ITBG20120050A1 - FUSION PROTEIN WITH PROTEASIC ACTIVITY AND SPECIFICITY OF ANTIBODIES - Google Patents
FUSION PROTEIN WITH PROTEASIC ACTIVITY AND SPECIFICITY OF ANTIBODIES Download PDFInfo
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- ITBG20120050A1 ITBG20120050A1 IT000050A ITBG20120050A ITBG20120050A1 IT BG20120050 A1 ITBG20120050 A1 IT BG20120050A1 IT 000050 A IT000050 A IT 000050A IT BG20120050 A ITBG20120050 A IT BG20120050A IT BG20120050 A1 ITBG20120050 A1 IT BG20120050A1
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Description
FUSION PROTEIN WITH PROTEASIC ACTIVITY AND ANTIBODY SPECIFY FUSION PROTEIN WITH PROTEASIC ACTIVITY AND ANTIBODY SPECIFY
PROTEINA DI FUSIONE CON ATTIVITÀ PROTEASICA E SPECIFICITÀ DEGLI ANTICORPI FUSION PROTEIN WITH PROTEASIC ACTIVITY AND ANTIBODY SPECIFICITY
CAMPO DI APPLICAZIONE FIELD OF APPLICATION
La presente invenzione si riferisce ad una proteina di fusione, creata mediante ingegneria genetica, destinata ad essere usata come farmaco, in saggi analitici e in diversi processi industriali. Tale proteina di fusione può essere impiegata in tutte le situazioni ove ci sia necessità di degradare parzialmente o totalmente una certa proteina in modo specifico. The present invention refers to a fusion protein, created by genetic engineering, intended to be used as a drug, in analytical assays and in various industrial processes. This fusion protein can be used in all situations where there is a need to partially or totally degrade a certain protein in a specific way.
STATO DELLA TECNICA STATE OF THE TECHNIQUE
Sono noti diversi farmaci che impiegano anticorpi contro uno specifico bersaglio, solitamente una proteina o peptide. Tra questi anticorpi vi è l'esempio della proteina di fusione etanercept, farmaco contro la proteina bersaglio TNF, che funziona come inibitore. L'etanercept è ottenuto dall'unione della parte costante (Fc) dell' immunoglobulina IgGl con il recettore due solubile del TNF, conferendo all' etanercept un'attività di legame con il TNF maggiore rispetto agli altri recettori solubili. Altri esempi di anticorpi utilizzati in terapia sono il Bevacizumab, Trastuzumab Adalimumab ed altri. In generale tutti questi farmaci attuano il loro compito o inibendo il legame di un certo fattore alla proteina bersaglio oppure, se sono coniugati a sostanze tossiche per la cellula, vengono internalizzati dalla cellulare alterata e quindi ne provocano la morte o ne compromettono il metabolismo cellulare (Elbakri, Nelson, & Abu Odeh, 2010) (Beck, Wurch, Bailly, & Corvaia, 2010). Several drugs are known that employ antibodies against a specific target, usually a protein or peptide. Among these antibodies is the example of the fusion protein etanercept, a drug against the target protein TNF, which functions as an inhibitor. Etanercept is obtained from the union of the constant part (Fc) of the IgG1 immunoglobulin with the soluble TNF receptor two, giving etanercept a greater TNF-binding activity than the other soluble receptors. Other examples of antibodies used in therapy are Bevacizumab, Trastuzumab Adalimumab and others. In general, all these drugs carry out their task either by inhibiting the binding of a certain factor to the target protein or, if they are conjugated to substances toxic to the cell, they are internalized by the altered cell and therefore cause its death or compromise cellular metabolism ( Elbakri, Nelson, & Abu Odeh, 2010) (Beck, Wurch, Bailly, & Corvaia, 2010).
Recentemente sono state sviluppate piccole proteine di fusione derivate dagli anticorpi denominati single-chain variable fragment antibody, o più semplicemente scFv antibody, abbreviato come scFv. Gli scFv derivano dalla fusione del dominio variabile pesante (VH) delle immunoglobuline con il dominio variabile leggero (VL) delle stesse. Recently, small fusion proteins derived from antibodies called single-chain variable fragment antibody, or more simply scFv antibody, abbreviated as scFv, have been developed. The scFvs arise from the fusion of the heavy variable domain (VH) of immunoglobulins with the light variable domain (VL) of the same.
