Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Catalytic antibody
Reexamination Certificate
2000-06-15
2003-06-24
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Catalytic antibody
Reexamination Certificate
active
06582945
ABSTRACT:
BACKGROUND OF THE INVENTION
Alzheimer's disease is a progressive and ultimately fatal form of dementia that affects a substantial portion of the elderly population. Definitive diagnosis at autopsy relies on the presence of neuropathological brain lesions marked by a high density of senile plaques. These extracellular deposits are found in the neo-cortex, hippocampus and amygdala as well as in the walls of the meningeal and cerebral blood vessels. The principal component of these plaques is a 39 to 43 residue &bgr;-amyloid peptide. Each plaque contains approximately 20 fmole (80 picograms) of this 4 kDa peptide (Selkoe et al.,
J. of Neurochemistry
46: 1820 (1986)). Apolipoprotein E and neurofibrillary tangles formed by the microtubule-associated tau protein are also often associated with Alzheimer's disease.
&bgr;-amyloid is proteolytically cleaved from an integral membrane protein called the &bgr;-amyloid precursor protein. The gene which codes for this protein in humans is found on chromosome 21 (St George-Hyslop et al.,
Science
235: 885 (1987), Kang et al.,
Nature
325: 733 (1987)). Numerous cultured cells and tissues (eg. brain, heart, spleen, kidney and muscle) express this &bgr;-amyloid precursor protein and also secrete the 4 kDa &bgr;-amyloid fragment into culture media, apparently as part of a normal processing pathway.
While it is difficult to establish an absolute causal relationship between &bgr;-amyloid or the plaques it forms and Alzheimer's disease, there is ample evidence to support the pathogenic role of &bgr;-amyloid. For example, patients with Down's syndrome have an extra copy of the &bgr;-amyloid precursor protein gene due to trisomy of chromosome 21 (St George-Hyslop et al.,
Science
235: 885 (1987), Kang et al.,
Nature
325: 733 (1987)). They correspondingly develop an early-onset Alzheimer's disease neuropathology at 30-40 years of age. Moreover, early-onset familial Alzheimer's disease can result from mutations in the &bgr;-amyloid precursor protein gene which fall within or adjacent to the &bgr;-amyloid sequence (Hardy, J.,
Nature Genetics
1: 233 (1992)). These observations are consistent with the notion that deposition of &bgr;-amyloid as plaques in the brain are accelerated by an elevation in its extracellular concentration (Scheuner et al.,
Nature Med
. 2: 864 (1996)) The finding that &bgr;-amyloid is directly neurotoxic both in vitro and in vivo (Kowall et al.,
Proc. Natl. Acad. Sci
. 88: 7247 (1991)), suggest that soluble aggregated &bgr;-amyloid, not the plaques per se, may produce the pathology.
Observations have indicated that amyloid plaque formation may proceed by a crystallization type mechanism (Jarrett et al.,
Cell
73: 1055 (1993)). According to this model, the seed that initiates plaque nucleation is an &bgr;-amyloid which is 42 or 43 amino acids long (A&bgr;
1-43
). The rate-determining nucleus formed by A&bgr;
1-43
or A&bgr;
1-42
allows peptides A&bgr;
1-40
or shorter to contribute to the rapid growth of an amyloid deposit. This nucleation phenomenon was demonstrated in vitro by the ability of A&bgr;
1-42
to cause the instantaneous aggregation of a kinetically stable, supersaturated solution of A&bgr;
1-40
. That finding has led to the possibility that A&bgr;
1-40
might be relatively harmless in the absence of the nucleation peptides A&bgr;
1-42
or A&bgr;
1-43
. Indeed, elevated levels of these long peptides have been found in the blood of patients with familial Alzheimer's disease (Scheuner et al.,
Nature Med
. 2: 864 (1996)). Moreover, A&bgr;
1-42
or A&bgr;
1-43
was found to be the predominant form deposited in the brain plaques of many Alzheimer's disease patients (Gravina et al.,
J. of Biol. Chem
. 270: 7013 (1995)).
