Studies on polymer conjugation of therapeutic proteins and peptides

Since the introduction of recombinant insulin in 1979 and thanks to the advances in pharmaceutical biotechnology, therapeutic proteins and peptides have gained significant importance in the treatment of many diseases and after they represent the only therapeutic option. However, such growing success is not devoid of potential pitfalls like short half-life, easy enzymatic degradation and immunogenicity. PEGylation, namely the attachment of polyethylene glycol (PEG) chain(s) to a bioactive molecule, is nowadays one of the most commonly used strategies to overcome the inherent limits of therapeutic proteins. The studies reported in this thesis are focused on the design and preparation of conjugates of therapeutic proteins and peptides with optimized polymeric carriers, in order to obtain improved performances in both pharmacokinetic and pharmacodynamic profiles of a conjugated biologic. The proteins and the peptide used are recombinant human granulocyte colony-stimulating factor (G-CSF), recombinant human growth hormone (hGH) and KR14, an anti-HIV triazole peptide (PT). G-CSF, hGH and KR14, owing to their low molecular weights, are rapidly excreted from the body and are subject to proteolytic degradation, resulting in low bioavailability and therefore requiring frequent administration schedule. The first part of this PhD thesis was focused on: i) transglutaminase-mediated conjugation of common non charged or new multicarboxylic PEGs to G-CSF, ii) characterization and stability studies of the conjugates, iii) pharmacokinetic studies in animals, to allow the comparison between the conjugates and with the reference G-CSF. In this study, the aim was to test multicarboxylic PEG derivatives for evaluating the contribution of negative charges on protein half-life prolongation thanks to the charge-selective property of the glomerular filtration barrier. PEGylation with high molecular weight PEGs, by increasing the hydrodynamic volume of a protein, decreases the glomerular filtration rate, but may constitute a steric hindrance and reduce the binding affinity between the therapeutic protein and its target. The charge selectivity concept of the glomerular filtration barrier can be exploited with the new polyanionic polymers, which with low molecular weights, can still extend the PK profile of the protein and achieve also a better preservation of protein activity. The investigated polyanionic PEG derivatives were i) H2N-PEG5k-(COOH)7, a heterobifunctional PEG that was derivatized on one end with β-glutamic acid units to contain 7 carboxyl groups and ii) H2N-PEG3.4k-b-PLE50, a linear polyanionic polymer that consists of a PEG linked to a PLE with the pendant ϒ-carboxyl groups of glutamic acid monomers. In addition to these two multicarboxylic PEGs, two classic uncharged PEGs with molecular weights of 5 kDa and 20 kDa were also used. After PEGylation, all the G-CSF-PEG conjugates were characterized by MALDI-TOF mass spectrometry, SDS-PAGE and circular dichroism and the pharmacokinetic studies were carried out in rats. The second part of this PhD thesis focused on the comparison of the biological activity (stimulation of somatic growth) of three hGH conjugates differing for the polymeric carrier (multicarboxylic or neutral) and for the site of polymer conjugation. The aim of this study was to evaluate if the protein stability and the protein/receptor recognition were influenced by the site of conjugation of the polymer and to assess the impact of polyanionic carriers on the pharmacodynamic behaviour of the protein. Conjugation of the multicarboxylic polymer, poly(L-glutamic acid), resulted in an increase of the final negative charge of the conjugate. Based on the charge selectivity concept of glomerular filtration, polyanionic polymers having relatively low molecular weights may provide a half-life extension of a given protein, similar to that provided by higher molecular weight neutral polymers. Two different site-specific mono-PEGylated forms of hGH were prepared exploiting an enzymatic PEGylation (PEG-Gln141-hGH) via transglutaminase (TGase) and a chemical N-terminal PEGylation (PEG-Nter-hGH), using neutral 20kDa PEGs. Interestingly the conjugates showed an increased thermal stability and the ability to refold after thermal denaturation. The third monoconjugate of hGH was prepared by chemical N-terminal conjugation, using poly(L-glutamic acid) with 50 glutamic acid units and with an aldehyde function at one end (PLE50ald). The glutamic acid monomers confer to this polymer a polyanionic characteristic. hGH-PLE50ald was characterized by MALDI-TOF mass spectrometry, SDS-PAGE, circular dichroism and the pharmacokinetic studies were carried out in rats. The bioactivities of a single dose of hGH-PLE50ald, PEG-Gln141-hGH and PEG-Nter-hGH were similar or even better to that of a daily injection of hGH, by testing the somatic growth in hypophysectomized rats. The third section of this work has dealt with: synthesis and characterization of KR14 peptide and its conjugation with PEG20kDa-Mal, pharmacokinetic studies of the conjugate in mice and SPR affinity binding experiments. A part of this work was developed at the Department of Biochemistry and Molecular Biology of the Drexel University College of Medicine in Philadelphia, under the supervision of Professor Irwin Chaiken, whose laboratory is focused on the design and realization of peptide drugs that inhibit the entry of HIV-1 in the target cells. This initial step of HIV-1 entry is characterized by the binding of envelope glycoprotein gp120 on to the cellular receptor CD4 and co-receptor. The triazole peptide KR14 derive from a previously known family of PT’s that has great therapeutic potential as it exhibits high affinity binding to gp120 and inhibits interactions of gp120 with both CD4 and the co-receptor surrogate mAb17b. KR14 was designed to contain a free thiol group at position 16 that has been exploited to achieve selective PEGylation with a PEG-maleimide of 20 kDa. The identity and purity of the conjugate were confirmed by SEC-HPLC and MALDI-TOF. Pharmacokinetic studies in mice demonstrated that PEGylation extended the t1/2 of the peptide from 44 min to 289 min. Even more remarkably the conjugate did not show the typical loss in bioactivity, common to most PEGylated drugs. In fact SPR studies demonstrated that PEG-KR14 is only about 1.5, 1.6 fold less active than KR14, a relevant result. In conclusion the studies of this PhD thesis further demonstrated the potential of PEGylation in the delivery of protein and peptides. Although PEGylation is already a mature technology that yielded several products in clinical use, we demonstrated that there are still reasons for implementation and new discoveries by pursing also the renal filtration charge selectivity and not only the size selectivity as done so far in this field

