and Experimental Methods
[0201] The following materials and experimental methods were
employed while reducing the present invention to practice as is
further demonstrated in the Examples that follow:
General Recombinant DNA and Phage Techniques
[0202] Standard recombinant DNA techniques were performed
essentially as described (Sambrook et al., 1989). General protocols
for antibody-phage display technology are from the Pharmacia
Biotech (Uppsala, Sweden) Recombinant Phage Antibody System
(RPAS).
Construction of 508 scFv on the Phage Display
[0203] The 508 IgM hybridoma used as the source for antibody
variable-region sequences was generated from splenocytes of a mouse
that had been immunized with a peptide corresponding to the 16
amino terminal residues of .beta.AP conjugated to keyhole limpet
hemocyanin, used as a carrier. mRNA extraction, first strand cDNA
synthesis, PCR amplification of variable heavy (V.sub.H) and
variable light (V.sub.L) sequences, and assembly of scFv cassettes,
were done according to protocols essentially as described
(Pharmacia Biotech RPAS manual). Assembled 508 scFv DNA was
digested with SfiI and NotI, and 100 ng were ligated with 150 ng of
vector DNA prepared by digestion of phagemid pCC-Gal6(Fv)
(Berdichevsky Y et al., J Immunol Methods.,
31;228(1-2):151-62,1999) with SfiI and NotI. This phage-display
system is designed to express the scFv in frame fusion protein with
cellulose binding domain (CBD) derived from Clostridium
thermocellum (Morag E et al. Appl Environ Microbiol., 61(5):1980-6,
1995). Ligated DNA was introduced into XL-1 Blue cells (Stratagene,
La Jolla, Calif.) by transformation and transformants were plated
onto 2.times.YT Agar plates containing 100 .mu.g/ml ampicillin and
1% glucose for overnight growth at 37.degree. C.
Selection of .beta.-amyloid Binding scFv-CBD Fusion Proteins
[0204] Individual clones were picked and grown, each in 5 ml
2.times.YT, 1% glucose, 100 .mu.g/ml Ampicillin overnight at
30.degree. C. IPTG was added at 1 mM for a 3 hr induction period.
Soluble scFv-CBD fusion proteins were isolated from each clone by
sonication of induced cell pellets. In order to identify functional
soluble 508(Fv) from non-functional ones, 250 ng/well
.beta.-amyloid peptide were covalently bound to epoxy-coated
microtiter plates for 16 hr at 4.degree. C. (Solomon, B., et al,
Proc. Natl. Acad. Sci. USA., 93:452-455,1996). The plates were
washed with PBS/0.05%--Tween 20 (PBST), is and blocked with a
mixture of 3% bovine serum albumin and milk powder in PBS for 16 hr
at 4.degree. C. The plates were then washed and incubated with the
soluble scFv-CBD recovered from the clones for 1 hr at 37.degree.
C. The bound antibody was detected with a rabbit anti CBD antiserum
followed by HRP-conjugated goat anti rabbit antibodies. Plates were
developed with the peroxidase chromogenic substrate ABTS and the
signal was recorded with an ELISA microtiter plate reader at 405
nm. Positive phage clones (pCC-508(Fv)) were propagated and their
DNA was sequenced using an automated model 373A DNA sequencer
(Applied Biosystems, USA).
Production of 508(Fv)-CBD Fusion Proteins in E. coli
[0205] For high level expression in E. coli, wild type (wt) and
mutated 508(Fv) derivatives were cloned into the pFEKCA3 vector as
described (Berdichevsky Y et al, Protein Expr Purif., 17(2):249-59,
1999). This vector utilizes the strong T7 promoter for expression,
where the T7 RNA polymerase gene is carried as a lac repressor
controlled-IPTG inducible gene in E. coli BL21 (DE3) (Studier, F.
W., et al., Methods Enzymol., 85, 60-89, 1990). Upon IPTG
induction, 508(Fv)-CBD proteins accumulated as insoluble inclusion
bodies. They were recovered by the cellulose-assisted refolding
method as previously described (Berdichevsky Y et al, Protein Expr
Purif., 17(2):249-59, 1999). SDS polyacrylamide gel electrophoresis
(SDS/PAGE) was used to separate proteins according to their
molecular weight under denaturing conditions (Laemmli, U.K., Nature
227:263-270,1970).
Stability Assay of the Purified 508(Fv)-CBD Protein
[0206] The activity of purified 508(Fv)-CBD protein was checked
before and after storage at 4.degree. C. for 7 days. 250 ng/well
.beta.-amyloid peptide was covalently bound to epoxy-coated wells
of microtiter plates for 16 hr at 4.degree. C. (Solomon B. et al.,
Proc. Natl. Acad. Sci. USA. 94, 4109-4112, 1997). Wells were
blocked with a mixture of 3% bovine serum albumin and bovine
hemoglobin in PBS for 2 hr at 37.degree. C., then washed and
incubated with the 508(Fv)-CBD protein (0.5 .mu.g/ml or as
otherwise specified) for 1 hr at 37.degree. C. Bound antibody
fragments were detected by incubation with HRP-conjugated rabbit
anti-mouse antibodies (BioMakor, Rehovot, Israel), diluted 1:5,000
and rabbit anti CBD diluted 1:10,000 in PBST for 1 hr at 37.degree.
C. The bound antibody fragments were monitored as described
above.
Construction of a Phage Library for the Isolation the 508(Fv)
.beta.AP Binding Mutants
[0207] Splicing overlap extension (SOE) PCR technique (Lefenbrve,
B., et al., Biotechniques, 19:186-188, 1995) was used to replace
V.sub.L cysteine codon 96 of 508(Fv) with other codons. pCC-508(Fv)
DNA was used as template. In a first step, the template DNA was
amplified with the following primers:
[0208] The antisense primer 508-mut-FOR: 5'-CCCCCCTCCGAAC
GTSNATGGGTAACTcgatcgCTGATGGCAGTA-3' (SEQ ID NO: 10) inserts a PvuI
restriction site (underlined), where S represents nucleotides C or
G and N represents A, C, T or G. This primer was used for the
replacement of cysteine codon 96 with phenylalanine (F), leucine
(L), serine (S), tyrosine (Y) or tryptophan codons. The primer SfiI
5'BACK: 5'-ATCTATGCggcccagccggccATG-3' (SEQ ID NO: 11) inserts an
SfiI site at the 5' end of the scFv. The resulting PCR product
(SfiI-508 mut) corresponds to the 5' half of 508(Fv)-CBD. In the
second PCR step, the complete 508(v)-CBD was re-assembled by
amplifying pCC-508(Fv) DNA with the SfiI-508mut PCR product from
step 1 serving as the 5' end primer and CBD(BX):
5'-GTGGTGCTGAGTggatccta TACTACACTGCCACCGGG-3' (SEQ ID NO: 12) as
the 3' end primer. The final PCR product (SfiI-508mut-BX) is a
complete 508(Fv)-CBD cassette with replacements at V.sub.L codon 96
and an engineered PvuI restriction site as a silent mutation for
analysis. SfiI-508mut-BX DNA was digested with SfiI, PvuI and NotI
and ligated in a three fragment ligation with SfiI and NotI
linearized pCC-Gal6(Fv) DNA which is a phagemid vector used to
display an anti E. coli .beta.-galactosidase scFv (Berdichevsky Y
et al, J Immunol Methods., 31;228(1-2):151-62, 1999). The resulting
ligated phagemid DNA was introduced into E. coli XL-1-Blue cells by
electroporation. Cultures of E. coli were used to produce
displaying phage by rescue with M13KO7 helper phage (Pharmacia
Biotech, Uppsala, Sweden).
