MXPA00001109A - Catalytic monoclonal antibodies with protease activity for the selective lysis of the protein component of plaques and aggregates related to pathological conditions - Google Patents

Abstract

Catalytic monoclonal antibodies (abzymes) with selective protease activity in the pathologies characterized by the presence of plaques and fibrillar aggregates with protein component;methods for the preparation thereof and the use thereof as medicaments in the treatment of pathologies such as Alzheimer's disease, amyloidosis, atherosclerosis, prions diseases.

Description

CATALYTIC MONOCLONAL ANTIBODIES WITH ACTIVITY OF PROTEASE FOR THE SELECTIVE LYSIS OF THE COMPONENT OF PROTEIN OF PLATES AND AGGREGATES RELATED TO PATHOLOGICAL CONDITIONS FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION Alzheimer's disease Alzherimer's disease (AD) is a degenerative disease that affects the central nervous system, mainly those areas related to intellectual functions, causing the necrosis of neuronal cells and, consequently, the progressive loss of cognitive faculties. , ls and mnemonics of the patients involved with inevitable fatal outcome.

The EA, which can be definitively diagnosed only by autopsy, is characterized by pathological structures that can be distinguished into: - senile or amylaceous plaques, located in the extracellular space, which are deposited in the brain and on the walls of the vessels cerebral blood - nerve nerves located within the cells. The formation of these structures causes a remarkable loss of neurons in the neocortex, the hippocampus and other related structures, with a great reduction in the concentration of neurotransmitters. These defects are due to the toxicity determined both directly and indirectly by the aforementioned neuropathological structures and the results of the death of the neurons, in the progressive loss of cognitive abilities. The anatomopathological structures mentioned above consist of the specific components: 1.- β-amyloid peptide (Aβ-, A4-, A4β-, β, β-peptide), which is derived from the processing of the amylase precursor protein (βAPP), and is a mixture of a small group of peptides, from 28 to 43 amino acids in length, arranged in the flat leaf structures. 2- Apolipoprotein E (Apo E). 3- Tau protein The Aß-peptide is the main component of the amyloid plaques; There are at least two different forms of plates, which are likely to represent two subsequent steps of the Aß-polymerization process: a) Diffuse or pre-macular plates, consisting of amorphous non-congophilic deposits of insoluble Aβ, with few amylaceous deposits, which contain few astrocytes or reactive microglia; these are usually located in the gray matter of the brain and do not apparently cause noticeable effects in adjacent tissues; b) senile or neurotic plaques that consist of a nucleus of fibrillar congofílicos deposits of Aß, that contain astrocitos or effective microglias and surrounded by dystrophic degenerative neurites. The tau protein associated with microtubules, in the hyperphosphorylated form, is the main component of neurofibrillary tangles. These are usually formed by paired helical filaments (FHP) which, in turn, are derived from proteins associated with microtubules (PAMs) and consist of an abnormal accumulation in the degenerative neurons of cytoskeletal proteins with specific biochemical and antigenic properties. Under normal conditions, PAMs probably regulate movement and stabilize the arrangement of neurons during the growth of axons and dendrites. ApoE is present in combination with the amylaceous plaques, with the neurofibrillary forms and with the amylaceous deposits of the cerebral vessels. ApoE could play a biochemical role in the development of AD, related to its ability to bind to Aß. ApoE plays supposedly the role of molecular carrier and could help to abduct Aß in the plaques. Early genetic analysis of AD revealed mutations of some genes in different chromosomes. In correlation with the characteristic presence of Aβ, a mutation of the gene encoding ßAPP in chromosome 21 has been identified at the levels of colors 717 and 670/671. A punctate mutation at these levels can change the processing of ßAPP, preventing the physiological segmentation in non-aggregating peptides and favoring, on the contrary, the amyloidogenic pathway. It should be noted, however, that only a small percentage of early cases of familial AD (4-5%) have been associated with mutations on chromosome 21. A second mutation has been identified that is strictly related to early familial AD, on the long arm of chromosome 14. On the gene involved, in unit S182, at least 15 different mutations were detected, related to familial AD, appearing such mutations in 80% of the early cases of AD. The S182 gene product is an integral membrane protein, whose function has not yet been clarified. A third gene, whose mutation is related to approximately 15% of familial early AD cases, is located on chromosome 1 and is called STM2. The function of the protein encoded by said gene is not yet known, but it seems to cause an increase in β-amyloid production. As far as the senile EA is concerned, this could be the result of oligogénic mutations. It has been observed to be related to mutations on chromosome 19, in particular against the gene encoding ApoE. ßAPP is a transmembrane glycoprotein (695-770 aa) that results mostly in the extracellular space. The physiological processing of ßAPP consists of segmentation by a-secretase enzyme within the Aβ sequence, immediately outside the transmembrane region (aa 16), with the formation and release of soluble amylaceous forms (AAPs) in the extracellular fluid. The action of a-secretase, therefore, includes the formation of Aβ. The amino acid sequence of ßAPP corresponding to Aβ is located partly in the extracellular space and partly in the membrane (the 28 aa of the amino-terminal domain to the single transmembrane domain of the precursor, minus 11-15 first residues of the transmembrane domain) . The expression of ßApp and the release of APP are modulated by neurotrophic factors and by cytokines. The expression of ßAPP increases when neuronal differentiation takes place and APPs can affect the growth of neurites and the survival of neurons in cell culture. The function of ßAPP is unclear: it can apparently play an adhesive / receptor role and be involved in synaptic plasicity. APPs control [Ca2 +], and modify the Ca2 + response of glutamate. APPs are jsensed along the axon and are then released at the synapses by the cones of actions that are growing and by the axon terminals. In summary, the functions of ßAPP and APP are: - regulation of the proliferation of cells in neuronal cells, - cell adhesion, - promotion of survival of neurons, - protection against excitotoxicity or against ischemic damage, - regulation of the growth of neurons, - regulation of intracellular calcium levels. When degradation of ßAPP does not occur physiologically, either due to the presence of the punctiform mutation or to the attack of enzymes other than a-secretase or to excessive production of ßAPP, form amyloidogenic fragments, ie insoluble fragments of Aβ, with a flat structure in ß sheet, aggregate in more or less complex fibrous formations until insoluble extracellular amyloid plaques form (Cummings, Neuroscience 48: 763 (1992); Neurobiol Aging 14: 547-560 (1993)). In the nucleus of neuritic plaques, a frequent Aβ form of 42 amino acids in length has been identified, namely Aβ? -2 (Rohrer, Proc.Nat.Acid.Sci.90: 10836-10840 (1993); Gravina, J. Biol. CHEM 270 7013-7016 (1995); Motter, Ann. Neurol., 38: 643-648 (1995); Cummings, # _. "', g? á JÉÜife .. ^ áife.