A Non-Covalent Antibody Complex for the Delivery of anti-cancer drugs

Antibody drug conjugates (ADCs), which are obtained by coupling a potent cytotoxic agent to a monoclonal antibody (mAb), are traditionally bound in a random way to lysine or cysteine residues, with the final product's heterogeneity having an important impact on their activity, characterization, and manufacturing. A new antibody drug delivery system (ADS) based on a non-covalent linkage between a Fc-binding protein, in this case Protein A or Protein G, and a mAb was investigated in the effort to achieve greater homogeneity and to create a versatile and adaptable drug delivery system. Recombinant staphylococcal Protein A and streptococcal Protein G were chemically PEGylated at the N-terminus with a 5 kDa and a 20 kDa PEG, respectively, yielding two monoconjugates with a mass of 48 50 and 48 45 kDa. Circular dichroism studies showed that both conjugates preserved secondary structures of the protein, and isothermal titration calorimetry experiments demonstrated that their affinity for mAb was approximately 107 M-1. Upon complexation with a mAb (Trastuzumab or Rituximab), in vitro flow-cytometry analysis of the new ADSs showed high selectivity for the specific antigen expressing cells. In addition, the ADS complex based on Trastuzumab and Protein G, conjugated with a heterobifunctional 20 kDa PEG carrying the toxin Tubulysin A, had a marked cytotoxic effect on the cancer cell line overexpressing the HER2/neu receptor, thus supporting its application in cancer therapy

Improving the Therapeutic Potential of G-CSF through Compact Circular PEGylation Based on Orthogonal Conjugations