Affinity Selection of .beta.-amyloid Binding 508mut-(Fv) Displaying
Phage Clones
[0209] A sample containing rescued phage particles was subjected to
one round of affinity selection (biopanning) and amplification. For
the selection cycle, 0.5 .mu.g/ml biotinylated .beta.-amyloid 1-16
amino acid peptide (.beta.AP(1-16), acids 1-16 OF SEQ ID NO: 3) in
a total volume of 1 ml were used. The phages were pre-incubated
with the biotinylated peptide for 2 hr at room temperature, and the
reaction mixture was then layered on streptavidin-coated 30 mm
polystyrene Petri dishes and incubated for 20 min at room
temperature. Unbound phages were removed by extensive washing with
PBST. The bound phages were eluted with 0.3 ml of 0.1 M HCl
titrated to pH 2.2 with glycine. The eluate was neutralized with 80
.mu.l of 0.5 M Tris (HCl) pH 10, and used to infect E. coli
XL-1-Blue cells. Individual bacterial colonies containing amplified
phage particles were used as a template for colony PCR (Novagen
Madison, USA) with primers SfiI5'Back and CBD(BX). The PCR product
of about the size of an intact scFv-CBD fragment (about 1250 bp)
was digested with the restriction enzyme PvuI and analyzed by
agarose gel electrophoresis.
ScFv Binding to Biotinylated .beta.AP(1-16)
[0210] Binding of scFv to .beta.AP(1-16) was analyzed by ELISA.
Coated plates with 50 .mu.l of 1 .mu.g/ml streptavidin in 0.1 M
NaHCO.sub.3, pH 9.6, were washed three times with PBST and 50 .mu.l
of 6 ng/.mu.l biotinylated .beta.AP(1-16) were then added to the
wells and incubated for 30 min at 37.degree. C. Wells were blocked
with a mixture of 3% bovine serum albumin and bovine hemoglobin in
PBS for 2 hr at 37.degree. C., then washed and incubated with the
scFv (0.5 .mu.g/ml or as otherwise specified) for 1 hr at
37.degree. C. For inhibition experiments, peptides were
pre-incubated with the antibody for 30 min at 37.degree. C. before
their addition to peptide coated wells. After washing, bound
antibody fragments were detected as described above. .beta.AP
specific binding phage clones were propagated and their DNA was
isolated and sequenced as described above.
Cell Culture and .beta.AP Cytotoxicity Assay
[0211] Rat phenochromocytoma PC12 cells were cultured in DMEM
supplemented with 5% horse serum, 10% fetal calf serum, 2 mM
L-glutamine, and 100 units/ml penicillin/streptomycin and incubated
at 37.degree. C. under 5% CO.sub.2. For the neurotoxicity assay,
cultured PC12cells were seeded into a 96-well plate at a density of
10.sup.4 cells/100 .mu.l/well in a serum-free medium supplemented
with 2 M of insulin. The effect on the prevention of the
neurotoxicity of .beta.A was measured as follows: 0.12 mM
.beta.-amyloid that was incubated for a week at 37.degree. C. for
the generation of fibrils, and further incubated in the presence of
508F(Fv)-CBD or with the unrelated Gal6(Fv)-CBD at a molar ratio of
.beta.AP to scFv of 15:1 or 30:1 for 24 hr. The .beta.A/antibody
mixture was added to the wells containing PC12 cells. The plates
were incubated at 37.degree. C. for 2 days, after which cell
viability was assessed by measuring cellular redox activity with
3-(4,5-dimethylthiazol-2-yl)-2,5-d- iphenyl tetrazolium bromide
(MTT), as described (Sladowski, D. et al., J. Immunol. Methods.,
157:203-207,1993). The plates were incubated overnight at
37.degree. C. MTT reduction was determined colorimetrically using
an ELISA microtiter plate reader set at 550 nm.
Aggregation of .beta.-amyloid peptide Measured by Thioflavin T
(ThT) Fluorimetry
[0212] Aggregation of .beta.-amyloid peptide was measured by the
Thioflavin T (ThT) binding assay, in which the fluorescence
intensity reflects the degree of .beta.-amyloid fibril formation.
ThT characteristically stains amyloid-like deposits (Levine, H.
III, Protein Sci., 2: 404-410,1993) and exhibits enhanced
fluorescence emission at 485 nm upon excitation at 435 nm when
added to the suspension of aggregated .beta.-sheet preparations.
Aqueous solutions of 0.12 mM .beta.AP in 0.1 M Tris (HCl) pH 7.1
were incubated at 37.degree. C. for 1 week and further incubated in
the presence of 508F(Fv)-CBD or with the unrelated Gal6(Fv)-CBD at
a molar ratio of .beta.AP to scFv of 15:1 or 30:1 for 24 hr. The
fluorescence was measured after addition of 1 ml of ThT (2 .mu.M in
50 mM Glycine, pH 9) with an LSB-50 Perkin Elmer Ltd., UK,
spectrofluorimeter.
Preparations of Phage Delivery System
[0213] 12 Balb/c-female mice were divided to four groups of 3 mice
per group. One group was used as control. Following a single dose
of 10.sup.11 phage particles (fd phage, taken from a 15-mer phage
peptide library which was provided by George P. Smith, University
of Missouri, Columbia, Mo.) administered intranasally, mice were
sacrificed in intervals of 1, 14 and 28 days in each group and
their brains were taken for further analysis.
Ability of Phage Carrying scFv to Enter/Remove .beta.AP Fragment
from the Brain
[0214] ScFv-508F fusion to filamentous minor coat gpIII were used
in order to investigate the ability of .beta.AP anti-aggregating
scFv to be carries by a fillamentous phage display system directly
into the CNS.
[0215] This scFv was prepared from anti-aggregation hybridoma 508
as described above and preserved its specific binding activity.
Nine Balb/c mice divided into three groups were treated as follows:
Mice of a first group were treated with 0.2 ml of 10.sup.-3 M
biotin .beta.A(1-16) alone. Mice of a second group were treated
with a mix of 10.sup.10 phage carrying 508F-scFv which were pre
incubated with 0.2 ml of 10.sup.-3 M biotin .beta.A(-16) for 1 hr.
Mice of a third group were used as control. Following a single dose
applied intranasally, mice were sacrificed in each group in
intervals of 1, 14 and 28 days and their brains were taken for
further analysis.
Preparations of Tissue Sections
[0216] Immediately following decapitation, brains were removed and
cut into two halves along the mid-sagittal sinus. Randomly, one
half-brain was fixed by immersion in 4% paraformaldehyde solution
in 0.1 M phosphate buffer for two hours in 4.degree. C. and then
immersed for cold protection in 4.5% sucrose in 0.1 M PBS over
night. The sections were then moved to 30% sucrose for 2 hr in
4.degree. C. Sections of coronal blocks containing the olfactory
and hippocampus were put in OCT and cut with thick-nesses of 6
.mu.m with a cryostat at -20.degree. C., and then taken up on glass
slides. Slides were kept at -70.degree. C. These slides were used
for phage detection using an immunofluorescence technique.