Neurobiol. Aging. 17: 653-659 (1998), This fragment, besides being the main component of the amylaceous plaques, is, among the several fragments, the one with the highest amyloidogenic characteristics, that is, it is highly capable of associating in more or less aggregates. complexes and form fibrils. A more hydrophobic domain on the molecule of Aβ? -42 has been perceived, which seems critical for the assembly of the amylaceous fibrils, because it increases the speed of aggregation thereof (Pike, J. Neuroscience 13: 1676 ( 1993); Aß? -42 could therefore play a more important role than shorter fragments in the formation of plaques.After the formation of the first fibrillar aggregates consisting of Aβ1-2, other shorter fragments are also added. the plates, Aß? -42 would therefore serve as a nucleation center for the aggregation.Aß .- 2 is deposited at the beginning and selectively in the senile plaques and this is a constant characteristic of all forms of AD. Aß? -42 in the fibrils and, already at this stage, ie before the fibrils are deposited in the amylaceous plaques, the fibrils themselves can begin a neurodegenerative process and also induce hyperphosphorylation of the tau protein The C-terminal amino acid is apparently critical for peptide aggregation (Jarrett, Biochemistry 32: 4693-4697 (1993). There is a direct relationship between the aggregation of the peptide and the neurotoxic potential. Aß has, in fact, a direct toxic effect on human neurons in vitro and such toxicity is directly proportional to the t state of aggregation. Aß accumulates in the plasma membrane or above it (it is incorporated in the double lipid layer) where it forms selective flow channels for Ca2 +, thus changing a structural change in the plasma window, with the formation of specific channels that alter the permeability of Ca2 +. The channel activity of the ionic conduits would therefore be in the lower part of the neurotoxic effect of Aβ. The effects of Aß can be summarized as follows: - Impaired neurite growth, - increased vulnerability of neurons to excitotoxicity, - destabilization of neuronal calcium homeostasis, - consequent toxicity of segregation, - pore formation with the transport of Ca2 + in the membrane, - formation of proinflammatory cytokine release. Currently, there are no pharmacological therapies against AD. The only medication marketed that has such indication is Tacrine (INN), an inhibitor of acetylcholinesterase, whose pharmacological activity is based on the obstruction of the catabolism of the neurotransmitter acetylcholine. The common therapeutic procedures based on β-peptide use different strategies: - inhibition of the indicated enzymes in the amyloidogenic pathway (β-secretase), - products that either obstruct the Aß-induced neurotoxicity or stabilize the neuronal cells preventing their sensitization to intracellular calcium, - Obstructive modifiers of the ionic conduits of Aß, - compounds that inhibit the aggregation of Aβ, modifying even the hydrophobic domain that apparently causes the amyloidogenic properties.

