Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0007—Nervous system antigens; Prions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

• composition for the production of anti-amyloid beta-peptide antibodies with D-peptides Composition for the production of anti-amyloid beta-peptide antibodies with D-peptides
• the invention relates to a composition containing D-peptides or antibodies for use as a therapeutic in the treatment of diseases in which aberrant protein aggregation or multimerization plays a role. Furthermore, the invention relates to a method for producing this composition and its use.
• Protein aggregation diseases or amyloid degeneration is a heterogeneous group of clinical conditions with the common criterion of often but not exclusively extracellular (systemic or local) deposition of a specific protein in the ordered conformation of beta-sheet structure.
• Alzheimer's disease also falls under the group of protein aggregation diseases or protein misfolding diseases.
• Alzheimer's disease (Latin Alzheimer's disease) occurs as a neurodegenerative disease in its most common form in people over the age of 65 years.
• the pathogenesis is characterized by a deterioration in cognitive performance, which is usually accompanied by a decrease in daily activities, with behavioral abnormalities and neuropsychological symptoms. Patients unlearn advanced skills and do not recognize nearby persons.
• Alzheimer's disease The life expectancy after an Alzheimer's diagnosis is statistically on average about seven to ten years. Ferri et al. show that in 2005 about 24 million people are affected by dementia, of which about 60% is due to Alzheimer's disease (Ferri et al., Lancet 366, No. 9503, 2005, pp. 2112-7). Alzheimer's disease is currently the most common dementia disease for which there is no causal therapy to date (October 2008).
• a pathological hallmark of Alzheimer's disease which can be determined even before the first clinical symptoms, is plaques (so-called Alzheimer's disease).
• Fibrils These protein aggregates consist mostly of incorrectly folded amyloid beta peptide (also called abeta peptide or amyloid beta peptide) and are deposited in the brain of Alzheimer's patients (Walsh and Selkoe, 2007, Journal of Neurochemistry 101: 1172-1184) and are the consequence of an increased accumulation of amyloid beta-peptide in the brain.
• amyloid beta-peptide fibrils are only the final stage of a process that begins with the elimination of monomeric amyloid beta-peptide from APP (amyloid precursor protein), then forms neurotoxic amyloid beta-peptide oligomers, and then with amyloid beta-peptide fibrils ends.
• APP amyloid precursor protein
• Experimental therapies are performed in in vitro models of amyloid beta-peptide aggregation, in cell models of amyloid beta peptide production, as well as in transgenic mouse models that form amyloid beta-peptide aggregates in the brain.
• active and passive immunization were performed as therapy in the transgenic mouse model of amyloid beta-peptide aggregation (Schenk et al., Nature, 1999; Bard et al., 2003).
• full-length L-amyloid beta-peptide was used Immunogen used and led in a mouse model of Alzheimer's disease in the context of immunization to a clearance of the plaques (Schenk et al., Nature, 1999).
• Geylis et al. describe cell lines obtained from healthy humans that synthesize antibodies that bind to amino acids 1-16 of the amyloid beta-peptide and discuss its use in passive immunization.
• Lee et al. (2005; American Neurological Association 58: 430-435) compared the antibody sera from humans who suffered from meningeal encephalitis after amyloid beta-peptide immunization and did not become ill. As a result of this study, the antibodies were mainly directed against amino acids 1-8 of the amyloid beta peptide.
• the object of the present invention is therefore to provide a composition for use as a therapeutic in the preventive treatment or therapy of protein aggregation / misfolding disorders.
• the composition should be able to be used in a method for active immunization against Alzheimer's disease and other protein agregiogenic diseases. In this case, the selective evocation of a B cell immune response is to be achieved while avoiding a T cell immune response.
• compositions containing D-peptides or antibodies for use as a therapeutic and / or for disease prevention wherein a) the D-peptides interact with an amyloid beta-peptide and b) the antibodies to the D-peptide from a ) and additionally bind to the amyloid beta peptide from a).
• the composition thus contains a D-peptide immunogen which interacts with an amyloid beta-peptide and antibody production of anti-amyloid beta-peptide antibodies.
• the composition may also contain an amyloid beta-peptide interacting antibody, which antibody is capable of binding to the aforementioned D-peptide and additionally to the amyloid beta-peptide.
• the "interaction" between the D-peptide and the amyloid beta-peptide is a protein-protein interaction.
• composition of the invention may be, for example, a vaccine, a medicament (eg in tablet form), an injection solution, a dietary or nutritional supplement
• composition according to the invention may consist only of D-peptides or only of antibodies , combined with the auxiliaries required for the respective application, such as, for example, salts, such as aluminum salts, buffers or solvents.
