U.S. patent application number 10/958211 was filed with the patent office on 2012-02-23 for methods and compositions comprising supramolecular constructs.
Invention is credited to Ruth Greferath, David Hickman, Yves Claude Nicolau.
Application Number | 20120045463 10/958211 |
Document ID | / |
Family ID | 34915784 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120045463 |
Kind Code |
A9 |
Nicolau; Yves Claude ; et
al. |
February 23, 2012 |
Methods and compositions comprising supramolecular constructs
Abstract
The present invention comprises novel compositions and methods
for eliciting high immune responses, of great specifity yielding
conformationally sensitive antibodies. These antibodies recognize
specific epitopes on a wide variety of antigens including but not
limited to, amyloid protein, prion protein, P.sub.170 glycoprotein.
The novel compositions of the invention comprise supramolecular
antigenic constructs generally comprising a peptide sequence,
covalently attached to pegylated lysine resulting in modified and
enhanced peptide presentation. The unique modification methodology
of the present invention is applicable to a variety of peptides and
can ultimately be employed in therapeutic formulations and vaccines
for diseases and disorders such as Alzheimer's disease.
Inventors: |
Nicolau; Yves Claude;
(Legier, CH) ; Greferath; Ruth; (Kehl, DE)
; Hickman; David; (Strasbourg, FR) |
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20060073158 A1 |
April 6, 2006 |
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Family ID: |
34915784 |
Appl. No.: |
10/958211 |
Filed: |
October 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10783975 |
Feb 20, 2004 |
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10958211 |
Oct 4, 2004 |
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60449573 |
Feb 21, 2003 |
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Current U.S.
Class: |
424/185.1 ;
424/450 |
Current CPC
Class: |
C07K 16/18 20130101;
A61P 25/00 20180101; C07K 14/4711 20130101; C07K 2317/70 20130101;
C07K 2317/34 20130101; A61P 25/28 20180101; A61P 35/00 20180101;
A61K 2039/6093 20130101; A61P 37/04 20180101; A61K 39/385 20130101;
A61K 2039/505 20130101; A61K 39/0007 20130101; A61K 2039/55555
20130101; C07K 2317/10 20130101; A61K 9/127 20130101 |
Class at
Publication: |
424/185.1 ;
424/450 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 9/127 20060101 A61K009/127 |
Claims
1. A composition comprising supramolecular antigenic
constructs.
2. The composition of claim 1, wherein the supramolecular antigenic
constructs comprises: a peptide sequence, covalently attached to
polyethylene glycol.
3. The composition of claim 1, wherein the supramolecular antigenic
constructs comprises: a peptide sequence, covalently attached to
pegylated lysine--one at each terminus; wherein the free PEG
terminus is covalently attached to a molecule of
phosphatidylethanolamine.
4. The composition of claim 2, wherein the antigenic constructs is
reconstituted in liposomes consisting of phospholipids and
cholesterol.
5. The composition of claim 1, further comprising a pharmaceutical
carrier.
6. The composition of claim 1, wherein the supramolecular antigenic
constructs may be used to treat disorders comprising Alzheimer's
disease, multidrug resistance in cancer cells, or prion
diseases.
7. The composition of claim 2, wherein the peptide sequence
comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, or SEQ ID NO: 6.
8. A method for inducing an immune response comprising the
administration of supramolecular antigenic constructs.
9. The method of claim 8, wherein the wherein the supramolecular
antigenic constructs comprises: a peptide sequence, covalently
attached to polyethylene glycol.
10. The method of claim 8, wherein the supramolecular antigenic
constructs comprises: a peptide sequence, covalently attached to
pegylated lysine- one at each terminus; wherein the free PEG
terminus is covalently attached to a molecule of
phosphatidylethanolamine.
11. The method of claim 8, wherein the supramolecular antigenic
construct is reconstituted in liposomes consisting of phospholipids
and cholesterol.
12. The method of claim 8, further comprising a pharmaceutical
carrier.
13. The method of claim 8, wherein the supramolecular antigenic
constructs may be used to treat disorders comprising Alzheimer's
disease, multidrug resistance in cancer cells, or prion
diseases.
14. The composition of claim 9, wherein the peptide sequence
comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, or SEQ ID NO: 6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. patent
application Ser. No. 10/783,699 filed Feb. 20, 2004 and U.S.
Provisional Patent Application Ser. No. 60/489,732 filed Jul. 24,
2003. The present application is related to U.S. Provisional Patent
Application Ser. No. 60/446,840 filed Feb. 12, 2003.
FIELD OF THE INVENTION
[0002] The present invention is related to methods and compositions
for eliciting high immune responses. In particular, the present
invention includes novel compositions and methods for yielding
conformationally sensitive antibodies.
BACKGROUND OF THE INVENTION
[0003] Humoral immunity is mediated by serum antibodies which are
proteins secreted by the B cell compartment of the immune response.
Antibodies are a heterogeneous mixture of serum globulins, all of
which share the ability to bind individually to specific antigens.
All serum globulins with antibody activity are referred to as
immunoglobulins.
[0004] All immunoglobulin molecules have common structural features
which enable to do two things: 1) recognize and bind specifically
to a unique structural entity on an antigen, and 2) perform a
common biologic function after combining with the antigen.
Basically, each immunoglobulin molecule consists of two identical
light chains and two identical heavy chains linked by disulfide
bridges.
[0005] A continuing need in the field of immunology and infectious
disease, concerns the availability of highly specific and highly
effective antibodies.
[0006] What is needed are effective methods and compositions for
generation of highly specific and highly effective antibodies.
Preferably such antibodies would recognize specific epitopes on
various antigens such as amyloid protein, prion protein or
P.sub.170 glycoprotein.
SUMMARY OF THE INVENTION
[0007] The present invention includes novel methods and
compositions for eliciting highly specific and highly effective
antibodies. Unlike currently available products the present
invention provides unique methods and compositions resulting in
antibodies having the ability to recognize specific epitopes from a
range of antigens.
[0008] The present invention satisfies the long felt need for
compositions that enable the generation of antibodies that
specifically recognize epitopes such as those of amyloid protein,
prion protein or P.sub.170 glycoprotein.
[0009] The present invention comprises unique antigen presentation
that results in enhanced exposure and ultimately antibodies with a
higher degree of conformational sensitivity. In one embodiment the
invention includes compositions comprising supramolecular antigenic
constructs comprising a peptide sequence, covalently attached to
pegylated lysine--one at each terminus.
[0010] Accordingly, it is an object of the present invention to
provide methods and compositions for eliciting specific and
effective immune responses.
[0011] It is another object of the present invention to provide
methods and compositions for treating and preventing the occurrence
or spread of infectious disease.
[0012] It is a further object of the present invention to provide
methods and compositions for preventing, treating or reducing
disease by eliciting an active cellular and humoral response in the
host.
[0013] Another object of the present invention is to provide
methods and compositions for reducing and preventing the occurrence
of hyperproliferative disorders.
[0014] It is yet another object of the present invention to provide
methods and compositions for vaccinating a human or animal against
selected infectious organisms.
[0015] It is yet another object of the present invention to provide
methods and compositions for passively immunizing a human or animal
against selected infectious organisms.
[0016] Another object of the present invention is to provide
supramolecular construct compositions that are antigenic and elicit
an immune response against infectious organisms in humans or
animals.
[0017] Another object of the present invention is to provide
vaccine compositions comprising supramolecular antigenic constructs
that are non-immunogenic in a human or animal to be immunized with
the composition; and carriers wherein the antigenic peptide is
uniquely presented on the surface of the carrier such that
resulting antibodies are highly specific and have a greater degree
of conformational sensitivity when administered into the human or
animal.
[0018] Yet another object of the present invention is to provide
methods and compositions comprising modified antigenic moieties to
increase an individual's response to infectious disease.
[0019] It is yet another object of the present invention to provide
immunogenic compositions wherein the carrier for the antigenic
peptide comprises modified liposomes.
[0020] It is another object of the present invention to provide
immunogenic compositions wherein the carrier for the antigenic
peptide comprises a colloidal metal.