: In this study, a circular conjugate of granulocyte colony-stimulating factor (G-CSF) was prepared by conjugating the two end-chains of poly(ethylene glycol) (PEG) to two different sites of the protein. For the orthogonal conjugation, a heterobifunctional PEG chain was designed and synthesized, bearing the dipeptide ZGln-Gly (ZQG) at one end-chain, for transglutaminase (TGase) enzymatic selective conjugation at Lys41 of G-CSF, and an aldehyde group at the opposite end-chain, for N-terminal selective reductive alkylation of the protein. The cPEG-Nter/K41-G-CSF circular conjugate was characterized by physicochemical methods and compared with native G-CSF and the corresponding linear monoconjugates of G-CSF, PEG-Nter-G-CSF, and PEG-K41-G-CSF. The results demonstrated that the circular conjugate had improved physicochemical and thermal stability, prolonged pharmacokinetic interaction, and retained the biological activity of G-CSF. The PEGylation strategy employed in this study has potential applications in the design of novel protein-based therapeutics

Poly(L-glutamic acid)-co-poly(ethylene glycol) block copolymers for protein conjugation

Poly(L-glutamic acid)-co-poly(ethylene glycol) block copolymers (PLE-PEG) are here investigated as polymers for conjugation to therapeutic proteins such as granulocyte colony stimulating factor (G-CSF) and human growth hormone (hGH). PLE-PEG block copolymers are able to stabilize and protect proteins from degradation and to prolong their residence time in the blood stream, features that are made possible thanks to PEG's intrinsic properties and the simultaneous presence of the biodegradable anionic PLE moiety. When PLE-PEG copolymers are selectively tethered to the N-terminus of G-CSF and hGH, they yield homogeneous monoconjugates that preserve the protein's secondary structure. During the current study the pharmacokinetics of PLE10-PEG20k-G-CSF and PLE20-PEG20k-G-CSF derivatives and their ability to induce granulopoiesis were, respectively, assessed in Sprague-Dawley rats and in C57BL6 mice. Our results show that the bioavailability and bioactivity of the derivatives are comparable to or better than those of PEG20k-Nter-G-CSF (commercially known as Pegfilgrastim). The therapeutic effects of PLE10-PEG20k-hGH and PLE20-PEG20k-hGH derivatives tested in hypophysectomized rats demonstrate that the presence of a negatively charged PLE block enhances the biological properties of the conjugates additionally with respect to PEG20k-Nter-hGH

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer

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P09.03 Hyaluronic acid as a new immunologic adjuvant in cancer: design of effective preventive and therapeutic vaccination strategies for HER2/neu-positive breast tumors

Background The use of proteins as immunogens is attractive for the development of vaccines, but requires efficient adjuvants to overcome their weak immunogenicity. Recently, we investigated the potential of the TLR2/4 agonist hyaluronan (HA) as an immunological adjuvant for protein-based vaccines.1 2 Conjugation of HA to antigens strongly increased their immunogenicity and promoted their rapid translocation to draining lymph nodes, resulting in robust and long-lasting humoral responses.1 On these bases, we investigated the potentiality of HA-based technology in the design of cancer vaccines. To this aim, HA was conjugated to the extracellular domain of rat HER2/neu (rHER2/neu) and validated in the preventive and therapeutic vaccination settings. Materials and Methods Female BALB/c or BALB-neuT mice were immunized with rHER2/neu-HA. In vivo depletion of CD4+, CD8+ T and B cells was performed, and sera and spleens were collected to characterized antigen-specific humoral and cellular responses. Vaccinated BALB/c mice were challenged and re-challenged with rHER2/neu-overexpressing TUBO cells to assess the protective or therapeutic activity of rHER2/neu-HA vaccination strategy, as well as immunological memory. Results HA performed efficiently as robust and long-lasting humoral (IgG1, IgG2a, and IgG2b) and cellular responses were detected using very low antigen doses and number of boosters. Outstandingly, at 1-year post-vaccination, anti-rHER2/neu specific antibodies showed even improved effector functions (maturation of affinity for the receptor and increased complement-derived cytotoxicity functions). HA vaccination turned out effective in both the prophylactic (100% mice survived) and therapeutic (tumor regression in 2/12 mice) settings, and broke tolerance against rHER2/neu, delaying spontaneous tumor growth in BALB-neuT mice. Both humoral and cellular responses contributed to the success of HA-based vaccination, but CD8+ T cells played only a marginal role. Conclusions Cancer vaccines have not yet achieved significant clinical efficacy due to their poor immunogenicity, and the validation of more effective adjuvants occurred sometimes at the expense of safety. HA combines the unique immunomodulatory features of a TLR agonist with the tolerability of a fully natural polymer, proving to be a promising adjuvant for the creation of effective and safe cancer vaccines with the potential for rapid clinical translation. References Dalla Pieta A, Carpanese D, et al. Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines. Cell Mol Immunol 2021;18(5):1197–1210. Rosato A, Montagner IM, Carpanese D, Dalla Pieta A. Hyaluronic acid as a natural adjuvant for protein and peptide-based vaccines. 30.04.2020. WO/2020/084558, PCT/IB2019/059122. Disclosure Information D. Carpanese: None. I. Montagner: None. A. Dalla Pieta: None. V. Rossi: None. A. Penna: None. G. Zuccolotto: None. G. Pasut: None. A. Grigoletto: None. A. Rosato: None

