Note: Descriptions are shown in the official language in which they were submitted.
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1
COMPLEMENT MEDIATED ASSAYS FOR
IN VIVO AND IN VITRO METHODS
FIELD OF THE INVENTION
The present invention is directed to methods and compositions for detecting
pathological conditions. In particular, the invention comprises methods and
compositions
using biological factors, such as complement components, for detecting
pathological
conditions.
BACKGROUND OF THE INVENTION
Diagnostics has traversed a broad range of disciplines from an initial
foothold in
serologic diagnostics to DNA molecular diagnostics, such as those using PCR.
Problems
with many current diagnostic technologies include the inability to directly
detect species
specific mRNA and proteins, and many also lack specificity and sensitivity.
The problems
of detection of molecular cancer metastasis, detection of residual disease,
the early
detection of HIV and other viral agents, sensitive carcinogen detection,
sensitivity in
detection of pathologic proteins or cells in normal tissue, and the need for
heightened
specificity and sensitivity in the determination of the precancerous state of
dysplasia,
illustrate the need for more accurate, sensitive and specific assays.
Furthermore, most of
these assays fail in detection of very low numbers of antigen or analyte
targets, such as low
number DNA, mRNA, protein or cellular targets in the presence of a large
amount of non-
specific material such as genomic DNA, mRNA, protein, or cells.
Other attempts at measuring infectious agents include the tests for prions.
Creutzfeld-Jakob disease (CJD) of humans and bovine spongiform encephalitis
(BSE) and
scrapie of animals are neurodegenerative diseases caused by prion proteins.
The infectious
prion is an abnormal disease producing isoform of the nornial prion protein
(PrP) called
PrPs~. Brain damage in prion disease occurs when abnormal prion protein
molecules, as a
consequence of ingestion gain entry to the brain and cause normal PrPs to take
on the
disease causing PrPs~ form.
Currently a labeled mouse monoclonal antibody 3F4 has been shown to bind
prions
with a sensitivity of binding of the antibody of 5 picograms per ml. This
indicates that
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billions of prion protein molecules or greater would be necessary to be
present to support
detection of binding of the antibody to the aberrant prion. Furthermore, the
assay is
complex, requiring selective precipitation of PrPsC by sodium
phosphotungstate. This
inability to more sensitively detect the presence of the prions in TSEs has
hampered an
understanding of the disease, attempts to determine if a cure scenario is
feasible, and
development of a vaccine.
What is needed are methods and compositions that will detect the prion protein
in a
large sample of plasma and concentrate and collect the normal and the aberrant
forms of
the prion in a small volume. Furthermore, methods are needed for diagnostic
assays that
will detect the presence of the aberrant prion protein with high levels of
sensitivity in the
presence of a large amount of non-specific or normal prion.
What is needed are methods and compositions that recognize the presence of
very
low numbers of infectious or other targets in an excess of non-specific, non-
target or
normal material. The target may be nucleic acid, such as DNA and RNA,
cellular, or
protein, in nature. Ideally, these methods and compositions comprise
diagnostic
technology that supports high levels of specificity and sensitivity in testing
procedures.
Preferred methods and compositions comprise diagnostic tests that are
configured for early
detection of the pathologic agent or other target in the sample by examining
the DNA,
RNA, cell, or soluble protein in solution, to detect the pathologic target
earlier in the
infection time-course.
SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions for detecting
pathological conditions. In particular, the invention comprises methods and
compositions
using biological factors, such as complement components, for detecting
pathological
conditions. Particularly described are assays for non-specific target
elimination that allow
for detection of low copy number targets in a large field of nontarget
material. Such assays
comprise methods comprising CMSA and MACMSA, which preferably comprise
detection
of complement proteins and components. The assays of the presentinvention can
be used
for detection of changes in cellular molecules or nucleic acids that are part
of disease states
or infections, or can be used for detection of molecules in the environment.
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Not only do the methods and compositions of the present invention comprise
detection of nucleic acid and other molecular targets, but the methods and
compositions of
the present invention comprise diagnostics at supramolecular levels to confirm
the presence
of the pathologic or other cellular targets in tissues. The present invention
comprises the
analysis of only the cell subset of interest in a very large cell specimen and
has the, ability
to compartmentalize and assay each cell component for the analyte of interest.
Other
embodiments comprise target analyte sorting and separation from non-specific
analyte for
increased sensitivity of detection. CMSA comprises the fixation and activation
of
complement by interactions between cell subset specific surface membrane
proteins, and
monoclonal or other antibodies. The initiation of the complement fixation
process results
in the production of the C3a peptide in quantities directly proportional to
the extent of
complement fixation.
One embodiment of CMSA, called MACMSA, comprises use of a soluble
immunogen found in the cytoplasm or released into the cellular environment.
These
methods and compositions are used to diagnose the presence of pathologic or
other specific
soluble immunogens in the cytosol or those released into the surrounding
media. The
diagnostic assays of the present invention are able to accurately diagnose the
presence of
the disease state and also determine the position of the patient in the time-
course of the
disease or other process.
DETAILED DESCRIPTION
The present invention comprises methods and compositions for the detection of
low
copy number targets of interest in diagnostic specimens in the presence of a
large excess of
normal material. The present invention can be used for diagnostic tests and
has the
capability to analyze specimens at the molecular, cellular, and tissue levels.
Methods and compositions of the present invention comprise non-specific target
elimination, NTE. NTE detects targets and supports high limits of specificity
and
sensitivity. Embodiments of NTE include the Haystack Processing technologies
such as
TPA (Target Protection Assay), RFTA (Restriction Fragment Target Assay), EAD
(Enzyme Assisted Diagnostics) and CPA (Cutter Probe Assays),and other
compositions
and methods as described in U.S. Patent numbers 5,962,225, and 6,100,040, and
U.S.
Patent Application Nos. 09/633,848; 09/992,128; 08/739,80; 09/569,504;
09/443,633;
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09/702,066; 10/055732; 09/705,067; and other detection methods such as those
disclosed
in U.S. Patent Application Nos. 09/776,568; and 09/933307; eachof which is
incorporated
herein in its entirety. The present invention is directed to methods and
compositions
including NTE which comprise Selective Target Monitoring technologies (STM)
with
Complement Mediated Signal Amplification (CMSA) and MACMSA (Membrane
Associated Complement Mediated Signal Amplification).
Not only do the methods and compositions of the present invention comprise
detection of nucleic acid and other molecular targets, but the methods and
compositions of
the present invention comprise diagnostics at supramolecular levels to confirm
the presence
of the pathologic or other cellular target in tissues. STM functions on a
cellular or nuclear
level to negate the presence of normal cells or nuclei in the sample by the
analysis of only
the cell subset of interest in a very large cell specimen and has the ability
to
compartmentalize and assay each cell component for the analyte of interest.
These low
copy number analytes are detected at low copy numbers by generating a signal
from the
specific analyte of interest, while no signal occurs from the normal or non-
specific analytes
present in the compartment. Other embodiments of STM comprise target analyte
sorting
and separation from non-specific analyte for increased sensitivity of
detection. STM on
a cellular level comprises CMSA. CMSA comprises the fixation and activation of
complement by interactions between cell subset specific surface membrane
proteins, and
monoclonal or other antibodies. The initiation of the complement fixation
process results
in the production of the C3a peptide in quantities directly proportional to
the extent of
complement fixation.
CMSA is used for detection of target cells and supports NTE in any sample,
particularly biological samples including, but not limited bo, all body
fluids, disaggregated
cells, such as those derived from tissue samples, lymph nodes and fine needle
aspirates,
and environmental samples. An embodiment of CMSA analysis on a cellular level
is
taught in U.S. Patent Application 09/776,568, U.S. Provisional Nos. 60/218,460
and
60/226,825, all of which are incorporated herein in their entirety. For
example, the intact
cell, or cell membrane ghost, or nucleus is treated with a monoclonal antibody
specific for
a surface protein of interest, thereby forming an Ab/Ag complex that fixes
complement. In
the presence of all the complement components, complement is activated to
produce C3a
peptides, whose quantity is directly proportional to the number of target
cells present. The
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target analyte comprises any cell subset, an HIV infected T-cell, a dysplastic
cell, and a
neoplastic cell or may also be a cell membrane or cell nucleus, as well as an
immunogenic
carcinogen or pathologic prion protein molecule.
C3a peptides are produced due to the interactive presence of a lipid membrane
containing a unique surface protein (irnmunogen), a monoclonal or polyclonal
antibody,
and the complement cascade components. The presence and quantification of the
C3a
peptide produced may be achieved by any number of methods known to those
skilled in the
art and discussed herein or in related documents. The key to CMSA is the
presence of a
lipid membrane that functions to amplify production of the C3a peptide by the
complement
cascade components. The present invention contemplates the use of lipid
membranes
found within the sample or lipid membranes that are added to the sample.
The methods and compositions comprising Membrane Associated Complement
Mediated Signal Amplification (MACMSA) are used to for sensitive soluble
protein
analysis. In an embodiment of this method, RBC sensitized stroma, comprising
antibody to
the unique protein attached to a RBC lipid membrane, interacts with the target
analyte
molecules present in the sample. Presence of the specific target analyte
causes an Ag/Ab
reaction to occur at the surface of the lipid RBC membrane, which in the
presence of the
complement components results in the full amplification of C3a peptide
production and
sensitive confirmation of the presence of the immunogenic target analyte.
MACMSA is
capable of molecular confirmation of a cellular diagnostic result as is taught
in U.S. Patent
Application 09/776,568, U.S. Provisional Nos. 60/218,460 and 60/226,825, all
of which are
incorporated herein in their entirety.
Soluble protein or peptide targets or other immunogenic molecules, whether
pathologic or not, can be analyzed by STM on a soluble cytoplasmic molecular
level that is
monitored by use of MACMSA. MACMSA can also sensitively detect protein/peptide
targets in any body fluid or other liquid sample. Another function of MACMSA
is to
detect and monitor non-protein chemicals in solution that are immunogenic
thereby fixing
and activating complement via the classical pathway, and to detect and monitor
polysaccharides or other related molecules that fix and activate complement
via the
alternative pathway. MACMSA is used for detection of soluble target molecules
in any
biological or environmental fluid sample including, but not limited to, all
body fluids, any
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soluble protein fluid suspension, environmental fluids, and chemical and
material
processing fluids containing the soluble, immunogenic target analyte.
Unique pathologic proteins or other immunogens at low molecule number in a
vast
excess of normal proteins are identified, using STM with high specificity and
sensitivity.
The specificity comes from the use of multiple specificity steps, and the
sensitivity is
supported by the minimization of signal background by non-specific target
elimination in
the fluid samples, either extracellular or intracellular, and generation of
signal from all
target molecules either intracellular or of exogenous target in a large sample
of analyte.
DESCRIPTION OF TABLES
Table I represents the stoichiometry of C3a peptide production by fixation and
activation of complement by both the classical and alternate pathways, which
mediates
target signal amplification in the MACMSA complement fixation assay.
Table II presents the TSE time-course and its requirements with references.
Table III represents some of the interactions between plasminogen (PG") and
plasma proteins, catalogued to delineate the PGn role in the BSE/TSE infection
time.
course.
Table IV depicts some of the in vivo interactions and functions of
plasminogen,
cataloglxed to delineate the PG" function in the BSE/TSE infection time-
course.
Table V is the Meta Screen Bovine Multiplex Diagnostic Assay, depicting the
three
MACMSA complement fixation assays.
Table VI is the Meta Screen Bovine Multiplex Diagnostic Assay, the molecular
direct automated analysis of RNA.
SELECTIVE TARGET MONITORING (STM)
STM cellular diagnostic technologies function on a cellular or nuclear
membrane
level to diagnose the presence of a pathologic or other cellular target,
usually a cell or
nuclear subset. A preferred embodiment comprises use of CMSA methods for
signal
amplification for the sensitive detection of the pathologic cell or nucleus.