[0021] Another object of the present invention is to provide
immunogenic compositions wherein the carrier for the antigenic
peptide comprises a baculovirus-derived vesicle.
[0022] It is still another object of the present invention to
provide immunogenic compositions in combination with
pharmaceutically acceptable adjuvants to stimulate the immune
response.
[0023] Yet another object of the present invention is to provide
immunogenic compositions that may be administered intramuscularly,
intravenously, transdermally, orally, or subcutaneously.
[0024] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiment and the
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 provides a schematic showing chemically modified
.beta.-amyloid antigen.
[0026] FIG. 2 provides representative schematic showing liposome
reconstituted with a chemically modified amyloid-antigen.
[0027] FIG. 3 provides a schematic showing a multiple P.sub.170
antigen.
[0028] FIG. 4 provides synthetic peptides, homologous to different
segments of PrP.sup.c used to investigate their influence on the
viability of primary rat hippocampal neurons.
[0029] FIG. 5 provides a schematic of the peptides derived from the
A.beta. sequences 4-11 (SEQ ID NO: 2), 1-16 (SEQ ID NO: 5), 22-35
(SEQ ID NO: 3) and 29-40 (SEQ ID NO: 4).
[0030] FIG. 6 provides a schematic showing the general synthetic
approaches to antigens derived from peptides sequences with or
without internal His or Lys residues.
[0031] FIG. 7 provides the results of ELISA conducted with 1:5000
diluted sera from pegylated amyloid/liposomes lipid A immunized
C57BL/6 mice. PEG-A.beta..sub.1-16 (- -black),
PEG-A.beta..sub.1-16+ALUM (- -grey), PEG-A.beta..sub.4-11 ( grey).
Means of the values of 10 mice per antigen; means of values from 2
mice are shown for A.beta..sub.1-16+ALUM. As a control mean values
of 12 palmitoylated A.beta..sub.1-16 (- - bright grey) injected
animals are shown (published 2002).
[0032] FIG. 8 provides the results of assays evaluating
solubilization of A.beta..sub.1-42 fibers by sera of
PEG-A.beta..sub.4-11 immunized C57BL/6 mice. Thioflavin
fluorescence emission intensity correlates with the amount of
fbrillar amyloid present in solution. A.beta..sub.1-42 fibers
formation during 7 days at 37.degree. C. in PBS, pH=7.1. Sera were
added on day 7 and incubated for 24 hrs. Bars 1-9 represent
solubilization experiments made with sera of vaccinated animals.
Means of 4 samples+SD are shown.
[0033] FIG. 9 provides the results of solubilization assay of
A.beta..sub.1-42 fibers by supernatants of hybridoma clones from
palm.-A.beta..sub.1-16 immunized C57BL/6 mice. A.beta..sub.1-42
fibers formation during 7 days at 37.degree. C. in PBS, pH=7.1.
Supernatants were incubated for 24 hrs. sfr medium=medium without
FCS. The hybridoma clones were grown in serum free medium for 1
day. Means of 4 samples+SD are shown.
[0034] FIG. 10 provides .sup.13C-.sup.13C correlation spectrum of
amyloid fibres made of the amyloid .beta.-peptide labeled at
.sup.10Tyr and .sup.12Val.
[0035] FIG. 11 provides projection of the .sup.13C-.sup.13C
correlation spectrum of A.beta.-peptide fibers (A) and after
incubation with the antibody for 12 days (B).
DETAILED DESCRIPTION
[0036] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims. The entire text
of the references mentioned herein are hereby incorporated in their
entireties by reference including U.S. patent application Ser. No.
10/783,699.
[0037] We report here a method of eliciting high immune responses,
of great specifity yielding conformationally sensitive antibodies.
These antibodies recognize specific epitopes on a wide variety of
antigens including but not limited to, amyloid protein, prion
protein, P.sub.170 glycoprotein.
Definitions
[0038] The terms "polypeptide", "peptide", and "protein", as used
herein, are interchangeable and are defined to mean a biomolecule
composed of two or more amino acids linked by a peptide bond.
[0039] The term "peptides," are chains of amino acids (typically
L-amino acids) whose alpha carbons are linked through peptide bonds
formed by a condensation reaction between the carboxyl group of the
alpha carbon of one amino acid and the amino group of the alpha
carbon of another amino acid. The terminal amino acid at one end of
the chain (i.e., the amino terminal) has a free amino group, while
the terminal amino acid at the other end of the chain (i.e., the
carboxy terminal) has a free carboxyl group. As such, the term
"amino terminus" (abbreviated N-terminus) refers to the free
alpha-amino group on the amino acid at the amino terminal of the
peptide, or to the alpha-amino group (imino group when
participating in a peptide bond) of an amino acid at any other
location within the peptide. Similarly, the term "carboxy terminus"
(abbreviated C-terminus) refers to the free carboxyl group on the
amino acid at the carboxy terminus of a peptide, or to the carboxyl
group of an amino acid at any other location within the
peptide.
[0040] Typically, the amino acids making up a peptide are numbered
in order, starting at the amino terminal and increasing in the
direction toward the carboxy terminal of the peptide. Thus, when
one amino acid is said to "follow" another, that amino acid is
positioned closer to the carboxy terminal of the peptide than the
preceding amino acid.
[0041] The term "residue" is used herein to refer to an amino acid
that is incorporated into a peptide by an amide bond. As such, the
amino acid may be a naturally occurring amino acid or, unless
otherwise limited, may encompass known analogs of natural amino
acids that function in a manner similar to the naturally occurring
amino acids (i.e., amino acid mimetics). Moreover, an amide bond
mimetic includes peptide backbone modifications well known to those
skilled in the art.
[0042] The phrase "consisting essentially of" is used herein to
exclude any elements that would substantially alter the essential
properties of the peptides to which the phrase refers. Thus, the
description of a peptide "consisting essentially of . . . "
excludes any amino acid substitutions, additions, or deletions that
would substantially alter the biological activity of that
peptide.
[0043] Furthermore, one of skill will recognize that, as mentioned
above, individual substitutions, deletions or additions which
alter, add or delete a single amino acid or a small percentage of
amino acids (typically less than 5%, more typically less than 1%)
in an encoded sequence are conservatively modified variations where
the alterations result in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. The following six groups each contain amino acids that are
conservative substitutions for one another: [0044] 1) Alanine (A),
Serine (S), Threonine (T); [0045] 2) Aspartic acid (D), Glutamic
acid (E); [0046] 3) Asparagine (N), Glutamine (Q); [0047] 4)
Arginine (R), Lysine (K); [0048] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and [0049] 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W).
[0050] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany it as found in its native state. Thus, the
peptides described herein do not contain materials normally
associated with their in situ environment. Typically, the isolated,
immunogenic peptides described herein are at least about 80% pure,
usually at least about 90%, and preferably at least about 95% as
measured by band intensity on a silver stained gel.
[0051] Protein purity or homogeneity may be indicated by a number
of methods well known in the art, such as polyacrylamide gel
electrophoresis of a protein sample, followed by visualization upon
staining. For certain purposes high resolution will be needed and
HPLC or a similar means for purification utilized.
[0052] When the immunogenic peptides are relatively short in length
(i.e., less than about 50 amino acids), they are often synthesized
using standard chemical peptide synthesis techniques.
[0053] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
a preferred method for the chemical synthesis of the immunogenic
peptides described herein. Techniques for solid phase synthesis are
known to those skilled in the art.
[0054] Alternatively, the immunogenic peptides described herein are
synthesized using recombinant nucleic acid methodology. Generally,
this involves creating a nucleic acid sequence that encodes the
peptide, placing the nucleic acid in an expression cassette under
the control of a particular promoter, expressing the peptide in a
host, isolating the expressed peptide or polypeptide and, if
required, renaturing the peptide. Techniques sufficient to guide
one of skill through such procedures are found in the
literature.
