Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Catalytic antibody
Reexamination Certificate
2000-02-14
2002-05-14
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Catalytic antibody
C435S346000, C424S094100, C530S324000
Reexamination Certificate
active
06387674
ABSTRACT:
The present invention relates to catalytic monoclonal antibodies, in particular with protease activity, for the selective lysis of the protein component of plaques and aggregates related to pathological conditions.
Alzheimer's Disease
Alzheimer's disease (AD) is a degenerative disease which affects central nervous system, mainly at the level of those areas related to the intellectual functions, causing necrosis of the neuronal cells and, as a consequence, the progressive loss of cognitive, mental and mnemonic abilities of the concerned patients with inevitably fatal outcome.
AD, which can be diagnosed definitely only by autopsy, is characterized by pathological structures which can be distinguished in:
senile or amyloid plaques, localized in the extracellular space, which deposit in the brain and in the walls of cerebral blood vessels;
neurofibrillar tangles localized inside the cells.
The formation of said structures causes a remarkable loss of neurons in neocortex, hippocampus and other related structures, with a great reduction of the neurotransmitter concentration. Said effects are due to the toxicity determined both directly and indirectly by the cited neuro-pathological structures and the neuron death results, in its turn, in the progressive loss of the cognitive capacities.
The anatomic-pathological structures cited above consist of specific components:
1—&bgr;-amyloid Peptide (A&bgr;, A4, A4&bgr;, &bgr;,&bgr;-peptide), which derives by the processing of the amyloid precursor protein (&bgr;APP), and is a mixture of a small group of peptides, 28 to 43 amino acid long, arranged in planar sheet structures.
2—Apolipoprotein E (Apo E)
3—Protein tau.
A&bgr; peptide is the main component of amyloid plaques; at least two different forms of plaques exist, which are likely to represent two subsequent steps of the A&bgr; polymerization process:
a) diffused or preamyloid plaques, which consist of amorphous, non Congo-philic deposits of insoluble A&bgr;, with few amyloid depositions, containing few reactive astrocytes or microglia; these are usually localized in brain's gray matter and apparently do not cause remarkable effects on the adjacent tissues;
b) senile or neuritic plaques which consist of a core of fibrillar, Congo-philic deposits of A&bgr;, containing reactive astrocytes or microglia and surrounded by degenerative, dystrophic neurites
Protein tau associated with microtubules, in the hyperphosphorylated form, is the major component of the neurofibrillar tangles. These are usually formed by paired helical filaments (PHF) which, in their turn, derive from microtubule associated: proteins (MAPs) and consist of an abnormal accumulation in the degenerative neurons of cytoskeletal proteins with specific biochemical and antigenic properties. Under normal conditions MAPs probably regulate movement and stabilize the arrangement of neurons during the growth of axons and of dendrites.
ApoE is present in combination with the amyloid plaques, with the neurofibrillar forms and with the amyloid deposits of cerebral vases. ApoE could play a biochemical role in the development of AD, related to its capability of binding A&bgr;. ApoE supposedly plays the role of molecular carrier and it could assist in sequestrating A&bgr; in the plaques.
Early AD genetic analysis revealed mutations of some genes on different chromosomes.
In correlation with the characteristic presence of A&bgr;, a mutation of the gene which codifies for &bgr;APP has been identified on chromosome 21, at the levels of codons 717 and 670/671. A punctiform mutation at said levels can change the &bgr;APP processing, preventing the physiological cleavage in non-aggregating peptides and favouring, on the contrary, the amyloidogenic pathway. It should, however, be noted that only a small percentage of early cases of familial AD (4-5%) has been related to mutations on chromosome 21.
A second mutation strictly related with familial, early AD, has been identified on the long arm of chromosome 14. On the involved gene, named S182, at least 15 different mutations were detected, related with familial AD, said mutations appearing in 80% of the early AD cases. The product of gene S182 is a membrane integral protein, whose function has not yet been clarified.
A third gene, whose mutation is related with about 15% of the cases of familial early AD, is localized on chromosome 1 and is named STM2. The function of the protein codified by said gene is not yet known, but it seems to cause an increase in the production of &bgr; amyloid.
As far as senile AD is concerned, this could be the result of oligogenic mutations. This has been observed to be related to mutations on chromosome 19, in particular against the gene encoding for ApoE.
&bgr;APP is a transmembrane glycoprotein (695-770 aa) which for the most part protrudes in the extracellular space. The physiological processing of &bgr;APP consists in the cleavage by the enzyme &agr;-secretase within the A&bgr; sequence, immediately outside the transmembrane region (aa 16), with formation and release of (AAPs) amyloid soluble forms in the extracellular fluid. The action of &agr;-secretase, therefore, prevents the formation of A&bgr;. The amino acid sequence of &bgr;APP corresponding to A&bgr; is localized in part in the extracellular space and in part in the membrane (the 28 aa from the amino-terminal to the transmembrane single domain of the precursor, plus the first 11-15 residues of the transmembrane domain). The &bgr;APP expression and APPs release are modulated by neurotrophic factors and by cytokines. The expression of &bgr;APP increases when the neuronal differentiation takes place and APPs can affect the neurite growth and neuron survival in cell cultures. The function of &bgr;APP is not clear: apparently it can play an adhesive/receptor role and be involved in synaptic plasticity. APPs control [Ca
2+
]
i
and modify the Ca
2+
response to glutamate. APPs are transported along the axon and are then released in the synapses by the growing axon cones and by the axon terminals.
In short, the functions of &bgr;APP and of APPs are:
regulation of cell proliferation in non-neuronal cells,
cellular adhesion,
promotion of neuron survival,
protection from excito-toxycity or from ischemic damages,
regulation of neuron growth,
regulation of calcium intracellular levels.
When the degradation of &bgr;APP does not take place physiologically, either due to the presence of the punctiform mutation or to the attack by enzymes different from &agr;-secretase or to an excessive production of &bgr;APP, amyloidogenic fragments form, i.e. insoluble fragments of A&bgr;, with a planar &bgr;-sheet structure, aggregate in more and more complex fibrous formations until insoluble extracellular amyloid plaques are formed (Cummings, Neuroscience 48:763 (1992); Kuo, Neurobiol. Aging 14:547-560 (1993)).
In the core of neuritic plaques, a prevalent A&bgr; form 42 amino acid long has been identified, namely A&bgr;
1-42
(Rohrer, Proc. Natl. Acad. Sci. 90:10836-10840 (1993); Gravina, J. Biol. Chem. 270: 7013-7016 (1995); Motter, Ann. Neurol. 38: 643-648 (1995); Cummings, Neurobiol. Aging. 17: 653-659 (1996). This fragment, besides being the major component of amyloid plaques, is, among the various fragments, the one with the highest amyloidogenic characteristics, i.e. it is highly capable of associating in more and more complex aggregates and of forming fibrils. A more hydrophobic domain has, in fact, been evidenced on the A&bgr;
1-42
molecule which seems to be critical for the assemblage of amyloid fibrils, in that it increases the aggregation rate thereof (Pike, J. Neuroscience 13:1676 (1993); A&bgr;
1-42
could therefore play a more important role than the shorter fragments in the formation of plaques. Following the formation of the first fibrillar aggregates consisting of A&bgr;
1-42
, other shorter fragments also aggregate in the plaques; A&bgr;
1-42
would therefore serve as a nucleation core for the aggregation.
A&bgr;
1-42
is deposited early and selectively in the senile plaques an
Rosini Sergio
Trasciatti Silvia
Abiogen Pharma S.p.A.
Nixon & Vanderhye P.C.
Patterson Jr. Charles L.
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