CMSA is based
upon the activation and fixation of complement by addition to the target cell
of an antibody
specific to a cell surface or nuclear membrane protein. In eucaryotic cells,
the classical
complement activation pathway is activated and the extent and target presence
monitored
by production of the C3a peptide. In prokaryotic cells, surface carbohydrates
similarly
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participate by activation of the alternate complement fixation pathway also
resulting in the
production of the C3a peptide. One embodiment of CMSA, called MACMSA,
comprises
use of a soluble immunogen found in the cytoplasm or released into the
cellular
environment. These methods and compositions are used to diagnose the presence
of
pathologic or other specific soluble immunogens in the cytosol or those
released into the
surrounding media. The diagnostic assays of the present invention are able to
accurately
diagnose the presence of the disease state and also determine the position of
the patient in
the time-course of the disease or other process.
Signal amplification in STM on a cellular or nuclear level is directly
proportional to
the extent of complement fixation and activation. The cell surface membrane
and nuclear
membrane protein markers react with the specific monoclonal or other antibody
to the
immunogens resulting in fixation and activation of complement. Also cell
surface
polysaccharides and other materials fix and activate complement via the
alternative
pathway. The extent of complement fixation may be monitored as a function of
the
number of C3a peptides produced upon activation of fixed complement molecules,
known
to those skilled in the art.
MEMBRANE ASSISTED COMPLEMENT MEDIATED SIGNAL AMPLIFICATION
(MACMSA) AND TARGET SIGNAL AMPLIFICATION
The methods and compositions comprising MACMSA comprise embodiments that
function at the molecular level by using compositions comprising attachment of
an
antigenic epitope or a peptide comprised of numerous epitopes to an
oligonucleotide that
acts as a reporter probe in nucleic acid assays. One embodiment of MACMSA
comprises
using a single epitope to produce increased numbers of C3a molecules after
binding of
antibody sensitized RBC stroma to the epitope in the presence of complement
followed by
complement fixation and activation.
The extent of complement fixation and activation is influenced by many
factors.
These factors include avidity of the epitope and monoclonal antibody, and
concentration of
key intermediates in the complement cascade. For example, spiking native
complement
with additional C3 will increase the numbers of C3a produced by the presence
of a single
epitope in the assay. Other factors are determined by the method of complement
fixation
employed, either the classical or alternate pathway and the relative effect of
C3 spiking on
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complement fixation by each; and the use of sensitized RBC stroma used to
amplify the
C3a production signal from a soluble immunogen, and methods of quantification
of the
resulting C3a peptide. The factors influencing C3a production in MACMSA, when
optimized, can provide significant C3a peptide production per single target.
MACMSA AND SINGLE TARGET PROTEIN DETECTION
To achieve the full signal amplification effect of a soluble protein target in
STM, a
preferred embodiment requires the introduction of a lipid membrane to the
assay namely
antibody sensitized red blood cell stroma.
PRODUCTION OF SENSITIZED RBC STROMA
A preferred embodiment for production of RBC sensitized stroma employs the
production of an IgG antibody pair, more preferably each IgG antibody has a
different
specificity. For example, one IgG of the pair is an IgG anti~h monoclonal
antibody used
to attach the antibody pair to the RBC surface, without any need for chemical
modification
of the RBC. The second IgG of the pair is an IgG anti-epitope monoclonal
antibody used
to bind the epitope present on the reporter probe and to fix and activate
complement.
The red blood cells carrying the Rh determinants allow attachment of the
antibody
pair to the RBC membrane. A benefit of using the Rh determinant is that the
Rh/antrRh
complex is known to not fix complement. Any other Ag/Ab pair could also be
employed
in the methods and compositions of the present invention. RBCs with Ab pairs
are referred
to as sensitized.
In place of the anti D antibody attachment, any other site could be used that
is
expressed homogeneously on the RBC surface. In another embodiment an Fab or
(Fab)2
fragment specific for the alternate attachment site may be used due to the
absence of the Fc
region on the attachment antibody fragment used.
The sensitized RBCs are washed and lysed in a hypotonic buffer solution and
the
resulting membrane material is referred to as stroma. Other embodiments call
for the use
of digitonin to produce a more intact RBC stroma or any method, which is known
to those
skilled in the art. The stroma is washed to remove RBC contents and
resuspendedin a
suitable buffer. The stroma may now be used as a reagent.
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Addition of stroma, the reporter probe with epitope and fresh complement and
cofactors allows maximal C3a production. The solution may now be assayed for
C3a
peptide production by use of any procedure known by those skilled in the art,
such as
ELISA and sensitized RBC lysis or any other method known by those skilled in
the art.
SIGNAL AMPLIFICATION OF SOLUBLE PROTEIN TARGETS IN MACMSA
In these embodiments of STM, complement fixation and activation is quantified
by
a novel method, namely detection of production of the inactive complement
peptides (ICP),
C3a. Detection of the ICPs, preferably C3a, is achieved by assays for proteins
or peptides
that are known to those skilled in the art, including but not limited to,
competitive and
sandwich immunoassays such as ELISA assays, immunoMTRF or assays included in
the
present invention such as complement mediated signal amplification (CMSA) and
lysis of
sensitized RBCs, and lysis of liposomes containing fluorescence and quencher
molecules.
Complement is a group of at least 25 glycoproteins with varying
electrophoretic
mobilities. Most circulate in the blood in an inactive precursor form and have
effects in the
body only after activation. Two major functions of complement in vivo are to
promote the
inflammatory response and to alter biological membranes to cause direct cell
lysis or
enhanced susceptibility to phagocytosis. Cell lysis occurs when antibody-
mediated
complement is fixed and activated by sequential interaction of the entire
complement
cascade. Most of these interactions result in the cleavage of an inactive
protein with the
release of small peptides in the complement response. In vitro, these peptides
have no
function, or are called inactive complement peptides (ICPs). The peptides that
do not
participate in a direct complement response, meaning the lysis of cells or the
opsonization
of cells, are referred to herein as inactive complement peptides (ICPs). These
inactive
complement peptides (ICPs) have multiple in vivo functions: chemotaxis,
enhancement of
phagocytosis, alteration of vascular permeability, and stability of cell
membranes (platelets
and granulocytes). In a few instances, inactive proteins aggregate resulting
in an active
protein.
THE CLASSICAL COMPLEMENT PATHWAY CASCADE:
The first complement component C1, attaches to the Fc portion of
immunoglobulin
molecules that have the appropriate binding site in the CH2 domain of the
heavy chain. All
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mu (0) chains have this site, and most gamma (y) chains. C1 is composed of 3
subunits:
Clq, Clr, and Cls held together by calcium ions. If IgG is the type of
antibody used, two
adjacent protein antigenic sites must each bind an antibody molecule to form a
doublet
arrangement to provide the specific conformation for binding of the C1
complex. One
IgM pentamer can bind the C1 complex. Clq binding to the FC region of the
antigen/antibody complex undergoes a conformational change that activates Clr,
which in
turn activates Cls, and fixes complement.
The following represent the steps in complement fixation and activation
resulting in
the production of the ICPs (C2a, C4a, C3a, and CSa).
Each molecule of Clq bound or fixed to the target membrane will produce at
least
an equivalent number of C3 convertase molecules and the ICPs, C2a, C4a, C3a,
and CSa.
At least one C3 convertase molecule is formed per one Clq molecule initially
bound.
Thousands of surface membrane proteins are expressed on a single cell, thus
activation of
complement fixed by multiple sites on a single cell or nuclear membrane can
produce
thousands of C2a, C4a, C3a, and CSa ICPs.
C 1 s propagates the complement sequence by cleaving C4 into C4a and C4b and
cleaving C2 to uncover a labile binding site. C4b contains a binding site and
attaches to the
cell membrane. C4a is released into the solution in vivo to stimulate
anaphylaxis by
stimulating mast cell degranulation and histamine release, thereby increasing
vascular
permeability. This released peptide may be used in the present invention to
amplify the
signal from a target.
C2 attaches to the C4b molecule on the cell membrane. The larger fragment C2b
combines with C4b to produce C4b2b, called C3 convertase, which possesses
enzymatic
activity. Each initial C4b2b molecule (C3 convertase) can generate attachment
of hundreds
of additional C4b2b (C3 convertase) active complexes to the cell membrane in
proximity to
the Clq binding site (the lipid structure is a requirement for this event),
and in doing so,
releases additional C4a and C2a ICPs which can be used for signal
amplification methods
in the present invention.
The third step, also an amplification reaction, is based on the function of
all the
bound C3 convertase molecules (C4b2b) to each cleave hundreds of C3 molecules
in
solution resulting in release of additional C3a peptide fragments into the
solution. This
peptide has anaphylatoxin activity in viva, and will be exploited as a signal
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marker method in vitro. The C3b larger fragment binds to the cell membrane
complex or
decays in solution. C3b fragments by themselves are not active catalytically
and do not
promote cell lysis but do increase phagocytosis upon attachment to the cell
(opsonin
activity in vivo . The importance here is the additional production and
release of C3a into
the solution in vitro and plasma in vivo.
Some C3b molecules join the extensive numbers of C3 convertase attached to the
entire cell membrane forming C4b2b3b5b or CS convertase releasing the CSa ICP
into the
solution.
In the presence of CSb, molecules of C6, C7, and C8 and a variable number of
C9
molecules, assemble themselves into aggregates in the presence of Zn+2 called
the
membrane attack complex (MAC). The complex compromises the integrity of the
cell
membrane by altering permeability of the membrane and results in cell lysis.
It must be noted that C3a generation in CMSA and MACMSA is only one
embodiment of a complement fixation assay, wherein C3a is the measure of the
extent of
complement fixation and activation. In related applications, it has been
stated that any
other peptide produced or any other complex MAC complex formation, etc.,
characteristically formed during complement fixation and activation can be
used as a
measure of complement fixation and activation. This is relevant to any
produced stable
product or stable complex formed by the classical pathway as well as the
alternate pathway
during complement fixation and activation.
THE ALTERNATE PATHWAY COMPLEMENT CASCADE
Cleavage of C3 and subsequent activation of the remainder of the complement
cascade occurs independently of complement fixing antibodies. Cell surface
particulate
polysaccharide and lipopolysaccharide molecules, endotoxin, trypsin-like
enzymes, and
Ag/Ab complexes of IgA, and IgG4, that do not activate Cl, all function to
activate the
alternate pathway. °The activation is mediated by the cleavage of C3
into (~3a which is
released in solution and C3b. This molecule would be rapidly degraded in the
fluid phase
(classical pathway), but in the alternate pathway, C3b becomes stabilized by
binding to the
surface of a particulate activator of the alternate pathway called factor B,
forming a stable
C3b-factor B complex, itself interacting with a serum protease (factor D),
cleaving factor B
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to produce C3bBb, that functions as a C3 convertase, again catalytically
producing many
additional C3a peptides.
The alternate complement activation pathway is activated by all viruses,
bacteria,
yeast or any other microbe containing polysaccharide or lipopolysaccharide
elements in its
exterior cell wall.
One embodiment of the present invention, the novel in vitro use of the
complement
cascade and the generation of the ICPs in the amplification of a signal to
detect very low
copy number of targets, is described herein.
SIGNAL AMPLIFICATION IN STM
The present invention comprises novel and sensitive methods for signal
amplification, called CMSA and MACMSA. Activation of the complement cascade
results
in the production of millions of inactive complement peptides (ICPs). Analysis
of the
sample for the detection and quantification of the ICPs results in the
generation of 40
million ICPs per pathologic cell membrane, or nucleus (CSMA), and generation
of 40,000
ICPs per soluble protein target or immunogenic epitope with the involvement of
complement fixing Ag/Ab reactions in proximity to a lipid matrix (MACMSA).
Table I summaries the production of the ICPs and theoretical quantification
provided by CMSA.