Asteroesclerosis Atherosclerosis is an extremely complex and variable pathology in which some components of different nature are deposited in the plaques that adhere to the intima of the blood vessels. Such formations cause local damage to the vessel and an increase in circulatory resistances with a consequent increase in pressure, as well as a decrease in blood flow to organs and tissues and therefore functional decompensation. The histological characterization of the atherosclerotic plaques has not yet been fully defined: the parameters responsible for the evolution of the plaque, the mechanisms of formation and the stages of evolution are still partially unclear and vary depending on strict factors such as age, sex, diet and environment. The study of the morphology and the composition of complex atheromatous plaques, extracted from patients undergoing peripheral surgery, revealed a remarkable heterogeneity in the chemical / biological composition of the aggregates: some formations consist mainly of lipoproteins and lipids, others in cells in formation, others in fibrous capsules, others, more complex, in neoformed vessels. On the other hand, the initial formation stages of the atheromatous plaques apparently have some common characteristics: in particular, the presence of LDL group lipoproteins (Hoff HR et al., J. Lipid Res 34: 789-798 (1993)).; Srinivasan SR et al Atherosclerosis 38: 137-147 (1981); Piotrowski JJ Life Sci. 58: 735-740 (1996)) seems to represent a common structural feature of atheromatous plaques at the initial stage of sedimentation. Westermark and colleagues (Am. J. Pathol., 147: 1186-1192 (1995) reported that amyloid deposits in the intima of the aorta are common in combination with atherosclerotic pathologies and aging.These researchers purified a fibrillar protein present in deposits. extracellular of patients with atherosclerosis: this protein is the N-terminal fragment of 69 amino acids in length of apolipoprotein A1 Currently, there are no effective pharmacological therapies for the treatment of the initial phases of the formation of atheromatous plaques. strategy of choice when complex evolved plaques are present, patients who are known to be prone to atherosclerosis can not receive adequate preventive therapies.

Amyloidosis Monoclonal gammopathies or plasma-cell dyscrasias are a group of clinically and biochemically different diseases, characterized by the abnormal proliferation of a cellular clone normally involved in the synthesis of immunoglobulins. The immunochemical characteristic of these diseases is the presence of structurally and electrophoretically homogeneous (monoclonal) immunoglobulins or of polypeptide subunits thereof in the patient's serum or urine. Some of these conditions are asymptomatic and apparently stable; others, such as multiple myeloma and amyloidosis, are progressive and fatal. The etiology of monoclonal gammopathies is unknown. Amyloidosis consists of a group of biochemical and chemically heterogeneous conditions, which is usually characterized by the deposition of proteins in fibrillar form in tissues. Such accumulation causes functional damage to the organs involved with an often deadly result. More particularly, AL amyloidosis is due to the sedimentation of fibrils formed by fragments of immunoglobulin light chains. This amyloidosis is distinguished in primary amyloidosis, if the plasma-cell clone is not very large and if amyloidosis is associated with multiple myeloma. Í2 AL amyloidosis is one of the most serious plasma-cell dyscrasias for which there are no effective treatments. The progress of the disease is rapid and fatal, with an average survival of only 12 months.

