Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-12-03
2003-03-04
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S009100, C424S130100, C424S133100, C424S134100, C424S137100, C424S138100, C424S139100, C424S141100, C424S145100, C436S501000, C436S513000, C436S536000, C436S547000
Reexamination Certificate
active
06528269
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a method for identifying prion protein which is involved in various transmissible neurological disorders of the central nervous system (CNS) in both humans and animals. Specifically, the method is based on the use of hybridomas or monoclonal antibodies (Mabs) and/or epitope binding fragments thereof prepared against prion protein in order to detect the presence of prion diseases. These antibodies or fragments thereof are suitable for use in highly sensitive immuno-assays for demonstrating the presence of prion protein. Additionally, the invention is also directed to pharmaceutical compositions containing the antibodies or fragments thereof.
BACKGROUND OF THE INVENTION
A prion is a small infectious protein. It is believed to be the cause of a number of degenerative neurological diseases. These prion caused diseases are collectively hereinafter referred to as “prion diseases”.
Prions were formerly called “slow viruses” but are now known to be devoid of nucleic acids and are, therefore, neither viruses nor viroids. The name prion is a contraction of the terms protein and infection. Prions are resistant to inactivation by procedures that modify nucleic acids.
The membrane glycoprotein, now called prion protein (PrP), is involved in the pathogenesis of prion diseases. However, the normal function of PrP and its precise role in disease is not fully understood. It is believed that prion diseases are associated with alterations in PrP. The PrP gene is generally expressed at high levels in neuronal cells of the brain and at lower levels in other tissue such as the heart, lung and spleen. Furthermore, studies indicate that prion diseases are associated with a build up of PrP in and around cells of the brain.
Normal cellular prion protein is encoded by one single gene which can exist in multiple glycoforms with molecular masses existing between 27-40 K. Daltons. The glycoprotein is attached to the cell membrane of mammalian cells by a glycosyl phosphatidyl inositol (GPI) anchor.
Prion caused diseases, or transmissible spongiform encephalopathies (TSE), are neurodegenerative disorders that affect both humans and animals. Prion diseases are referred to as sponiform encephalopathies due to the characteristic of forming holes or pores in cranial tissue.
Development of prion disease may be the result of mutations in the PrP gene. Inherited prion diseases include; Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker syndrome or disease (GSS) in humans. Prion diseases can also be contracted by an infectious mechanism. This group of diseases includes iatrogenic CJD and a new variant of CJD, which may be the result of transmission of bovine spongiform encephalopathy (BSE, also referred to as “Mad Cow” disease) from cattle to humans.
The majority of the prion diseases are sporadic disorders. The causes of these sporadic disorders are currently unknown (Parchi, et al.,
Neurol
., 8, 286-293 1995).
The prion diseases present in humans are generally present as a progressive dementia (impairment of intellectual functions) or ataxia (defective muscular coordination). In contrast, scrapie of sheep and bovine spongiform encephalopathy (BSE) are generally manifested as ataxia illness. Sheep and cattle possessing these forms of prion diseases lose their coordination and subsequently have to be destroyed.
The neuropathology of transmissible spongiform encephalopathies (TSE) typically comprises vacuolation of neuronal soma and of neurites and neuronal loss accompanied by reactive astrogliosis. The prognosis of patients having prion diseases is fatal.
Furthermore, the recent discovery of the transmission of bovine spongiform encephalopathy (“Mad Cow” disease) to humans raises an important health issue concerning the potential spreading of these fatal pathogens from domestic animals to humans. Therefore, there is a strong need for diagnostic agents to detect prion diseases and for therapeutic agents to inhibit infection.
Accumulated evidence suggests that the causative agent underlying prion diseases is a proteinaceous infectious pathogen that lacks nucleic acid (Prusiner,
Science
. 216, 136-144 1982). Prions differ from conventional bacteria, viruses, and viroids by their unique structure and properties. All prion diseases are believed to share the same basic pathogenic mechanism that involves the conversion of the normal cellular prion protein (PrP
c
or PrP
sen
) into a form that is infectious, insoluble in non-ionic detergents and partially resistant to proteases (PrP
res
or PrP
sc
). As mentioned above, PrP
c
is a cell surface protein anchored to the membrane by a GPI anchor.
Along these lines, PrP
c
and PrP
res
share an identical amino acid sequence. The conversion of PrP
c
to PrP
res
may involve a conformational change of PrP
c
from a predominantly alpha-helical form to a beta-sheet structure (Pan, et al.,
Proc. Natl. Acad. Sci. USA
, 90, 10962-10966 1993). As a result, the difference between the normal form of PrP and the form associated with diseases may be solely conformational. (Notwithstanding the above, the accumulation within the central nervous system (CNS) of these abnormal PrP
res
in the brain is a cardinal feature of the prion disease pathology.)
The strongest evidence suggesting that PrP
c
is essential in the development of prion disease came from studies using PrP
c
“knock-out” mice which are devoid in PrP
c
and resistant to prion infection (Bueler, et al., Cell. 73, 1339-1347 1993). However, the conditions that trigger and determine the conversion of PrP
c
to PrP
res
remain unclear.
Experimental models of inherited prion diseases offer one approach to the study of the PrP
c
to PrP
res
conversion. Since many of the pathogenic mutations of the PrP gene (PrP
M
) have high penetrance, it is likely that the change in PrP
M
metabolism plays an important role in determining the conversion of PrP
M
into PrP
res
. Detailed studies on cell models of inherited prion diseases have underlined the complexity and the diversity of the metabolic changes affecting PrP (Peterson, et al.,
J. Biol. Chem
., 271, 12661-12668 1996).
The concept that TSEs are solely mediated by an infectious proteinaceous agent is not accepted by all investigators. It has been suggested that PrP functions as a cofactor and the development of prion diseases requires another infectious agent (e.g., a virus). Alper, et al.,
Nature
. 214: 764-766 1967. Narang,
Proc. Soc. Exp. Biol.Mod
. 212: 208-224 1996. The prion hypothesis is also difficult to reconcile with two well established observations: one is the strain specificity of the prion protein and the other is the species restriction of disease transmission. The recent demonstration of the transmission of BSE to primates and mice raises the possibility that at least in some situations the infectious agent is able to surmount the species barrier. Lasmezas, et al.,
Nature
. 381, 743-744 1996. Fraser, et al.,
Vet. Rec
. 123: 472-477 1988. These observations suggest that the transmission of prion disease is a complex process which is still not fully understood.
One of the most puzzling observations in prion infected humans or animals is the lack of a robust inflammatory response during the progression of the disease. Neither humoral nor cell mediated immune responses against the prion protein have been detected in infected humans or animals. Earlier studies suggested that prion infection may result in suppression of the host immune function. Garfin, et al.
J. Immunology
. 120: 1986-1990 1978. The reasons that prions can evade recognition by the host immune system are not known. Accumulated evidence suggested that host animals may be tolerant to PrP
c
and PrP
res
derived from that species. In contrast to many other self proteins, the state of unresponsiveness to the prion protein is not overcome either by infection or by immunization with prion protein in Complete Freund's Adjuvant (CPA).
A few studies have provided indirect evidence that the host may
Gambetti Pierluigi
Sy Man-Sun
Case Western Reserve University
Fay Sharpe Fagan Minnich & McKee LLP
Swartz Rodney P
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