[0055] Once expressed, recombinant peptides can be purified
according to standard procedures, including ammonium sulfate
precipitation, affinity columns, column chromatography, gel
electrophoresis and the like. Substantially pure compositions of
about 50 to 95% homogeneity are preferred, and 80 to 95% or greater
homogeneity are most preferred for use as therapeutic agents.
[0056] One of skill in the art will recognize that after chemical
synthesis, biological expression or purification, the immunogenic
peptides may possess a conformation substantially different than
the native conformations of the constituent peptides. In this case,
it is often necessary to denature and reduce the antiproliferative
peptide and then to cause the peptide to re-fold into the preferred
conformation. Methods of reducing and denaturing proteins and
inducing re-folding are well known to those of skill in the
art.
[0057] Antigenicity of the purified protein may be confirmed, for
example, by demonstrating reaction with immune serum, or with
antisera produced against the protein itself.
[0058] The terms "a", "an" and "the" as used herein are defined to
mean "one or more" and include the plural unless the context is
inappropriate.
[0059] The terms "detecting" or "detected" as used herein mean
using known techniques for detection of biologic molecules such as
immunochemical or histological methods and refer to qualitatively
or quantitatively determining the presence or concentration of the
biomolecule under investigation.
[0060] By "isolated" is meant a biological molecule free from at
least some of the components with which it naturally occurs.
[0061] The terms "antibody" or "antibodies" as used herein include
monoclonal antibodies, polyclonal, chimeric, single chain,
bispecific, simianized, and humanized antibodies as well as Fab
fragments, including the products of an Fab immunoglobulin
expression library.
[0062] The term "antigen" refers to an entity or fragment thereof
which can induce an immune response in a mammal. The term includes
immunogens and regions responsible for antigenicity or antigenic
determinants.
[0063] As used herein, the term "soluble" means partially or
completely dissolved in an aqueous solution.
[0064] Also as used herein, the term "immunogenic" refers to
substances which elicit or enhance the production of antibodies,
T-cells and other reactive immune cells directed against an
immunogenic agent and contribute to an immune response in humans or
animals.
[0065] An immune response occurs when an individual produces
sufficient antibodies, T-cells and other reactive immune cells
against administered immunogenic compositions of the present
invention to moderate or alleviate the disorder to be treated.
[0066] The term "carrier" as used herein means a structure in which
antigenic peptide or supramolecular construct can be incorporated
into or can be associated with, thereby presenting or exposing
antigenic peptides or part of the peptide to the immune system of a
human or animal. The term "carrier" further comprises methods of
delivery wherein supramolecular antigenic construct compositions
comprising the antigenic peptide may be transported to desired
sites by delivery mechanisms. One example of such a delivery system
utilizes colloidal metals such as colloidal gold.
[0067] In addition, the term "carrier" further comprises delivery
mechanisms known to those skilled in the art including, but not
limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin
(BSA) and other adjuvants. It is also to be understood that the
supramolecular antigenic construct compositions of the present
invention can further comprise adjuvants, preservatives, diluents,
emulsifiers, stabilizers, and other components that are known and
used in vaccines of the prior art. Any adjuvant system known in the
art can be used in the composition of the present invention. Such
adjuvants include, but are not limited to, Freund's incomplete
adjuvant, Freund's complete adjuvant, polydispersed .beta.-(1,4)
linked acetylated mannan ("Acemannan"), TITERMAX.RTM.
(polyoxyethylene-polyoxypropylene copolymer adjuvants from CytRx
Corporation), modified lipid adjuvants from Chiron Corporation,
saponin derivative adjuvants from Cambridge Biotech, killed
Bordetella pertussis, the lipopolysaccharide (LPS) of gram-negative
bacteria, large polymeric anions such as dextran sulfate, and
inorganic gels such as alum, aluminum hydroxide, or aluminum
phosphate.
[0068] Carrier proteins that can be used in the supramolecular
antigenic construct compositions of the present invention include,
but are not limited to, maltose binding protein "MBP"; bovine serum
albumin "BSA"; keyhole lympet hemocyanin "KLH"; ovalbumin;
flagellin; thyroglobulin; serum albumin of any species; gamma
globulin of any species; syngeneic cells; syngeneic cells bearing
Ia antigens; and polymers of D- and/or L-amino acids.
[0069] Further, the term "effective amount" refers to the amount of
antigenic/immunogenic composition which, when administered to a
human or animal, elicits an immune response. The effective amount
is readily determined by one of skill in the art following routine
procedures.
[0070] For example, supramolecular antigenic construct compositions
may be administered parenterally or orally in a range of
approximately 1.0 .mu.g to 1.0 mg per patient, though this range is
not intended to be limiting. The actual amount of the composition
required to elicit an immune response will vary for each individual
patient depending on the immunogenicity of the composition
administered and on the immune response of the individual.
Consequently, the specific amount administered to an individual
will be determined by routine experimentation and based upon the
training and experience of one skilled in the art.
[0071] The compositions of the present invention are used to
produce antibodies directed against antigenic peptides. Resulting
antibodies are administered to individuals to passively immunize
them against a variety of diseases or disorders, including but not
limited to, Alzheimer's disease or prion disease.
[0072] The immunogenic compositions of the present invention
comprise liposomes made by reconstituting liposomes in the presence
of purified or partially purified or modified antigenic peptides.
Additionally, peptide fragments may be reconstituted into
liposomes. The present invention also includes antigenic peptide
fragments modified so as to increase their antigenicity. For
example, antigenic moieties and adjuvants may be attached to or
admixed with the peptide. Examples of antigenic moieties and
adjuvants include, but are not limited to, lipophilic muramyl
dipeptide derivatives, nonionic block polymers, aluminum hydroxide
or aluminum phosphate adjuvant, and mixtures thereof.
[0073] The present invention further encompasses antigenic peptides
modified with hydrophobic moieties, such as palmitic acid, that
facilitate insertion into the hydrophobic lipid bilayer of a
carrier. Hydrophobic moieties of the present invention may be fatty
acids, triglycerides and phospholipids wherein the fatty acid
carbon back bones has at least 10 carbon atoms. Most preferable are
lipophilic moieties having fatty acids with a carbon backbone of at
least approximately 14 carbon atoms and up to approximately 24
carbon atoms. The most preferred hydrophobic moieties have a carbon
backbone of at least 14 carbon atoms. Examples of hydrophobic
moieties include, but are not limited to, palmitic acid, stearic
acid, myristic acid, lauric acid, oleic acid, linoleic acid, and
linolenic acid. The most preferred hydrophobic moiety is palmitic
acid.
[0074] The supramolecular antigenic construct compositions of the
present invention are administered to a human or animal to induce
immunity to antigenic agents such as infectious organisms. The
immunized human or animal develops circulating antibodies against
the infectious organism, thereby reducing or inactivating its
ability to stimulate disease.
[0075] The supramolecular antigenic construct compositions of the
present invention are also used to produce a panel of monoclonal or
polyclonal antibodies that are specific for various disorders,
including for example, Alzheimer's disease. Antibodies are made by
methods well known to those of ordinary skill in the art.
[0076] The compositions of the present invention are administered
to a human or animal by any appropriate means, preferably by
injection. For example, a modified antigenic peptide reconstituted
in liposomes is administered by subcutaneous injection. Whether
internally produced or provided from external sources, the
circulating antibodies bind to antigen and reduce or inactivate its
ability to stimulate disease.
[0077] Liposomes that can be used in the compositions of the
present invention include those known to one skilled in the art.
Any of the standard lipids useful for making liposomes may be used.
Standard bilayer and multi-layer liposomes may be used to make
compositions of the present invention. While any method of making
liposomes known to one skilled in the art may be used, the most
preferred liposomes are made according to the method of Alving et
al., Infect. Immun. 60:2438-2444, 1992, hereby incorporated by
reference. The liposome can optionally contain an adjuvant. A
preferred adjuvant is detoxified lipid A, such as monophosphoryl or
diphosphoryl lipid A.