Anti-HER2 Super Stealth Immunoliposomes for Targeted-Chemotherapy

: Liposomes play an important role in the field of drug delivery by virtue of their biocompatibility and versatility as carriers. Stealth liposomes, obtained by surface decoration with hydrophilic polyethylene glycol (PEG) molecules, represented an important turning point in liposome technology, leading to significant improvements in the pharmacokinetic profile compared to naked liposomes. Nevertheless, the generation of effective targeted liposomes - a central issue for cancer therapy - has faced several difficulties and clinical phase failures. Active targeting remains a challenge for liposomes. In this direction, we designed a new Super Stealth Immunoliposomes (SSIL2) composed of a PEG-bi-phospholipids derivative that stabilizes the polymer shielding over the liposomes. Furthermore, its counterpart, conjugated to the fragment antigen-binding of trastuzumab (Fab'TRZ -PEG-bi-phospholipids), is firmly anchored on the liposomes surface and correctly orients outward the targeting moiety. Throughout this study, the performances of SSIL2 are evaluated and compared to classic stealth liposomes and stealth immunoliposomes in vitro in a panel of cell lines and in vivo studies in zebrafish larvae and rodent models. Overall, SSIL2 shows superior in vitro and in vivo outcomes, both in terms of safety and anticancer efficacy, thus representing a step forward in targeted cancer therapy, and valuable for future development. This article is protected by copyright. All rights reserved