• the use of small amounts of the composition is sufficient because the immunogen is degraded with delay - the immunogen is easy to modify or combine.
• composition according to the invention after immunization with D-peptides leads to the generation of amyloid beta-peptide-specific antibodies.
• This immunization with amyloid beta peptide-foreign immunogen from D-peptide is superior to the amyloid beta-peptide immunogen because an antibody response to the amyloid beta peptide is evoked without the concomitant side-effect anti-amyloid beta peptide T cell immune response ,
• Immunization is meant generating an immune response against a defined antigen for the purpose of eliminating, neutralizing and / or otherwise rendering harmless to the organism Immunization: introduction of an antigen (usually a peptide sequence) such that an endogenous immune response is generated which results in the elimination of the antigen, and especially related antigens.
• an antigen usually a peptide sequence
• bypassive immunization is meant the introduction of antibodies parenterally resulting in the elimination of the antigen.
• amyloid beta-peptide multimers can be prevented by binding an antibody to the multimerizing domain of the amyloid beta peptide, which antibody is formed after immunization with a D-peptide.
• Amyloid beta-peptide multimers in the context of the invention means the stable assembly of several amyloid beta-peptide molecules with the acquisition of new functions ("gain fo function").
• multimerization domain defines those domains of the amyloid beta peptide that are involved in the interaction of the amyloid beta peptides with one another
• amino acids 10-42 of the amyloid beta peptide fulfill this function.
• Alzheimer's disease diabetes mellitus and other amyloid diseases, as well as diseases in which the homo-multimerization of a protein is crucial, such.
• Parkinson's disease frontotemporal dementia, amyotrophic lateral sclerosis, cystic fibrosis, certain forms of epilepsy.
• the invention further relates to a composition in which the D-peptide a) contains a retro-inverse sequence of the amyloid beta peptide or amyloid beta-peptide partial fragments and consists entirely of D-amino acids and / or b) to the multimerization domain of the Amyloid beta peptide binds and / or c) contains the sequence SEQ ID NO: 1 or SEQ ID NO: 2 and consists entirely of D-amino acids and / or d) contains D-peptides having the sequence SEQ ID NO: 1 or SEQ ID NO: 2, wherein the D - Peptides with the sequence SEQ ID NO: 1 or SEQ ID NO: 2 partially contain L-amino acids and / or e) homologous sequences to SEQ ID NO: 1 or SEQ ID NO: 2 contains
• D-peptides in one variant consist of a retro-inverse sequence to the amyloid beta-peptide or amyloid beta-peptide subfragments and all of D-amino acids.
• a "partial fragment” consists of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more amino acids homologous to the amino acid sequence of the amyloid beta peptide.
• the D-peptides according to the invention bind to the multimehs mecanicsdomäne of the amyloid beta-peptide.
• the D-peptides have the sequence SEQ ID NO: 1 or SEQ ID NO: 2 and consist entirely of D-amino acids.
• the D-peptides have the sequence SEQ ID NO: 1 or SEQ ID NO: 2, and partially contain L-amino acids.
• the D-peptides have homologous sequences to SEQ ID NO: 1 or SEQ ID NO: 2.
• D-peptide is meant a peptide which is composed of amino acids in the D-form.
• L-amino acids Containing "partial" L-amino acids means that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids homologous to the amino acid sequence of the D-amino acid D-peptide by the same amino acid in each case the L-conformation are replaced.
• Homologous sequences in the context of the invention means that an amino acid sequence has an identity with SEQ ID NO: 1 or SEQ ID NO: 2 of at least 70%, 75%, 80%, particularly preferably 85%, 90%, in particular 91%. . 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
• Homologous sequences to the sequences of the invention consisting of D-amino acids may also partially contain L-amino acids. Instead of the term “identity”, the terms “homologous” or “homology” are used to the same extent in the present description.
• the identity between two nucleic acid sequences or polypeptide sequences is determined by comparison with the aid of the program BESTFIT based on the algorithm of Smith, TF. and Waterman, M.S. (Adv. Appl. Math. 2: 482-489 (1981)) calculated by setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameter for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3.
• the identity between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the entire sequence length, as determined by comparison with the aid of the GAP program based on the algorithm of Needleman, SB and Desire, CD. (J. Mol. Biol. 48: 443-453) is calculated by setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acid gap creation penalty: 50 and gap extension penalty: 3.