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TABLE I
ICP Characterization and Quantification in CMSA
10' sites to Nature
fig C'
per membrane of Number ICPs produced based
on
surface ICP binding of each Cls molecule
(First Amplificationgenerated
Step)
C 1 q, C 1r, NONE NONE
C 1 S
C4b C4a 10 /cell or nuclear membrane
C4b2b3b C3a 10 /cell or nuclear membrane
C4b2b3b5b CSa 10 /cell or nuclear membrane
C6,7,8,9 NONE NONE
(Second 200 fold
increase
in ICPs
Amplification
Step)
C 1 q, C 1 NONE NONE
r, C 1 s
200 x 10'
C4b C4a
2 x 105 per Cls bound
C4b2b3b C3a 2 x lOJ
C4b2b3b5b CSa 2 x 10
C6,7,8,9 NONE NONE
(Third Amplification200 fold
increase
in ICPs
Step)
C 1 q, C 1 NONE NONE
r, C 1 s
C4b NONE NONE
C4b2 C3a 200 x 2 x lOJ o~
b3b
, 4 x 10 oY 40 Million (theoretical)
Not amplified here (same
value as
C4b2b3b5b CSa amplification step number
2) NONE
C6,7,8,9 NONE NONE
SUMMARY: ICP
numbers produced
in CMSA based
on the presence
of a
single pathologic
target cell
Primary Amplification
Step
3 x 103 ICPs
Secondary Amplification
Step
6 x 105 ICPs
Tertiary Amplification
Step
4 x 10 ICPs
Total ICPs
generated
per single
pathologic
target
4.0603 x 10
ICPs 40 Million
It is theorized that the above results are achieved by reducing the ICP
generation
1000-fold, due to the ability of a single immunogenic epitope to fix 1000-fold
less
complement than an average cell membrane.
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A preferred ICP is the peptide fragment C3a, because it is found in very high
numbers after complement fixation. Production of other ICPs (C4a, C2a, and
CSa) may
also be detected although they provide less than one percent of the total
signal generated by
the detection of a single pathologic cell, nucleus, or nucleic acid species.
In general, the novel in vitro use of the complement cascade to quantify the
presence of a pathologic cell or nucleus is based upon monitoring the extent
of complement
fixation and activation as a function of the number of inactive complement
peptides (ICPs)
that are produced. Basically, each target cell fixes thousand of complement
molecules after
addition of antibodies specific for the target cell surface protein and the
subsequent
reaction with the complement cascade. The initial complement molecules that
are fixed
can themselves exert an additional 200-fold amplification effect. These
complement
molecules also provide for another 200-fold signal amplification effect later
in the course
of the complement cascade. This results in the following theoretical total
signal
amplification profile in CMSA
a) Multiple cell surface protein markers on the dysplastic cell each fixing
complement, yielding 1000-fold amplification per pathologic target,
b) Primary 200-fold amplification during early stages of complement fixation,
c) , Secondary 200-fold amplification at a later step in the complement
cascade.
Total 40 million ICPs produced per target.
In MACMSA, the following represents the total signal amplification profile:
a) A single soluble protein or reporter immunogenic epitope fixes one
complement molecule.
b) Primary 200-fold amplification effect during early stages of complement
fixation that is lipid membrane dependent requiring the use of the RBC
sensitized stroma reagent.
c) Secondary 200-fold amplification at a later step in the complement cascade
(membrane independent). Total 40,000 ICPs produced per target.
SIGNAL AMPLIFICATION
Methods of signal amplification using the classical complement pathway employ
methods of CMSCA and MACMSA. Signal amplification methods for the alternate
pathway is similarly initiated by a step wherein a thioester on native C3 beds
to
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polysaccharide, such as a polysaccharide on the surface of an organism. Next,
the binding
of Factor B and its subsequent activation stabilizes the complex:
C3Ha0 + Factor B + Factor D = C3bBb + C3a
C3bBb = activated Factor B or C3 convertase
The first signal amplification step occurs by the convertase cleaving numerous
native C3 molecules producing numerous C3a peptides and additional C3b
molecules that
attach to the complex to form additional C3 convertase, that release
additional C3a in the
solution.
The C3 convertase (C3bBb) cleaves hundreds of C3 molecules generating
additional C3b molecules, which attach to the complex and amplifies its
activity. Cleavage
of the C3 mediates release of hundreds of C3a ICP molecules to mediate
amplificationin
vivo of the immune response and in vitro signal amplification.
The second level of signal amplification employs the aggregation on the
surface of
a microorganism or a protein aggregate of numerous C3b units, Factor B, and
Properdin
(stabilizing protein) acts as a potent CS convertase producing hundreds of CSa
(ICPs), thus
cleaving CS to an active CSb and release of a CSa into the solution. The
remainder of the
complement cascade is identical to later steps in the classical pathway. Thus,
the ICPs,
generated by complement fixation of the classical complement pathway, or the
alternate
complement pathway are used for in vitro signal amplification target detection
strategies.
STOCHIOMETRY OF ICP PRODUCTION VIA THE CLASSICAL COMPLEMENT
PATHWAY (See Table I)
In Table I, it is represented that three levels of C3a production or signal
amplification occurs based upon CMSA treatment of a cell or nuclear membrane.
First, thousands of surface protein molecules on a single cell or a single
nuclear
membrane fix thousands of C1 molecules producing a minimum of thousands of C3a
peptides post complement fixation.
Second, there exists a 200-fold amplification per each of the C 1 a molecules
fixed
due to the presence of the membrane proximity component for complement
activation.
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Third, there exists an additional 200-fold amplification per each of the 2 x
105
bound C3 convertase (C4b2b3b) molecules by cleavage of additional solution C3
and
formation of additional C3a peptide for a total of 4 x 107 or 40 million
molecules of C3a
peptide generated per target membrane.
THEORETICAL STOCHIOMETRY OF ICP PRODUCTION VIA THE ALTERNATE
COMPLEMENT PATHWAY
C3a production by the alternate pathway must be empirically determined.
Similar
signal amplification quantification can be configured based on the cyclic C3
convertase
enzymatic complexes formed. Though not wishing to be bound by any particular
theory, it
is believed that the absolute numbers of ICPs produced in the alternate
pathway are on the
same order of magnitude or greater than that observed by complement fixation
and
activation via the classical pathway.
DETECTION AND QUANTIFICATION ASSAYS FOR THE ICPS
(C4a, C2a, C3a, CSa)
Many assay strategies are available to determine the presence and
quantification of
the individual or combined ICPs. The present invention comprises assays for
measuring
the presence and number of individual or combined ICPs and is not limited to
the assays
and embodiments disclosed herein. The individual ICPs can be quantified by
assays for
proteins, including but not limited to sandwich ELISA assays, or similar
assays that use a
capture antibody bound to a solid support and a different labeled reporter
antibody both
specific for different epitopes on each ICP (C4a, C2a, C3a, CSa).
For example, an embodiment of the C3a sandwich ELISA assay is configured using
a biotinylated anti-C3a reporter antibody and is followed by addition of an
IgG anti-biotin
alkaline phosphatase polymer conjugate to facilitate signal generation per C3a
molecule by
introduction of the substrate, 1,2-dioxetanes. Any other enzyme known to those
skilled in
the art may be used to quantify the number of C3a molecules. The enzyme may
provide a
color signal, a fluorescent signal, or a chemiluminescent signal, all known to
those skilled
3 0 in the art.
A preferred embodiment of the signal generated by the C3a peptide molecules is
mediated by the use of an anti biotin alkaline phosphatase polymer, known to
generate 4
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logs of signal per polymer molecule. The polymer is then reacted with a
chemiluminc-scent
substrate generating a stable light signal. One such substrate is the
l,~Dioxetanes, which
have been shown to detect 0.01 attomole quantities of alkaline. phosphatase
enzyme,
translating to a ten-fold increased level of target detection by the enzyme
polymer. This
detection system will support unprecedented high levels of target detection
and, due to the
nature of antibody conjugates to enzymes, will provide a relatively low
background in the
negative controls
Such methods may also be automated. An example is shown below.
Step I. Prepare a magnetic bead with a covalently bound IgG anti-C3a capture
antibody. The binding can be achieved by any chemistry known to those skilled
in the art
such as covalently linking a carboxylated magnetic bead to the primary amine
on the n-
terminal end of the antibody molecule, or any other chemistry known to those
skilled in the
art.
Step II. The magnetic bead is washed to remove non-bound capture probes and
Step III. Conjugated beads are added to a sample containing the C3a peptide in
solution,
which is mixed and incubated.
Step IV. The magnetic beads are washed to remove non specific bound materials
Step V. Addition of another antibody, IgG anti-C3a, which has reporter
function and is
specific for a different epitope on the C3a peptide molecule. This antibody
possesses an alkaline phosphatase (AP) polymer covalently attached to it. This
may be generated by any method known to those skilled in the art, the
preferred
one being attachment antibody N-terminal amine ofthe maleimide derivative of
the AP polymer, which results in covalent bond formation. Any other chemistry
may also be employed.
Step VI. Wash to remove unbound reporter probe. The number of washes and the
wash
buffer may be critical in resolving non-specific signal from unbound reporter
enzyme.
Step VII. Addition of the magnetic beads to a solution containing the 1,2-
Dioxetane
substrate and incubate under conditions foi the production of a stable
chemiluminescent signal.
The reporter antibody, and hence the target, is detected by the activation of
a
chemiluminescent substrate to produce light by enzymatic catalysis.
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The reporter antibody can also be detected using immuno MTRF methods as
disclosed in U.S. Patent Application No. 09/443,633 or by conjugating a label,
such as a
single molecule of fluoroscein isothiocyanate, to each ICP reporter antibody.
Another method of the present invention for C3a quantification comprises steps
to
identify and quantify the specific ICP of interest using sensitized RBCs
conjugated with
anti-specific ICP antibodies, which will only react with the free-floating
ICPs in solution.
In this embodiment RBCs linked to anti-ICP monoclonal antibodies will in the
presence of
complement undergo complement-mediated immunoerythrocyte lysis, releasing
hemoglobin for quantitation.
The extent of RBC lysis is directly proportional to the quantity of ICPs
produced
and targets present.
Another method for assay of C3a production would be the use of IgG anti-C3a
antibody imbedded on the surface of a liposome containing fluorescence and
quencher
molecules in close proximity, so that no fluorescent signal can be detected.
Introduction of
a C3a peptide to the antibody-sensitized liposome, in the presence of the
complement
components will result in complement mediated lysis of the liposome, releasing
the
fluorescence and quencher molecules into the solution. Their release and
separation can be
monitored by the detection of a fluorescent signal. The extent of liposome
lysis is directly
proportional to the quantity of ICPs produced and targets present.
GENERATION OF SENSITIZED RBCs FOR C3a ASSAY: RBC ENZYME
TREATMENTS
One embodiment of the present invention comprises methods to identify and
quantify specific ICPs of interest comprising use of sensitized RBCs that are
conjugated
with specific anti-ICP antibodies that will only react with the free-floating
ICPs in solution
and in the presence of fresh complement, result in red blood cell lysis upon
binding of free
ICPs with subsequent complement fixation and red blood cell lysis.
The sensitized or immuno RBCs can be generated by stripping the RBCs with a
proteolytic enzyme such as bromelain, ficin, or papain and by other methods
known to
those skilled in the art, that attach the ICP specific antibodies to the RBC
surface,
producing sensitized immunoerythrocytes which bind the free floating ICP in
solution.
This attachment of an antibody to the stripped RBC surface by simple exposure
of the
1~
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antibody to the erythrocyte provides a non-covalent attachment of the antibody
molecule,
and is sufficient for some applications. Due to the fact that chemical
modification of the
RBC surface involves increased fragility of the modified RBC, which may result
in the
spontaneous release of hemoglobin and make quantification of the ICP peptides
difficult,
other methods are also contemplated by the present invention.