Studies on polymer conjugation of therapeutic proteins and peptides

Since the introduction of recombinant insulin in 1979 and thanks to the advances in pharmaceutical biotechnology, therapeutic proteins and peptides have gained significant importance in the treatment of many diseases and after they represent the only therapeutic option. However, such growing success is not devoid of potential pitfalls like short half-life, easy enzymatic degradation and immunogenicity. PEGylation, namely the attachment of polyethylene glycol (PEG) chain(s) to a bioactive molecule, is nowadays one of the most commonly used strategies to overcome the inherent limits of therapeutic proteins. The studies reported in this thesis are focused on the design and preparation of conjugates of therapeutic proteins and peptides with optimized polymeric carriers, in order to obtain improved performances in both pharmacokinetic and pharmacodynamic profiles of a conjugated biologic. The proteins and the peptide used are recombinant human granulocyte colony-stimulating factor (G-CSF), recombinant human growth hormone (hGH) and KR14, an anti-HIV triazole peptide (PT). G-CSF, hGH and KR14, owing to their low molecular weights, are rapidly excreted from the body and are subject to proteolytic degradation, resulting in low bioavailability and therefore requiring frequent administration schedule. The first part of this PhD thesis was focused on: i) transglutaminase-mediated conjugation of common non charged or new multicarboxylic PEGs to G-CSF, ii) characterization and stability studies of the conjugates, iii) pharmacokinetic studies in animals, to allow the comparison between the conjugates and with the reference G-CSF. In this study, the aim was to test multicarboxylic PEG derivatives for evaluating the contribution of negative charges on protein half-life prolongation thanks to the charge-selective property of the glomerular filtration barrier. PEGylation with high molecular weight PEGs, by increasing the hydrodynamic volume of a protein, decreases the glomerular filtration rate, but may constitute a steric hindrance and reduce the binding affinity between the therapeutic protein and its target. The charge selectivity concept of the glomerular filtration barrier can be exploited with the new polyanionic polymers, which with low molecular weights, can still extend the PK profile of the protein and achieve also a better preservation of protein activity. The investigated polyanionic PEG derivatives were i) H2N-PEG5k-(COOH)7, a heterobifunctional PEG that was derivatized on one end with β-glutamic acid units to contain 7 carboxyl groups and ii) H2N-PEG3.4k-b-PLE50, a linear polyanionic polymer that consists of a PEG linked to a PLE with the pendant ϒ-carboxyl groups of glutamic acid monomers. In addition to these two multicarboxylic PEGs, two classic uncharged PEGs with molecular weights of 5 kDa and 20 kDa were also used. After PEGylation, all the G-CSF-PEG conjugates were characterized by MALDI-TOF mass spectrometry, SDS-PAGE and circular dichroism and the pharmacokinetic studies were carried out in rats. The second part of this PhD thesis focused on the comparison of the biological activity (stimulation of somatic growth) of three hGH conjugates differing for the polymeric carrier (multicarboxylic or neutral) and for the site of polymer conjugation. The aim of this study was to evaluate if the protein stability and the protein/receptor recognition were influenced by the site of conjugation of the polymer and to assess the impact of polyanionic carriers on the pharmacodynamic behaviour of the protein. Conjugation of the multicarboxylic polymer, poly(L-glutamic acid), resulted in an increase of the final negative charge of the conjugate. Based on the charge selectivity concept of glomerular filtration, polyanionic polymers having relatively low molecular weights may provide a half-life extension of a given protein, similar to that provided by higher molecular weight neutral polymers. Two different site-specific mono-PEGylated forms of hGH were prepared exploiting an enzymatic PEGylation (PEG-Gln141-hGH) via transglutaminase (TGase) and a chemical N-terminal PEGylation (PEG-Nter-hGH), using neutral 20kDa PEGs. Interestingly the conjugates showed an increased thermal stability and the ability to refold after thermal denaturation. The third monoconjugate of hGH was prepared by chemical N-terminal conjugation, using poly(L-glutamic acid) with 50 glutamic acid units and with an aldehyde function at one end (PLE50ald). The glutamic acid monomers confer to this polymer a polyanionic characteristic. hGH-PLE50ald was characterized by MALDI-TOF mass spectrometry, SDS-PAGE, circular dichroism and the pharmacokinetic studies were carried out in rats. The bioactivities of a single dose of hGH-PLE50ald, PEG-Gln141-hGH and