[0217] The other mid-sagittal half-brain was used for preparing
paraffin tissue section for histology. The section were fixed in 4%
paraformaldehyde for 2 hr, then transferred to 10% formalin saline
for 2 days in room temperature, followed by embedding in paraffin,
and cut with thick-nesses of 4 .mu.m on a microtom and then taken
up on glass slides. The slides were kept at room temperature until
used.
Detection of Antigen in Brain Sections
[0218] Immunofluorescence: Sections were blocked with 3% bovine
serum albumin in PBS for 30 min and then incubated with rabbit
polyclonal serum anti fd (1:100) or Streptavidin coupled with PE
(sigma) for 1 hr at 37.degree. C. Slides were then washed three
times, 5 min each, in PBS, treated again with the blocking buffer
for 5 minutes at room temperature, and then reacted with secondary
r Cy.sup.TM 3 donkey anti-rabbit IgG (for phage detection) at 1:400
(sigma) or, with streptavidin coupled to PE, 1:50 dilution, for 1
hr at room temperature. Finally, the preparations were washed three
times in PBS, observed using a fluorescence microscope at a final
magnification of .times.10, and recorded on film or using a
Hamamatsu digital camera (C4742) and Metamorph Universal Imaging;
West Chester, Pa.) computer software.
[0219] Histology: Six-micrometer sections were stained with
hematoxylin and eosin. The stained sections were examined and
photographed at a final magnification of .times.40. Finally, the
preparations were washed three times in PBS, observed on a
microscope, and recorded on film.
Immunization with f3-YYEFRH
[0220] Immunizations were performed with a genetically engineered
fd phage carrying the peptide YYEFRH (SEQ ID NO: 7) fused to its
minor coat gpIII. Doses of 10.sup.10 phages per injection were used
to immunize at 14-day intervals, through intraperitoneal
injections. Mice were injected the phages with or without Freund's
complete adjuvant (Difco) for the first injection and Freund's
incomplete adjuvant (Difco) for the second injection. Following 7
days of each injections, the mice were bled and their serum were
tested by ELISA for antibody IgG reactivity for both phage coat
proteins and for .beta.A.
Epitope Libraries
[0221] The 15-mer phage-peptide library used in this study was
provided by George P. Smith (University of Missouri, Columbia,
Mo,). The library consists of about 1.9.times.10.sup.9 phage
particles and comprises a random peptide repertoire of 15 amino
acid residues fused to coat glycoprotein VIII of the fd phage.
Experiments with this library were carried out according to
instructions of the provider (George P. Smith University of
Missouri, Columbia, Mo.).
Biotinylation of Antibodies
[0222] For antibody biotinylation, 100 .mu.g of each antibody in
0.1 M NaHCO.sub.3, pH 8.6, was incubated for 2 hr at room
temperature with 5 .mu.g of biotinamidocoproate
N-hydroxysuccinimide ester (Sigma, B 2643) from a stock solution of
1 mg/ml in dimethylformamide and dialyzed at 4.degree. C. against
phosphate-buffered saline (PBS; 0.14M NaCl/0.01 M phosphate buffer,
pH 7.4) overnight.
Isolation of Phage Presenting Epitopes from Peptide Library
[0223] A library sample containing 10.sup.9 infectious phage
particles was subjected to three rounds of selection (biopanning)
and amplification. For each selection cycle a biotinylated
monoclonal antibody (1 .mu.g/.mu.l) in a total volume of 25 .mu.l
was used. The phage clones were pre incubated with the biotinylated
antibody overnight at 4.degree. C., and the reaction mixtures were
then layered in 1 ml of PBS containing 0.5% Tween 20 on
streptavidin-coated 30 mm polystyrene Petri dishes and incubated
for 20 min at room temperature. Unbound phages were removed by
extensive washing (10 times for 10 min each) in PBS/0.05% Tween 20.
The bound phages were eluted with 0.3 ml of 0.1 M HCl titrated to
pH 2.2 with glycine. The eluate was neutralized and used to infect
E. coli K91 cells. After three rounds of panning individual
bacterial colonies containing amplified particles were grown on a
microtiter plate and the selected phages were tested by ELISA for
their ability to bind to the studied antibody, as described
below.
Antibody Binding to Isolated Phage
[0224] Binding of antibodies to phage was analyzed by ELISA. Wells
of microtiter plates (Maxisorb, Nunc) were coated with 50 .mu.l (at
dilution of 1:1000 in 0.1 M NaHCO.sub.3, pH 8.6) of rabbit
anti-phage serum and incubated overnight at 4.degree. C. The wells
were blocked with a mixture of 3% bovine serum albumin and
hemoglobin at a ratio of 1:1 (in PBS) for 2 hr at 37.degree. C.
Coated plates were washed three times with PBS/0.05% Tween 20, and
50 .mu.l of enriched phage clones containing 10.sup.10 phage
particles were added to the wells and incubated for 1 hr at
37.degree. C. After washing, the studied antibody was added (1
.mu.g/ml or as otherwise specified) and allowed to bind to the
coated plate overnight at 4.degree. C. and the binding constant
thereof was measured. Positive phage clones were propagated and
their DNA were sequenced in the insert region at the Sequencing
Unit of the Weizmann Institute of Science (Rehovot, Israel) by
using Applied Biosystem Kit (United States, Applied Biosystem).
[0225] fd gpVIII Phage Display of .beta.A(1-16)
[0226] Coat glycoprotein VIII of filamentous phage is presented in
approximately 2700 copies on the phage coat. The following
oligonucleotides were prepared: sense--5'-agctccGATGCTGAATTCGG
TGATAGCGGCTACGAAGTGCATCATCAGAAAcctgcag-3' (SEQ ID NO: 13); and
antisense--5'-ggTTTCTGATGATGCACTTCGTAGC
CGCTATCATGACGAAATTCAGCATCgg-3' (SEQ ID NO: 14). These
oligonucleotides were used to form a duplex (68-70.degree. C., 10
minutes, followed by slow cool to room temperature) which encodes
for amino acids 1-16 of human .beta.AP and contain a silent
mutation of a specific restriction site (EcoRI) which is useful for
further analysis. The duplex was phosphorylated and lygated into
HindIII/PstI linearized f88-4 phagemid, which is a vector used to
display fusion peptides on gpVIII of filamentous phage. The
resulting ligated phagemid DNA was introduce into E. coli K91K
cells by transformation and transformants were plated onto
2.times.YT Agar plates containing 10 .mu.g/ml tetracycline and 1%
glucose for overnight growth at 37.degree. C. Individual bacterial
colonies containing phage particles were used to inoculate 2YT
medium containing 10 .mu.g/ml tetracycline for overnight growth at
37.degree. C. for amplification. The DNA phagemid product obtained
from each colony was analyzed by EcoRI. Positive clones were
further amplified for antigen preparation.
Immunization with f88-EFRH
[0227] Immunizations were performed with a genetically engineered
fd phage carrying the peptide VHEPHEFRHVALNPV (SEQ ID NO: 8) fused
to its major coat glycoprotein VIII. Doses of 10.sup.10 phages per
injection were used to immunize at 14-day intervals, through
intraperitoneal injections. Mice were injected the phages with or
without Freund's complete adjuvant (Difco) for the first injection
and Freund's incomplete adjuvant (Difco) for the second injection.