A novel process for production of antibody sensitized RBCs is mediated by the
use
of an IgG antibody pair. The characterization of the molecule is as follwvs:
1. Two IgG molecules are attached to each other by any method known to those
skilled in the art, where the attachment does not interfere with the antibody
binding sites.
2. One antibody must be specific to any of the ICP peptides for assay; for
example, the IgG anti-C3a antibody used in the C3a peptide assay. Other
embodiments require this antibody to be specific for any immunogenic epitope
on the target.
3. The other antibody is specific for an antigen on the RBC. A most preferred
embodiment comprises use of an antibody specific for the Rh determinant. The
Rh determinant extensively covers the RBC membrane with thousands of
molecules and this is the site at which the antibody pair binds to the
erythrocyte.
This antigen/antibody reaction does not fix complement. This is important in
light of the use of this immunoerythrocyte in the presence of fresh complement
to monitor attachment of the C3a peptide to the complement fixing anti-C3a
antibody in close proximity to the RBC surface. Any interactive
antigen/antibody interaction that does not fix complement may also be
employed.
4. The Rh determinants on the RBC surface are responsible for bringing the
antibody to the C3a and other peptides in close proximity to the lipid
membrane
surface without altering the stability of the immunoerythrocyte.
The sensitized immunoerythrocyte in the presence of the corresponding peptide
and
fresh complement will undergo lysis by the membrane attach complex and
hemoglobin will
be released.
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THE ANTIBODY PAIR METHOD FOR IN VIVO NEUTRALIZATION OF A
PATHOLOGIC ANALYTE BY SENSITIZED RBCs
Another embodiment for use of the antibody-pair molecule may involve its use
in
vivo to neutralize the activity of a pathologic analyte. This analyte may be a
viral particle,
antibody molecule, dysplastic or cancer cell, and even an immunogenic
environmental
carcinogen. Attachment of the IgG anti Rh-IgG anti pathologic analyte antibody
pair to the
RBC surface would facilitate the immediate attachment and neutralization of
the pathologic
analyte to any of the RBCs that have been sensitized.
Neutralization of the activity of the pathologic analyte would immediately
block its
reactive effect and would initiate its removal from the body mediated by
macrophage
phagocytosis or the function of another clearance system in the spleen and
liver. It is
known to those skilled in the art that RBCs possessing immune complexes on
their surface
are rapidly cleared by these body systems.
PRODUCTION OF SENSITIZED RBC STROMA FOR USE IN MACMSA
MACMSA requires the interaction of a lipid/antibody complex with a soluble
protein or reporter probe immunogenic epitope. The preferred embodiment for
production
of this complex is the sensitization of the RBCs by the aforementioned method
with
subsequent lysis of the sensitized RBCs in a hypotonic buffer solution
resulting in the
production of antibody attached lipid membrane (RBC stroma) that will exert
the full
signal amplification effect of the immunogenic epitope or soluble protein by
the MACMSA
process. Stroma production is achieved by placement of the immunoerythrocytes
in a
hypotonic buffer resulting in RBC lysis and membrane ghost formation. The
stroma is
then washed in buffer and resuspended in buffer for use as a reagent. Any
other method
known to those skilled in the art may be used.
STMIPRION SORTING IN SOLUBLE PROTEIN SAMPLES
One embodiment of prion sorting in a protein sample can be achieved by
attachment of a monoclonal antibody at its C-terminal end to a magnetic bead.
The
magnetic beads are placed in a large volume solution such as plasma or brain
biopsy
extract or any other and mixed. The epitopes available for interaction on the
C-terminal
end of the prion molecule remain exposed in both the normal and aberrant prion
molecule.
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The magnetic beads are collected with a magnet and washed in buffer. All the
prion present in the sample will be separated from all other soluble proteins.
The C-
terminal antibody will capture the normal and pathologic prions due to the
continued
accessibility of the epitopes of both forms even after the transition has
occurred.
In this embodiment the prion sorted magnetic bead is treated with a monoclonal
antibody available to the N-terminal end of the prion molecule (the (3 sheet
isoforni end)
that is labeled with an alkaline phosphatase polymer or any label known to
those skilled in
the art. Both are incubated and washed in buffer.
The N-terminal end of the pathologic prion has undergone a transition from an
a, to
a (3 sheet form. During this transition, epitopes, normally found on the N-
terminal end are
covered and new epitopes exposed. To date no monoclonal antibodies have been
found
that successfully discriminate the a sheet form (normal prion) from the (3
sheet form of the
aberrant prion.
Monoclonal antibodies specific for the N-terminal end of the pathologic prion
are
necessary for use in these sensitive diagnostic assay embodiments.
Next, the magnetic beads are exposed to a chemiluminescent substrate, such as
the
1,2-Dioxetanes, which would be able to detect 0.01 attomoles quantities of
alkaline
phosphatase enzyme. Theoretically, supporting increased sensitivity than that
achieved by
prion precipitation by sodium phosphotungstate and time resolved fluorescence
previously
mentioned.
CURRENT PrPs~ DIAGNOSTIC ASSAY: PROBLEMS WITH SPECIFICITY AND
SENSITIVITY
To date there has only been one report of a monoclonal antibody specific for
the
aberrant prion that would not react with normal prion. This report was non-
substantiated
resulting in the following strategy for post mortem brain assay.
First, since one could not differentiate between the normal and aberrant
prions, one
needed to rely on the protease (proteinase K) resistance of PrPs~ post enzyme
treatment,
not observed with PrP° or PrP enzyme treatment.
Once the post mortem sample is treated by proteinase K then the surviving
prions
are reacted with the non-discriminatory prion monoclonal antibodies currently
used. 'The
rationale is that the only surviving prion must be an aberrant or (3 sheet
conformation prion.
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It is known to those skilled in the diagnostic assay design that this type of
assay is fraught
with sensitivity problems, and that the extensive sample manipulations, and
improper
protease treatment could also result in specificity problems.
STM MACMSA SENSITIVE DETECTION OF PATHOLOGIC PRIONS
STRATEGY FOR A BLOOD BASED EARLY DIAGNOSTIC FOR BSE
Current post-mortem brain diagnostic assays currently are based upon selective
destruction of normal prion in a sample by protease and guanidinium treatment
followed by
ELISA assay for identification of the resistant prion, which is assumed to be
the PrPsc
aberrant form. Also, Western blot has been shown to be of value in this area.
The
antibody in this assay recognized both normal and aberrant prion with no
ability to
discriminate between either. The success of these assays can be attributed to
their
insensitivity, namely, the lack of specificity of the anti prion antibody and
the insensitivity
of the Western blot.
For the post-mortem diagnostic assay to function, large samples of obviously
diseased brain must be obtained, which will assure a positive result even with
the use of
such insensitive methods. Furthermore, these diagnostics are costly and time
consuming.
What is needed is a more sensitive and rapid diagnostic for the postmortem
assay.
Consideration that this assay would only provide disease (BSE) confirmation as
a cause of
death, an even greater challenge in diagnostic process design would be to
develop a
sensitive, rapid, and inexpensive early infection diagnostic assay for BSE and
other TSEs.
It is known to those skilled in the art that early in the infection, blood
plasma levels
contain 10 to 1000 aberrant prions (PrPsC) per milliliter (closer to the
former earliest in the
infection time-course). These early BSE assays could take the form of blood or
other body
fluid assays where a sample can be acquired by a non-invasive (urine sample
acquisition)
or a minimally invasive (blood draw) technique, or any other sterile sample
taken for
diagnostic purposes known to those skilled in the art.
Remembering that no antibody currently exists that exclusively and selectively
discriminates the normal a sheet from the abnormal (3 sheet prion conformation
poses
difficulties in early diagnostic disease assay design.
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The approach used in this embodiment of the invention was developed by
thorough
analysis of all the facts related to BSE and other TSE diseases, such as
scrapies, where TSE
animal model systems abound.
It is known to those skilled in the art that scrapies infection can be blocked
by
inhibition of B cell formation, T cell formation, and complement depletion
(C1q deficient
host). The disease inhibition can be reversed by reestablishment of T and B
cell production
and complement activity (reversal of Clq deficiency).
Table II presents a number of conclusions drawn relative to the TSE time-
course
and its requirements (appropriate references are indicated).
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SOME CONCLUSIONS REGARDING TSEs
TABLE II
Remark Reference
Replication of infectivity occurs Eklund, et al.
in lymphoid organs 1967
Infectivity found in components of Aguzzi, A.,
the lymphoreticular et al. 1998
system (LRS), lymph nodes and Peyer's
plaques in the
small intestine where replication
of infectivity can be
demonstrated almost immediately following
oral
administration of prion preparations.
Follicular dendritic cells (FDC) may Aguzzi, A.,
be the main et al. 1998
population of cells involved in LRS
replication of prion.
All mutations that disrupted the differentiationAguzzi, A.,
and et al. 1998
response of B-cells prevented the
development of clinical
disease.
Spongifonn encephalopathy developed Aguzzi, A.,
after peripheral et al. 1998
inoculation in mice expressing immunoglobulin
that was
exclusively of the M subclass and
without detectable
specificity for the normal form of
the prion PrP~ in mice
with B-cells but no functional FDC.
Differentiated B-cells are crucial Aguzzi, A.,
for neuroinvasion by et al. 1998
spongiform encephalopthy regardless
of the specificity of
their receptors.
If prions were delivered to the CNS Aguzzi, A.,
spongiform et al. 1998
encephalopthy pathogenesis and prion
expansion in the
brain proceeded without any detectable
influence of the
immune status of the host
B-cells may transport prions from Aguzzi, A.,
lymphoid organs to et al. 1998
nervous tissue
The apparent protection of B-cell Aguzzi, A.,
deficient mice from et al. 1998
prion administered intraperitoneally
may result in the
absence of immunoglobulins.
Complexing of PrPJ'' with antibodies Eigen, M. 1996
may favor
nucleation (a process proposed to
underlie the formation of
prion infectivity)
Com~lexing of PrP"' with antibodies Aguzzi, A.,
may opsonized et al. 1998
s
and enhance access to lymphoid sites
of abnormal
PrP
prion expansion
One cannot exclude the possibility Aguzzi, A.,
that IgMs below the et al. 1998
threshold of detectability, or indirect
effects of antibodies
may be involved in spongiform encephalopathy
pathogenesis.
The TSE infected B-cells carrying Aguzzi, A.,
PrP"' are the et al. 1998
bottleneck of disease promulgation.
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Remark Reference
Interaction exists between the B-cellsAguzzi, A.,
(carriers of et al. 1998
infectivity) and the T-cells (unable
to propagate
infectivity). Spleen mice contain
both T cells and B-cells,
and upon infection of the B-cells,
a B-cell mediated
secondary infection of the spleen
mice's T cells takes
place.
TSE infected B-cells are predictive Aguzzi, A.,
of the pathological et al. 1998
outcome and progression of disease
In mouse scrapie, depletion of B lymphocytesKlein, M.A.
prevents et al.
neuropathogenesis after intraperitoneal1997;
inoculation
probably due to impaired lymphotoxin-dependentKlein, M.A.
et al.
maturation of FDCs, which are a major1998
extracerebral
prion reservoir.
FDCs trap immune complexes with Fcy Klein, M.A.
receptors and et al.
C3d/C4b-opsonized antigens with CD21/CD351997;
complement receptors. Klein, M.A.
et al.
1998
Depletion of circulating immunoglobulinsKlein, M.A.
or of individual et al.
Fcy receptors had no effect on scrapie1997;
pathogenesis if B-
cell maturation was unaffected Klein, M.A.
et al.