PEG-Nter-hGH were similar or even better to that of a daily injection of hGH, by testing the somatic growth in hypophysectomized rats. The third section of this work has dealt with: synthesis and characterization of KR14 peptide and its conjugation with PEG20kDa-Mal, pharmacokinetic studies of the conjugate in mice and SPR affinity binding experiments. A part of this work was developed at the Department of Biochemistry and Molecular Biology of the Drexel University College of Medicine in Philadelphia, under the supervision of Professor Irwin Chaiken, whose laboratory is focused on the design and realization of peptide drugs that inhibit the entry of HIV-1 in the target cells. This initial step of HIV-1 entry is characterized by the binding of envelope glycoprotein gp120 on to the cellular receptor CD4 and co-receptor. The triazole peptide KR14 derive from a previously known family of PT’s that has great therapeutic potential as it exhibits high affinity binding to gp120 and inhibits interactions of gp120 with both CD4 and the co-receptor surrogate mAb17b. KR14 was designed to contain a free thiol group at position 16 that has been exploited to achieve selective PEGylation with a PEG-maleimide of 20 kDa. The identity and purity of the conjugate were confirmed by SEC-HPLC and MALDI-TOF. Pharmacokinetic studies in mice demonstrated that PEGylation extended the t1/2 of the peptide from 44 min to 289 min. Even more remarkably the conjugate did not show the typical loss in bioactivity, common to most PEGylated drugs. In fact SPR studies demonstrated that PEG-KR14 is only about 1.5, 1.6 fold less active than KR14, a relevant result. In conclusion the studies of this PhD thesis further demonstrated the potential of PEGylation in the delivery of protein and peptides. Although PEGylation is already a mature technology that yielded several products in clinical use, we demonstrated that there are still reasons for implementation and new discoveries by pursing also the renal filtration charge selectivity and not only the size selectivity as done so far in this field.Sin dall’introduzione dell’insulina ricombinante, nel 1979 e grazie ai progressi della biotecnologia farmaceutica, proteine e peptidi terapeutici hanno raggiunto una significativa importanza nel trattamento di molte patologie e rappresentano l’unica opzione terapeutica. Questo crescente successo non è comunque, privo di potenziali ostacoli come una breve emivita, una facile degradazione enzimatica e l’immunogenicità. La PEGhilazione, ovvero il legame di catene di polietilenglicole (PEG) a molecole bioattive, è oggigiorno una delle più comuni strategie usate per superare i limiti inerenti alle proteine terapeutiche. Gli studi riportati in questa tesi di dottorato sono focalizzati sulla progettazione e preparazione di coniugati di proteine e peptidi terapeutici con carrier polimerici ottimizzati, in modo da ottenere migliori prestazioni dell’entità coniugata, sia dal punto di vista del profilo farmacocinetico che farmacodinamco. Le proteine e il peptide usati in questo lavoro sono G-CSF, o fattore di crescita umano ricombinante stimolante la formazione di colonie granulocitiche, hGH, o ormone della crescita umano ricombinante e KR14, un peptide anti-HIV. G-CSF, hGH e KR14, a causa del loro basso peso molecolare, sono rapidamente eliminati dall’organismo e sono soggetti a degradazione proteolitica, con conseguente bassa biodisponibilità e necessità di somministrazioni frequenti. La prima parte di questa tesi di dottorato è focalizzata su: i) PEGhilazione enzimatica via transglutaminasi di G-CSF a PEG non carichi e a nuovi PEG multicarbossilati, ii) caratterizzazione e studi di stabilità dei coniugati ottenuti, iii) studi farmacocinetici in ratti, per avere un confronto tra i coniugati e la proteina nativa come riferimento. Lo scopo di questo studio è stato quello di testare i PEG multicarbossilati per valutare il contributo delle cariche negative sul prolungamento dell’emivita della proteina, grazie alle proprietà di selezione della carica della barriera di filtrazione del glomerulo. La PEGhilazione con PEG ad alto peso molecolare, aumentando il volume idrodinamico della proteina, riduce la velocità di filtrazione glomerulare, ma può costituire un impedimento sterico e ridurre l’affinità di legame tra la proteina terapeutica e il suo target. Il concetto di selettività della carica della barriera di filtrazione glomerulare può essere sfruttato con i nuovi polimeri polianionici, che, con un basso peso molecolare, possono mantenere un profilo farmacocinetico della proteina esteso e raggiungere anche una migliore conservazione dell’attività della proteina. I PEG polianionici usati per questo lavoro sono stati i) H2N-PEG5k-(COOH)7, un PEG eterobifunzionale che è stato derivatizzato ad un’estremità con unità di acido β-glutammico e