Following 7 days of each injections, the mice were bled and their
serum were tested by ELISA for antibody IgG reactivity for both
phage coat proteins and for .beta.A.
Inhibition of Antibody Binding to .beta.-amyloid Peptide
[0228] The inhibition of antibody binding to .beta.AP(1-16) by
various small peptides was performed using 250 .mu.g/well
biotinylated .beta.-amyloid peptides (1-16) bound covalently to
ELISA plates as previously described. The plates were washed with
PBS/0.05% Tween 20 and blocked with a mixture of 3% bovine serum
albumin and hemoglobin, ratio 1:1 (in PBS) for 2 hr at 37.degree.
C. The peptides were pre incubated with 1:3000 dilution of serum
after third immunization with f88-EFRH for 30 min at 37.degree. C.
before their addition to .beta.AP-coated wells and were left
overnight at 4.degree. C. therein. After washing, bound antibody
was detected by incubation with HRP-conjugated rabbit anti-mouse
immunoglobulin, as described above. The results were used to derive
the IC.sub.50, which is the half molar concentration of peptide
that fully inhibits antibody binding. Peptides were synthesis by
Applied Biosystems Synergy Model 430A in the Unit for Chemical
Services of The Weizmann Institute of Science by solid-phase using
Fmoc chemistry.
Cell Culture and Cytotoxicity Assay
[0229] Rat phenochromocytoma PC12 cells were cultured in DMEM
supplemented with 5% horse serum, 10% fetal calf serum, 2 mM
L-glutamine, and 100 units/ml penicillin/streptomycin and incubated
at 37.degree. C. under 5% CO.sub.2. For the neurotoxicity assay,
cultured PC12 cells were seeded into a 96-well plate at a density
of 10.sup.4 cells/100 .mu.l/well in a serum-free medium
supplemented with 2 M of insulin. The effect on the prevention of
the neurotoxicity of .beta.A was measured as follows: 0.12 mM
.beta.-amyloid that was incubated for a week at 37.degree. C. for
the generation of fibrils, and further incubated in the presence of
serum of EFRH-phage immunized mice and serum of a non-relevant
phage immunized mice at dilutions of 5:1 and 10:1 for 24 hr. The
.beta.A antibody mixture was added to the wells containing PC12
cells. The plates were incubated at 37.degree. C. for 2 days, after
which cell viability was assessed by measuring cellular redox
activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-d- iphenyl
tetrazolium bromide (MTT), as described (Sladowski, D. et al., J.
Immunol. Methods., 157:203-207,1993). The plates were incubated
overnight at 37.degree. C. MTT reduction was determined
colorimetrically using an ELISA microtiter plate reader set at 550
nm.
Preparation of Monoclonal Antibodies Against PrP 106-126
[0230] Mice immunized with synthetic peptide corresponding to the
sequence of human PrP 106-126 (SEQ ID NO: 25)(obtained from Chiron
Technologies, Claton Victoria, Australia) coupled to the larger
carrier KLH were used for generating monoclonal antibodies
following the fusion techniques of Kohler and Milstein (Kohler and
Milstein 1975).
[0231] Hybridomas were tested for the production of
peptide-specific antibodies by ELISA, as follows: Peptide PrP
106-126 was covalently attached to the epoxy groups of Eupergit-C
coated 96 well plates (Solomon et al. 1992, 1993). The residual
epoxy groups were blocked by incubating the plates with 3% skim
milk (blocking solution). Undiluted hybridoma supernatants were
applied for 1 hour at 37C. Wells were excessively rinsed (as in
each step of the procedure) and further incubated with horseradish
peroxidase (RP) labeled goat-anti-mouse antibodies specific for
mouse IgG or IgM (diluted in blocking solution). After washing,
antibody binding was visualized using ortho-phenyldiamine as
substrate for HRP. Optical density was measured at 492 nm. Selected
monoclonal antibodies were scaled-up and purified according to
published procedures: IgG molecules on a protein A column (Harlow
et al. 1988). and IgM on KaptiveM coloumn. Two mAbs, namely 2-40
and 3-11, were used for further studies. The mAb 3F4 was purchased
from Senetek, Calif. USA.
Search for Antibody's Epitope Location Using Phage Display
Library
[0232] The antibodies 3-11 (IgM) and 2-40 (IgG) were biotinylated.
The following libraries (provided by G. P. Smith) were searched to
find the epitope of the antibodies studied, as previously described
(Frenkel et al. 1998). 1. fUSE5/15-mer library where foreign 15-mer
are displayed on all 5 copies of pIII. 2. f884/6-mer library where
foreign 6-mer are displayed on up to .about.300 copies of pVIII
(recombinant gene VIII encoding the peptide is inducible with
IPTG)
[0233] The biopanning of the phages to find the antibodies' epitope
was performed as previously described (Frenkel et al. 1998).
Competitive Inhibition of Antibodies Bound to PrP by Peptide
NMKH
[0234] The ELISA competitive binding of the above antibodies to
covalently bound PrP peptide was performed as described above. The
antibodies were preincubated with peptide NMKH at equimolar ratio
before adding to the wells.
PrP 106-126 Aggregation and Immunocomplexation
[0235] In vitro aggregation of peptide 106-126 was induced by
incubation of an aqueous solution of PrP 106-126 (10 mg/ml) for
various time intervals at 37.degree. C. The aggregated peptide was
incubated either with monoclonal antibodies 2-40, 3-11 or 3F4 at
conditions specified later.
Cytotoxicity Assay of PrP 106-126 Using PC12 Cells
[0236] Rat pheochromocytoma PC12 cells were cultured in DMEM
supplemented with 8% horse serum, 8% fetal calf serum, 2 mM
L-glutamine and 100 units/ml penicillin/streptomycin and incubated
at 37.degree. C. under 5% CO.sub.2. For the neurotoxicity assay,
cultured PC12 cells were seeded on 96-well plates at a density of
2.times.10.sup.4 cells/100 .mu.l/well in a serum-free medium
supplemented with 2 .mu.M of insulin. Cells were treated for 3-5
days with 100 .mu.M PrP 106-126 preincubated 4-7 days at 37.degree.
C. The cell viability was assessed by the MTT assay which measures
the activity of mitochondrial enzymes responsible for the
conversion of the tetrazolium salt,
3-(4,5-dimethylthiazol-2-yl)-2,5-diph- enyl-tetrazolium bromide
(AT) to a formazan product in viable cells (Hansen et al. 1989).
MTT was added to the wells to a final concentration of 1 mg/ml and
incubated with the cells for an additional 3 hours at 37.degree. C.
Cell lysis buffer (20% wt/vol SDS in a solution of 50%
dimethylformamide, pH 4.7) was added, and the plate was incubated
overnight at 37.degree. C. MTT reduction was determined
colorimetrically by measuring the optical density (OD) at 550
nm.
Prevention of PrP 106-126 Neurotoxicity
[0237] The effect of mAbs on the inhibition of PrP 106-126
neurotoxicity was determined as follows: PrP 106-126 (10 mg/ml) was
incubated for 7 days at 37.degree. C. to induce maximal aggregation
of the peptide. Monoclonal antibodies 3-11, 2-40 and 3F4 were added
for 1 hour to samples of 1 mM of the already aggregated peptide.