1998
Mice deficient in C3, Clq, Bf/C2 and Botto, M., et
combinations al. 1998;
thereof or complement receptors were Kuhn, S.E.,
partially or fully Taylor,
protected against spongiforni encephalopathyR.P., et a1.1998;
i.p.upon
exposure to limiting amounts of prions.Molina, H.,
et al. 1996
Splenic accumulation of prion infectivityKlein, M.A.
in complement et al.
deficient mice and PrPs~ accumulation1997;
was delayed,
indicating that activation of specificKlein, M.A.
complement et al.
components is involved in the initial1998
trapping of prions in
lymphoreticular organs early after
infection
Lymphotoxin beta (LT beta) aids FDC Montrasio, F.et
in neuroinvasion
a1.2000
Sympathetic nerve fibers transport Aguzzi, A.,
the rogue prions et al. 1998
through the peripheral nervous system
into the CNS
Plasminogen has a neurotrophic factor-likeNakajima, K.,et
effect on
cultured neurons a1.1994
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CONSENSUS OF OPINION ON EARLY TSE TIMECOURSE EVENTS
It is, herein, proposed that these documented observations can be interpreted
as
follows. This predicted chronology of events that define the early stages of
disease will
support the presented early specific and sensitive diagnostic processes to
bedetailed herein.
The T cells may function in antigen (aberrant prion) display, in order for B
cell
production of a weak initial antibody response (IgM initially). Complexation
of the anti-
priori antibody and the aberrant priori, will fix and activate plasma
complement thereby
depositing C3b and other complement fragments on the aberrant priori antibody
complex.
The C3b may then interact with the M cell (in intestinal Peyer's patches of
man) surface
C3b or other complement receptor, resulting in ingestion via phagocytosis of
the Ag/Ab
C3b complex. The infection of BSE by ingestion of tainted meat supports the
intestinal site
of entry into the body. Furthermore, prior to phagocytosis, an exposeds lysine
on the fold
of the ~3 sheet aberrant priori binds a serum plasminogen molecule at its
I~ringle 1-3 domain
(Fischer, 2000). The role of this bound plasminogen is not well understood
however;
plasminogen has been definitively implicated in a number of documented
activitiesin vivo.
Table III represents some of the documented interactions between plasminogen
and
plasma proteins. It is herein stated that the plasminogen Kringles, regions
may bind n or c
terminal lysine molecules as well as s lysine residues present in the internal
regions of the
peptide/protein when accessible to the I~ringle region.
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TABLE III
BIOLOGICALLY RELEVANT PLASMINOGEN (PLASMII~- PROTEIN
INTERACTION INVOLVES ANCHORING OF THE ZYMOGEN THROUGH LYSINE
BINDING SITES IN THE KRINGLE DOMAINS.
PG" or Plasmin binding Function Reference
to:
C terminal Lysine of Stimulate Christensen, U. 1985
fibrinogen and
involved: enhance clot
lysis
C terminal lysine residuesInhibition Hortin,G.L., et al,
of of 1988
PGn and plasmin treatedplasmin
clots
Other proteins with Binding to Hamanoue,M, et al,
C terminal a-enolase 1994
lysine
Other proteins with Inhibition Sugiyama, N., et
C terminal of PGn al, 1988
lysine
Internal Lysine involved:
s-Lysine of PrPs~ Undetermined Fischer, M.B., et
al, 2000
s-aminocaproic acid Inhibition Sugiyama, N., et
of PG" al, 1988
To understand the possible function of the plasminogen on the PrPsC molecule,
Table IV
depicts some of the known documented in vivo interactions and functions of
plasminogen,
catalogued to delineate the PG" function in theBSE/TSE infection time-course.
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TAELE IV
PLASM1NOGEN/PLASMIN FUNCTIONS
Function Example Reference
Thrombolytic activityClot lysis Miyato, T.
1982
Cell movement Macrophage invasion in Unkeless,
J.C.
inflammation 1973
Mammary cell involution Ossowski,
after L.
lactation 1979
Breakdown of follicular Reich, R.
wall for 1985
ovulation
Trophoblast invasion Strickland,
into S.
endometrium during embryogenesis1976
Angiogenesis Gross, J.L.
1983
Keratinocyte accumulationMorioka,
after S. 1991
wound healing
Mediator in pathologicTumor cell growth, invasionOssowski
process of L. &
of cell migration surrounding tissues, Reich, E.
metastasis 1983
Ability of cell bound Mullin D.E.
plasmin to &
directly degrade extracellularRohrlich,
matrix S.T.
proteins: 1983
Proteoglycans Edmonds-Act,
X.
1980
Fibronectin Jilek, F.
1977
Laminin Schlechte,
W.
1989
Type IV collagen MacKay, A.R.
1990
Indirectly responsible Stricklin,
for lysis of G.P.
matrix proteis through 1977
activation of
metalloprotease proenzymes
Activation of transformingRegulation of inflammationKhalil, N.
1996
growth factor (31(TGFfI)
to its
active form
Extracellular or Classic complement pathwayAgostoni,A.1994
pericellular
protease causing Alternate complement Brade, V.
proenzyme pathway 1974
conversion Contact phase of blood Cochrane,C.G.
coagulation
1974
Proinsulin-insulin conversionVirgi,M.A.G.
1980
Bradykinin generation Habal, F.M.
from 1976
kininogen
Proteolytic y-globulin Janeway,
C.A.
activationdestruction 1968
of
2~
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Function Example Reference
activation/destructionCoagulation factor V Lee C.D.
of &
plasma proteins Mann, K.G.
1989
Coagulation factor VII Denson K.W.
&
Redman, C.W.
1980
Anticoagulation protein Varadi, K.
C 1994
Neuronal excitotoxicityBinding of PG" to neuronalHamanoue,
cells
M.1994
Interaction _in ormal prion PrP release
vivo with _in vivo
plasma platelets
Plasminogen functions presented herein, pose a strong case for the requirement
of
plasminogen on the PrPs~ molecule for the invasiveness of the prion in early
stage disease.
This includes normal prion accumulation and invasion of the aberrant prion
into tissues
necessary at some stage of the infectious PrPsC time-course.
PrPs~ entry at the site of the M Cell in the intestinal Peyers patches in man
is
supported by 1) an understanding of mucosal immunology (Mucosal Immunology,
1999),
which is the efFector site for vaccines in the body, 2) acceptance of the
PrPsC infection
requirement for active B cells and T cells, and 3) the presence of the C3b
complement
receptor on the M Cell macrophage (fixed monocyte) surface.
It is not to be interpreted that this is the only mechanism of PrPSO entry
into the
body, but only one of a number of mechanisms, some of which involve non-
specific
pinocytosis of digestive fluid by intestinal epithelial cells (IEC), uptake by
the FDCs,
uptake by B-cells, and other purported mechanisms.
Upon formation of the PrPsc/PG"/antibody/C3b the PrPso complex enters the
lymphatic system by some method, where it travels to all lymphoid organs
including the
tonsils and ultimately the brain. Movement of the PrPsC complex in the
lymphatic system
may afford the complex protection from phagocytosis by the C3b receptors
present on
numerous phagocytic cell surface in phagocytic departments that are not
involved in the
infection and may even limit the infection by disintegrating the PrPs~
complex.
Remembering that C3b receptors are found on all polymorphonuclear granulocytic
macrophages in the circulation and all fixed monocytic macrophages in the
body, some
protective effect would be required.
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An alternate sequence of events for PrPsc infection could entail PrPsc entry
to the
blood, post ingestion, followed by complex formation (host IgM
antibody/primary antibody
response, complement, and plasminogen addition), and transport to the brain,
whereupon
glial fixed monocytic cells possessing C3b receptors could phagocytize and
internalize the
PrPsc complex and further the disease course.
Whatever events occur during the early staging of the BSE infection, the
existence
of the PrPsc complex as presented herein is indicated.
TARGETS FOR EARLY PrPsc DETECTION
Peripheral infection, in particular, oral uptake of TSE infected material is
epidemiologically most relevant for BSE, Sheep Scrapie, I~uru, and "~CJD.
Acceptance of this rationale provides a number of targets that may be of value
in an
early disease time-course, blood based pre-mortem PrPsc assay. They are:
1. Presence of plasminogen on the (i sheet conformation, PrPsc
2. Presence of a host primary response antibody (IgM) or other antibody that
may be
found to bind to the PrPsc
3. Presence of C3b or other complement cascade products such as C3d and C4b or
other complement fragments on the PrPsc molecule
4. Presence of PrPsc, only possible for use as a target if a monoclonal
antibody is
produced that will discriminate PrPsc from PrPc or PrP (not as yet known to be
in
existence).
CHARACTERISTICS OF AN EARLY BLOOD DIAGNOSTIC ASSAY FOR THE
PRESENCE OF THE ABERRANT PRION PROTEIN, PrPsc
It is preferable that early diagnostic assays for a disease be highly
specific, highly
sensitive and capable of automation, as well as cost efficient, to assess the
state of the
animal or human, as well as for monitoring residual disease, once a treatment
or cure is
available. The high specificity of the assay, defined as the lack of false
positive results, can
be provided by a mufti-step procedure that is designed to limit the generation
of signal by
non-infectious target analyte (prions).
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The high sensitivity of the assay, defined as the lack of false negative
results, can be
provided by the ability to screen large amounts of sample analyte, namely
prion from a
large volume of plasma, for the aberrant prion.
Ease of use of the assay, such as by automation, allows for widespread use in
limiting epidemics by monitoring the spread of disease and maintaining
surveillance of the
state of the animal or human. Furthermore, combined manual and automated
assays are
readily achieved with the ability to screen volumes of plasma sample.
In related applications, the concept of non-specific target elimination (NTE)
was
presented. Basically, NTE are process designs that limit the generation of
signal or abolish
the generation of signal from non specific targets. On a molecular level the
Haystack
Processing inventions TPA and RFTA, previously presented, achieve such by the
use of
enzymes to degrade non-specific targets resulting in the generation of signal
exclusively
from the target analyte. These diagnostic processes function to detect very
low copy
number DNA and RNA targets. On a supramolecular level (whole cells,
intracellular and
extracellar proteins, and other immunogens), the Haystack Processing invention
Selective
Target Monitoring (STM, previously presented), mediated by Complement Mediated
Signal Amplification (CMSA) and the Membrane Assisted Complement Mediated
Signal
Amplification (MACMSA), also achieve such by the use of a signal generation
process
initiated by an immunogenic protein or other immunogenic target and permits no
signal to
be generated by a non-specific target analyte. All Haystack Processing
strategies result in
direct analysis of previously non analyzable large samples of analyte with the
ability to
detect the presence of very low copy number DNA, RNA, cell subsets, and
protein aid
other immunogenic targets of interest.
The MACMSA process provides a complement fixation assay for immunogenic
targets. The MACMSA assay has been presented earlier and is based upon C3a
generation
as a result of complement fixation and activation, wherein the increasing
number of targets
produces increasing levels of C3a.
The TSE META SCREEN ASSAY: TARGETS FOR PrPs~ DETECTION IN BLOOD
AND OTHER HOST FLUIDS EARLY IN The INFECTIOUS TIM~COURSE
As previously presented, the targets for PrPsC presence, are indirect in
origin, due to
the lack of an antibody specific to the aberrant prion. Meta Screen Assay
functions by
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detection of plasminogen, host primary antibody (IgM) and a complement
fixation product
C3b, C3d, C4b, or other complement cascade product or fragment on a PrPs~
molecule, not
found on the PrP~ or PrP prion molecule.