• These homologous D-peptides bind to either the amyloid beta-peptide and / or the amyloid beta-peptide multimerization domain. They may be derived from the amyloid beta-peptide (homologous) and are functionally defined, i. able to evoke an immune response identical at least to the amyloid beta peptide. However, they are not the amyloid beta peptide itself and produce an antibody response in mice to the amyloid beta peptide and / or amyloid beta peptide multimers.
• the invention further provides a composition comprising antibodies, wherein the antibody binds to the amyloid peptide or amyloid beta peptide and a) binds to a retro-inverse sequence of the amyloid beta peptide or amyloid beta peptide subfragments and / or b) binds to the multimerization domain of the amyloid beta peptide and also binds to the amyloid beta peptide and / or c) to SEQ ID NO: 1 or SEQ ID NO: 2 or homologous sequences thereof.
• the antibodies have properties of the amyloid beta peptide itself and compete with the multimerization of the amyloid beta peptide when used to generate antibodies peptides that bind to the amyloid beta peptide multimembrane domain.
• Another object of the invention is a process for the preparation of the composition according to the invention, are obtained by immunization of animals or animal cells and fusion of animal cells with myeloma cells and subsequent selection and cultivation antibody-producing hybrid cells and antibodies are isolated and purified.
• Antibody purification can be done, for example, with the in in "Monoclonal antibodies"
• an immunization strategy may be as follows: the composition may be injected subcutaneously (eg, three times, day 0: complete Freund's adjuvant, day 21 and 22: incomplete Freund's adjuvant; Blood sample approx. 100 ⁇ l on day 31).
• mice are anesthetized on day 50, decapitated, and the spleen is removed.
• the spleen cells (splenocytes) obtained therefrom are mixed with mouse myeloma cells in the ratio 1: 5 and fused by addition of 50% polyethylene glycol (PEG) (8 minutes, room temperature). Thereafter, the cells are washed and cultured overnight.
• PEG polyethylene glycol
• Another object of the invention is a use of a composition according to the invention for the prevention of amyloid beta-peptide multimers.
• D-peptides or antibodies which bind to the multimerization domain of the amyloid beta-peptide can be used
• the composition contains 10-1000 ⁇ g of immunogen. In a further variant, the composition contains 20-900, 25-750, 30-600, 40-500, 50-400, 50-300 or 50-250 ⁇ g immunogen. A composition which contains less than 10 ⁇ g of immunogen or more than 1000 ⁇ g of immunogen would also be conceivable.
• immunogen is understood to mean an antigen leading to immunity
• An immunogen is a substance capable of eliciting an immune response, and immunogens differ from antigens derived from antigens be recognized by an antibody, but not all of which alone can trigger an immune response.
• immunological is understood to mean the altered reactivity of the immune system towards antigens (such as viruses, bacteria or foreign protein) caused by immunization and characterized by the occurrence of specific antibodies and / or cells.
• antigens such as viruses, bacteria or foreign protein
• Another object of the invention is a use of the composition of the invention in the prevention and / or treatment of Alzheimer's disease.
• D-peptides that bind to the multimerization domain of the amyloid beta peptide may be used to prepare a medicament for the prevention and / or therapy of Alzheimer's disease.
• Another object of the invention is a use of the inventive composition containing D-peptide as a therapeutic and / or disease prevention of Alzheimer's disease, wherein the D-peptide a) from a retro-inverse sequence to amyloid beta-peptide or amyloid beta-peptide partial fragments contains and consists entirely of D-amino acids and / or b) binds to the multimerization domain of the amyloid beta peptide and / or c) contains the sequence SEQ ID NO: 1 or SEQ ID NO: 2 and consists entirely of D-amino acids and / or d) contains the sequence SEQ ID NO: 1 or SEQ ID NO: 2, wherein the D-peptides having the sequence SEQ ID NO: 1 or SEQ ID NO: 2 partially contain L-amino acids and / or e) homologous sequences SEQ ID NO: 1 or SEQ ID NO: 2 contains.
• Another object of the invention is a use of a composition according to the invention containing antibodies as a therapeutic and / or disease prevention or for the diagnosis of Alzheimer's disease, wherein the antibodies a) bind to a retro-inverse sequence of the amyloid beta peptide or amyloid beta peptide subfragments and / or b) bind to the multimerization domain of the amyloid beta peptide and also to the amyloid beta peptide and / or c) to SEQ ID NO: 1 or SEQ ID NO: 2 or homologous sequences thereof.