Tra il dominio VHe VLvi può essere la presenza di una sequenza peptidica, chiamata peptide linker, che permette un ottimale unione tra di essi favorendo la loro attività. Il peptide linker più usato è (G ly4Ser)3. Il peptide linker oltra alla formula citata può avere anche diversa lunghezza, composizione e struttura secondaria come α-elica o βfoglietto (George & Heringa, 2002). Gli scFv conservano l'attività di legame (ma di valenza uno) di un anticorpo intero ma sono molto più piccoli. Dato che gli scFv non possiedono la porzione costante non sono in grado di indurre l'azione effettrice tipica di ogni isotipo (H olliger & Fludson, 2005; Janeway, Travers, Walport, & Shlomchik, 2007; Weisser & Fiali, 2009). Negli ultimi anni quindi sono state sperimentate e sviluppate diverse proteine di fusione che sfruttano le caratteristiche degli scFv appena descritte. Un esempio di proteina di fusione con domini scFv è MG7-scFv/SEB descritta da Tong et al. Tale proteina di fusione è stata progettata per combattere il carcinoma gastrico, Γ enterotossina stafilococcale B (SEB) è stata fusa con la l'scFv anti MG7 (MG7-scFv) ossia specifico per l'antigene MG7 espresso dalle cellule gastriche cancerose. Il dominio scFv quindi si lega preferibilmente alle cellule del carcinoma che espongono MG7 mentre SEB attua la funzione citotossica uccidendole (Tong, Liu, Lu, Shu, & Wang, 2010). I principali problemi nell'uso degli anticorpi monoclonali in terapia riguarda il costo di produzione, il processo di produzione, allergie e vari effetti collaterali soprattutto a livello cardiovascolare ( FHansel, Kropshofer, Singer, Mitchell, & George, 2010). Per superare questi problemi ultimamente si sono progettate delle proteine di fusione con un dominio scFv e un dominio Fc, un notevole esempio di applicabilità industriale di questa metodica che permette di risolvere questi problemi è stata da descritta da Wang et al. (2012). Between the VHe VLvi domain there may be the presence of a peptide sequence, called peptide linker, which allows an optimal union between them, favoring their activity. The most used linker peptide is (G ly4Ser) 3. In addition to the above formula, the linker peptide can also have different length, composition and secondary structure such as α-helix or β sheet (George & Heringa, 2002). The scFvs retain the binding (but valence one) activity of a whole antibody but are much smaller. Since scFvs do not possess the constant portion they are unable to induce the effector action typical of any isotype (Holliger & Fludson, 2005; Janeway, Travers, Walport, & Shlomchik, 2007; Weisser & Fiali, 2009). In recent years, therefore, various fusion proteins have been tested and developed that exploit the characteristics of the scFv just described. An example of a fusion protein with scFv domains is MG7-scFv / SEB described by Tong et al. This fusion protein was designed to combat gastric cancer, Γ staphylococcal enterotoxin B (SEB) has been fused with the anti MG7 scFv (MG7-scFv) ie specific for the MG7 antigen expressed by cancerous gastric cells. The scFv domain then preferably binds to the carcinoma cells that expose MG7 while SEB implements the cytotoxic function by killing them (Tong, Liu, Lu, Shu, & Wang, 2010). The main problems in the use of monoclonal antibodies in therapy relate to the cost of production, the production process, allergies and various side effects especially at the cardiovascular level (FHansel, Kropshofer, Singer, Mitchell, & George, 2010). To overcome these problems, fusion proteins with an scFv domain and an Fc domain have recently been designed, a remarkable example of industrial applicability of this method that allows to solve these problems has been described by Wang et al. (2012).
Negli ultimi anni sono state sviluppate proteine artificiali analoghe agli scFv, i nanobody. I nanobody sono proteine artificiali derivate dalla parte variabile degli anticorpi degli animali della famiglia tassonomica Cameliade (ad esempio Camelus dromedarius, Camelus bactrianus, Lama giamo, Lama guanoco, Lama alpaca and Lama vicugna) (Aetselier & Evets, 2002; Cortez-Retamozo, 2004; De Clercq et al., 2012; Hulstein et al., 2005; Muyldermans et al., 2009; Stijlemans et al., 2004). I nanobody oltre ad essere più piccoli, sono a singolo dominio perciò non necessitano di peptide linker rispetto agli scFv. I nanobody sono in grado di riacquisire il folding corretto anche dopo denaturazione completa, sono altamente solubili, anche in microorganismi procariotici. I nanobody sono in sperimentazione per terapia in alcune patologie, come la sindrome di Willebrand (Hulstein et al., 2005). In alcune proteine di fusione ad uso terapeutico si è preferito usare domini nanobody invece che scFv per la formazione di proteina di fusione (Tijink et al., 2008). In recent years, artificial proteins analogous to scFvs, the nanobodies, have been developed. Nanobodies are artificial proteins derived from the variable part of the antibodies of animals of the taxonomic family Cameliade (e.g. Camelus dromedarius, Camelus bactrianus, Lama giamo, Lama guanoco, Lama alpaca and Lama vicugna) (Aetselier & Evets, 2002; Cortez-Retamozo, 2004 ; De Clercq et al., 2012; Hulstein et al., 2005; Muyldermans et al., 2009; Stijlemans et al., 2004). In addition to being smaller, nanobodies are single-domain and therefore do not require linker peptides compared to scFvs. The nanobodies are able to reacquire the correct folding even after complete denaturation, they are highly soluble, even in prokaryotic microorganisms. Nanobodies are being tested for therapy in some pathologies, such as Willebrand syndrome (Hulstein et al., 2005). In some fusion proteins for therapeutic use it has been preferred to use nanobody domains instead of scFv for the formation of fusion protein (Tijink et al., 2008).
Il legame anticorpo-molecola bersaglio, come anche scFv-bersaglio o nanobodybersaglio, solitamente non inibisce irreversibilmente l'attività del bersaglio (es un recettore), in altri casi vi è IMnternalizzazione del bersaglio nella cellula o la fagocitazione. Quindi la primaria funzione di queste proteine è quello di opsonizzare il bersaglio, quindi un singolo dominio variabile inibisce un bersaglio alla volta (Elbakri et al., 2010) (Janeway et al., 2007). The antibody-target molecule binding, as well as scFv-target or target nanobody, usually does not irreversibly inhibit the activity of the target (eg a receptor), in other cases there is internalization of the target in the cell or phagocitation. Hence the primary function of these proteins is to opsonize the target, thus a single variable domain inhibits one target at a time (Elbakri et al., 2010) (Janeway et al., 2007).
Per ovviare a tali inconvenienti e favorire il processo di inibizione irriversibile in modo diretto del bersaglio ed ottenere altri ed ulteriori vantaggi, il richiedente ha studiato la presente invenzione. To obviate these drawbacks and favor the irreversible inhibition process directly of the target and obtain other and further advantages, the applicant has studied the present invention.
ESPOSIZIONE DELL'INVENZIONE EXPOSURE OF THE INVENTION
La presente invenzione è espressa e caratterizzata nella rivendicazione principale. The present invention is expressed and characterized in the main claim.
Altre caratteristiche della presente invenzione sono espresse nelle rivendicazioni secondarie. Other features of the present invention are expressed in the secondary claims.