This is achieved by use of an antibody-sensitized red blood cell (Rh
Pos/R2R2),
stromal cocktail, represented as follows:
TARGET Molecule Pair to Sensitize Rh Pos Stroma for MACMSA
Plasminogen (PG") IgG anti PG" - IgG anti-D
Antibody to Heterologous IgG anti bovine IgM (or other
PrPs~ Ab)-IgG anti D
(low levels)
ex. bovine
assay
IgG anti C3b - IgG anti-D
Complement IgG anti C3d - IgG anti-D
fragment on IgG anti C4b - IgG anti-D
PrPsC
IgG anti other complement component -
IgG anti-D
TION OF RBCs BY THE MOLECULE PAIR METHOD
This novel method for sensitization of RBCs uses the D antigen expressed
homogeneously on the entire RBC surface to attach the molecule pair. The D
antigen was
selected due to its inabili to fix complement upon complexation with the anti-
D antibody
of the molecule pair. In related applications, other sites on the RBC may also
be used,
however, since complement fixations must not occur, an Fc deficient Fab or
(Fab)2
fragment to another homogeneously surface expressed RBC protein or other
similarly
functioning moiety may be used in place of IgG anti-D.
The method for sensitization of the intact RBCs by the molecule pair has been
presented in related applications. Sensitized RBC stromal may be produced from
sensitized RBCs by any method known to those skilled in the art. One preferred
example
may involve the use of digitonin to produce high quality strorna.
STRATEGIC APPROACHES TO THE SENSITIVE EARLY INFECTION DETECTION
OF PrPs~ 1N BLOOD
STRATEGY I: This process utilizes the existence of a monoclonal antibody
specific
for the PrPsC that exhibits no reactivity to normal prion (PrP). To date, no
monoclonal
antibody has been found to selectively discriminate the aberrant (PrPs~) from
normal prion
(PrP° or PrP). The steps of the process are:
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Step I: Isolation of all the prion in a sample (PrP and PrPs~) by attachment
to a magnetic bead coated with a charged moiety known to those skilledin the
art, that will
specifically bind this prion protein subset.
Step II: Addition of the monoclonal antibody specific to PrPsC in the form of
sensitized RBC stroma (IgG anti PrPs~-IgG anti-D is the sensitization molecule
pair).
Step III: Addition of the immune complement reagent and incubation under
conditions to generate the amplified signal, namely, the C3a peptide.
Step IV: Assay for the C3a peptide by any method known to those skilled in
the art.
STRATEGY II: This process eliminates the need for a discriminatory monoclonal
antibody for PrP and PrPs~. It has been reported that the PrPs~ molecule binds
plasminogen, the inactive zymogen proteolytic enzyme, while the PrP does not
bind the
zymogen. Furthermore, documented observations, previously discussed herein,
also
support the attaclnnent of an IgM antibody to the aberrant prion. This IgM
antibody would
be produced as a low titer (possibly currently undetectable by current ELISA
assay)
primary, antibody response to a protein antigen, similar to a protein vaccine.
Also, the
model presented herein calls for complement fixation or C3b or other
complement
fragments to be attached to the prion/3 target complex presented. The steps of
this process
are:
Step I: Isolation of all the prion in a sample (PrP and PrPs~) by attachment
to a magnetic bead coated with a charged moiety known to those skilled in the
art, that will
specifically bind this prion protein subset.
Step II: Addition of the monoclonal antibody specific for any non-Kringle
region site on the PG" molecule.
It should be noted that plasminogen is known by those skilled in the art to
participate in fibrinolysis, metastasis of cancer cells into tissue, and
release of normal prion
proteins from blood platelets, thereby insuring that the PrPsC molecule
possesses invasive
potential as well as securing the flow of blood platelets and providing the
continuous
supply of PrP substrate for conversion to PrPs~ and the resultant production
of the amyloid
fibrils and plaques in brain tissue continuous in the disease time-course.
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One must also consider that plasminogen is prevalent in blood at high
concentration
and PrPsC upon introduction to the body, due to reaction kinetics, based on
concentrations
of the reactants, should be spontaneously bound to the PrPs~ molecules
present.
Furthermore, antibody (monoclonal) specific to the host IgM and another
antibody
(monoclonal) specific to C3b may also be added. All three antibodies should
indirectly
detect the early time-course PrPs~/PG"/host IgM/C3b complex.
Step III: Addition of the immune complement reagent with cofactors and
incubation under conditions to generate the amplified signal, namely, C3a
peptide
production.
Step IV: Assay for the C3a peptide by any method known to those skilled in
the art.
STRATEGY III: This process eliminates the need for use of the immune
complement
reagent, while still focusing on detection of the plasminogen moiety attached
to the PrPsc
molecule, absent from the PrP molecule.
Note: The activation peptide of the plasminogen post PrPs~ binding must be
sterically
available for removal by any activation molecule known to those skilled in the
art.
Step I: Isolation of all the prion in a sample (PrP and PrPs~) by attachment
to a magnetic bead coated with a charged moiety known to those skilled in the
art, that will
specifically bind this prion protein subset.
Step II: Addition of an activation molecule under conditions that will convert
the plasminogen zymogen proenzyme to the active enzyme plasmin, known by those
skilled in the art.
Step III: Addition of another zymogen or proenzyme that the plasmin will
activate, or addition of a substrate that the plasmin will catalytically
modify, both steps
initiate generation of a signal. Any zymogen, cascade, or any enzymatic
reactionknown to
those skilled in the art may be used. The preceding is referred to as Zyrnogen
Mediated
Signal Amplification or ZMSA and was presented previously (provisional patent
number
60/226,823).
Step IV: Addition of a substrate to the second zyrnogen (proenzyme) to
catalytically generate a detectable signal.
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Step V: Detection of the amplified signals, generated in Steps III and IV, by
any method known to those skilled in the art.
STRATEGY IV: This process comprises use of steps involving serine proteases
activating immune complement. The PrPsC plasrninogen complex is treated by any
method
lrnown to those skilled in the art resulting in the formation of the active
enzyme, plasmin.
The resulting plasmin should activate the complement pathway by cleavage of C3
into C3a
pathway resulting in the increasing generation of the C3a signal peptide.
Step I: Isolation of all the prion in a sample (PrP and PrPsC) by attachment
to a magnetic bead coated with a charged moiety known to those skilled in the
art, that will
specifically bind this plasma protein subset.
Step II: Addition of any activation molecule, known by those skilled in the
art, under conditions that will convert the plasminogen zymogen proenzyme to
the active
enzyme plasmin.
Step III: Addition of immune complement reagent and cofactors under
conditions that will allow the activation of complement, resulting in the
generation of the
amplified signal, the C3a peptide.
Step IV: Assay for the C3a peptide by any method known to those skilled in
the art.
STRATEGY V: Monoclonal antibodies currently available that bind, PrPs~, PrP~
and
PrP can be attached to magnetic bead and used to remove all prion proteins
from the
sample. This will support large sample testing and will allow thorough removal
by
washing of non-specific moieties associated with the prion fraction, such as
plasma
plasminogen, plasma IgM, and plasma C3b.
This strategy will. be fully presented as the Meta Screen Assay and will be
automated on robotics platform.
ADDITIONAL STRATEGIES FOR PrPs~ ASSAY DEVELOPMENT
The embodiments for the sensitive detection of PrPso presented, herein, are
only
meant to indicate a direction in assay development or treatments for humans or
animals.
Any similar strategy is herein included, and any other moiety, such as a
different enzyme
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or another type of molecule that has specificity for PrPs~ and not PrP is also
included
herein. The present invention is illustrated by the examples included herein
which are not
to be construed in any way as imposing limitation. The present invention is
further
illustrated by the following examples, which are not to be construed in any
way as
imposing limitations upon the scope thereof. It will be clear to one of skill
in the art that
various other modifications, embodiments, and equivalents thereof exist which
do not
depart from the spirit of the present invention and/or the scope of the
appended claims.
THE MULTIPLEX BOVINE METASCREEN ASSAY
The sensitive C3a complement fixation MACMSA assay can not only detect low
copy number aberrant prions, but may also include detection of other diseases
of the host.
One embodiment in cattle may provide early and sensitive multiplex detection
for the
following:
DISEASE
MULTIPLEX DISEASES AGENT
PrP (BSE) Prion
Vesicular Stomatitis Virus (VSV) Virus
Foot and Mouth Disease (FMD) Virus
Mycobacteria Species Bacterium
Brucellosis Species Bacterium
The main requirement for these and additional disease screening in this
sensitive
C3a complement fixation system is the production of the molecule pair
sensitized RBC
stroma. The following correlates the disease with the specific molecule pair
sensitizing
antibody:
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DISEASE TARGET SENSITIZING MOLECULE PAIR
BSE PrP IgG anti PG" - IgG anti-D
BSE PrP Mouse IgG anti bovine IgG - IgG
anti-D
BSE PrP IgG anti C3b - IgG anti-D
BSE PrP IgG anti C3d - IgG anti-D
BSE PrP IgG anti C4b - IgG anti-D
VSV Protein IgG anti VSV coat protein-IgG anti-D
Coat
FMD Protein IgG anti FMD coat protein - IgG
Coat anti-D
MycobacteriaCell SurfaceIgG anti mycobacterium surface
Protein protein-IgG anti-D
Brucellosis,Cell SurfaceIgG anti Brucella surface protein
etc. Protein - IgG anti-D
It need be understood that these diseases include any number of and all known,
and
the targets selected may be any known, as long as one possesses an
accompanying
antibody, antibody fragment, or other molecule with similar function.
This antibody specific for the target becomes one part of an antibody pair, a
molecule pair; the other antibody of the pair is any antibody and related
moieties
previously discussed that functions to attach the molecular pair to any
surface on the red
blood cell or stroma to be sensitized. In a preferred embodiment this
attachment site is the
Rh POS (R2R2) surface protein on the RBC.
THE BOVINE METASCREEN ASSAY (see Table V)
The targets of interest must interact with their respective sensitized RBC
stroma to
assure complement fixation and activation, monitored by C3a production.
Step I
A: Isolation Of All Prions
The first step involves isolation of all prion in the plasma sample by
attachment of
both PrPs~ and PrP° (PrP) to antibody coated magnetic beads. The
antibody
removes all prion from the plasma and allows washing of the prion material
(the
Mab is specific to the C terminal end of all prion). Washing removes non-
specifically bound plasminogen bovine IgM, and exogenous C3a, C3b, C3d and
C4b from the plasma attached to the prion coated magnetic beads.
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It must be remembered that plasminogen, endogenous host antibody (IgM) and
C3a, C3b, C3d and C4b is found in normal plasma, necessitating washing all
bound
prions to remove these non-specific moieties.
B: Plasma Aliquot Secured For Analysis
The remaining plasma contains bovine pathogenic viruses and bacteria, which is
analyzed separately from the prions, but in parallel. This will be later
described in
detail.
The prions, after thorough washings, bound to the magnetic beads, are treated
with
the three RBC sensitized stromas previously mentioned. An aliquot of the
remaining plasma is directly added to the four RBC sensitized stromas that
relate to
the specific virus and bacteria presence that were previously mentioned. These
microorganisms may fix complement by either the classical pathway or the
alternate pathway. Plasma containing intact microorganisms, upon introduction
to
the corresponding sensitized stroma (anti-microbe antibody is present), will
result
in complement fixation by the classical pathway; however, carbohydrate present
on
the microorganism may similarly fix complement via activation of the alternate
pathway (note: activation of the alternate pathway also leads to C3a
production).
Step II
A: Release of Bound Prion Into A Suspension Of Sensitized RBC Stroma
The total prion content on the magnetic bead is released by any method known
to
those skilled in the art (ex. Chemical, acidity, alkalinity, treatment
followed by bead
removal and solution neutralization, etc.) into a solution containing the
three
sensitized stroma (specific to plasminogen, host IgM, C3b, C3d, and C3b to
screen
selectively for PrPs~ in a complement fixation MACMSA assay where PrP~ and
PrP are transparent to the assay.
B: Addition of Plasma Aliquot To A Suspension Of T'he Corresponding Sensitized
RBC Stroma
This direct addition of a large plasma aliquot will allow the presence of the
microorganism at low numbers to directly interact with the sensitized stroma
and to
directly interact with the sensitized stroma and to fix complement via the
classical
pathway, produce C3a, and be detected.