Given the central role played by &bgr;-amyloid, it has become increasingly important to understand the interrelationship between the different pools of these molecules in the body. Free &bgr;-amyloid present in the blood most likely arises from peptide released by proteolytic cleavage of &bgr;-amyloid precursor protein present on cells in the peripheral tissues. Likewise most of the free &bgr;-amyloid found in the brain and cerebrospinal fluid is probably derived from peptide released by secretase cleavage of &bgr;-amyloid precursor protein expressed on brain cells. The peptides are identical regardless of origin, and the results from several studies suggest an intercommunication between these pools.
SUMMARY OF THE INVENTION
One aspect of the present invention is an antibody which catalyzes hydrolysis of &bgr;-amyloid at a predetermined amide linkage. In one embodiment, the antibody preferentially binds a transition state analog which mimics the transition state adopted by &bgr;-amyloid during hydrolysis at a predetermined amide linkage and also binds to natural &bgr;-amyloid with sufficient affinity to detect using an ELISA. In another embodiment, the antibody preferentially binds a transition state analog which mimics the transition state adopted by &bgr;-amyloid during hydrolysis at a predetermined amide linkage, and does not bind natural &bgr;-amyloid with sufficient affinity to detect using an ELISA. Antibodies generated are characterized by the amide linkage which they hydrolyze. Specific antibodies include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of &bgr;-amyloid.
Another aspect of the present invention is a vectorized antibody which is characterized by the ability to cross the blood brain barrier and is also characterized by the ability to catalyze the hydrolysis of &bgr;-amyloid at a predetermined amide linkage. In one embodiment, the vectorized antibody is a bispecific antibody. Preferably, the vectorized antibody has a first specificity for the transferrin receptor and a second specificity for a transition state adopted by &bgr;-amyloid during hydrolysis. Specific vectorized antibodies include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of &bgr;-amyloid.
Another aspect of the present invention is a method for sequestering free &bgr;-amyloid in the bloodstream of an animal by intravenously administering antibodies specific for &bgr;-amyloid to the animal in an amount sufficient to increase retention of &bgr;-amyloid in the circulation. Therapeutic applications of this method include treating patients diagnosed with, or at risk for Alzheimer's disease.
Another aspect of the present invention is a method for sequestering free &bgr;-amyloid in the bloodstream of an animal by immunizing an animal with an antigen comprised of an epitope which is present on &bgr;-amyloid endogenous to the animal under conditions appropriate for the generation of antibodies which bind endogenous &bgr;-amyloid. Therapeutic applications of this method include treating patients diagnosed with, or at risk for Alzheimer's disease.
Another aspect of the present invention is a method for reducing levels of &bgr;-amyloid in the brain of an animal by intravenously administering antibodies specific for endogenous &bgr;-amyloid to the animal in an amount sufficient to increase retention of &bgr;-amyloid in the circulation of the animal. In one embodiment, the antibodies are catalytic antibodies which catalyze hydrolysis of &bgr;-amyloid at a predetermined amide linkage. The antibodies may be either monoclonal or polyclonal. In one embodiment, the antibodies specifically recognize epitopes on the C-terminus of &bgr;-amyloid
1-43
.
Another aspect of the present invention is a method for reducing levels of &bgr;-amyloid in the brain of an animal, by immunizing the animal with an antigen comprised of an epitope which is present on endogenous &bgr;-amyloid under conditions appropriate for the generation of antibodies which bind endogenous &bgr;-amyloid. In one embodiment, the antigen is a transition state analog which mimics the transition state adopted by &bgr;-amyloid during hydrolysis at a predetermined amide linkage. In a preferred
Atwood Pierce
Boston Biomedical Research Institute
Farrell Kevin M.
Patterson Jr. Charles L.
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