La proteina di fusione oggetto della presente invenzione è formata da uno o più domini scFv uniti ad uno o più domini con attività proteolitica. In alcune forme di realizzazione della presente invenzione i domini sono legati tramite peptidi linker. Il dominio scFv della proteina di fusione oggetto della presente invenzione conferisce specificità di legame alla molecola target mentre il dominio proteolitico degrada il bersaglio. The fusion protein object of the present invention is formed by one or more scFv domains joined to one or more domains with proteolytic activity. In some embodiments of the present invention the domains are linked via linker peptides. The scFv domain of the fusion protein object of the present invention confers binding specificity to the target molecule while the proteolytic domain degrades the target.
Detti domini scFv della proteina di fusione oggetto della presente invenzione, possono essere sostituiti da domini nanobody. I nanobody rispetto agli scFv sono di minori dimensioni, quindi in grado di legare epitopi detti criptici, inoltre possiedono maggior stabilità. In generale i domini scFv o nanobody della proteina di fusione oggetto della presente invenzione svolgono la stessa funzione, ossia riconoscono e legano di un epitopo specifico. Said scFv domains of the fusion protein object of the present invention can be replaced by nanobody domains. Compared to scFv, nanobodies are smaller in size, therefore able to bind epitopes called cryptic, and also have greater stability. In general, the scFv or nanobody domains of the fusion protein object of the present invention perform the same function, ie they recognize and bind to a specific epitope.
Scopo della presente invenzione è una proteina di fusione in grado di inibire totalmente o parzialmente uno specifico bersaglio attraverso la sua totale o parziale degradazione proteolitica, ossia tale proteina di fusione scinde almeno un legame peptidico della proteina bersaglio. Tale attività proteolitica posseduta da questa proteina di fusione permette l'utilizzo di minori dosi di farmaco rispetto agli altri farmaci ad esempio quelli sopra elencati, e provocando anche minori effetti collaterali, infatti la proteina di fusione oggetto dell'invenzione è in grado di inibire irreversibilmente il bersaglio. Una singola proteina di fusione quindi inibisce direttamente e irreversibilmente più proteine bersaglio, mentre in altre strategie (come anticorpi o scFv da soli) si inibisce una proteina alla volta e non in modo reversibile. The object of the present invention is a fusion protein capable of totally or partially inhibiting a specific target through its total or partial proteolytic degradation, ie such fusion protein cleaves at least one peptide bond of the target protein. This proteolytic activity possessed by this fusion protein allows the use of lower doses of drug than the other drugs, for example those listed above, and also causing fewer side effects, in fact the fusion protein object of the invention is able to irreversibly inhibit the target. A single fusion protein thus directly and irreversibly inhibits multiple target proteins, while in other strategies (such as antibodies or scFv alone) it inhibits one protein at a time and not reversibly.
ILLUSTRAZIONE DEI DISEGNI ILLUSTRATION OF DRAWINGS
Queste ed altre caratteristiche della presente invenzione appariranno chiare dalle seguenti descrizioni e disegni, forniti a titolo esemplificativo, non limitativo, e con descrizione maggiormente approfondita nel resto del testo in riferimento ad alcune forme realizzazione della presente invenzione. In rifermento alle tavole di disegno: These and other characteristics of the present invention will become clear from the following descriptions and drawings, provided by way of non-limiting example, and with a more detailed description in the rest of the text with reference to some embodiments of the present invention. In reference to the drawing tables:
- La figura A illustra, in modo stilizzato e semplificato, una forma di realizzazione della proteina di fusione oggetto della presente invenzione che interagisce con una proteina bersaglio. - Figure A illustrates, in a stylized and simplified way, an embodiment of the fusion protein object of the present invention which interacts with a target protein.
- La figura B illustra, in modo stilizzato e semplificato, la catena polipeptidica di una proteina di fusione oggetto della presente invenzione in assenza delle strutture secondarie, terziare e quaternarie tipiche della proteina. - Figure B illustrates, in a stylized and simplified way, the polypeptide chain of a fusion protein object of the present invention in the absence of the secondary, tertiary and quaternary structures typical of the protein.
- La figura C illustra, in modo stilizzato e semplificato, una forma di realizzazione della proteina di fusione oggetto della presente invenzione senza peptidi linker che interagisce con una proteina bersaglio. - Figure C illustrates, in a stylized and simplified way, an embodiment of the fusion protein object of the present invention without linker peptides which interacts with a target protein.
- La figura D illustra, in modo stilizzato e semplificato, una forma di realizzazione della proteina di fusione oggetto della presente invenzione con un peptide linker tra il dominio nanobody e il dominio proteasico. - Figure D illustrates, in a stylized and simplified way, an embodiment of the fusion protein object of the present invention with a linker peptide between the nanobody domain and the protease domain.
DESCRIZIONE DETTAGLIATA DI ALCUNE FORME DI REALIZZAZIONE PREFERENZIALE DEL DETAILED DESCRIPTION OF SOME FORMS OF PREFERENTIAL REALIZATION OF THE
TROVATO FOUND
Nella presente domanda di brevetto per la parola "dominio", "domain" e le loro declinazioni, ci si riferisce ad una porzione discreta dal punto di vista funzionale e/o strutturale della proteina (pagina 44, Glossary of Judicial Claim Constructions in thè Chemical , Pharmaceutical and Biotechnology Arts, 2010). In the present patent application for the word "domain", "domain" and their declensions, we refer to a discrete portion from the functional and / or structural point of view of the protein (page 44, Glossary of Judicial Claim Constructions in the Chemical , Pharmaceutical and Biotechnology Arts, 2010).
Nella presenta domanda di brevetto per la parola proteasi e le sue declinazioni, ci si riferisce a proteine che sono in grado di scindere il legame peptidico, sono comprese nella definizione di proteasi le endoproteasi, esoproteasi, enzimi deubiquitinanti, peptidasi, endopeptidasi e endopeptidasi. In the present patent application for the word protease and its declensions, we refer to proteins that are able to cleave the peptide bond, the endoproteases, exoproteases, deubiquitinating enzymes, peptidases, endopeptidases and endopeptidases are included in the definition of protease.