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Step III: Incubate And Add Human Complement
Initial formation of the antigen/antibody-stromal complex upon addition of
complement and cofactors results in C3a peptide formation. This includes use
of washed
prion with its respective three molecule pair sensifized stromas as a separate
sample. Also
included is the use of plasma aliquot and complexation with the appropriate
anti-microbial
sensitized RBC stromas as another separately analyzed sample.
THE META SCREEN BOVINE DIAGNOSTIC ASSAY
The Meta Screen Bovine diagnostic assay is presented in Table V and Table VI.
The diagnostic process is a multiphase approach in a multiplex diagnostic
assay. Herein,
the Meta Screen Assay presented will involve bovine diagnostic multiplex assay
to early
detect a number of targets responsible for a number of disease states early in
the bovine
infection time-course. Similar Meta Screen Assays may be configured for a
human
multiplex diagnostic (including a target responsible for Creutzfeld Jacob
Disease (TSE in
humans), such as that responsible for the anti prion antibody to "~CJD, as
well as other
targets such as pre-cancerous (dysplasia), cancer, metastatic cancer, residual
disease and
any other applicable and similar target.
The Meta Screen Assay may also be converted into an environmental panel
wherein, immunogenic chemical pollutants may be multiplex assayed along with
other
targets such as microbial contaminants, carcinogenic molecules, and any other
conceivable
and applicable target that meets the immunogenic criterion of the process, the
immunogenic property of the target.
Meta Screen diagnostic panels may be configured for all biological organisms
and
their diseases that can be determined to have an immunogenic target and a
monoclonal
antibody specific for that target.
THE MACMSA DIAGNOSTIC PROCESS: A COMPLEMENT FIXATON ASSAY
(Table V and Table VI)
As a complement fixation assay based on a number of targets, MACMSA would
reflect the extent of complement fixation known to those skilled in the art
and as such
allow quantification of the extent of signal amplification. These amplified
products of
complement fixation and activation could include all peptides produced by
fixation and
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activation of the cascade as well as the formation of any of the cascade
structures, or any
other moiety produced by this fixation and activation.
It was recognized that an effective method to perform a complement fixation
reaction, namely MACMSA would be to use a molecule pair comprised of two
attached
antibodies to sensitize RBC stroma as a mechanism to assure maximal complement
fixation
and activation of a single immunogenic target to be detected. It is known by
those skilled
in the art that a lipid matrix is required to assure maximal complement
fixation and
activation of a single imrnunogenic molecule.
The molecule pair, in one embodiment is a monoclonal IgG antibody specific to
the
target, coupled by known chemistries to another IgG antibody specific to the
Rh D protein
on the RBC, the site for attachment of the molecule pair and other possible
attachment
sites. The attachment moiety may also be an antibody fragment, or other
attachment
molecule specific for any homogeneously expressed protein on the surface of
the RBC.
The D site of molecule pair attaclnnent was selected due to the knowninabili
of
the D/IgG anti-D to fix complement. Thus molecule pair attachment will
sensitize the
RBC without chemical modification and without complement fixation or
activation, along
with the antibody fragments Fab or (Fab)Z, and similar functioning molecules
can be used,
providing complement is not fixed or activated.
The theoretical extent of amplification or complement fixation employing this
system has been calculated to be 40,000 C3a peptides produced per single
immunogenic
molecule target in MACMSA, described earlier herein. Theoretically delivering
a BSE
diagnostic assay sensitive to 100 aberrant prion molecules (4 million C3a
molecules are
readily detectable by any method known to those skilled in the art).
DESCRIPTION OF THE BOVINE META SCREEN ASSAY: PHASE I (Table ~
The goal of the assay is to determine the presence of the predicted
prion/protein
complex that would be found early in the infection. This complex as stated is
PrPso/plasminogen/host IgM antibody/C3b, C3d, and/or C4b.
Confirmation of any component of this complex will provide a sensitive
diagnostic
result.
Step I: The Sample (Aliquot I)
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Plasma from a single animal or pools of plasma from several animals may be
used
as a sample. An aliquot of 100 ~.1 can be assayed on a ~obotic analyzer device
The ability
to analyze a large volume sample is known by those skilled in diagnostics to
assure the
detection of the pathologic target in the earliest stages of the host
infection time-course.
If necessary for early BSE detection, a SOcc or larger plasma sample can be
manually prepared and the concentrated prions introduced into the robotic
platform for
automated PrPsC complex target detection. To achieve this, magnetic beads
would be
added to the large volume plasma sample.
Step II: Magnetic Bead Addition
Magnetic beads are added that are coated with an IgG monoclonal antibody that
or
any other molecule known to bind PrPsC, PrP°, and PrP, namely all prion
forms, known by
those skilled in the art.
Step III: Wash
Wash the magnetic beads to remove non-specific proteins including plasminogen,
human antibody (or any type, specifically IgM in the circulation or other
intestinal
antibody.
Step IV: Dissociate the Prions
Any method known by those skilled in the art may be used to dissociate the
prions
from the magnetic beads, place them in a separate tube and remove the stripped
magnetic
beads.
Step V: Sensitized Stroma Addition
Add molecule pair sensitized Rh Pos (R2R2) sensitized stroma. The R2R2 Rh Pos
type was selected for its greatest RBC surface expression and strongest
interaction with the
anti-D moiety of the molecule pair. Any other Rh Pos type may , also be used.
The
molecule pairs are:
IgG anti PGn - IgG anti-D
IgG anti IgM (or other Ab) - IgG anti-D
IgG anti C3b - IgG anti-D
IgG anti C3d - IgG anti-D
IgG anti C4b - IgG anti-D
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The attachment site of the molecule pair should be homogeneously expressed on
the
RBC surface. The above are the preferred embodiment but all other similarly
functioning
attachment molecules have been discussed.
The molecule pairs presented will detect the aberrant prion protein complex
(PrPsC/plasminogen/bovine IgM/any complement fragment attached) found early in
the
BSE time-course.
Step VI: Incubate To Allow The Antigen/Antibody Reaction To Occur
Incubate 37°C for 30 to 60 minutes at pH 7.2.
Step VII: Complement and Cofactor Addition
Complement, calcium and magnesium are added to the prion-stroma mixture. One
embodiment calls for use of human complement due to the fact that the C3a
peptide ELISA
used to quantify C3a is composed of monoclonal antibodies (capture and
reporter) specific
to the human C3a peptide. Calcium and magnesium are each added at a
concentration of
mM.
15 Step VIII: Incubate at Room Temperature
Incubate at 24°C for 30 to 60 minutes to allow complement fixation
by the
target/stroma complex and activation resulting in increased or amplified
levels of C3a
production.
Step IX: Assay for C3a Peptide
20 The prion/stroma/complement mix is mixed by periodic aspiration up and down
in
the pipette tip and, upon completion of the incubation, the stroma is allowed
to settle to the
well bottom; an aliquot of supernate is removed of 50 to 100 ~1 to quantify
C3a peptides
present.
Quidel Corporation possesses a C3a (human) sandwich ELISA using horseradish
peroxidase to generate a color substrate signal.
Another C3a quantification assay embodiment currently under development is a
C3a sandwich ELISA using magnetic beads coated with the capture antibody IgG
anti C3a
and the reporter antibody another IgG anti C3a labeled with biotin (for
example), and
subsequent use of a streptavidin alkaline phosphatase conjugate, which,
coupled with a
sensitive chemiluminescent substrate (1,2 Dioxetane) known to those skilled in
the art,
provides a more sensitive assay for C3a quantification.
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DESCRIPTION OF THE BOVINE META SCREEN ASSAY: PHASE II (Table V)
The goal, herein, is to detect the presence of microbes, bacteria, fungi,
virus, etc. in
the sample. The complement fixation reaction involves the presence of
immunogenic
proteins found on the microbe surface and requires the possession of the IgG
anti-microbe
surface protein molecule attached to the IgG anti-D (the molecule pair) used
to sensitize the
stroma.
Step I. The Sample (Aliquot II)
Plasma from a single animal or pools of plasma from several animals may
be used as a sample. Currently, an aliquot of 100 ~l can be assayed on a
robotic analyzer
devise. The ability to analyze a large volume sample is known by those skilled
in
diagnostics to assure the detection of the pathologic target in the earliest
stages of the
infection time-course.
If necessary for early BSE detection, a SOcc or larger plasma sample can be
manually prepared and the microbes present can be concentrated by any method
known to
those skilled in the art. The concentrated microbes are nextprocessed by
direct addition to
the various sensitized stromas.
Step II: Sensitized Stroma Panel Addition
The sensitized stroma panel, in this embodiment, will detect the presence of
Vesicular Stomatitis Virus (VSV), Foot and Mouth disease virus (FMD),
Mycobacterial
s ep ties, Brucella species and even the Nemavirus that some believe to be the
etiologic
agent for BSE (Narang, 1998). The molecule pairs used to sensitize the Rh Pos
(R2R2)
RBC stroma are:
IgG anti VSV coat protein - IgG anti-D
IgG anti FMD coat protein - IgG anti-D
IgG anti any Mycobacteria surface protein - IgG anti-D
IgG anti any Brucella surface protein - IgG anti-D
IgG anti any Nemavirus surface protein - IgG anti-D
The sensitized stromal panel will support the complement fixation reactions to
detect the presence of the intact microbe.
Step III: Incubate To Allow 'The Microbe/Antibody Reaction To Occur
Incubate 37°C for 30 to 60 minutes at pH 7.2.
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Step IV: Complement and Cofactor Addition
Complement, calcium, and magnesium are added to the microbe-strorna mixture.
One embodiment calls for use of human complement due to the fact that the C3a
peptide
ELISA used to quantify C3a is composed of monoclonal antibodies (capture and
reporter)
specific to the human C3a peptide. Calcium and magnesium are each added at a
concentration of 20 mM.
Heterologous complement from other species may be used, only if the two anti-
C3a
monoclonals for the species specific C3a peptide are in hand.
Step V: Incubate at Room Temperature
Incubate the mixture at 24°C for 30 to 60 minutes to allow complement
fixation by
the target/strorna complex and activation resulting in increased or amplified
levels of C3a
production.
Step VI: Assay for C3a Peptide
The rnicrobe/stroma mix is mixed by periodic aspiration and, upon ccrnpletion
of
the incubation, the stroma is allowed to settle to the well bottom; an aliquot
of supernate is
removed of 50 to 100 ~,l to quantify C3a peptides present.
Quidel Corporation possesses a C3a peptide sandwich ELISA using horseradish
peroxidase to generate a color substrate signal.
Another C3a quantification assay embodiment currently under development is a
C3a sandwich ELISA using magnetic beads coated with the capture antibody IgG
anti C3a
and the reporter antibody another IgG anti C3a labeled with biotin (for
example), and
subsequent use of a streptavidin alkaline phosphatase conjugate, which,
coupled with a
sensitive chemiluminescent substrate (1,2 Dioxetane) known to those skilled in
the art,
provides a more sensitive assay for C3a quantification.
DESCRIPTION OF THE BOVINE META SCREEN ASSAY: PHASE III (Table V)
The goal, herein, is to confirm detection of the presence of a microbe in the
sterile
plasma of the cow. This approach is based on the complement activation
produced by the
Alternate Complement Pathway in the absence of the antibody on the sensitized
stroma. It
is known by those skilled in the art that intact microbes possess carbohydrate
surface
moieties, which when exposed to complement, with its cofactors activates
complement and
produces C3a peptides proportionate to the number of microbes present in the
sample.
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This internal confirmation can detect low copy microbial targets in any fluid,
such
as plasma, urine, cerebrospinal, and amniotic.
Step I: The Sample (Aliquot III)
Plasma from a single animal or pools of plasma from several animals may be
used
as a sample. An aliquot of 100 ~,1 can be assayed on a robotic analyzer
devise. The ability
to analyze a large volume sample is known by those skilled in diagnostics to
assure the
detection of the pathologic target in the earliest stages of the infection
time~course.