The antibody-peptide mixtures, as well as the aggregated peptide
alone, were applied to the cells to a peptide final concentration
of 100 .mu.M. Cell viability following a 3 day incubation at
37.degree. C. with the aforementioned reaction mixture, was
assessed as described above. 100% viability was defined as the
value of MTT assay for untreated cells.
Modulation of PrP Conformation Followed by Thioflavin T (ThT)
Fluorimetry Assay
[0238] Increasing amounts of PrP 106-126 peptide (0-0.8 mg/ml) were
incubated for 7 days at 37.degree. C. Prion amyloid fibril
formation was measured by the Thioflavine T (ThT) binding assay.
The binding of ThT to amyloid fibrils of certain origins generates
a specific fluorescent signal: a 114 nm red shift in the excitation
peak from 336 nm of excitation spectrum of the free dye in solution
to a new excitation peak,at 450 nm of the bound dye. Additionally
the bound dye has an enhanced emission at 482 nm (Naiki et al.
1989, LeVine 1993). The aggregation of the prion peptide was
followed using samples of PrP 106-126 (0.3 mg/ml) in 0.1M Tris/HCl
pH-7.1 incubated for 7 days at 37.degree. C., either with or
without mAbs 3-11, 2-40 and 3F4 at various dilutions.
Disaggregation of already formed prion amyloid fibrils was measured
using samples of PrP 106-126 that were incubated for 7 days at
37.degree. C. and then supplemented with the mAbs for an additional
24 hours. Fluorescence (emission at 482 nm after excitation at 435
nm) was measured after an addition of the samples to ThT (2 .mu.M
in 50 mM glycine, pH-9).
Experimental Results
[0239] Examples 1-6 below relate to the production of a single
chain version of the anti aggregating monoclonal antibody. Examples
7-8 below relate to delivery of peptide or antibody displaying
phage to the brain. Examples 9-14 below relate to the production of
high titers of anti-aggregating polyclonal antibodies by direct
immunization with beta amyloid antigens displayed on a phage, and
to characterization of these antibodies.
EXAMPLE 1
Generation of an IgM Hybridoma 508
[0240] Immunization of a mouse with a 16 amino acid peptide of
beta-amyloid (acids 1-16 of SEQ ID NO: 3) conjugated to KLH (SEQ ID
NO: 9) was carried out as described hereinabove. Repetitious
immunization eventually produced a low but measurable antibody
titer against beta-amyloid. Subsequent splenectomy of the immunized
mouse facilitated preparation of IgM hybridoma 508 expressing
scFvAb with specificity to beta-amyloid. RNA was subsequently
extracted from this hybridoma. The IgM 508 hybridoma showed
specific activity to A.beta. in preventing its toxic affects on
PC12 cells (Anavi, S. 1998, M.Sc. thesis from the department of
Molecular Microbiology and Biotechnology of the Tel-Aviv
University, I).
EXAMPLE 2
Cloning of the Variable Domains of the 508 IgM Hybridoma as a
scFv
[0241] MAb 508 showed specific recognition of .beta.-amyloid and
prevented its toxic affects on PC12 cells (Anavi S., 1998, ibid).
For cloning the 508 antibody as a scFv in a phage display vector,
RNA was extracted from 10.sup.8 508 hybridoma cells and was used as
a source for antibody variable region coding sequences. RT-PCR was
used to amplify the variable domains that were cloned into the
phage display vector pCC-Gal6(Fv), as described in Materials and
Methods. When hybridoma derived antibodies are cloned as scFvs,
some of the clones may contain aberrant sequences that are not
functional. Therefore, to identify phagemid clones carrying
functional .beta.-amyloid binders from the generated clones, 10
individual clones were picked at random and soluble scFv-CBD fusion
protein was produced thereby. FIG. 2 shows a physical map of
plasmid pCC-508 which was used to express the 508-scFv. The CBD
domain serves as an immunological detection of soluble scFv protein
or as a novel approach in refolding of soluble scFv protein
inclusion bodies of overexpressed protein (Berdichevsky Y et al,
Protein Expr Purif., 17(2):249-59, 1999). The plasmid used for
508-scFv over expression is shown in FIG. 3. The soluble scFv-CBD
from the selected clones was incubated in wells of an ELISA plate
that has been coated with .beta.-amyloid peptide. Of the analyzed
clones, 50% showed specific binding to .beta.AP. FIGS. 1a-e
demonstrate and illustrate the preparation of 508 scFv from IgM
antibody. FIG. 4 shows .beta.AP binding by the scFv-CBD produced by
a positive clone that was chosen for further analysis. PCR analysis
was used to characterize its DNA insert. It was found that the
positive clone (designated pCC-508(Fv)) contained an intact DNA
insert (FIG. 5). DNA sequencing of pCC-508(Fv) confirmed that the
clone expresses an intact scFv fragment (see, a FIGS. 11a-b and SEQ
ID NOs: 5 and 6, for nucleic and amino acid sequences,
respectively, modified as further described below).
EXAMPLE 3
Site Directed Mutagenesis of 508-(Fv) Antibody
[0242] The DNA sequencing analysis of pCC508-(Fv) revealed the
unusual appearance of a cysteine residue at the position 96 of
V.sub.L CDR3 (Kabat, E. A. et al., Sequences of proteins of
immunological interest, 5th Ed., 1991). The deduced amino acid
sequence of V.sub.L CDR3 is: H.sup.89QRSSYPCT.sup.97 (SEQ ID NO:
15). The presence of an unpaired cysteine residue in a scFv may
reduce its folding yield and also decrease its stability in
solution and its storage half life. Therefore, 508(Fv) was
subcloned into an expression vector and produced in E. coli as
described in Materials and Methods. FIG. 6 summarizes the
production process of 508(Fv)-CBD by the cellulose-assisted
refolding method (Berdichevsky Y et al, Protein Expr Purif.,
17(2):249-59, 1999). Although 508(Fv)-CBD could be purified to near
homogeneity (FIG. 6 lane 7) by this method, it refolded relatively
poorly and was unstable upon storage at 4.degree. C. (FIG. 7). It
was assumed that substitution of the cysteine with a different
residue may increase the production yield and stability of the
soluble scFv without having an adverse affect on its affinity
(Kirpriyanov, M. S. et al., Protein Engineering, 10:445-453,
1997).
[0243] For the replacement of the 508 V.sub.L cysteine 96 codon SOE
PCR was used, which enabled the replacement of Cys 96 with
phenylalanine, leucine, serine, tyrosine or tryptophan codons. In
addition, the PCR scheme employed permits the persistence of the
cysteine residue at that position. 508(Fv) mutants were cloned into
the pCC-Gal6(Fv) phage vector, resulting in the generation of a
micro library (having 6 potential variant). The replacing residues
chosen are generally acceptable at that CDR3 position, as they are
found in various antibodies in the Kabat database (Kabat, E. A. et
al., Sequences of proteins of immunological interest, 5th Ed.,
1991). However, different replacements may vary in their effect on
.beta.AP binding. To test which replacement maintains .beta.AP
binding, a single cycle of affinity selection was performed on the
508(Fv)-Mut micro phage library using biotinylated .beta.AP(1-16)
as a capturing antigen. PCR amplification and restriction analysis
was used to monitor the enrichment of library clones after the
affinity selection cycle. When the 508Mut-(Fv)-CBD DNA is digested
with PvuI, a typical restriction pattern is obtained upon
agarose-gel electrophoresis and ethidium-bromide staining. The
lower 750 and 500 bp fragments represent the 508Mut-(FV)-CBD DNA,
while an intact 1250 bp fragment represent scFv-CBD from the
pCC-Gal6(Fv) DNA which was used as a vector. It was found that
before affinity selection the library was heavily contaminated with
the pCC-Gal(Fv) vector DNA. This is evident from the fact that the
DNA of 18/19 randomly picked library clones was not cleaved at the
PvuI site engineered adjacent to 508 V.sub.L position 96. Only one
of the 19 analyzed clones showed the expected restriction pattern
associated with a mutation (FIG. 8a). However, after affinity
selection, 5/11 randomly selected clones showed the expected
restriction pattern is (FIG. 8b). This indicates an enrichment
factor of about 10 fold, which demonstrates the ability of 508 scFv
mutants to bind the .beta.AP(1-16) epitope. The DNA sequences of
the 5 mutants were determined and are shown in Table 1 below.