[0078] When the vesicles are liposomes, the antigenic peptide
generally has a hydrophobic tail that inserts into the liposome
membrane as it is formed. Additionally, antigenic peptides can be
modified to contain a hydrophobic tail so that it can be inserted
into the liposome. For example, antigenic peptide may be exposed on
the surface of previously formed liposomes by chemical attachment
or electroinsertion.
[0079] The antibodies provided herein are monoclonal or polyclonal
antibodies having binding specificity for infectious organisms or
antigenic peptides representative of various disorders such as
Alzheimer's disease, multi drug resistant cancer and prion
diseases.
[0080] The monoclonal antibody is prepared by immunizing an animal,
such as a mouse or rabbit, with supramolecular antigenic construct
compositions of the present invention. Spleen cells are harvested
from the immunized animals and hybridomas generated by fusing
sensitized spleen cells with a myeloma cell line, such as murine
SP2/O myeloma cells (ATCC, Manassas, Va.). The cells are induced to
fuse by the addition of polyethylene glycol. Hybridomas are
chemically selected by plating the cells in a selection medium
containing hypoxanthine, aminopterin and thymidine (HAT).
[0081] Hybridomas are subsequently screened for the ability to
produce monoclonal antibodies against specific diseases or
disorders. Hybridomas producing antibodies of interest are cloned,
expanded and stored frozen for future production. The preferred
hybridoma produces a monoclonal antibody having the IgG isotype,
more preferably the IgG1 isotype.
[0082] The polyclonal antibody is prepared by immunizing animals,
such as mice or rabbits with supramolecular antigenic construct
compositions of the present invention described above. Blood sera
is subsequently collected from the animals, and antibodies in the
sera screened for binding reactivity against target agents.
[0083] Either the monoclonal antibody or the polyclonal antibody,
or both may be labeled directly with a detectable label for
identification a target agent in a biological sample as described
below. Labels for use in immunoassays are generally known to those
skilled in the art and include enzymes, radioisotopes, and
fluorescent, luminescent and chromogenic substances including
colored particles, such as colloidal gold and latex beads. The
antibodies may also be bound to a solid phase to facilitate
separation of antibody-antigen complexes from non-reacted
components in an immunoassay. Exemplary solid phase substances
include, but are not limited to, microtiter plates, test tubes,
magnetic, plastic or glass beads and slides. Methods for coupling
antibodies to solid phases are well known to those skilled in the
art.
[0084] Alternatively, the antibody may be labeled indirectly by
reaction with labeled substances that have an affinity for
immunoglobulin, such as protein A or G or second antibodies. The
antibody may be conjugated with a second substance and detected
with a labeled third substance having an affinity for the second
substance conjugated to the antibody. For example, the antibody may
be conjugated to biotin and the antibody-biotin conjugate detected
using labeled avidin or streptavidin. Similarly, the antibody may
be conjugated to a hapten and the antibody-hapten conjugate
detected using labeled anti-hapten antibody. These and other
methods of labeling antibodies and assay conjugates are well known
to those skilled in the art.
[0085] In a preferred embodiment, the antibody is labeled
indirectly by reactivity with a second antibody that has been
labeled with a detectable label. The second antibody is preferably
one that binds to antibodies of the animal from which the
monoclonal antibody is derived. In other words, if the monoclonal
antibody is a mouse antibody, then the labeled, second antibody is
an anti-mouse antibody. For the monoclonal antibody to be used in
the assay described below, this label is preferably an
antibody-coated bead, particularly a magnetic bead. For the
polyclonal antibody to be employed in the immunoassay described
herein, the label is preferably a detectable molecule such as a
radioactive, fluorescent or an electrochemiluminescent
substance.
Formulations
[0086] The naturally occurring or synthetic protein, peptide, or
protein fragment, containing all or an active portion of an
immunogenic protein or peptide can be prepared in a physiologically
acceptable formulation, such as in a pharmaceutically acceptable
carrier, using known techniques. For example, the protein, peptide
or protein fragment is combined with a pharmaceutically acceptable
excipient to form a therapeutic composition.
[0087] Alternatively, the gene for the protein, peptide, or protein
fragment, containing all or an active portion of the immunogenic
peptide, may be delivered in a vector for continuous administration
using gene therapy techniques. The vector may be administered in a
vehicle having specificity for a target site, such as a tumor.
[0088] The compositions of the present invention may be
administered in the form of a solid, liquid or aerosol. Examples of
solid compositions include pills, creams, and implantable dosage
units. Pills may be administered orally. Therapeutic creams may be
administered topically. Implantable dosage units may be
administered locally, for example, at a tumor site, or may be
implanted for systematic release of the therapeutic composition,
for example, subcutaneously. Examples of liquid compositions
include formulations adapted for injection intramuscularly,
subcutaneously, intravenously, intra-arterially, and formulations
for topical and intraocular administration. Examples of aerosol
formulations include inhaler formulations for administration to the
lungs.
[0089] The compositions may be administered by standard routes of
administration. In general, the composition may be administered by
topical, oral, rectal, nasal or parenteral (for example,
intravenous, subcutaneous, or intramuscular) routes. In addition,
the composition may be incorporated into sustained release matrices
such as biodegradable polymers, the polymers being implanted in the
vicinity of where delivery is desired, for example, at the site of
a tumor. The method includes administration of a single dose,
administration of repeated doses at predetermined time intervals,
and sustained administration for a predetermined period of
time.
[0090] A sustained release matrix, as used herein, is a matrix made
of materials, usually polymers which are degradable by enzymatic or
acid/base hydrolysis or by dissolution. Once inserted into the
body, the matrix is acted upon by enzymes and body fluids. The
sustained release matrix desirably is chosen by biocompatible
materials such as liposomes, polylactides (polylactide acid),
polyglycolide (polymer of glycolic acid), polylactide co-glycolide
(copolymers of lactic acid and glycolic acid), polyanhydrides,
poly(ortho)esters, polypeptides, hyaluronic acid, collagen,
chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,
polysaccharides, nucleic acids, polyamino acids, amino acids such
phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene, polyvinylpyrrolidone and silicone. A preferred
biodegradable matrix is a matrix of one of either polylactide,
polyglycolide, or polylactide co-glycolide (co-polymers of lactic
acid and glycolic acid).
[0091] The dosage of the composition will depend on the condition
being treated, the particular composition used, and other clinical
factors such as weight and condition of the patient, and the route
of administration.
[0092] The composition may be administered in combination with
other compositions and procedures for the treatment of diseases.
For example, unwanted cell proliferation may be treated
conventionally with surgery, radiation or chemotherapy in
combination with the administration of the composition, and
additional doses of the composition may be subsequently
administered to the patient to stabilize and inhibit the growth of
any residual unwanted cell proliferation.
Supramolecular Antigenic Constructs
[0093] The supramolecular antigenic constructs of the present
invention generally comprise a peptide sequence, covalently
attached to pegylated lysine- one at each terminus. The length of
the PEG (polyethylenglycol) chain may vary from 8 to 150000. The
free PEG terminus is covalently attached to a molecule of
phosphatidylethanolamine (where the fatty acid can be: myristic,
palmitic, stearic, oleic etc. or combination thereof). This
supramolecular structure is reconstituted in liposomes consisting
of phospholipids and cholesterol (phosphatidylethanol amine,
phosphatidyl glycerol, cholesterol in varied molar ratios. Other
phospholipids can be used. Lipid A is used at a concentration of
approximately 40 .mu.g/pmole of phospholipids.
[0094] In certain embodiments, the supramolecular antigenic
constructs comprise a peptide having the amino acid sequence of
f3-amyloid. In certain other embodiments, the supramolecular 1
antigenic construct comprises peptide sequences that are the
extracellular loops 1, 4 and 6 of the P170 glycoprotein. In certain
other embodiments, the supramolecular 1 antigenic construct
comprises peptide sequences that comprise amino acid sequences
109-129 of the prion protein.
[0095] The present invention further comprises monoclonal
antibodies raised against a supramolecular structure reconstituted
in liposome, wherein, for example, the peptide sequence comprises
an amino acid sequence from amyloid protein. Additionally,
monoclonal antibodies raised against supramolecular structures
wherein the peptide sequence is an/or several amino acid sequences
from the P-glycoprotein (P.sub.170) extracellular loops are also
included in the present invention.