If necessary for early BSE detection, a SOcc or larger plasma sample can be
manually prepared and the microbes present can be concentrated by
centrifugation,
dialysis, or any other method known to those skilled in the art. The
concentrated microbes
are next processed by direct addition of complement.
The goal of this phase of the assay is to confirm the presence of the target
microbe
by another signal amplification method, comprising direct activation of the
Alternate
Complement Pathway by the direct presence of the microbe, in the sterile
sample, resulting
in increased C3a peptide production
Step II: Complement and Cofactor Addition
Complement, containing properdin and Factor B as cofactors, is directly added
to
the plasma sample for analysis. Interaction of the microbe and complement will
activate
complement and result in C3a peptide production. One embodiment of the assay
calls for
the use of human complement, due to the fact that the C3a peptide ELISA, used
to quantify
C3a is composed of monoclonal antibodies (capture and reporter) specific to
the human
C3a peptide.
Step III: Incubate at Room Temperature
Incubate the mixture at 24°C for 30 to 60 minutes to allow complement
activation
(no fixation due to lack of Ag/Ab complex formation) resulting in increased or
amplified
levels of C3a production.
Step IV: Assay for C3a Peptide
The microbe/stroma composite is mixed by periodic aspiration up and down in
the
pipette tip and, upon completion of the incubation, the stroma is allowed to
settle to the
well bottom; an aliquot of supernate is removed of 50 to 100 ~l to quantify
C3a peptides
present.
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Quidel Corporation possesses a C3a peptide sandwich ELISA using horseradish
peroxidase to generate a color substrate signal.
Another C3a quantification assay embodiment currently under development is a
C3a sandwich ELISA using magnetic beads coated with the capture antibody IgG
anti C3a
and the reporter antibody another IgG anti C3a labeled with biotin (for
example), and
subsequent use of a streptavidin alkaline phosphatase conjugate, which,
coupled with a
sensitive chemiluminescent substrate (1,2 Dioxetane) known to those skilled in
the art,
provides a more sensitive assay forC3a quantification.
DESCRIPTION OF THE BOVINE META SCREEN ASSAY: PHASE IV
In related applications, an automated analysis technique has bee presented for
automated RNA isolation and direct RNA analysis using RP-TFO RNA TPA
diagnostic
analysis process.
It is the goal of this phase of the assay to confirm on a molecular level
(mRNA) the
presence of the organism and even determine the serotype, and determine the
antibiotic
resistance of the microbe, to initiate a successful treatment course.
This RP-TFO diagnostic process works well with single stranded mRNA or other
single stranded RNA targets, such as found in VSV and FMD infections.
Furthermore,
mRNA can be detected for any derepressed gene in the DNA of the microbe, for
example,
2.0 the mRNA, which would establish antibiotic resistance to the microbe.
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THE META SCREEN BOVINE MULTIPLEX
DIAGNOSTIC ASSAY
TABLE VI
DIRECT RNA ANALYSIS
PHASE IV
Plasma (Aliquot 4)
Automated Magnetic Bead RNA Extraction
Perform RP-TFO RNA TPA)
Add
RP-TFO Specific for RNA of:
VSV
FMD
Perform signal amplification
MTRF:
Sensitive signal
Signal amplification process
Create a clzezzzilunziziescezzt signal
Assay for chemiluminescence
Disease Screen:
Vesicular Stomatitis Virus,
Foot and Mouth Disease
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THERAPEUTIC STRATEGY IN LIMITING AND RESOLVING BSE INFECTION
ROLE OF THE MOLECULE PAIR 1N VIVO: DISEASE ERR.ADICATION
HOST ANTIBODY RESPONSE TO PrPsc AND ITS CONSEQUENCE
With the presented evidence that B cells and T cells participate in an early
TSE
(BSE) infection event (described and referenced herein), one scheme for BSE
and PrPsc
infection would involve antigen (PrPsc) DISPLAY by the appropriate T
lymphocyte and
subsequent presentation to a B cell subset that would produce an antibody
specific to
PrPsc.
This antibody would be required to fix complement, a requirement also found in
an
early infection event in scrapies (described and referenced herein). It could
be an IgM or
other antibody that is characterized as a single molecule that alone can fix
complement.
Any other similar single antibody molecules fixing complement are included in
embodiments of the invention.
The complement requirement would place complement fragments C3b, C3d, C4b
and other molecules on the PrPsc/plasminogen/host antibody complex.
It is known by those skilled in the art that all phagocytic cells possess one
of a
number of complement fragment receptors, primarily functioning in opsonized
clearance of
antigen/antibody complexes and microbial organisms, which are both opsonized
in vivo.
The following chart presents phagocytic cell types, all possessing the C3b
receptor for
internalization of the antigen, which in this discussion is the
PrPsc/plasminogen/host
antibody/complement fragment complex:
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Phagocytic Cell Location
Type Of
(Different Phagocytic PhagocyticInvolvement Of Tissue
In BSE
compartments) Type Activity Infection Time-Course
PolymorphonuclearPolys Peripheral
or Not postulated
Granulocyte PMNs blood
MATURE Monocyte Spleen Spleen B-cells in the spleen
(fixed monoc
tes)
y '
Kupfer cells, fix
Liver Liver ed tissue monocyte
expressing C3b
M-Cell , a fixed tissue
monocyte
IntestineIntestineexpressing C3b receptor;
M cells/
vaccine effector site
Brain Brain filial Cells or fixed
tissue monocyte
expressing C3b
IMMATURE MonocytePeripheralPeripheralMay occur to a minor
extent
blood blood
Ingestion of the PrPsC complex would carry the target complex throughout the
lymphatic system, the tonsils, and the other lymphoid organs to the brain.
Now that the PrPs~ complex has left the intestine and traveled to the brain
via the
lymphatic system, amyloid fibril formation begins to occur and plaques begin
to form
characteristic of the disease.
The singular point of disruption of the proposed time-course is the phagocytic
event
itself, no matter where it occurs. It is known that some viruses, such as HIV,
forni low
grade infections in monocytes (fixed tissue and immature circulating types),
survive and
advance the disease state. It is also understood that a similar virus
phagocytized and
internalized by a different phagocytic cell subset namely the circulating
polymorphonuclear
granulocytes would be destroyed. This is a function of different digestive
vesicles and
enzymes being present in different phagocytic compartments.
In this BSE infection scenario and this invension, it is stated that the C3b
mediated
PrPs~ complex internalization is a critical point to disrupt the infection and
limit and
resolve it.
It is proposed that a shift in the phagocytic cell compartment for the PrPSO
complex
may result in its internalization and destruction. To achieve this, a method
is needed to
direct the PrPsC complex away from the fixed monocytes in tissues to the
circulating
polymorphonuclear granulocytes in the peripheral blood.
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USE OF A MOLECULE PAIR SENSITIZED BOVINE RED BLOOD CELL TO
CHANGE THE PHAGOCYTIC COMPARTMENT WHICH INTERNALYZES THE
PrPsC COMPLEX
A therapeutic embodiment of the molecule pair has been presented in related
applications where it has been proposed that immunogens in the circulation can
be
removed from the circulation mediated by a method for attachment of the
immunogen in
vivo to the molecule pair sensitized RBCs and that the nature of this
attachmentexclusively
predisposes the phagocytosis by the polymorphonuclear granulocytes in
circulation as
opposed to the fixed tissue monocytes regulated primarily by the dispersal of
the
sensitizing antibody on the RBC surface, represented by the following chart:
Molecule Pair
Attachment Sites on
Sensitized RBC
Surface Mechanism Site of Ingestion
Discrete CRI CRl exchange
Patches
around surface reaction Fixed monocytes spleen/liver
of RBC
(Taylor,
1998)
Homogeneous PMN Circulating PMNs
over
entire surface phagocytosis
of RBC
DESIGN OF THE MOLECULE PAIR FOR A STRATEGY TO DIRECT THE PrPsc
COMPLEX TO A DIFFERENT PHAGOCYTIC COMPARTMENT TO RESULT IN ITS
DESTRUCTION
The molecule pair is characterized as being a pair of monoclonal antibody
molecules covalently attached, one being specific for the target, namely the
PrPSC complex
and the other specific for a site that is homogeneously expressed on the
bovine RBC
surface.
It is known that the life expectancy of a bovine RBC is approximately four
months
and a number of proteins are homogeneously expressed on the surface. The
molecule pair
attachment site would be any one that is homogeneously expressed over the
entire surface.
The attachment antibody cannot fix complement, and as such, an antibody
fragment
(Fab) or (Fab)2 devoid of the Fc region would be used to avoid complement
fixation and
sensitized RBC clearance by the circulatory system. The antibody recognizing
the PrPsC
complex would be the IgG anti complement fragment antibody, identical in
function to the
CA 02447892 2003-11-19
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complement receptors on the phagocytic cell surface. The molecule pairs ofthe
preferred
embodiment of the invention would be: IgG anti C3b - (Fab)2 anti bovine RBC
antigen,
anti C3d - (Fab)2 anti bovine RBC antigen, anti C4b - (Fab)a anti bovine RBC
antigen, and
similar for any other complement fragment attached to the PrPsC complex.
PrPs~ CLEARANCE FROM THE BLOODSTREAM
The stated molecule pair is used to in vivo sensitize the bovine RBCs (4 month
life
expectancy). The IgG anti complement fragment antibody attached to the RBC
surface
will compete with the monocytic phagocytic cells for the
PrPsC/plasminogen/host
antibody/complement fragment complex and understanding that the sensitized
RBCs would
greatly outnumber the fixed tissue monocytes, one can prevent the PrPS~
complex
internalization by the "normal" route and attach the complex to sensitized
RBCs via the
molecule pair. This will interrupt the nornial BSE infection course.
It is also known to those skilled in the art that polymorphonuclear
granulocytic
phagocytes also express Fc receptors on their surface, which aid in
in~rnalization of any
complex containing the Fc region. This should further assist in interruption
of the normal
infection course of the PrPs~ complex. In reality, this may not play an
important role in
phagocytic compartment redirection probably do to the presence of Fc receptors
in the
fixed monocytes of the spleen and liver, which are responsible for destruction
of antibody
coated RBCs (Fc regions present).
Next, the RBCs with attached PrPs~ complex are phagocytized as the complexes
bind to the molecule pair sensitized RBCs. The zipper mechanism known for. PMN
phagocytosis attaches the PMN to the first PrPsC complex which remains intact
in the
circulation to mop up or attache additional PrPsC complexes, causing more
complete
phagocytosis until a certain threshod of PrPs~ complexes is met, which
supports the total
engulfinent and destruction of the sensitized RBC and all the PrPs~ complexes
that are
attached.
It is important to note that, one, the PrPs~ complex is effectively
neutralized upon
binding to the sensitized RBC and no downside exists for its delayed
destruction, and two,
binding of the PrPs~ complex to the molecule pair sensitized RBC will not fix
additional
complement and release toxic hemoglobin into the circulatory system, due to
the presence
of decoy acceleration factors (DAF) found on blood elements and body tissues
that prevent
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CA 02447892 2003-11-19
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formation of the membrane attack complex (MAC), a group of late complement
cascade
proteins that form a hole in the cell membrane. DAF on the RBC membrane
inhibitsMAC
formation.
The mechanism of action of DAF involves the acceleration of destruction of
classical and alternate pathway C3 and CS convertases (Nicholson-Weller, 1982;
Medof,
1984; Pangburn, 1986; Fujita, 1987).
This new phagocytic compartment would prevent the PrPs~ complex from pursuing
its "normal" course and the PrPSC complex internalization in the granulocytic,
phagocytic
compartment would destroy the complex and limit resolve the infection, as well
as any
other different compartment involved in internalization and digestion of the
PrPsC complex.
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