che possiede 7 gruppi carbossilici e ii) H2N-PEG3.4k-b-PLE50, un polimero polianionico lineare costituito da PEG legato a PLE, il quale possiede i gruppi pendenti ϒ-carbossilici dei monomeri dell’acido glutammico. Oltre a questi due PEG multicarbossilati, sono stati usati anche due PEG non carichi, di peso molecolare 5 e 20 kDa. Dopo PEGhilazione, tutti i coniugati di G-CSF sono stati caratterizzati tramite MALDI-TOF, SDS-PAGE e dicroismo circolare e gli studi farmacocinetici sono stati condotti in ratti. La seconda parte di questa tesi di dottorato si focalizza sul confronto dell’attività biologica (stimolazione della crescita somatica) di tre coniugati dell’ormone della crescita che si differenziano per il carrier polimerico (multicarbossilato o neutro) e per il sito di coniugazione del polimero. Lo scopo di questo studio è stato valutare se la stabilità della proteina e il riconoscimento proteina/recettore sono stati influenzati dal sito di coniugazione del polimero e verificare l’impatto di carrier polianionici sul profilo farmacodinamico della proteina. La coniugazione del polimero multicarbossilato, acido (poli)glutammico, risulta in un aumento della carica negativa netta del coniugato. Sulla base del concetto di selettività della carica della filtrazione glomerulare, polimeri polianionici aventi pesi molecolari relativamente bassi, possono fornire un’estensione dell’emivita di una proteina simile a quella ottenuta con polimeri neutri di alto peso molecolare. Due diverse forme di hGH mono-PEGhilato in maniera sito-specifica sono state preparate sfruttando una PEGhilazione enzimatica (PEG-Gln141-hGH) via tranglutaminase (TGase) e una PEGhilazione chimica all’N-terminale (PEG-Nter-hGH), usando PEG neutri di peso molecolare 20 kDa. Interessante è il fatto che i coniugati hanno mostrato una stabilità termica aumentata e la capacità di refold dopo denaturazione termica. Il terzo monoconiugato di hGH è stato preparato tramite coniugazione all’N-terminale, usando un acido (poli)glutammico con 50 unità di acido glutammico e con una funzione aldeidica ad un’estremità (PLE50ald). I monomeri di acido glutammico conferiscono al polimero la caratteristica di polianionico. hGH-PLE50ald è stato caratterizzato tramite MALDI-TOF, SDS-PAGE, dicroismo circolare e gli studi farmacocinetici sono stati condotti in ratti. La bioattività di una dose singola di hGH-PLE50ald, PEG-Gln141-hGH e PEG-Nter-hGH si è dimostrata simile o migliore rispetto a iniezioni giornaliere di hGH, misurando la crescita somatica in ratti ipofisectomizzati. La terza parte di questo lavoro tratta di: sintesi e caratterizzazione di KR14 e coniugazione di KR14 a PEG20kDa-Mal, studi farmacocinetici del coniugato in topi e studi di affinità di legame tramite SPR. Una parte di questo lavoro è stata sviluppata al Dipartimento di Biochimica e Biologia Molecolare presso Drexel University College of Medicine a Philadelphia, sotto la supervisione del Professor Irwin Chaiken, il cui laboratorio è focalizzato sulla progettazione e realizzazione di farmaci peptidici che inibiscono l’entrata del virus HIV-1 nelle cellule target. Questo passaggio iniziale di entrata del virus è caratterizzato dal legame della glicoproteina di membrana gp120 al recettore cellulare CD4 e al co-recettore. Il peptide KR14 proviene da una famiglia già nota di peptidi, modificati con triazolo, che ha un considerevole potenziale terapeutico, esibendo alta affinità di legame per la gp120 e inibendo le interazioni di gp120 con CD4 e con il surrogato del co-recettore mAb17b. KR14 è stato progettato in modo da comprendere un gruppo tiolico libero in posizione 16, che è stato utilizzato per la PEGhilazione selettiva con un PEG-maleimide di 20 kDa. L’identità e la purezza del coniugato sono state confermate tramite SEC-HPLC e MALDI-TOF. Gli studi farmacocinetici in topi hanno dimostrato che la PEGhilazione ha prolungato l’emivita del peptide da 44 minuti a 289 minuti. Ancora più interessante, il coniugato non ha mostrato la tipica perdita di bioattività, comune alla maggior parte dei farmaci PEGhilati. Infatti gli studi SPR hanno dimostrato che PEG-KR14 è solo 1,5, 1,6 volte meno attivo di KR14, un risultato rilevante. In conclusione gli studi di questa tesi di dottorato hanno dimostrato ulteriormente il potenziale della PEGhilazione nella veicolazione di proteine e peptidi. Anche se la PEGhilazione è già una tecnologia matura che ha portato diversi prodotti in uso clinico, abbiamo dimostrato che ci sono ancora motivi per l’implementazione e le nuove scoperte, perseguendo anche la selettività di carica della filtrazione renale e non solo la selettività per dimensioni, come fatto finora in questo campo

Efficacy of PEGylated ciliary neurotrophic factor superagonist variant in diet-induced obesity mice

: Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules