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Method for detecting peptides comprising a cross-b structure

Method for detecting peptides comprising a cross-b structure description/claims

The invention relates to the field of compositions comprising a protein, more specifically to pharmaceutical compositions. More specifically, the invention relates to the detection and/or removal of conformationally altered proteins and/or molecules comprising a cross-β structure from a pharmaceutical composition or any of its constituents comprising a protein.

Pharmaceutical compositions are in general suitable for administration to a subject, said subject being an animal or a human. Many pharmaceutical compositions are available that are either manufactured or purified by processes in which proteins or peptides are involved, or are based on protein and/or polypeptide and/or peptide or amino-acid compositions, including compositions with amino-acid derivatives. Important categories of nowadays pharmaceutical compositions comprising a protein or a proteinaceous compound as an active substance include, but are not limited to hormones, enzymes, vaccines and antigens, cytokines and antibodies. In addition to the above-mentioned proteinaceous pharmaceutical compositions, a large number of pharmaceutical compositions are manufactured with the help of a production and/or purification step comprising proteins. For example, many pharmaceutical compositions comprise one or more proteins as a stabilizing agent.

Safety aspects are of great concern with any pharmaceutical composition. Drug stability during production and storage, and after administering to the body, attracts much effort during development of new active compounds, and thereafter. Market withdrawals of initially successful pharmaceutical compositions are sometimes necessary because of the occurrence of unforeseen and undesired side effects. For example: plasma, erythropoietin, insulin, antibodies, aprotinin, albumin, thrombopoietin, interferon α, factor VIII, have all caused unwanted side effects after administration in individuals. These examples underline that continuous improvement of the current safety testing methodologies is necessary to reduce the risk for unforeseen, unwanted and/or deleterious side effects after administering pharmaceutical compositions to a human or animal.

Health problems related to the use of pharmaceutical compositions are for example related to the fields of haematology, fibrinolysis and immunology. An incomplete list of observed side-effects after administration of pharmaceutical compositions comprises for example fever, anaphylactic responses, (auto)immune responses, disturbance of haemostasis, inflammation, fibrinolytic problems, including sepsis and disseminated intravascular coagulation (DIC), which can be fatal. Said side effects can be caused by either an alteration of a protein or a proteinaceous compound present in said pharmaceutical composition, or by added diluents or carrier substances of said pharmaceutical composition. A proteinaceous compound in this specification means any compound which comprises a peptide, polypeptide, or protein, and/or altered or degraded forms thereof. Alteration of the proteinaceous compound of a pharmaceutical composition comprises for example denaturation, multimerization, proteolysis, acetylation, glycation, oxidation or unfolding of proteins.

An increasing body of evidence shows that unfolding of initially properly folded native proteins leads to the formation of toxic structures in said proteins.

The present invention discloses that said toxic structures are cross-β structures, The present invention further discloses methods and means for detecting cross-β structures in pharmaceutical composition and/or any of its constituents comprising a protein.

In this specification, the terms “cross-β structure conformation” and “cross-β structure” are synonymous and are interchangeably used herein.

A cross-β structure is defined as a part of a protein or peptide, or a part of an assembly of peptides and/or proteins, which comprises an ordered group of β-strands, typically a group of β-strands arranged in a β-sheet, in particular a group of stacked or layered β-sheets also referred to as “amyloid”. A typical form of stacked β-sheets is in a fibril-like structure in which the β-sheets may be stacked in either the direction of the axis of the fibril or perpendicular to the direction of the axis of the fibril. The term peptide is intended to include oligopeptides as well as polypeptides, and the term protein includes proteins with and without post-translational modifications, such as glycosylation. It also includes lipoproteins and complexes comprising proteins, such as protein-nucleic acid complexes (RNA and/or DNA), membrane-protein complexes. Different fluorescent light scattering profiles of amyloid dyes, such as for example Congo red or Thioflavin T in staining various amyloid-like aggregates indicate that different cross-β structures occur. Said cross-β structures are for example found in glyeated proteins and in fibrils1. Such fibrillar aggregates accumulate in various tissue types and are associated with a variety of degenerative diseases. The term “amyloid” is being used to describe fibrillar deposits (or plaques)2. In literature, an amyloid fibril is preferably defined as an aggregate that is stained by Congo red and/or Thioflavin T, that appears as fibrils under an electron microscope, and that contains an increased amount of β-sheet secondary structure. Additionally, the presence of β-sheet rich structures can be defined with X-ray fibre diffraction techniques and/or Fourier transform infrared spectroscopy. A common denominator of amyloid-like structures is the presence of the cross-β structure structural element. Peptides or proteins with amyloid-like structures are cytotoxic to cells 3-6. Diseases characterized by amyloid are referred to as conformational diseases or amyloidoses and include for example Alzheimer's disease (AD), light-chain amyloidosis, type II diabetes and spongiform encephalopathies like for example Bovine Spongiform Encephalopathy (BSE) and Creutzfeldt-Jakob's disease.

In addition, deleterious effects of aggregated proteins are not solely mediated by said amyloid fibrillar depositions of proteins, but also by soluble oligomers of aggregates with amyloid-like properties and by diffuse amorphous aggregates 3,5. The recent finding that toxicity is an inherent property of misfolded proteins implies a common mechanism for said conformational diseases 1,3,6.

We showed that tissue-type plasminogen activator (tPA) and factor XII (FXII) are specifically activated by many polypeptides, once they have adopted the cross-β structure conformation7. This led us to recognize that a ‘cross-β structure pathway’ exists that regulates the recognition and clearance of unwanted proteins1. Polypeptides can refold spontaneously, at the end of their life cycle, or refolding can be induced by environmental factors such as pH, glycation, oxidative stress, heat, irradiation, mechanical stress, proteolysis or contact with denaturing surfaces or compounds, such as negatively charged lipids, plastics or biomaterials. At least part of the polypeptide refolds and adopts the amyloid-like cross-β structure conformation. This cross-β structure containing conformation is then the signal that triggers a cascade of events that induces clearance and breakdown of the particle. When clearance is inadequate unwanted polypeptides can aggregate and form toxic structures ranging from soluble oligomers up to precipitating fibrils and amorphous plaques. Such cross-β structure containing structures underlie various diseases, depending on the polypeptide that accumulates and on the part of the body where accumulation occurs.

The presence of cross-β structures in proteins triggers multiple responses. As mentioned, cross-β structure comprising proteins can activate tPA and FXII, thereby initiating the fibrinolytic system and the contact system of haemostasis. Besides activation of the coagulation system through FXII, the cross-β structure conformation may induce coagulation, platelet aggregation and blood clotting via direct platelet activation and/or the release of tissue factor (Tf) by activated endothelial cells. In addition, the complement system is another example of a proteolytic cascade that is activated by cross-β structures. This system can be activated by the amyloid-β peptide associated with Alzheimer's Disease or by zirconium or aluminum or titanium. The latter being compounds that can induce cross-β structure conformation in proteins. The innate and adaptive immune systems are yet another example. Amyloid-β activates the innate and adaptive immune responses. β2-glycoprotein I is an auto-immune antigen only upon contact with a negatively charged lipid surface, such as cardiolipin9. We have now shown that cardiolipin induces cross-β structure conformation in β2-glycoprotein I (described in more detail elsewhere). Moreover, we have shown that ligands for Toll-like receptors that are implicated in the regulation of immunity induce cross-β structure conformation in proteins. These ligands include lipopolysaccharide and CpG oligodeoxynucleotides (ODN) (described in more detail elsewhere).

The β2-glycoprotein I protein (β2GPI), together with IgM antibodies, C1q and likely other proteins are all also acting in another way in the proposed cross-β structure pathway. It is assumed that a set of cross-β structure binding proteins bind specifically to sites of ‘danger’, e.g. negatively charged phospholipids, amyloid plaques, sites of ischemic injury, necrotic areas, all with its own specificity. Upon binding, the ‘dangerous’ condition is neutralized and for example excessive coagulation at negatively charged lipid surfaces will not occur. Secondly, the proteins bound to the ‘dangerous’ site undergo a conformational change resulting in the formation of the cross-β structure conformation. This fold then acts as a signal for cross-β structure binding proteins that are part of the ‘cross-β structure pathway’, leading to the clearance of the bound protein or protein fragment and removal of the ‘danger’.

The cross-β structure pathway also acts in yet another way. Proteins that circulate in complex with other proteins may comprise a shielded cross-β structure conformation. Once the protein is released from the accompanying protein, the cross-β structure becomes exposed, creating a binding site for cross-β structure binding proteins of the cross-β structure pathway. This then results in breakdown or clearance of the released protein. An example is factor VIII, which circulates in complex with von Willebrand factor (vWF). In this complex, factor VIII is prevented from clearance, so vWF covers the clearance signal that becomes exposed after the complex is dissociated. This clearance signal is the cross-β structure. Treatment of hemophilia patients with recombinant factor VIII (FVIII) may induce inhibitors (anti-FVIII autoantibodies) because the patients lack sufficient vWF to protect the clearance signal comprising the cross-β structure conformation. Excess exposure of FVIII comprising cross-β structure conformation may induce activation of the immune system and generation of anti-FVIII antibodies similar to the generation of anti-β2GPI autoimmune antibodies by β2GPI bound to negatively charged phospholipids and possibly autoimmune responses.

The compounds listed in Table 1 and the proteins listed in Table 2 all bind to polypeptides with a non-native fold. In literature, this non-native fold has been designated as protein aggregates, amorphous aggregates, amorphous deposit, tangles, (senile) plaques, amyloid, amyloid-like protein, amyloid oligomers, amyloidogenic deposits, cross-β structure, β-pleated sheet, cross-β spine, denatured protein, cross-β sheet, β-structure rich aggregates, infective aggregating form of a protein, unfolded protein, amyloid-like fold/conformation and perhaps alternatively. The common theme amongst all polypeptides with an amyloid-like fold, that are ligands for one or more of the compounds listed in Table 1 and 2, is the presence of a cross-β structure.

The compounds listed in Table 1 and 2 are considered to be only an example of compounds known to day to bind to amyloid-like protein conformations. The lists are thus non-limiting. More compounds are known today that bind to amyloid-like protein conformation. For example, in patent AU2003214375 it is described that aggregates of prion protein, amyloid, and tau bind selectively to polyionic binding agents such as dextran sulphate or pentosan (anionic), or to polyamine compounds such as poly (Diallyldimethylammonium Chloride) (cationic). Compounds with specificity for amyloid-like folds of proteins listed in this patent and elsewhere are equally suitable for methods and devices disclosed in this patent application. Moreover, also any compound or protein related to the ones listed in Table 1 and 2 are covered by the claims. For example, point mutants, fragments, recombinantly produced combinations of cross-β structure binding domains and deletion- and insertion mutants are part of the set of compounds as long as they are capable of binding to protein with cross-d structure conformation (i.e. as long as they are functional equivalents). Even more, also any newly discovered small molecule or protein that exhibits affinity for a protein and/or peptide with the cross-β structure conformation can be used in any one of the methods and applications disclosed here.

The compounds listed in Table 3 are also considered to be part of the ‘Cross-β structure pathway’, and this consideration is based on literature data that indicates interactions of the listed molecules with compounds that likely comprise the cross-β structure conformation but that have not been disclosed as such.

Generally, for the production of a proteinaceous pharmaceutical composition, a protein or proteinaceous molecule or compound is isolated from an animal or plant or is synthesized in vitro. Said protein or proteinaceous molecule or compound is subjected to a number of processes like for example a purifying or isolating process from an animal or plant source, or asynthesis process, such as for example a peptide synthesis process, or a synthesis in a plant cell, a yeast cell or a bacteria, or a synthesis in a eukaryotic cell, and/or a manufacturing process, like for example the coupling of chemical molecules to a peptide or protein, and/or an isolation procedure or a purification procedure, and/or concentrating process, like for example the isolation of recombinant protein from a bacterial production cell, or purification by a physical, or a chemical, or an immunological isolation method, and/or a formulation and/or a storage process, including for example a lyophilization process and/or the addition of a suitable stabilizer, a diluent and/or an adjuvant.

Any one of these processes affects the folding of a protein or a proteinaceous compound. Quality control in a manufacturing process preferably aims at identifying and/or minimizing the deleterious effects of each process step for said pharmaceutical composition, thereby increasing the activity of the composition in the final composition and/or decreasing the undesired side effects of the composition.

Alteration of a protein or proteinaceous composition is generally detected by measuring a specific binding site or a specific activity of said protein or proteinaceous composition, or an increase in size or multimerization state of said protein or proteinaceous composition, or a decrease in therapeutic activity of said proteinaceous composition.

As to the first of said methods, a partially unfolded or misfolded protein can still expose a specific binding site. Therefore, testing the quality of a pharmaceutical composition by only testing for a specific binding site is not always a reliable method, because the partial unfolding or degradation of said protein is not detected.

The second of said methods, the size-related detection method is based on the concept that denaturation leads to aggregation of proteins, thereby increasing the size of the proteinaceous molecule. One of several methods for detecting an increase in size of proteins is called size exclusion chromatography. Nowadays, size exclusion chromatography is widespread used as a method to analyse the contents of a protein drug. This technique is generally accepted for the testing of protein drug stability (http://etd.utmem.edu/WORLD_ACCESS/vmi/reviewofanalyticmethod.htm).

Because said detection method only detects the size of proteinaceous molecules, it cannot detect misfolded proteins or proteins with increased content of cross-β structure conformation that have not aggregated or increased in size. Therefore, quality control based on the above-described method of detecting an increase in size of the proteinaceous molecules, does not prevent undesired side effects caused by conformational changes such as for example cross-β structure conformation formed upon denaturation, proteolysis, chemical modification, or unfolding of proteins, in the absence of increased molecular size. Moreover, nowadays guidelines that determine the acceptable amounts of aggregates in proteinaceous drug solutions are based on technical limitations of the available purification methods, rather than on knowledge about expected undesired side effects of the aggregated proteins. Therefore a better quality control method is highly needed by scientists involved in development of proteinaceous compositions and/or pharmaceutically active compounds and formulations and for manufacturers of proteins or proteinaceous compositions and/or vaccines and/or pharmaceutical compositions and constituents thereof, comprising a protein.

The present invention discloses that unfolded and/or misfolded proteins or proteinaceous molecules like for example molecules that are proteolysed, denatured, unfolded, glycated, oxidized, acetylated, multimerized or otherwise structurally altered, adopt a cross-β structure conformation. Furthermore, the present invention discloses that unwanted and/or toxic side effects of pharmaceuticals are caused by proteins present in said pharmaceutical and adopting a cross-β structure conformation.

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