Prion diseases The term prion was introduced to define a class of particles responsible for the transmission of some neurodegenerative diseases, such as Creutzfeld-Jakob disease, kuru, Gerstmann-Straussier-Scheinker syndrome (GSS), familial mortal insomnia (IFF) , bovine spongiform encephalopathy (BSE). This type of pathology is characterized by the accumulation in the brain of a protease resistant protein (PrPres), derived from a protein sensitive to the protease (PrPsen) of 33-35 kDa. The amino acid sequence of the two proteins is the same and they differ in conformation. The clinical signs of the diseases coincide with the structural modification of PrPsen to PrPres, with accumulation of the latter in fibrillar structures. The histopathological characteristic of the nervous tissue of patients with prion diseases is, in fact, the presence of crystalline aggregates with a spherical structure located in the postsynaptic evaginations. In raw brain extracts in infected rodents, fibrillar structures with a well-defined morphology have been identified: there are two types of fibrils, formed by two or four helical sub-filaments; the progress of rotation of the subfilamypes as well as the space between them are regular. This characteristic microscopic structure allows to distinguish between the aggregates present in prion diseases and those that characterize other diseases with plaque accumulations, such as Alzheimer's disease. Prions are highly resistant to the action of the protease and are usually inactivated by chemical and physical means. Some monoclonal antibodies are capable of catalyzing chemical reactions that involve components recognizable by the antibody itself. Catalytic monoclonal antibodies are called abzymes and are usually prepared using as antigen the stable analog of the transition state of the reaction to be catalyzed by the abzyme itself. For the preparation of abzymes with protease activity, the analog of the transition state of the peptide bond between two selected amino acids, ie a dipeptide analog, is usually employed; on the other hand, an abzyme with specific protease activity is obtained in this way and any proteins containing said peptide in its own sequence can be the target of the abzyme. To increase the specificity of antibody, attempts have been made to use immunogens in which no more than 8 amino acids are present linked to the dipeptide analogue. In this case too, however, the resulting specificity and selectivity could not ensure the absence of cross reactions between the abzyme and other protein molecules other than the target.

In addition, abzymes have been produced up to now for the purpose of increasing reaction rates. On the contrary, no abzymes produced are known in order to exercise absent or deficient activities in the affected organism with one of the pathologies described above.

BRIEF DESCRIPTION OF THE INVENTION It has now been surprisingly discovered that catalytic monoclonal antibodies (hereinafter abzymes) with protease activity can be prepared using the stable analog of the transition state. Very unexpectedly, using as the immunogen the protein agent responsible or involved in pathologies characterized by plaques or aggregates with a protein component, abzymes with extremely selective protease activity can be obtained towards the protein component of the plaque or the characteristic aggregate of the pathology involved. It is therefore an object of the present invention an abzyme or a fragment thereof or a manipulated fragment thereof that participates in the segmentation reaction of protein molecules whose presence, either in the free form or in the aggregated form, It is related to pathologies of various devices and organs. A further object of the present invention is a process for the preparation of said abzyme.

A still further object of the present invention are the pharmaceutical compositions containing said abzyme or a mixture of abzymes and medicaments useful for the treatment or prevention of pathologies related to proteins in the free or aggregated form against various apparatuses or organs. These and other objects of the invention will be described, in the various embodiments thereof, below in more detail also by means of examples.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, the abzyme has protease activity against protein molecules whose presence, whether in the free form or in the aggregated form, is related to pathologies of various apparatuses and organs. As a general rule, according to the present invention, the abzyme is obtained by directly using its protein target as an immunogenic agent, without the need for a synthetic derivative of the analogue of the transition state. The procedure comprises: a) immunizing an animal for the purpose of the abzyme; b) obtain the hybridoma; c) selecting the hybridomas for their catalytic activity.

The preparation of the abzyme is carried out with conventional techniques for the preparation of monoclonal antibodies, such as immunization in vivo, in vitro immunization and phage display. The resulting antibody can be complete or can be isolated and used only in the part of the molecule that maintains the catalytic activity. The present invention also encompasses any modification resulting from handling procedures, such as variable single chain fragment (ScFv), or modification methods, such as binding of fragment antigens (Fab) and equivalent analogs. In a first mode, the abzyme is direct against Aβ1-42. Said abzyme selectively recognizes the Aß-i- ^ assembled in senile plaques and is capable of exerting peptidase catalytic activity specifically against it, segmenting it from smaller fragments, which can be easily metabolized and which do not show aggregating activities. Such a procedure would allow the treatment of patients in both early and late stages of the disease, although in the latter condition the neurodegenerative process is already advanced and irreversible. It is essential for the enzyme against Aβ-42 that the immunogen, i.e. the fragment itself of the β-amyloid fragment 1-42, be in sheet conformation in β. The intended use of the Aβ1-42 fragment as the inmonogenic agent is due to the fact that the center of the extracellular insoluble amyloid deposits consist mainly of Aβ1-2. Furthermore, Aβ - - - 42 is, among the various fragments, the one with the highest affinity, that is, it is highly capable of associating with more and more complex aggregates and assembling into fibers. Aß-i- ^ is, in fact, more hydrophobic than other fragments and its rate of aggregation is higher. It has been found that the secondary structure of the peptide is of extreme importance due to its neurotoxic activity (Simmos, Mol.Pharmacol. 45: 373-379 (1994) and fragment 1-42 exerts its action only after reaching the flat sheet conformation. In ß. Considering that Aß? ^ 2 has flat sheet conformation in ß also in the amylaceous plates, it is necessary to use the peptide in its secondary structure as the immunogen.The commercial (new) Aß? - 2 has a random spiral structure; the formation of leaf in ß can be obtained by the incubation of the new Aβ1-42 in aqueous solution for a few days (aging): this treatment determines a transition from the conformation of the random spiral to the leaf in ß (Simmons, 1994), obtained the aged Aß-i- ^. (The immunogen can optionally be linked to a protein carrier, when the immunogen itself is not of sufficient size to produce the appropriate immune response or if it is considered useful in the practice of the present invention. abzima against monoclonal immunoglobulins or their polypeptide subunits typical of monoclonal gammopathies, for example multiple myeloma and amyloidosis.

In a third preferred embodiment, the abzyme is directed against apolipoproteins, for example apo A and / or apo B, responsible for atherosclerosis. In a fourth preferred embodiment, the abzyme against prions is directed. A further aspect of the present invention relates to the method for the selection of hybridomas. Conventionally, hybridomas are selected with affinity techniques. Such techniques, however, do not select the abzymes based on the catalytic activity, so that the selected abzymes are sometimes not the ones that have the best activity. In accordance with the present invention, screening is carried out by contacting the hybridomas with a staining reagent which reveals that the catalytic reaction involved has taken place. The detection of the reacted species can be carried out with conventional methods, for example colorimetric or spectrophotometric techniques. The following example illustrates the invention more broadly.

EXAMPLE Abzima against The synthetic fragment of Aβ? -42 in aqueous solution, prepared according to a procedure illustrated below, has been used as an antigen to inject 8 weeks of age. Plates of patients with AD can also be used. After carrying out the immunization, according to conventional schemes, the splenocytes of the immunized animal are fused with mouse myeloma cells to produce hybridomas. The hybridomas resulting from the fusion are selected according to a spectrophotometric procedure described below. The Congo red dye (RC) binds selectively to β-amyloid, when it is in the flat sheet configuration in β; such structural conformation is related to the state of aggregation of the peptide itself in the amylaceous plaques. When RC is added to the solution containing added Aß (aged), the dye is retained by the aggregates and, once the sample has been centrifuged, remains trapped in the pellet, with a consequent decrease in its concentration in the supernatant . The difference in absorption at a wavelength of 485 nm (peak absorbance of RC) indicates the presence of the soluble peptide in the supernatant and is therefore an indicator of protease activity and the presence of the desired abzyme. A solution is prepared at 100 μM of RC. The aged Aß is prepared at 25μM in PBS. 100 μM of aged Aß are supplied in test tubes. 2Q 100 μM is taken at,. { ours, from each receptacle that contains the hybridomas, and they are supplied in the corresponding test tube, which is shaken and incubated at 37 ° C for 1 hour. The reaction is quenched in ice. The test tubes are kept on ice and 100 μM cold RC is added, then they are shaken, incubated on ice for 1 hour, then centrifuged at 14,000 g for 5 minutes.