Nella presenta domanda di brevetto per il termine "dominio proteasico" e le sue declinazioni, ci si riferisce alla porzione, o porzioni, della proteina di fusione oggetto della presente invenzione che possiede omologia strutturale o di sequenza di una proteasi o di un frammento, tale dominio proteasico possiede la capacità di funzionare come proteasi o di essere convertito in una proteasi attiva. In the present patent application for the term "protease domain" and its declensions, reference is made to the portion, or portions, of the fusion protein object of the present invention which possesses structural or sequence homology of a protease or fragment, such protease domain possesses the ability to function as a protease or to be converted into an active protease.
Nella presente domanda si brevetto per la parola "epitopo" e le sue declinazioni, ci si riferisce in alla regione delTantigene riconosciuta dall· anticorpo, dal scFv, dal nanobody, dal dominio scFv o dal dominio nanobody. Gli epitopi sono chiamati anche determinanti antigenici (Janeway et al., 2007). In the present patent application for the word "epitope" and its declensions, reference is made to the region of the antigen recognized by the antibody, the scFv, the nanobody, the scFv domain or the nanobody domain. Epitopes are also called antigenic determinants (Janeway et al., 2007).
Nella presenta domanda di brevetto per la parola "scFv" e le sue declinazioni, ci si riferisce a single-chain Fagment variable antibody, in italiano anticorpo a singola catena frammento variabile, una tipologia di proteina artificiale formata dai frammenti variabili degli anticorpi; tali domini variabili possono essere uniti indirettamente tramite peptide linker. Alcuni esempi di scFv sono riportati nei lavori di Holliger & Fludson, 2005 e di Weisser & Fiali, 2009. In the present patent application for the word "scFv" and its declensions, we refer to single-chain Fagment variable antibody, in Italian single-chain variable fragment antibody, a type of artificial protein formed by variable fragments of antibodies; such variable domains can be indirectly joined by peptide linkers. Some examples of scFv are shown in the works of Holliger & Fludson, 2005 and Weisser & Fiali, 2009.
Nella presente domanda di brevetto per i termini "dominio scFv", "domini scFv" e le loro declinazioni, ci si riferisce alla porzione, o alle porzioni, della proteina di fusione oggetto della presente invenzione che possiede omologia strutturale o di sequenza amminoacidica di un certo scFv, possedendo quindi la capacità di legare un epitopo specifico. In the present patent application for the terms "scFv domain", "scFv domains" and their declensions, reference is made to the portion, or portions, of the fusion protein object of the present invention which has structural homology or amino acid sequence of a certain scFv, thus possessing the ability to bind a specific epitope.
Nella presente domanda brevetto per la parola "nanobody" e le sue declinazioni, ci si riferisce a proteine artificiali derivate dalla porzione legante Tantigene delle proteine immunoglobuliniche del sistema immunitario di animali appartenenti alla famiglia tassonomica Cameliade (ad esempio Camelus dromedarius, Camelus bactrianus, Lama giamo, Lama guanoco, Lama alpaca and Lama vicugna); come descritto in letteratura (Aetselier & Evets, 2002; Cortez-Retamozo, 2004; De Clercq et al., 2012; Hulstein et al., 2005; Muyldermans et al., 2009; Stijlemans et al., 2004). In the present patent application for the word "nanobody" and its declensions, reference is made to artificial proteins derived from the Tantigen binding portion of the immunoglobulin proteins of the immune system of animals belonging to the taxonomic family Cameliade (for example Camelus dromedarius, Camelus bactrianus, Lama giamo , Lama guanoco, Lama alpaca and Lama vicugna); as described in the literature (Aetselier & Evets, 2002; Cortez-Retamozo, 2004; De Clercq et al., 2012; Hulstein et al., 2005; Muyldermans et al., 2009; Stijlemans et al., 2004).
Nella presente domanda di brevetto per i termini "domini nanobody", "domini nanobody" e le loro declinazioni, ci si riferisce alla porzione, o alle porzioni, della proteina di fusione oggetto della presente invenzione che possiede omologia strutturale o di sequenza amminoacidica di un certo nanobody, possedendo quindi la capacità di legare un epitopo specifico. In the present patent application for the terms "nanobody domains", "nanobody domains" and their declensions, reference is made to the portion, or portions, of the fusion protein object of the present invention which has structural or amino acid sequence homology of a certain nanobody, thus possessing the ability to bind a specific epitope.
Nella presenta domanda di brevetto per la parola "proteina" e le sue declinazioni, ci si riferisce ad un polimero di amminoacidi e non limitato ad una certa lunghezza. Polipeptidi, peptidi, oligopeptidi, dimeri, multimeri e simili sono inclusi nella definizione di proteina. In the present patent application for the word "protein" and its declensions, reference is made to a polymer of amino acids and not limited to a certain length. Polypeptides, peptides, oligopeptides, dimers, multimers and the like are included in the definition of protein.
Una prima forma di realizzazione della proteina di fusione oggetto della presente invenzione è mostrata in figura A: Essa 9) è composta da un dominio scFv 3) che interagisce, lega, la proteina bersaglio 1) attraverso Fepitopo specifico 2), tale dominio scFv è legato tramite peptide linker 4) ad un dominio con attività enzimatica di tipo proteolitico 5) legato a sua volta tramite un altro linker peptidico 6) ad un altro dominio scFv 7) che lega la proteina bersaglio 1) grazie al riconoscimento dell'epitopo corrispondente 8) differente dal precedente. Gli scFv possiedono il vantaggio, rispetto agli anticorpi monoclonali, di essere molto più piccoli in quanto vengono impiegati solo i domini variabili; inoltre essendo a singola catena possono essere prodotte anche da cellule di lievito o procarioti, in maggior quantità e minor costo economico. A first embodiment of the fusion protein object of the present invention is shown in figure A: It 9) is composed of a scFv domain 3) which interacts, binds, the target protein 1) through the specific epitope 2), this scFv domain is linked by peptide linker 4) to a domain with proteolytic enzymatic activity 5) linked in turn through another peptide linker 6) to another scFv domain 7) that binds the target protein 1) thanks to the recognition of the corresponding epitope 8 ) different from the previous one. The scFvs have the advantage, compared to monoclonal antibodies, of being much smaller as only the variable domains are used; moreover, since they have a single chain, they can also be produced by yeast cells or prokaryotes, in greater quantities and at a lower economic cost.
Una proteina di fusione oggetto della presente invenzione in un'alternativa forma di realizzazione può essere applicata ad esempio nel caso delle patologie provocate da alterazioni genetiche dove sia ha la fusione di due quadri di lettura aperti (open reading frame o ORF) di due geni. Ad esempio il carcinoma papillare alla tiroide è originato da traslocazioni, in particolare nel riarrgiamento RET RET/PTC2 vi è l'unione tra l'ORF di aRIPKA e RET (Zhu & Zhu, 2004); quindi la proteina di fusione oggetto della presente invenzione in una sua forma di realizzazione lega, attraverso i domini scFv un epitopo specifico per aRIPKA e un epitopo specifico per RET, il dominio proteolitico che si trova tra i due domini scFv attua il taglio proteolitico inattivando l'attività della proteina bersaglio RET/PTC2. L'inattivazione della proteina aberrante RET/PTC2 porta alla regressione della patologia. A fusion protein object of the present invention in an alternative embodiment can be applied for example in the case of pathologies caused by genetic alterations where there is the fusion of two open reading frames (ORF) of two genes. For example, papillary thyroid cancer originates from translocations, in particular in the RET RET / PTC2 rearrangement there is the union between the ORF of aRIPKA and RET (Zhu & Zhu, 2004); therefore the fusion protein object of the present invention in one of its embodiments binds, through the scFv domains, an epitope specific for aRIPKA and an epitope specific for RET, the proteolytic domain which is located between the two scFv domains implements the proteolytic cut by inactivating the activity of the RET / PTC2 target protein. Inactivation of the aberrant RET / PTC2 protein leads to regression of the disease.
Una proteina di fusione oggetto della presente invenzione in una possibile forma di realizzazione è rappresentata in modo stilizzata nella figura C: il dominio proteasico 14) della proteina di fusione 13) è collegato in modo diretto, data la mancanza di peptidi linker, ai due domini scFv 12) e 15); tali domini scFv legano epitopi diversi 11) e 16), portando vicino il dominio proteasico proteina bersaglio 10) tale da scindere almeno un legame peptidico del bersaglio. In questa configurazione è possibile è possibile impiegare la proteasi K o peptidasi K (classificazione PBD: EC 3.4.21.64 - Peptidase K.). La proteasi K ha attività a pH 7.5-12 e possiede una bassa specificità, dato che scinde il legame peptidico adiacente ad un residuo alifatico o aromatico (Ebeling et al., 1974) (Betzel, Teplyakov, Harutyunyan, Saenger, & Wilson, 1990). In questa forma di realizzazione la proteina di fusione impedisce la degradazione indiscriminata grazie all'ingombro sterico dato dai domini scFv attaccati, la presenza di molecole di glicole polietilenico covalentemente attaccate alla proteina di fusione aumenta notevolmente questo effetto. Allo stesso tempo però i domini scFv conferiscono specificità di azione alla proteasi, permettendo un sufficiente avvicinamento tra il sito catalitico della proteasi e il sito bersaglio solo se vi è la presenza degli epitopi specifici 11) e 16) e ad una precisa distanza tra di loro. A fusion protein object of the present invention in a possible embodiment is represented in a stylized way in figure C: the protease domain 14) of the fusion protein 13) is directly connected, given the lack of linker peptides, to the two domains scFv 12) and 15); these scFv domains bind different epitopes 11) and 16), bringing near the target protein protease domain 10) such as to cleave at least one peptide bond of the target. In this configuration it is possible to use protease K or peptidase K (PBD classification: EC 3.4.21.64 - Peptidase K.). Protease K has activity at pH 7.5-12 and possesses low specificity, since it cleaves the peptide bond adjacent to an aliphatic or aromatic residue (Ebeling et al., 1974) (Betzel, Teplyakov, Harutyunyan, Saenger, & Wilson, 1990 ). In this embodiment the fusion protein prevents indiscriminate degradation due to the steric hindrance given by the attached scFv domains, the presence of polyethylene glycol molecules covalently attached to the fusion protein greatly increases this effect. At the same time, however, the scFv domains confer specificity of action to the protease, allowing a sufficient approach between the catalytic site of the protease and the target site only if there is the presence of specific epitopes 11) and 16) and at a precise distance between them .
Nelle patologie provocate da placche amiloidi (Schenk, Basi, & Pangalos, 2012), la presente invenzione in una sua forma di realizzazione è in grado di degradare i peptidi delle placche β-amiloidi, grazie all'attività proteolitica posseduta dal dominio proteasico e dalla specifità, verso i peptidi delle placche β-amiloidi, conferitagli dai domini scFv. In pathologies caused by amyloid plaques (Schenk, Basi, & Pangalos, 2012), the present invention in one of its embodiments is able to degrade the peptides of β-amyloid plaques, thanks to the proteolytic activity possessed by the protease domain and by the specificity, towards the peptides of the β-amyloid plaques, conferred on it by the scFv domains.
La presente invenzione in una sua forma di realizzazione è composta da un dominio con attività proteolitica unito ad un dominio scFv che riconosce esclusivamente la catena ε delle immunoglobuline IgE, in questa forma di realizzazione la presente invenzione è in grado di inibire l'azione delle immunoglobuline IgE nelle patologie allergiche che possono essere debilitanti o fatali come ad esempio lo shock anafilattico (Janeway et al., 2007). The present invention in one of its embodiments is composed of a domain with proteolytic activity joined to an scFv domain which recognizes only the ε chain of IgE immunoglobulins, in this embodiment the present invention is able to inhibit the action of immunoglobulins IgE in allergic diseases that can be debilitating or fatal such as anaphylactic shock (Janeway et al., 2007).
La proteina di fusione oggetto della presente invenzione in una sua forma di realizzazione i domini scFv sono sostituiti da domini nanobody. Alcuni patogeni sfruttano la variazione degli epitopi per evitare la risposta immunitaria dell'ospite. Tra questi patogeni vi è Γ HIV, i virus dell'epatite, plasmodium e tripanosoma. Tali epiotpi solitamente sono in proteine situate in membrana o nella parete del patogeno e spesso sono altamente glicosilate. Queste proteine sono piuttosto ingombranti stericamente (grazie alla glicosilazione) e raramente permettono l'accesso degli anticorpi agli epitopi criptici sottostanti i quali sono molto meno vari. Per favorire l'esposizione al sistema immunitario di questi la proteina di fusione oggetto della presente invenzione in una forma di realizzazione è costituita da domini nanobody, la proteasi quindi degrada la parte del patogeno che nasconde i epitopi criptici rendendoli "visibili" al sistema immunitario. The fusion protein object of the present invention in one of its embodiments the scFv domains are replaced by nanobody domains. Some pathogens exploit epitope variation to avoid host immune response. Among these pathogens are Γ HIV, hepatitis viruses, plasmodium and trypanosome. Such epiotpes are usually in proteins located in the membrane or wall of the pathogen and are often highly glycosylated. These proteins are rather bulky sterically (thanks to glycosylation) and rarely allow antibodies to access the underlying cryptic epitopes which are much less varied. To favor the exposure of these to the immune system, the fusion protein object of the present invention in one embodiment consists of nanobody domains, the protease then degrades the part of the pathogen that hides the cryptic epitopes making them "visible" to the immune system.
Detti domini nanobody in un alternativa forma di realizzazione legano epitopi specifici criptici delle proteine dell· involucro del virus HIV (es HIV-1 e HIV-2). Nel caso particolare del virus HIV la quasi totalità dei casi gli scFv (e quindi anche gli anticorpi naturali dell'uomo e primati) legano epitopi della proteina gpl20, tale targeting attua una forte pressione selettiva al virus, inducendo ben presto alla selezione positiva di virus mutanti per gpl20. Tale selezione annulla il beneficio conferito datazione degli anticorpi (o dagli scFv) nel combattere Γ infezione (Novitsky et al., 2009). I nanobody essendo in grado di legare epitopi criptici induce una minore pressione selettiva rispetto a quella verso gpl20, concedendo al sistema immunitario maggiori possibilità di combattere l'infezione prima che si selezionino mutanti virali per tali epitopi criptici. In modo analogo i nanobody sono intensamente studiati per la loro capacità di legare epitopi criptici in tripanosoma (Stijlemans et al., 2004). In un sua forma di realizzazione la proteina di fusione 21) oggetto della presente invenzione è costituita da un dominio nanobody 18) che lega un epitopo criptico 17) del complesso proteico del patogeno 23) ed è collegato attraverso un peptide linker 20), al dominio proteasico 22). La proteina di fusione 21) permette la degradazione della regione più esterna del bersaglio 19) permettendo agli anticorpi endogeni di accedere agli antigeni criptici 17). Said nanobody domains in an alternative embodiment bind specific cryptic epitopes of the envelope proteins of the HIV virus (eg HIV-1 and HIV-2). In the particular case of the HIV virus, almost all cases the scFv (and therefore also the natural antibodies of humans and primates) bind epitopes of the gpl20 protein, this targeting implements a strong selective pressure on the virus, soon inducing the positive selection of viruses mutants for gpl20. This selection nullifies the benefit conferred by dating antibodies (or scFvs) in fighting Γ infection (Novitsky et al., 2009). Being able to bind cryptic epitopes, nanobodies induce a lower selective pressure than that against gpl20, giving the immune system a greater chance of fighting the infection before viral mutants are selected for these cryptic epitopes. Similarly, nanobodies are intensively studied for their ability to bind cryptic epitopes in trypanosomes (Stijlemans et al., 2004). In one of its embodiments, the fusion protein 21) object of the present invention consists of a nanobody domain 18) which binds a cryptic epitope 17) of the pathogen protein complex 23) and is connected through a peptide linker 20), to the domain protease 22). The fusion protein 21) allows the degradation of the outermost region of the target 19) allowing the endogenous antibodies to access the cryptic antigens 17).
Per quanto riguarda la cinetica di azione, e quindi l'attività della proteina di fusione oggetto della presente invenzione è necessario tenere presente alcuni elementi. In almeno sua forma di realizzazione, la proteina di fusione oggetto della presente invenzione è costituita da due domini di legame (che essi siano scFv o nanobody è indifferente) l'attività di legame alla proteina bersaglio è tipo cooperativo, ossia, una volta che si lega il primo di dominio viene favorito quella del secondo. Il processo di legame della proteina di fusione perciò procede in modo favorevole rispetto al suo distacco. Tale reazione di legame è airequilibrio e reversibile, quindi in un momento di tempo la proteina di fusione si distacca dal bersaglio. La velocità della reazione di legame è direttamente proporzionale alla concentrazione dei reagenti (proteina di fusione e bersaglio), alla temperatura, all'affinità di legame e inversamente proporzionale alla massa dei reagenti (proteina di fusione e bersaglio). Una volta legatasi la proteina di fusione attua il taglio proteolitico. Il taglio proteolitico sulla proteina bersaglio induce delle modificazioni in generale: la proteina bersaglio viene divisa in almeno due parti, e alcuni amminoacidi vicini (o anche gli stessi) agli epitopi riconosciuti dalla proteina di fusione vengono a mancare. La diminuzione di massa del bersaglio provoca un aumento della velocità traslazionale dato che a pari energia cinetica le molecole più grosse possiedono minore velocità. Quindi la maggior velocità e il temporaneo distacco del dominio di legame favorisce l'allontanamento degli epitopi. La prima separazione del dominio di legame è ulteriormente favorita se il taglio proteolitico provoca una denaturazione dell'epitopo o addirittura l'eliminazione (o in parte) dell' epitopo stesso. Il distacco dell'altro dominio di legame provoca il completo allontanamento della proteina di fusione che può quindi rilegare il bersaglio degradato o legare un nuovo bersaglio (più favorito). In alcune sue forme di realizzazione della presente invenzione la proteina di fusione possiede una cinetica di distacco dalla proteina bersaglio degradata molto sfavorita; ciò può essere dato da un affinità di legame molto alta o da una ridottissima variazione della struttura del bersaglio degradato. Per risolvere questo problema si possono impiegare, contemporaneamente alla proteina di fusione, degli scFv o nanobody liberi che legano lo stesso epitopo in modo competitivo alla proteina di fusione. As regards the kinetics of action, and therefore the activity of the fusion protein object of the present invention, it is necessary to keep in mind some elements. In at least its embodiment, the fusion protein object of the present invention consists of two binding domains (whether they are scFv or nanobody is indifferent) the binding activity to the target protein is cooperative, that is, once binds the first domain is favored that of the second. The binding process of the fusion protein therefore proceeds favorably with respect to its detachment. This binding reaction is non-equilibrium and reversible, so in a moment of time the fusion protein detaches from the target. The rate of the binding reaction is directly proportional to the concentration of the reactants (fusion and target protein), to the temperature, to the binding affinity and inversely proportional to the mass of the reactants (fusion and target protein). Once the fusion protein is bound, it carries out the proteolytic cut. The proteolytic cleavage on the target protein induces modifications in general: the target protein is divided into at least two parts, and some amino acids close (or even the same) to the epitopes recognized by the fusion protein are missing. The decrease in the mass of the target causes an increase in the translational speed since, at the same kinetic energy, the larger molecules have a lower speed. Therefore the greater speed and the temporary detachment of the binding domain favors the removal of the epitopes. The first separation of the binding domain is further favored if the proteolytic cleavage causes a denaturation of the epitope or even the elimination (or in part) of the epitope itself. Detachment of the other binding domain causes complete removal of the fusion protein which can then bind the degraded target or bind a new (more favored) target. In some of its embodiments of the present invention the fusion protein possesses a very disadvantaged detachment kinetics from the degraded target protein; this can be given by a very high binding affinity or by a very small variation of the structure of the degraded target. To solve this problem, it is possible to use, simultaneously with the fusion protein, free scFvs or nanobodies that bind the same epitope in a competitive way to the fusion protein.
Per ridurre la degradazione e l'immunogenicità della proteina di fusione oggetto dell'invenzione possono essere legati in modo covalente alla proteina di fusione una o più molecole di glicole polietilenico o PEG, come ad esempio H(OCH2CH2)nOH (Roberts, Bentley, & Harris, 2012). To reduce the degradation and the immunogenicity of the fusion protein object of the invention, one or more molecules of polyethylene glycol or PEG, such as for example H (OCH2CH2) nOH (Roberts, Bentley, & Harris, 2012).
La presente invenzione in alcune sue forme di realizzazione viene somministrata con agenti veicolanti per permettere l'entrata della proteina di fusione nella cellula, tra gli agenti veicolanti più usati vi sono le nanoparticelle, dendrimeri, liposomi, polimersomi e virus. The present invention in some of its embodiments is administered with carrier agents to allow the entry of the fusion protein into the cell, among the most used carrier agents are nanoparticles, dendrimers, liposomes, polymersomes and viruses.
Nell'industria sono largamente usate le proteasi, soprattutto quelle derivate da organismi basofili ma in generale essi mancano di specificità verso il substrato (Kumar & Takagi, 1999) (Gupta, 2005). Nell'industria tessile la seta grezza viene sottoposta ad un processo detto degommatura (in inglese degumming) per togliere la sericina in eccesso (Freddi, Mossotti, & Innocenti, 2003). Negli ultimi anni sono state studiate alcune proteasi, mediante screening, per l'impiego nel processo di degumming ma i risultati non discostano molto dai metodi tradizionali (Johnny & Chinnammal, 2012). In questo contesto la presente invenzione in alcune sue forme di realizzazione può essere creata per possedere specificità verso la sericina ma non verso la fibroina, permettendo un degumming specifico, dato che non si impiegano temperature alte, saponi e alcali è un processo delicato ed ecocompatibile. Proteases are widely used in industry, especially those derived from basophilic organisms but in general they lack substrate specificity (Kumar & Takagi, 1999) (Gupta, 2005). In the textile industry, raw silk is subjected to a process called degumming (in English degumming) to remove excess sericin (Freddi, Mossotti, & Innocenti, 2003). In recent years some proteases have been studied, by screening, for use in the degumming process but the results do not differ much from traditional methods (Johnny & Chinnammal, 2012). In this context, the present invention in some of its embodiments can be created to possess specificity towards sericin but not towards fibroin, allowing a specific degumming, since high temperatures, soaps and alkalis are not used, it is a delicate and environmentally friendly process.
In riferimento alla proteina di fusione oggetto della presente invenzione, la scelta dell'uso dei domini scFv piuttosto che dei domini nanobody è dettata dal bilanciamento tra le diverse caratteristiche, tra cui: With reference to the fusion protein object of the present invention, the choice of using the scFv domains rather than the nanobody domains is dictated by the balance between the different characteristics, including:
• Disponibilità di varietà anticorpale, la quale è attualmente maggiore per gli scFv. • Availability of antibody variety, which is currently greater for scFvs.
• Sistema di produzione e impiego industriale, i nanobody sono più stabili e si possono produrre facilmente in bioreattori di procarioti. • Industrial production and use system, nanobodies are more stable and can be easily produced in prokaryote bioreactors.
• Se impiegata come farmaco la presente invenzione non deve essere immunogenica. I nanobody dato che derivano dai camelidi potrebbero possedere epitopi che possono essere riconosciuti dal sistema immunitario umano; gli scFv invece possono già essere umanizzati. • If used as a drug, the present invention must not be immunogenic. Nanobodies derived from camelids may possess epitopes that can be recognized by the human immune system; the scFvs, on the other hand, can already be humanized.
• Tipologia di target. I nanobody riescono ad accedere a spazi altrimenti non accessibili da parte degli scFv. Inoltre alcuni target patologici sono situati in regioni anatomiche poco accessibili, come il sistema nervoso centrale, in questi casi si ha la necessità di minimizzare la dimensione della proteina di fusione, a questo scopo i nanobody sono la scelta migliore (Caljon et al., 2012). • Type of target. Nanobodies are able to access spaces otherwise not accessible by scFvs. Furthermore, some pathological targets are located in inaccessible anatomical regions, such as the central nervous system, in these cases it is necessary to minimize the size of the fusion protein, for this purpose nanobodies are the best choice (Caljon et al., 2012 ).
In riferimento alla proteina di fusione oggetto della presente invenzione, la scelta dell'uso della tipologia di attività del dominio proteasico è frutto del bilanciamento di alcune caratteristiche delle proteasi. Alcune delle caratteristiche più importanti da considerare sono: With reference to the fusion protein object of the present invention, the choice of the use of the activity typology of the protease domain is the result of the balancing of some characteristics of the proteases. Some of the most important features to consider are:
• Specificità dell' attività catalitica e struttura generale della proteina di fusione. • Dimensione del dominio proteasico. • Specificity of the catalytic activity and general structure of the fusion protein. • Size of the protease domain.
• L'immunogenicità. • Immunogenicity.
• Stabilità della struttura. • Stability of the structure.
• Solubilità. • Solubility.
Se una forma di realizzazione della proteina di fusione oggetto della presente invenzione viene ad essere impiegata in ambito industriale è preferibile impiegare domini proteasici che abbiano attività catalitica anche ad alte temperature e a pH lontani dalla neutralità. Viceversa se in un alternativa forma di realizzazione la proteina di fusione venisse impiegata come farmaco è estremamente importante che il dominio non sia immunogenico o ancora più grave allergenica, mentre la stabilità del folding non è così cruciale. Altresì importante è che la proteina di fusione non comprometta, data la sua attività proteasica, processi fisiologici, come la coagulazione sanguigna e il sistema del complemento. If an embodiment of the fusion protein object of the present invention is to be used in the industrial field, it is preferable to use protease domains which have catalytic activity even at high temperatures and at pH far from neutral. Conversely, if in an alternative embodiment the fusion protein is used as a drug it is extremely important that the domain is not immunogenic or even more severe allergenic, while the stability of the folding is not so crucial. It is also important that the fusion protein does not compromise, given its protease activity, physiological processes, such as blood coagulation and the complement system.
Per evitare la degradazione in trans o in cis tra le proteine di fusione in soluzione durante lo stoccaggio è opportuno miscelare anche degli inibitori reversibili delle proteasi. To avoid trans or cis degradation between fusion proteins in solution during storage, reversible protease inhibitors should also be mixed.
In alcune forme di realizzazione della la proteina di fusione oggetto della presente invenzione può essere fatta esprimere direttamente nella cellula, ad esempio impiegando vettori retrovirali. In some embodiments of the fusion protein object of the present invention can be made to express directly in the cell, for example by using retroviral vectors.
In alcune forme di realizzazione della proteina di fusione oggetto della presente invenzione i domini di legame possono legame anche epitopi uguali. In some embodiments of the fusion protein object of the present invention the binding domains can also bind equal epitopes.
In alcune forme di realizzazione della proteina di fusione oggetto della presente invenzione i domini di legame possono essere collegati, anche più di due, in cluster, uno dopo l'altro, con o senza peptidi linker. In some embodiments of the fusion protein object of the present invention the binding domains can be linked, even more than two, in clusters, one after the other, with or without linker peptides.
In molte forme di realizzazione della proteina di fusione oggetto della presente invenzione il bersaglio può essere inteso anche come insieme di proteine, ad esempio dimeri, trimeri ecc. quindi in questa configurazione la proteina di fusione solitamente lega epitopi di proteine diverse. In many embodiments of the fusion protein object of the present invention, the target can also be understood as a set of proteins, for example dimers, trimers, etc. therefore in this configuration the fusion protein usually binds epitopes of different proteins.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002047717A2 (en) * | 2000-12-14 | 2002-06-20 | Genencor International, Inc. | Targeted enzyme prodrug therapy |
| WO2004019878A2 (en) * | 2002-08-27 | 2004-03-11 | Compound Therapeutics, Inc. | Adzymes and uses thereof |
| WO2007144882A2 (en) * | 2006-06-14 | 2007-12-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Targeted chimeric proteins |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002047717A2 (en) * | 2000-12-14 | 2002-06-20 | Genencor International, Inc. | Targeted enzyme prodrug therapy |
| WO2004019878A2 (en) * | 2002-08-27 | 2004-03-11 | Compound Therapeutics, Inc. | Adzymes and uses thereof |
| WO2007144882A2 (en) * | 2006-06-14 | 2007-12-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Targeted chimeric proteins |
Non-Patent Citations (1)
| Title |
|---|
| KASTURIRANGAN S ET AL: "Targeted hydrolysis of Beta-amyloid with engineered antibody fragment", CURRENT ALZHEIMER RESEARCH, BENTHAM, UNITED ARAB EMIRATES, vol. 7, no. 3, 1 May 2010 (2010-05-01), pages 214 - 222, XP008162244, ISSN: 1875-5828, DOI: 10.2174/156720510791050876 * |
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