Suitable replacements of 508 L.sub.L cysteine 96 codon were found
to be phenylalanine, serine or tyrosine.
1TABLE 1 Lane (FIG. 8b) Amino Acid Sequence SEQ ID NO: 4
.sup.89HQRSSYP-C.sup.96-T 16 5 .sup.89HQRSSYP-F.sup.96-T 17 6
.sup.89HQRSSYP-Y.sup.96-T 18 8 .sup.89HQRSSYP-F.sup.96-T 19 11
.sup.89HQRSSYP-S.sup.96-T 20
EXAMPLE 4
The Recognition of .beta.AP(1-16) by scFv 508 Mutants
[0244] For further examination of mutated 508 scFv derivatives, the
mutated genes were subcloned into an expression vector and
overexpressed in E. coli, as described for the wild type protein
above. The interactions of the various mutated 508-(Fv) proteins
(Table 1) with .beta.AP(1-16) were tested in an ELISA assay. FIGS.
9a-b show that while the wild type 508-(Fv)-CBD binds at a half
maximum binding (HMB) of 10.sup.-5 M, all the mutants showed
improved binding to .beta.AP: the HMB of C96S and C96Y is
5.times.10.sup.-6 M and the HMB of C96F is 10.sup.-7 M. For further
examination the 508-scFv mutant that carries the C96F mutation
(508F(Fv)) was chosen, which show the higher affinity and shelf
stability (FIGS. 9a-b). The specificity of .beta.AP(1-16) binding
by 508F(Fv) was tested in a competitive ELISA. As is shown in FIG.
10, binding of the purified 508F(Fv)-CBD to .beta.AP was inhibited
by soluble .beta.AP(1-16) peptide serving as the competitor in the
liquid phase in a dose-dependent manner. Inhibition of 50% binding
was obtained at about 1 .mu.M competitor. Binding was not affected
by an irrelevant peptide (WVLD, SEQ ID NO: 4).
EXAMPLE 5
Prevention of the .beta.-amyloid Neurotoxic Effect by 508F(Fv)
[0245] In order to find out whether 508F(Fv) exhibits a protective
effect similar to the parental IgM antibody in preventing .beta.A
mediated neurotoxicity toward cultured cells, an in vitro test was
applied using rat phenochromocytoma PC12as described (Solomon B. et
al., Proc. Natl. Acad. Sci. USA., 94: 4109-4112,1997). Viability of
the cells exposed to .beta.A with or without antibody was measured.
As shown in FIG. 12, 508F(Fv) is prevented the neurotoxicity of
.beta.A (90% cell viability) at a molar ratio .beta.AP:scFv of
15:1, while the unrelated scFv showed no effect. Purified CBD or
the scFv alone had no affect on the cells.
EXAMPLE 6
Disaggregation of .beta.-amyloid Fibril by 508F(Fv)
[0246] To examine the effect of 508F(Fv) on disruption of the
.beta.A fibril (the toxic form of .beta.AP) the ThT reagent that
binds specifically to fibrillar structures (Levine, H. III, Protein
Sci., 2: 404-410,1993) was used. The interference with the already
formed .beta.A fibril was measured at the same molar ratio of
.beta.AP:scFv as in the .beta.A neurotoxic assay and was
quantitated by ThT fluorimetry. FIG. 13 shows that 508F(Fv)
incubated with pre-formed .beta.A fibrils disrupted the fibril
structure indicating extensive deterioration of fibril morphology,
as is evidenced by a substantial (62%) decrease in ThT
fluorescence.
EXAMPLE 7
Ability of Filamentous Phage to Enter the CNS via Olfactory
Track
[0247] Female Balb/c mice were treated with phage vector f88-EFRH
via intranasal administration. The propose of this experiment was
to check the ability of filamentous phage to reach the hippocampous
region via olfactory tract. Since the phage is not carrying any
specific molecule for targeting neuron cells, it should be vanished
without causing any harm after several day following the
administration. In order to investigate the appearance of phage in
the olfactory bulb and the hippocampous region double labeling of
antibodies was used as follows: Rabbit polyclonal antibody anti
filamentous phage and mouse monoclonal antibody against EFRH
epitope fused to glycoprotein VIII of the phage surface. One day
following a single intranasal administration of 10.sup.11 phages
animals showed such phages in their olfactory bulb and hippocampous
(FIGS. 14a-d). Seven days after the administration phages were
detected in the olfactory bulb of only one mouse of the three
tested, whereas no phages were revealed in the hippocampous. No
evidence of phages was detectable 28 days following administration
(FIGS. 15a-d). As shown in FIGS. 16a-d, no evidence of change in
the neuron population of the brain of treated mice was evident.
EXAMPLE 8
Filamentous Phage are Suitable Vehicle for Carrying Active Antibody
Fragment to the CNS
[0248] To check whether a filamentous phage can carry an antibody
to the CNS via the olfactory track and still preserve the activity
against .beta.-amyloid a filamentous phage displaying 508F was
incubated with 10.sup.-3 M biotinlated .beta.AP(1-16), in order to
form antibody antigen immunocomplex. Balb/c mice were divided into
two groups and were administrated intranasally with two different
antigens: 508F-.beta.AP(1-16) immunocomplex and for comparison
biotinlated .beta.AP(1-16) alone. Following a single dose the mice
were sacrificed and brain sections thereof prepared and reacted
with streptavidin coupled to a fluorescent agent. Fluorescence was
detected only in brain sections of mice that were administered with
508F-.beta.AP(1-16), but not, or to a much lesser extent, in brain
sections of mice that were administered with .beta.AP(1-16) alone
(FIGS. 17a-d). No histological findings characterized treated mice
(FIGS. 18a-d).
[0249] It is therefore assumed that the phage act as an inert
vehicle of antibody to the brain, carrying the .beta.AP(1-16)
molecule into the brain.
EXAMPLE 9
[0250] Raising Anti-aggregating .beta.AP Antibody Through
Immunization of Mice with f3-EFRH Phage
[0251] The anti-aggregating epitope within .beta.AP (EFRH, SEQ ID
NO: 1) map to positions 3-6 of the amino acid sequence of .beta.AP.