In vitro tests The aged Aß was incubated at 25μM in PBS, with the supernatant in the clone of selected hybridomas. After centrifugation at 14,000 g, the precipitate was resuspended. A 1% solution of Congo red in distilled water was added dropwise to a test tube containing the resuspended pellet. After 1 hour of incubation on ice, a culture was carried out on a slide that was observed under the optical microscope.

Claims (22)

1. NOVELTY OF JA INVENCIÓN CLAIMS 1. - An abzyme or fragment thereof or a manipulated fragment thereof promoted to proteins in an aggregated form, which are involved in the selected pathologies of Alzheimer's disease, atherosclerosis, amyloidosis, prion disease.
2. 2. An abzyme or a fragment thereof or a manipulated fragment thereof according to claim 1, further characterized in that the aggregated protein is Aβ? -42.
3. 3. An abzyme or a fragment thereof or a manipulated fragment thereof according to claim 1, further characterized in that the aggregated proteins are monoclonal immunoglobulins or the typical polypeptide subunits of the monoclonal gammopathies.
4. 4. An abzyme or fragment thereof or a manipulated fragment thereof according to claim 1, further characterized in that the aggregated proteins are apolipoproteins.
5. 5. An abzyme or a fragment thereof or a manipulated fragment thereof according to claim 1, further characterized in that the added proteins are prion proteins. 6. - An abzyme according to any of claims 1-5, further characterized in that the derivative between ScFv and Fab is selected. 7. A process for the preparation of the abzyme according to claims 1-6, which comprises: a) immunizing an animal for the purpose of the abzyme, b) obtaining the hybridoma, c) selecting the hybridomas for its activity , further characterized in that the selection of the point (c) is carried out by contacting the hybridomas with staining reagent which reveals that the catalytic reaction involved has taken place. 8. A method according to claim 7, further characterized in that the immunization is performed with synthetic Aβ1- 2 added in sheet conformation in ß 9.- Pharmaceutical compositions containing an effective amount of the abzyme in accordance with claims 1 -6. 10. Pharmaceutical compositions containing an effective amount of a mixture of abzymes. 11. The use of an abzyme in accordance with claims 1-6, for the preparation of a medicament useful for the treatment or prevention of pathologies against various organs and organs related to proteins in the aggregate form. 12. The use of an abzyme according to claims 1 or 2 or 6, for the preparation of a medicament useful for the treatment or prevention of Alzheimer's disease. 13. The use of an abzyme according to claims 1 or 3 or 6, for the preparation of a medicament useful for the treatment or prevention of amyloidosis. 14. The use of an abzíma according to claims 1 or 4 or 6, for the preparation of a drug useful for the treatment or prevention of atherosclerosis. 15. The use of an abzyme according to claims 1 or 5 or 6, for the preparation of a medicament useful for the treatment or prevention of prion diseases. 16. The use according to any of claims 11-15, wherein the derivative between ScFv and Fab is selected. 17. The use of for the preparation of the abzyme according to claim 2. 18. The use of aggregated monoclonal immunoglobulins, or their polypeptide subunits typical of monoclonal gammopathies, for the preparation of the abzyme in accordance with the claim 3. The use of aggregated apolipoproteins for the preparation of the abzyme according to claim 4. 20. The use of aggregated prions for the preparation of the abzyme according to claim 5. 21. The use of apolipoproteins for the preparation of the abzyme according to claim 4. 22. - The use of prions for the preparation of the abzyme according to claim 5.