[0096] Also included in the present invention are monoclonal
antibodies raised against a supramolecular structure wherein the
peptide sequences comprise an amino acid sequence selected from a
protein of interest. More specifically, for example, the invention
includes monoclonal antibodies raised against a supramolecular
structure reconstituted in liposome wherein the peptide sequence is
an amino acid sequence selected from 13-amyloid protein (4-10, or
1-8, or 8-16, etc.) which does not induce cerebral bleeding in
transgenic mice for human Alzheimer's disease. The invention
further includes monoclonal antibodies sensitive to the
conformational characteristics of antigenic peptides.
Amyloid
[0097] The 7 amino acid sequence: FRHDSGY (SEQ ID NO:1) of
.beta.-amyloid was synthesized. One lysine was attached covalently
at each end of the sequence (1). The lysines, prior to attachment
to the above sequence were reacted with a chain of
Polyethylenglycol (PEG, n=8-2000). Polyethylenglycol chains bound
to lysine at one end are covalently attached to a molecule of
dioleyl-phosphatidyl choline ethanolamine (or any fatty
acid-phosphatidylcholine) as described (2). ##STR1##
Chemically Modified .beta.-Amyloid Antigen
[0098] The chemically modified antigen is then reconstituted in
liposomes consisting of phospholipids and cholesterol (3). Examples
of suitable liposomes include, but are not limited to, DOPG, DOPEA,
Chol. (Lipid A was at the concentration of 40 .mu.g/.mu.mole
phospholipid.) A representative schematic showing liposome
reconstituted with a chemically modified amyloid-antigen is shown
in FIG. 2.
[0099] The supramolecular antigenic constructs of the present
invention have vast advantages over the palmitoylated antigens,
reconstituted in liposomes. Primarily, the long PEG chains
(n=8-5000) enhance significantly the exposure and accessibility of
the peptide sequence. Antigen presentation becomes much more and
the conformation sensitivity of the elicited antibodies is
enhanced. Another advantage of the present invention is that
peptide sequences in different conformations may be used. The
increased distance between the sequence and surface of the liposome
makes sure that the surface does not interact with the sequence,
thus, possibly influencing its conformation. Also, antigenicity of
the construct becomes significantly higher than that of
palmitoylated sequences reconstituted in liposomes. High titers of
antibodies comprised between 1:5000 and 1:10000 are obtained in
mice, within a few weeks. Additionally, the affinity of the
antibodies for the antigen is significantly increased. In the case
of the amyloid sequence FRHDSGY (SEQ ID NO: 1), the antibody
elicited by ip or iv injection of the construct are efficiently
solubilizing A.beta..sub.1-40 and A.beta..sub.1-42fibers,
protecting in vitro PC12 cells against apoptosis and metabolic
inhibition (MTT reduction) induced by A.beta..sub.1-42 and
A.beta..sub.1-40 fibers.
[0100] In one embodiment of the present invention, the FRHDSGY (SEQ
ID NO: 1) sequence of the amyloid protein is used, however any
other amyloid protein sequence can be substituted. Monoclonal
antibodies obtained from mice immunized with the described
construct display, besides the in vitro properties mentioned above
for the polyclonal antibodies, biological activity in APP[V717I]
FVB transgenic mice for human Alzheimer's Disease. Significant
levels of memory restoration and of curiosity awakening in these
mice are observed. The mAb does not induce bleeding in the brain of
the immunized, transgenic mice.
[0101] Though not wishing to be bound by the following theory,
based on in vitro studies of the interaction of anti-amyloid mAb
(against the 1-16 sequence, generated by the methods of the present
invention) mainly of fiber solubilization and of CD spectra, it
appears that the antibodies bind preferentially to .beta.-amyloid
in its .alpha.-helix conformation. This would explain the amyloid
fiber solubilization effect in thermodynamic terms. Since the
antibody, by binding preferentially to the .alpha.-helix, removes
the .alpha.-helix amyloid from the equilibrium: [0102]
A.beta.(.alpha.-helix)A.beta.(.beta.-sheet) thereby increasing
amounts of .beta.-amyloid, in .beta.-sheet conformation undergo
conformational transition to the soluble .alpha.-helix form in
order to re-establish the equilibrium. The stochiometric
observations made, support the idea of the mAbs directly
influencing the conformation equilibrium.
[0103] As Selkoe (2002) has elaborated, Alzheimer's Disease appears
as a synaptic failure. In the earlier stages of the disease memory
loss may originate in such failure. It is thought that soluble
oligomers A.beta..sub.1-40, for example, might be able to block the
synapse. The monoclonal antibodies, generated by methods of the
present invention, bind to soluble oligomers A.beta..sub.1-40.
Measurement of conductivity of synapses in the presence and absence
of the antibodies permits the determination of the action of the
antibody on synapses, in the presence of soluble oligomers.
[0104] The inventors of the present invention checked the activity
of a number of mAbs obtained with the epitopes such as
A.beta..sub.4-11 (SEQ ID NO: 2), A.beta..sub.22-35 (SEQ ID NO: 3),
and A.beta..sub.29-40 (SEQ ID NO: 4) embedded in an supramolecular
construct (see FIG. 5). The sequence 4-11 was determined to be the
epitope to the mAb elicited by the palmitoylated A.beta.1-16
antigen. (A.beta..sub.1-16 (SEQ ID NO: 5)
[0105] According to the methods of the present invention, new and
uniquely modified peptide antigens were used in order to raise
mAbs: TABLE-US-00001 Residue 22-35: VGSNKGAIIGLM (SEQ ID NO:3)
[0106] Junctions between extracellular and Transmembrane.TM.
domains have been found to be targeted by inhibitory antibodies
(such as Herceptin-Trastuzumab anti-HER2/neu antibodies) and, in
multispanning.TM. proteins, to form pockets that are targeted by
small molecular weight inhibitors (Dragic et al., 2000). Though not
wishing to be bound by the following theory, this sequence is
likely to be crucial for the oligomerization capacity of
A.beta..sub.1-42 and A.beta..sub.1-40, as it represents the
transition between polar and hydrophobic regions (wherein the
phrase "extracellular sequence" is used to refer to the
extracellular sequences in the A.beta..sub.1-42 amyloidogenic
sequence). The sequence contains the first two GXXXGXXXG motifs of
the A.beta..sub.1-42 and A.beta..sub.1-40 sequences. GXXXG are key
inducers of oligomerization of hydrophobic sequences (Russ and
Engelmann, 2000). Interestingly, the first GXXXG motif is predicted
to be extracellular, while the following two are predicted to be
placed in the membrane. Though not wishing to be bound by the
following theory, it may be assumed, by analogy, that
oligomerization of A.beta. peptides is specifically triggered by
the GXXXG motifs. TABLE-US-00002 Residue 29-40: GAIIGLMVGGVV (SEQ
ID NO:4)
[0107] The hydrophobic sequence of A.beta..sub.1-42 and
A.beta..sub.1-40 contain the motif GXXXGXXXGG, which has been found
to induce strong oligomerization of hydrophobic sequences (Eilers
et al., 2002; Leeds et al., 2001; Lemmon et al., 1994; Russ and
Engelmann, 1999; Russ and Engelmann, 2000; Smith and Bormann,
1995). Therefore, this motif is viewed as a prime target for
therapeutic approaches; since it must play a major role in all
pathogenic processes that lead to A.beta..sub.1-42 and
A.beta..sub.1-40 formation, oligomerization and accumulation. In
the intact sequence of APP, it is likely that this motif caps the
downstream sequence that will need to unfold for .gamma.-secretase
to process, as was shown for the SREBP cleavage (Ye et al., 2000).
This sequence has not previously been identified by anybody as
being important for amyloid oligomerization. The supramolecular,
pegylated antigens have higher antigenicity and the antibodies
elicited by them have higher affinities. Beside A.beta..sub.1-16,
supramolecular constructs of the present invention also include
peptides represented by A.beta..sub.4-11 (SEQ ID NO: 2),
A.beta..sub.22-35 (SEQ ID NO: 3), A.beta..sub.29-40 (SEQ ID NO: 4)
for use in vaccines.