In order to generate specific immune response against .beta.AP,
mice were immunized with genetically engineered fd phage carrying
the peptide YYEFRH (SEQ ID NO: 7) fused to its minor coat gpIII
according to the immunization schedule shown in FIG. 19. Doses of
10.sup.10 phage particles per injection were used to immunize, at
14-day intervals, through intraperitoneal injection. Following 7
days of each injection, mice were bled and their sera tested by
ELISA for IgG antibody reactivity against wild type phage (not
bearing the peptide YYEFRH on its surface) and against .beta.AP
(FIGS. 20a-b). This route of administration a very high response
against .beta.AP (1:750) following the third injection.
Furthermore, it was found that injection through phage carrying
epitope is long lasting (FIG. 21), it is non-toxic and may be given
without adjuvant. The phage vector is found to be an immunogenic
tool to raise a high affinity immune response within 14 days from
the first injection. The immune response against the peptide YYEFRH
(SEQ ID NO: 7) is low, compared to the immune response against the
entire phage and could be explained by the low copy number of the
fusion gpIII on the phage envelope. Therefore, for further analysis
phages displaying the epitope through glycoprotein VIII were
employed.
EXAMPLE 10
Isolation of f88-EFRH Peptide-display Phage by an Anti-aggregating
mAb
[0252] To identify a disagreggating EFRH peptide epitope a
phage-epitope library was screened with biotinylated antibody.
After three cycles of panning and phage amplification, 90
individually isolated bacterial colonies were grown in microtiter
plates and their phages were assayed for antibody binding. ELISA
analysis revealed that of the phage-clones which were selected
followed by three biopanning cycles, most (above 80%) bound
specifically to anti-aggregating mAb, respectively. DNA from 6
positive clones was sequenced (Table 2). The sequence EFRH (SEQ ID
NO: 1) appeared in 4 clones, one additional clone had the sequence
EFRH (SEQ ID NO: 1), with only one residue replacement of proline
with phenylalanine. In one additional clone, the inserted peptide
bears the sequence of the three residues FRH (acids 2-4 of SEQ ID
NO: 1), lacking the glutamate residue.
2TABLE 2 Amino acid Sequence (name) SEQ ID NO: No. of Phages
VHEPHEFRHVALNPV (C3-II) 8 2 DTEFRHSSNNFSAVR (C7-II) 21 1
STEFRHQTTPLHPNS (C11-I) 22 1 KEPRHHIQHHERVIR (F8-II) 23 1
SAADFRHGSPPISAF (D3-I) 24 1
[0253] Binding of anti-aggregating mAb to the EFRH-bearing phage
was concentration dependent; half-maximal binding was obtained at
an antibody concentration of 100 ng/ml, corresponding to 10.sup.-9
M (FIG. 4) which resembles the level of binding of these antibodies
to the whole peptide. One specific f88-EFRH phage (termed C3-II,
table 2) showed higher level of avidity as is compared to the
others (FIG. 22). It may be due to higher level of EFRH epitope
exposure on its surface. Binding tests of f88-EFRH or f3-EFRH with
the same concentration of anti-aggregating antibody (1 .mu./ml)
demonstrated a higher number of EFRH epitope copies per phage which
may lead to higher serum titer via phage immune response (FIG.
23).
EXAMPLE 11
Raising Anti-aggregating .beta.AP Antibody Through Immunization of
Mice with f88-phage
[0254] In order to generate the same specific immune response
against .beta.AP, mice were immunized with genetically engineered
fd phage carrying the peptide VHEPHEFRHVALNPV (SEQ ID NO: 8) fused
to its major coat gpVIII. This phage was selected from a 15-mer
phage peptide library by an anti aggregating .beta.AP antibody and
is presenting the mAb epitope (underlined) within .beta.AP. This
phage was used to immunize mice as described. Following 7 days of
each injection with 10.sup.10 phage particles (without adjuvant)
the mice were bled and their sera tested by ELISA for IgG antibody
reactivity against wild type phage and against .beta.AP. The
results are summarized in FIGS. 24a-b. All animals showed a
measurable response of IgG antibody against the wild type phage,
and titers increased with the second and the third injection. This
route also gave the highest titer measurable responses against
.beta.AP (1:50,000) after the third injection (FIG. 24b).
EXAMPLE 12
Inhibition of Antibody Serum Binding to .beta.-amyloid Peptide
[0255] The interaction of mouse serum immunized by phage f88-EFRH
with .beta.AP was further assayed by competitive inhibition
experiments. FIG. 25 shows inhibition of mice serum antibody with
synthesized peptides derived from .beta.AP, each of which includes
the sequence EFRH) such as: DAEFRH (positions 1-6, SEQ ID NO: 3),
DAEFRHD (positions 1-7, SEQ ID NO: 3), DAEFRHDSG (positions 1-9,
SEQ ID NO: 3), and .beta.AP itself,
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (positions 1-40, SEQ ID
NO: 3).
[0256] FIG. 25 shows that all of the synthetic peptides which bear
the motive EFRH (SEQ ID NO: 1) similarly inhibited binding of mouse
serum antibody to the .beta.AP with IC.sub.50 values of about
5.times.10.sup.-6 M. These data indicate that the epitope of mouse
serum antibody within the .beta.AP molecule is composed of four
amino acid residues corresponding to positions 3-6 in the .beta.AP
which was found to act as a regulatory site controlling both the
solubilization and the disaggregation process of the .beta.A
molecule.
EXAMPLE 13
Prevention of the .beta.-amyloid Neurotoxic Effect by Serum
Antibody Raised against EFRH-phage
[0257] In order to find out whether serum of f88-EFRH immunized
mice exhibits a protective effect similar to the parental IgG
antibody in preventing .beta.A mediated neurotoxicity toward
cultured cells, the in vitro test using rat phenochromocytoma PC12
was applied as described (Solomon B. et al., Proc. Natl. Acad. Sci.
USA., 94: 4109-4112,1997). Viability of the cells exposed to
.beta.A with or without antibody was measured. As shown in FIG. 26,
diluted serum of 1:5 prevented the neurotoxicity of .beta.A (80%
cell viability), while the unrelated serum showed no effect.
EXAMPLE 14
Disaggregation of .beta.-amyloid Fibril by Serum of EFRH Immunized
Mice
[0258] To examine the effect of serum of f88-EFRH immunized mice on
disruption of the .beta.A fibril (the toxic form of .beta.AP) the
ThT reagent that binds specifically to fibrillar structures
(Levine, H. III, Protein Sci., 2: 404-410,1993) was used. .beta.AP
samples were incubated for a week at 37.degree. C. and then were
exposed to different dilutions mouse serum antibody. Fibril
formation was quantitated by the ThT fluorometry binding assay.
FIG. 27 shows that mouse serum, at dilution of 1:5 and 1:20,
disrupted the fibril structure of .beta.A with extensive
deterioration of fibril morphology, as indicated by a substantial
75% (1:5 dilution) and 50% (1:20 dilution) decrease in ThT
fluorescence. The unrelated serum used as control (serum from
non-immunized mouse), did not significantly inhibit fibril
formation as is compared to the immunized serum. This result
strongly emphasizes the ability of the EFRH epitope displayed by a
filamentous phage vector to evoke an immune response resulting in
anti-agreggation antibody.
EXAMPLE 15
[0259] The teachings of the present invention can also be applied
to prion related diseases which are also characterized by plaque
formation.