• Another object of the invention is a use of a composition containing antibodies for the diagnosis of Alzheimer's disease, for the detection or quantification of biomolecules or for the localization of biomolecules in tissues, cells or in the detection of specific cell types, wherein the antibody a) to a retro-inverse Sequence of the amyloid beta peptide or amyloid beta peptide subfragments and also binds to the amyloid beta peptide and / or b) to the multimerization domain of the amyloid beta peptide and / or c) to SEQ ID NO: 1 or SEQ ID NO: 2 or homologous sequences thereof and also binds to the amyloid beta peptide.
• Detection or quantification of biomolecules in the context of the invention is understood as meaning the determination of substances by antigen-antibody reactions (eg immunoassays).
• the advantage of the present invention is that a therapeutic antibody response is evoked without the D-peptide used thereby being processed by T cells.
• a method was invented, with the help of which immunization strategies against protein aggregation diseases or Protein conformational diseases can be developed.
• a method in the context of the therapy of Alzheimer's disease which immunizes with amyloid beta-peptide-interacting D-peptides in a subject an amyloid beta-peptide toxicity neutralizing and / or amyloid beta-peptide clearing humoral immune response be achieved.
• methods can be developed which is a disease other than Alzheimer's disease in which the aggregation or multimerization of proteins is also disease-relevant and in which D-peptides binding to multimerizing interfaces are used (CNS protein conformation disorders, diabetes etc.).
• clearing the removal of a particular exogenous or endogenous substance from a tissue as a specific performance of an organ (eg, renal clearance), a cellular component (eg, macrophages, microglia), or subcellular compartments (eg proteasome).
• organ eg, renal clearance
• cellular component eg, macrophages, microglia
• subcellular compartments eg proteasome
• a further advantage of the invention is that in addition the epitope space of the amyloid beta peptide is left, which in principle can limit the humoral immune response with high self-tolerance In the case of the use according to the invention of amyloid beta-peptide-binding D-peptides In addition, the T-cell immune response is suppressed because these peptides can not be degraded and presented.
• the invention also relates to antibodies which are directed against the amyloid beta-peptide multimerization domain and prevent the multimerization of the amyloid beta peptide. Then, by labeling with the antibody, amyloid beta-peptides or the accumulated amyloid beta-peptide deposits can be broken down by the body's own immune system. Further antibodies according to the invention are directed against the amyloid beta-peptide in general and not against the multimerization domain in particular. Examples
• Figure 1 shows a Western blot of synthetic amyloid beta-peptide (1) and CHO cells transfected with APP (3); irrelevant control (2). This Western blot shows that 9A6 does not recognize an APP or amyloid beta-peptide on the Western blot (left side), while the universal anti-amyloid beta-peptide antibody IC16 does (right side).
• Figure 2 shows a Western blot of immunoprecipitation of IC16 (right side) and 9A6 (right side) from the supernatant of permanently APP transfected CHO cells secreting the amyloid beta peptide. It can be seen that 9A6 recognizes amyloid beta-peptide oligomers.
• Figure 3 shows an immunohistochemical staining with 9A6 of a cortical sample of a patient with Alzheimer's disease. It can be seen that the 9A6 antibody recognizes weakly amyloid beta-peptide plaques.
• mice were immunized with D-peptides.
• the D3 peptide was used as well as a retro-inverse peptide of the amyloid beta peptide (riAbeta1 -16; retro-inverse-Abeta (1-16)).
• riAbeta1 -16 retro-inverse-Abeta (1-16)
• booster injections booster injections
• a fusion was performed to produce monoclonal antibodies by standard methods. It was possible to isolate monoclonal antibodies which bind to D3, hAbeta1 -16 and to the amyloid beta peptide.
• immunization with said D-peptides results in a protective immune response against the amyloid beta-peptide.
• "booster effect" is understood to mean an immunological secondary reaction or an enhanced immune response after a repeated contact with the antigen.
• the peptides used for immunization had the following sequences:
• D3 was covalently linked to KLH (keyhole limpet hemocyanin) and immunized with standard procedures to mice. These were then fused according to standard protocols with mouse myeloma cells. The cell culture supernatant of the resulting hybridoma cells was then tested for both the recognition of the D3 peptide and the amyloid beta peptide in the standard ELISA.
• KLH keyhole limpet hemocyanin
• the immunization with D3 has an antibody response to the amyloid beta peptide and 2. that there is a binding site on certain antibodies that can recognize both the amyloid beta peptide and D3.
• the exemplified antiD3 / anti-amyloid beta-peptide antibody 9A6 was further tested in the assays shown in Figure 1, Figure 2 and Figure 3.

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• 2008
  • 2008-11-19 DE DE102008037564A patent/DE102008037564A1/de not_active Withdrawn
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