[0108] Methodologies for the mono-pegylation of peptides at the
N-.alpha.-position are known and widely used. Site-specific
mono-pegylation at internal, N- or C-terminal amino-acid residues
of medium sized peptides has also been described following either
solid-phase or peptide-grafting approaches. However, solid-phase
synthetic approaches to di-pegylated peptides have been shown to be
severely hampered by steric hindrance and upon starting this
project no efficient synthetic methodologies were reported for such
compounds. Furthermore, peptides derivatised site-specifically at
the N- and C-termini with both a PEG and lipid moiety have not
previously been reported. Herein the present inventors describe a
novel methodology for the synthesis of such A.beta. peptide
conjugates.
[0109] In arriving at the present invention several approaches were
attempted most of which were unsuccessful. For example, the initial
approach to the synthesis focused upon the on-resin grafting of
lipid-PEG conjugates containing distal amine groups, to side-chain
protected peptides (A.beta..sub.4-11, 1-16, 22-35 and .sub.29-40)
containing terminal Glutamic acid residues. No coupling products
were observed under a wide variety of reaction conditions. As
described in Example 2 and shown in FIG. 5, the supramolecular
constructs described herein were generally synthesized using
standard Fmoc/tBu amino acid side-chain protections.
[0110] This novel approach to the synthesis of N- and C-terminal
lipid-PEG .beta.-amyloid antigens using protected peptides is
applicable to a wide variety of peptide sequences including for
example multi-drug resistance protein P-glycoprotein.
[0111] In an effort to evaluate the efficacy of the antigenic
peptides described herein, experiments were conducted to compare
the immunogenicity of PEGylated and palmitoylated antigens using
ELISA and disaggregation assays (see Example B, and FIG. 7). The
ELISA data showed that liposomal PEG-A.beta.1-16 is significantly
more immunogenic than palmitoylated A.beta..sub.1-16 Additional
ALUM did not enhance the immunogenicity of PEG-A.beta..sub.1-16 in
the mice. The antibody response induced by PEG-A.beta..sub.4-11 was
slower in comparison to PEG-In summary therefore, present invention
provides novel monoclonal antibodies against supramolecular
antigens exposing different amyloid sequences. In particular,
original synthetic pathways were devised in order to bind
covalently two polyethylene glycol (n=70) chains to selected
amyloid sequences. At the free end of the PEG chain, phosphatidyl
ethanol amine was covalently attached. Though not wishing to be
bound by the following theory, it is believed that its function is
to anchor the pegylated amyloid sequence in the bilayer of
liposomes. Pegylation is shown herein to increase the
immunogenicity of the antigens as compared to palmitoylation.
Affinity studies, epitope determination, induction of
conformational transition by these monoclonal antibodies are being
conducted presently in our laboratory. The unique modification
methodology of the present invention is applicable to a variety of
peptides and can ultimately be employed in therapeutic formulations
and vaccines for diseases and disorders including, but not limited
to Alzheimer's disease, cancer, and infectious disease.
Multidrug Resistance 1 (MDR 1) in Cancer Cells
[0112] Multidrug resistance 1 in cancer cells is caused by the
overexpression of the P-glycoprotein (P.sub.170), a membrane pump
which ejects a large variety of unrelated chemotherapy agents from
cancer cells.
[0113] Immunization with palmitoylated extracellular sequences of
P.sub.170, reconstituted in liposomes, led to restoration of the
sensitive phenotype in vitro in MDR1 L.sub.1210 mouse leukemia
cells (3). Further results have been obtained in vivo (Madoulet,
Tosi, Nicolau et al., 2002-unpublished results) indicating a 70%
increase of survival half-life in immunized mice, inoculated with
MDR cancer cells, undergoing chemotherapy.
[0114] The inventors of the present invention demonstrate herein
that an antigen consisting of the P.sub.170 extracellular sequences
1,4 and 6 constructed, according to the method of the present
invention is far more efficient in eliciting antibodies which
largely revert the MDR phenotype to the sensitive phenotype in
vitro and in vivo.
[0115] According to the methods of the present invention, peptides
corresponding to P.sub.170 extracellular loops 1, 4 and 6 were
synthesized and then attached to pegylated lysines--1 at each
end--which in turn were covalently attached to one dioleyl
phosphatylethanolamine molecule at each end. Any fatty acid,
myristic, palmitic, stearic or polyunsaturated fatty acids may be
used.
[0116] These 3 constructs were reconstituted in liposomes
consisting of PC-PEA-PG-Cholesterol (or any other phospholipid and
cholesterol combination). Lipid A was added at concentration of 40
.mu.g/.mu.mole of phospholipids. The ratio peptide:phospholipid was
1:200 (other ratios may be used).
[0117] The length of the polyethylenglycol chains varied: the
longer the peptide sequence, the higher the number of PEG molecules
in the chain needs to be. For the 3 sequences used, the PEG
chain-length varied from 10 to 5000. Other chain lengths can be
used. FIG. 3 provides a representative schematic showing a multiple
P.sub.170 antigen.
[0118] IP inoculation of this antigen, followed by three boostings
at 2 weeks interval elicited high titres of anti P.sub.17o
antibodies (1:5000-1:10000) capable of blocking the pumping
activity of P.sub.170, in vitro and in vivo.
Prion Diseases
[0119] Prions cause neurodegenerative diseases such as scrapie in
sheep, bovine spongiform encephalopathy in cattle and
Creutzfeldt--Jacob--Disease in humans. The only known component of
the particle is the scrapie isoform of the protein, PrP.sup.Sc.
Although prions multiply, there is no evidence that they contain
nucleic acid. PrP.sup.Sc is derived from the non-infectious,
cellular protein PrP.sup.c by a posttranslational process during
which PrP.sup.c undergoes a profound conformational change.
[0120] The scrapie protein, PrP.sup.Sc has a critical role in
neuronal degeneration and during disease development undergoes a
three stage transition as follows: (normal cellular isoform of
protein) PrP.sup.c--infectious form (scrapie isoform of protein)
PrP.sup.Sc_protein PrP27-30. Such a cascade of events occurs during
the development of Creutzfeldt--Jacob Disease (CJD), Kuru,
Gerstmann--Straussler-Scheinker Syndrome (GSS), fatal familial
insomnia in man, scrapie in sheep and goats, encephalopathy in mink
and bovine spongiform encephalopathy in cattle.
[0121] The cellular non-toxic protein (PrP.sup.c is a
sialoglycoprotein of MW 33-35 K that is expressed predominantly in
neurons. In the diseases mentioned above, PrP.sup.c is converted
into an altered form (PrP.sup.Sc), which is distinguishable from
its normal homologue by its relative resistance to protease
digestion. PrP.sup.Sc accumulates in the central nervous system of
affected animals and individuals and its protease-resistant core
aggregates extracellularly. The molecular basis of the pathogenesis
is not understood.
[0122] Very interesting observations were made concerning the
neurotoxicity of a fragment of the protein, which may have a
bearing on the understanding of the mechanism of nerve
cell-degeneration occurring in related encephalopaties.
[0123] On the basis of the observation, that the (J-amyloid
fragment responsible for the extracellular deposition of amyloid
fibrils and plaques in the Alzheimer Disease is neurotoxic, it was
hypothesized that neuronal death in related encephalopathies might
be due to toxic effects of abnormal extracellular accumulation of
PrP.sup.Sc and/or its degradation products.
[0124] Synthetic peptides, homologous to different segments of
PrP.sup.c were used to investigate their influence on the viability
of primary rat hippocampal neurons (FIG. 4)
[0125] The present inventors demonstrated that neuronal death
occurs from chronic exposure of primary rat hippocampal cultures to
micromolar concentrations of a peptide corresponding to residues
106-126 of the amino-acid sequence deduced from human PrP.sup.c
cDNA, in a concentration dependent manner (Example 1).
[0126] As detailed in Example 1, the inventors showed that the
neuronal death induced by PrP 106-126 occurred by apoptosis in a
dose dependent manner. In the terminal stages of subacute
encephalopaties, such as scrapie, PrP.sup.Sc reaches at whole brain
concentrations 10 to 20 times higher than PrP.sup.c, which
resembles strikingly the data listed in Table 1 for the 2
concentrations of PrP106-126.
[0127] The process of programmed cell death induced by PrP106-126
is associated, among others with the induction of the
testosterone--repressed prostate message--2 gene (TRPM-2). It is
not known whether apoptosis is activated in vivo during--related
encephalopaties, but the expression of the TRPM-2 mRNA is increased
10-fold in scrapie-infected hamsters.
[0128] It appears from these data, that a neurotoxic mechanism is
possibly responsible for neuronal cell loss in related
encephalopaties and could also be relevant in Alzheimer's
disease.
[0129] The possible mechanism of this neurotoxicity was
investigated in a model system aiming at detecting and analyzing
ionic channel formations upon the interaction of peptides or
proteins with lipid bilayers.
[0130] Low pH, which favors channel formation by PrP106-126,
converts also this peptide from ahelical to R-sheet conformation.
Whereas peptide mapping of PrP.sup.Sc with Edman sequencing and
mass spectrometry revealed no differences between its amino acid
sequence and that predicted from the PrP.sup.c gene sequence; no
chemical modifications where found that might distinguish
PrP.sup.Sc from PrP.sup.p; Fourier Transform infrared spectroscopy
and circular dichroism spectroscopy revealed however a significant
conformational difference between PrP.sup.Sc and PrP.sup.p.
[0131] PrP.sup.c is essentially .alpha.-helical with little or no
R-sheet, whereas PrP.sup.Sc has a high .beta.-sheet content and
less .alpha.-helical structure.
[0132] The sequence KTNMKHMAGAAAAGAVVGGLG (PrPI06-126) (SEQ ID NO:
6) is not only very hydrophobic but it converts also, at low pH to
.beta.-sheet conformation. Moreover, it can convert in solution,
other peptides to .beta.-sheet conformation.
[0133] Based upon these observations and upon techniques developed
by the inventors, a "vaccine" was developed against diseases by
eliciting a strong humoral and cellular immune response in mice to
the neurotoxic PrP106-126, and then challenge the immunized mice
with brain extracts from scrapie mice.
[0134] As in the previous examples, pegylated lysines were attached
covalently at each end of the PrP106-126 sequence. The length of
the PEG chain was 12-4000. The PEG chains were coupled each other
to one molecule of phosphatidyl ethanol amine and reconstituted in
PG-PEA-chol liposomes--lipid A.
[0135] Injected into mice these supramolecular antigenic constracts
elicited a strong humoral immune response, yielding antibodies with
high affinity for the PrP106-126 sequence, and having solubilizing
effects within.
[0136] It should be understood that the foregoing relates only to
preferred embodiments of the present invention, and that numerous
modifications or alterations may be made therein without departing
from the spirit and the scope of the invention as set forth in the
appended claims. The references cited throughout are hereby
incorporated by reference in their entireties.
EXAMPLE 1
[0137] The present inventors demonstrated that neuronal death
occurs from chronic exposure of primary rat hippocampal cultures to
micromolar concentrations of a peptide corresponding to residues
106-126 of the amino-acid sequence deduced from human PrPc cDNA, in
a concentration dependent manner. The data are shown in Table 1.
TABLE-US-00003 TABLE 1 Chronic treatment of hippocampal neurons for
9 days cell death % Peptide 20 .mu.m 80 .mu.m PrP 106-126 18 .+-. 8
100 .+-. 8 PrP 57-64 0 .+-. 5 3 .+-. 4 PrP 89-106 5 .+-. 2 2 .+-. 6
PrP 106-114 0 .+-. 3 12 .+-. 6 PrP 127-135 3 .+-. 6 15 .+-. 9 PrP
127-147 1 .+-. 7 18 .+-. 7 PrP 106-126 scrambled 3 .+-. 2 8 .+-.
3
[0138] The data are the means.+-.s.e. of 6-10 determinations and
are normalized to the toxic effect of PrP106-126 (designated 100%
response).
[0139] It was shown that the neuronal death induced by PrP 106-126
occurred by apoptosis in a dose dependent manner. In the terminal
stages of subacute encephalopaties, such as scrapie, PrP.sup.Sc
reaches at whole brain concentrations 10 to 20 times higher than
PrP.sup.c, which resembles strikingly the data listed in Table 1
for the 2 concentrations of PrP106-126.
[0140] The process of programmed cell death induced by PrP106-126
is associated, among others with the induction of the
testosterone--repressed prostate message--2 gene (TRPM-2). It is
not known whether apoptosis is activated in vivo during-related
encephalopaties, but the expression of the TRPM-2 mRNA is increased
10-fold in scrapie-infected hamsters.
EXAMPLE 2
Methods for Making Supramolecular Antigenic Constructs
[0141] The supramolecular constructs described herein were uniquely
synthesized using standard Fmoc/tBu amino acid side-chain
protections. Peptides which are modified at both the C- and
N-terminus by a PEG-lipid moiety have not previously been reported.
Typically, pegylation of peptides results in mixtures of
regioisomers. The inventors herein demonstrate a convenient method
for the site-specific attachment of a PEG-lipid conjugate to both
the C- and N-terminus of A.beta. using partially protected
peptides.
[0142] For those peptide sequences containing internal Lys or His
residues (4-11, 1-16, 22-35), an orthogonally protected Lys(ivDde)
was added to each termini. An additional Gly was added to the
C-terminal to facilitate synthesis. The Fmoc group was removed with
20% piperidine in DMF and N-acetylated using acetic anhydride.
Selective cleavage of the ivDde groups was achieved with 3%
hydrazine hydrate in DMF for one hour. The 2-chlorotrityl resin was
favored over the more widely used Wang resin since the former
proved to be much more resistant to hydrazinolysis. Furthermore,
the 2-chlorotrityl resin is extremely acid sensitive and thus,
unlike the Wang resin, enables the isolation of protected peptides.
Indeed, it was necessary to perform the coupling reaction in the
solution phase as coupling of the resin-bound peptide to the
pre-activated pegylated lipid reagent DSPE-PEG-SPA did not give
rise to any coupling product. Thus selective cleavage from the
resin under mild conditions (acetic
acid/trifluoroethanol/dichloromethane, 1:1:8, 1 h, rt) gave the
internally protected peptides (FIG. 5).
[0143] Solution-phase couplings were achieved successfully with the
peptides derived from sequences A.beta..sub.4-11 (SEQ ID NO: 2),
A.beta..sub.1-16 (SEQ ID NO: 5), A.beta..sub.22-35 (SEQ ID NO: 3),
to DSPE-PEG-SPA in DMSO and excess base (FIG. 6). The reactions
were then quenched by the addition of excess ethanolamine for 2 h
and the solution lyophilized. Purification by HPLC
(semi-preparative reverse-phase C.sub.4 column) gave between 50-70%
purity of the N- and C-terminal PEG-lipid conjugates whose
identities were confirmed by MALDI (matrix assisted laser
desorption ionization). Each sequence showed considerable variation
in the ease of the coupling reaction and conditions were adjusted
accordingly (temperature, number of molar equivalents DSPE-PEG-SPA,
time). Purification by HPLC proved excellent for the separation of
excess DSPE-PEG-SPA from the desired product, however since the
former shows no affinity to the column, separation of
mono-PEG-lipid (both N- and C-terminal) peptide products from the
desired product proved difficult. Attempts to separate these
products using size-exclusion chromatography also proved
unsuccessful, presumably due to their relatively large
polydispersities. Nevertheless the present inventors are using
cation-exchange chromotagraphy to separate the mono- and di-coupled
products before final side-chain deprotections. Subsequent peptide
side-chain deprotections and separation of the excess quenched
DSPE-PEG-SPA enables the isolation of the desired conjugates to
much higher purity.
EXAMPLE 3
Comparison of Immunogenicity of PEGylated and Palmitoylated
Antigens, ELISA and Disaggregation Assays
[0144] Liposomal antigens were prepared as described (Nicolau et
al., 2002, PNAS, 99, 2332-37). The sequences PEG-A.beta..sub.1-16,
-A.beta..sub.4-11 and -A.beta..sub.22-35 were reconstituted in a
construct consisting of liposomes made of dimyristoyl phosphatidyl
choline (DMPC), dimyristoyl phosphatidyl ethanolamine (DMPEA),
dimyristoyl phosphatidyl glycerol (DMPG) and cholesterol
(0.9:0.1:0.1:0.7 molar ratios) containing monophosphoryl lipid A
(40 mg/mM phospholipids).
ELISA
[0145] The antigens and palmitoylated A.beta..sub.1-16 were used
for the immunization in C57BL/6 mice in 2 week intervals. 10-12
animals were immunized with each antigen. Sera were taken 5 days
after the boostings and ELISA were conducted with several dilutions
of the sera. Comparative results showing the immunogenicity of the
different antigens are presented in FIG. 7.
[0146] The ELISA data showed that liposomal PEG-A.beta..sub.1-16 is
significantly more immunogenic than palmitoylated A.beta..sub.1-16.
Additional ALUM did not enhance the immunogenicity of
PEG-A.beta..sub.1-16 in the mice. The antibody response induced by
PEG-A.beta..sub.4-11 was slower in comparison to
PEG-A.beta..sub.1-16.
Disaggregation Assays
[0147] Nine sera (1:100 dilution) from the
liposomal-PEG-A.beta..sub.4-11 immunized animals were used in an
assay where pre-formed A.beta..sub.1-42 fibers were incubated with
the antisera. The assay was performed as described (Nicolau et al.,
2002).
[0148] Solubilization of A.beta..sub.1-42 fibers by the different
sera was observed with an incubation time of 24 hrs (FIG. 8) Some
of the sera solubilized the fibers to an extent of 75% (sera from
mouse 5 and 6). The spleen cells of these mice were used for the
production of monoclonal antibodies.
EXAMPLE 4
Solubilization Assay
[0149] From two palmitoylated A.beta..sub.1-16/liposomes/lipid
A-immunized animals, 25 supernatants were obtained from recently
generated hybridoma clones which were shown to be specific for
A.beta..sub.1-42 specific antibodies. They were tested in a
solubilization assay according to methods and protocols described
in PNAS 2002, 99, 2332-2337._The results are summarized in FIG.
9.
[0150] The supernatants of 5 hybridoma clones were found to be able
to solubilize .beta.-amyloid fibers in vitro to an extent of up to
75%. The two best clones 15 and 27 were chosen for the purification
of monoclonal antibodies. They are being used for further
investigations as positive control mAbs in vivo.
EXAMPLE 5
Investigation of the .alpha.-Sheet to .alpha.-Helix Transition of
the A.beta..sub.1-42-Peptide by Solid State NMR Spectroscopy
[0151] To avoid loss of .sup.13C-labelled amino acids the synthesis
of the A.beta..sub.1-42 by Fmoc peptide synthesis was verified by a
test-synthesis without labeled amino acids. The identity of the
obtained A.beta..sub.1-42 peptide could be verified by MALDI mass
spectroscopy and a purification procedure using HPLC with a
reversed phase column and an ammonia buffered acetonitrile water
gradient.sup.4 could be established.
[0152] The successful setup of a protocol for synthesis and
purification of the amyloid .beta.-peptide is followed by the
synthesis of the labeled peptide including a .sup.13C labeled
valine at position 12 (.sup.12val) and a .sup.13C labeled tyrosine
at position 10 (.sup.10tyr).
[0153] The labeled A.beta..sub.1-42 was used to generate fibers by
incubating the peptide solution in PBS buffer for one week at
37.degree. C. .sup.13C NMR spectra of the lyophilized fibers
confirm the .beta.-sheet structure and are in agreement with
published results. Incubation of the fibers with A.beta..sub.1-16
specific antibody for 2 days did not show a significant change
.sup.13C spectrum. First assessments of NMR measurements indicate a
change in the secondary structure (FIG. 10).
REFERENCES
[0154] 1. C. Nicolau, R. Greferath, T. S. Balaban, J. Lazarte and
R. Hopkins (2002). Proc. Natl. Acad. Sci. USA. 99,2332-2337. [0155]
2. Fluka A G (2002) Cat. # 79898. [0156] 3. P.-F. Tosi, D. Radu,
and C. Nicolau (1995). Biochem. Biophys. Res. Chem. 212, 494-500.
[0157] 4. Fukuda H, Shimizu T, Nakajima M, Mori H, Shirasawa T.
Bioorg. Med. Chem.Lett. 1999; 9: 953-956 [0158] 5. Petkova A T,
Ishii Y, Balbach J J, Antzutkin O N, Leapman R D, Delaglio F, Tycko
R. Proc. Natl. Acad. Sci. U.S.A 2002; 99: 16742-16747
Sequence CWU 1
1
6 1 7 PRT Artificial Sequence Synthetic 1 Phe Arg His Asp Ser Gly
Tyr 1 5 2 11 PRT Artificial Sequence Synthetic MISC_FEATURE
(3)..(3) Xaa =
Ng-(2,2,4,6,7-Pentamethyl-dihydrobenzofurane-5-sulfonyl)-L-argini
ne whereas the "g" in "Ng" is a superscript MISC_FEATURE (4)..(4)
Xaa = N-imidazol-trityl-L-histidine MISC_FEATURE (5)..(5) Xaa =
L-aspartic acid b-tert-butyl ester MISC_FEATURE (6)..(6) Xaa =
O-tert-butyl-L-serine MISC_FEATURE (8)..(8) Xaa =
O-tert-butyl-L-tyrosine MISC_FEATURE (9)..(9) Xaa = L-glutamic acid
b-tert-butyl ester 2 Lys Phe Xaa Xaa Xaa Xaa Gly Xaa Xaa Lys Gly 1
5 10 3 12 PRT Artificial Sequence Synthetic 3 Val Gly Ser Asn Lys
Gly Ala Ile Ile Gly Leu Met 1 5 10 4 12 PRT Artificial Sequence
Synthetic 4 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 1 5 10
5 18 PRT Artificial Sequence Synthetic MISC_FEATURE (2)..(2) Xaa =
L-aspartic acid b-tert-butyl ester MISC_FEATURE (4)..(4) Xaa =
L-glutamic acid b-tert-butyl ester MISC_FEATURE (6)..(6) Xaa =
Ng-(2,2,4,6,7-Pentamethyl-dihydrobenzofurane-5-sulfonyl)-L-argini
ne Wherein the "g" in "Ng" is superscript MISC_FEATURE (7)..(7) Xaa
= N-imidazol-trityl-L-histidine MISC_FEATURE (8)..(8) Xaa =
L-aspartic acid b-tert-butyl ester MISC_FEATURE (9)..(9) Xaa =
O-tert-butyl-L-serine MISC_FEATURE (11)..(11) Xaa =
O-tert-butyl-L-tyrosine MISC_FEATURE (12)..(12) Xaa = L-glutamic
acid b-tert-butyl ester MISC_FEATURE (14)..(14) Xaa =
N-imidazol-trityl-L-histidine MISC_FEATURE (15)..(15) Xaa =
N-g-Trityl-L-glutamine MISC_FEATURE (16)..(16) Xaa =
N-e-tert-butoxycarbonyl-L-lysine 5 Lys Xaa Ala Xaa Phe Xaa Xaa Xaa
Xaa Gly Xaa Xaa Val Xaa Xaa Xaa 1 5 10 15 Lys Gly 6 21 PRT
Artificial Sequence Synthetic 6 Lys Thr Asn Met Lys His Met Ala Gly
Ala Ala Ala Ala Gly Ala Val 1 5 10 15 Val Gly Gly Leu Gly 20
* * * * *