[0260] The possible involvement of the PrP protein in the
pathogenesis of nerve cell degeneration and glial cell reaction led
to the identification of a PrP sequences that play a role in the
amyloid formation. A fragment of PrP consisting of amino acids
106-126 was demonstrated to be toxic to rat hippocampal neurons
(Forloni et al. 1993), to mouse cortical and cerebellar cells
(Brown et al., 1994; 1997), and to be particularly highly
fibrillogenic (Selvaggini et al. 1993). The formed fibrils were
partially resistant to proteases digestion and exhibited properties
of in situ amyloid (Selvaggini et al. 1993, Tagliavini et al.
1993). Synthetic peptides corresponding to this region of PrP
exhibit conformational flexibility from -helix to a .beta.-sheet
conformation (Selvaggini et al. 1993) which was similar to
conformational changes from PrP.sup.C to PrP.sup.Sc. The
conformational plasticity of this region is further emphasized by
the findings that two distinct prion strains which exhibit
different sites of proteolytic cleavage within this region 03Bessen
and Marsh, 1994; Telling et al., 1996).
[0261] As is further described below, the inventors of the present
invention have generated mAbs specific to epitopes formed by amino
acids 106-126 of the PrP protein. Such antibodies are usefull in
studying plaque formation and morphology and as possible active
agents for treating or preventing prion generated plaque
diseases.
[0262] Preparation of Monoclonal Antibodies against PrP 106-126
[0263] Mice immunized with a synthetic peptide corresponding to the
amino acid sequence of human PrP 106-126 coupled to the larger
carrier KLH were used for generating monoclonal antibodies reactive
against epitopes on this peptide. Sera derived from the immunized
mice was subjected to ELISA and several positive clones composed
mainly of immunoglobulin M (IgM) molecules and to a lesser extent
IgG molecules were detected and isolated.
[0264] Two immunoglobulin clones that were isolated as described
above and designated mAbs 3-11 (IgM) and mAb 2-40 (IgG1) were
utilized in further studies.
EXAMPLE 16
Search for Epitope Location Using Phage Display Library
[0265] Phage display libraries displaying various peptide fragments
of the human PrP 106-126 polypeptide were generated as described
hereinabove in the method section. Clones reactive to mAbs 3-11 or
mAb 2-40 (for each library) were not detected following 6 cycles of
library biopanning (368 clones screened) raising the possibility
that epitopes that are recognized by these antibodies are of a
conformational nature.
EXAMPLE 17
Competitive Inhibition of Antibodies Bound to PrP by Peptide
NMKH
[0266] The above described anti-PrP 106-126 antibodies were
preincubated with a peptide of an amino acid sequence NMKH (SEQ ID
NO: 26) at equimolar ratio before reacting with the PrP 106-126
polypeptide in order to determine the ability of NMKH to compete
with the human PrP 106-126 for antibody binding. As shown in FIG.
28, this sequence is highly conserved between mice and human
sequences (and others) with the exception of two different amino
acids, M109 instead of L and M 112 instead of V. These two
differences probably contribute to antigenic differeces between the
mouse and human sequences, since the antibodies which were derived
from mice immunized with the human sequence corresponding to PrP
106-126 did not cross react with the mouse sequence corresponding
to peptide PrP 106-126.
[0267] Competition (as analyzed by ELISA) between the NMKH and
whole peptide was not detected, supporting the suggestion that the
epitope may depend upon three dimensional conformation and not
primary structure. In addition, co-reacting mAb 3F4 (recognizing
the sequence HI) and mAb 3-11 with PrP 106-126 did not produce an
additive response, suggesting that their epitopes may be adjacent
or overlapping (Solomon and Balas, 1991).
EXAMPLE 18
PrP Aggregation and Immunocomplex Formation
[0268] Incubation of PrP 106-126 at 37.degree. C. at different
concentrations led to a dose dependent fibrillar aggregation as
measured by the Thioflavin T binding fluorescence assay (FIG. 30).
A PrP 106-126 concentration of 0.3 mg/ml was selected for further
studies.
EXAMPLE 19
Cytotoxicity Assay of PrP 106-126 Using PC12 Cells
[0269] Since the major target organ for PrP.sup.Sc is the nervous
system, in vitro neuronal model systems were used to analyze PrP
toxicity. A clonal cell line which exhibits neuronal properties has
been established from rat central nervous system tumors (Schubert
et al. 1974). The rat pheochromocytoma PC12 cells--a neuron-like
cloned tumor cell line (Greene and Tischler, 1976)--has been shown
to enable in vitro replication of PrP.sup.Sc (Rubenstein et al.
1984). As such, this cell line was utilized by the present study as
a model for detecting molecules with the ability to prevent
toxicity induced by PrP fibrils.
[0270] Using this model system, it was uncovered that PrP 106-126
is toxic to PC12 cells in a dose dependant manner, which toxicity
is related to the conformational state of the peptide and to the
exposure time of the cells to aggregated peptide. Cell viability
considerably decreased (as detected by MTT assay), when cells were
incubated with PrP 106-126 for 5 days in comparison to a 3 day
exposure. Furthermore, preincubation of PrP 106-126 at 37.degree.
C. prior to its addition to the cells increased its toxicity
probably by increasing the amount of amyloid fibrils. Preincubation
of the peptide for 7 days resulted in a significantly higher
toxicity which was concentration dependent (FIG. 29). Under the
described conditions, cell viability was reduced to 10% at a
concentration of 100 .mu.M of PrP 106-126 as was measured by MTT
assay.
EXAMPLE 20
Prevention of PrP 106-126 Neurotoxicity
[0271] The cytoprotective effect of mAb 3-11 and 2-40 is shown in
FIG. 31. Mabs 3-11 and 2-40 inhibited cell death which was induced
by 100 .mu.M PrP 106-126. The viability of cells treated with a
mixture of either antibody and the peptide was 85-89%, as compared
to a 40% survival rate in cells treated with the peptide alone (The
antibodies without the peptide had no affect on cell viability).
The antibodies' protective effect was apparently related to the
specific epitope on the PrP molecule, since such protection was not
demonstrated with mAb 3F4 (44% viability).
EXAMPLE 21
Anti-aggregating Properties of Monoclonal Antibodies as Measured by
ThT Assay
[0272] The amyloid fibril formation of PrP 106-126 was detected by
a thioflavin-T (ThT) binding assay. Both mAbs 3-11 and 2-40 prevent
PrP 106-126 fibrillar aggregation and disaggregate the aggregated
form of this peptide. A significant decrease in amyloid fibril
formation (about 80% prevention) was observed when PrP 106-126 was
incubated at 37.degree. C. in the presence of mAbs 3-11 and 2-40.
When these antibodies were added to already formed aggregates of
PrP 106-126, more than 50% of the fibrils were disaggregated. In
contrast, mAb 3F4 had lower efficacy both in inhibiting amyloid
fibril formation and in inducing their disaggregation (FIG. 32).
Both the inhibition of amyloid fibril formation and the induction
of disaggregation were dependent on the antibody's concentration
and the epitope location (FIGS. 32-33).
[0273] The protective effect of mAbs 3-11 and 2-40 of the present
invention and of mAb 3F4 was calculated as shown in Equation 1
below. 1 % Protective effect = 100 - Emission 482 nm ( PrP106 - 126
incubated with Ab ) Emission 482 nm ( PrP106 - 126 incubated alone
) .times. 100 ( Equation 1 )
[0274] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications cited herein are incorporated by reference in their
entirety.
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Sequence CWU 0
0
* * * * *