RESUMO
Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETAâ³), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETAâ³, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETAâ³ was much better tolerated than Ec1-ETAâ³; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.
Assuntos
ADP Ribose Transferases/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Toxinas Bacterianas/química , Exotoxinas/química , Polietilenoglicóis/química , Pró-Fármacos/farmacologia , Fatores de Virulência/química , Proteases Virais 3C , Animais , Repetição de Anquirina , Antígenos de Neoplasias/química , Antineoplásicos/farmacocinética , Área Sob a Curva , Sítios de Ligação , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Química Click , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Molécula de Adesão da Célula Epitelial , Feminino , Meia-Vida , Humanos , Camundongos Endogâmicos C57BL , Pró-Fármacos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required. Mol Cancer Ther; 16(5); 879-92. ©2017 AACR.
Assuntos
Antraciclinas/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/tratamento farmacológico , Ado-Trastuzumab Emtansina , Aminoaciltransferases/química , Animais , Antraciclinas/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Brentuximab Vedotin , Linhagem Celular Tumoral , Cisteína Endopeptidases/química , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Antígeno Ki-1/química , Antígeno Ki-1/imunologia , Maitansina/análogos & derivados , Maitansina/química , Maitansina/imunologia , Camundongos , Neoplasias/imunologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/química , Trastuzumab/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I-truncated Pseudomonas Exotoxin A (PE40/ETAâ³) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETAâ³ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETAâ³ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETAâ³, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.
Assuntos
ADP Ribose Transferases/química , Antígenos de Neoplasias/química , Antineoplásicos/química , Toxinas Bacterianas/química , Moléculas de Adesão Celular/química , Exotoxinas/química , Fatores de Virulência/química , ADP Ribose Transferases/genética , Animais , Antígenos de Neoplasias/genética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Toxinas Bacterianas/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Click , Eletroforese em Gel de Poliacrilamida , Molécula de Adesão da Célula Epitelial , Exotoxinas/genética , Feminino , Células HT29 , Humanos , Células MCF-7 , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Polietilenoglicóis/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Ressonância de Plasmônio de Superfície , Carga Tumoral/efeitos dos fármacos , Fatores de Virulência/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Exotoxina A de Pseudomonas aeruginosaRESUMO
INTRODUCTION: The epithelial cell adhesion molecule (EpCAM) is abundantly expressed in epithelial tumors, on cancer stem cells and circulating tumor cells. Together with its role in oncogenic signaling, this has sparked interest in its potential for tumor targeting with antibodies and drug conjugates for safe and effective cancer therapy. Recent advances in protein engineering, linker design and drug formulations have provided a multitude of EpCAM-targeting anticancer agents, several of them with good perspectives for clinical development. AREAS COVERED: This article reviews the biological, therapeutic and technical aspects of EpCAM-targeted drug delivery for cancer therapy. The authors discuss seminal findings, which distinguish EpCAM as a target with oncogenic function and abundant expression in epithelial tumors. Moreover, recent trends in engineering improved anti-EpCAM antibodies, binding proteins that are not derived from immunoglobulins and drug conjugates derived from them are highlighted and their therapeutic potential based on reported preclinical and clinical data, originality of design and perspectives are critically assessed. EXPERT OPINION: EpCAM has shown promise for safe and efficient targeting of solid tumors using antibodies, alternative binding molecules and novel drug conjugates. Among the myriad of EpCAM-targeting drug delivery systems investigated so far, several could demonstrate therapeutic benefit, other formulations engineered to become tailor-made missiles are on the brink.
Assuntos
Antineoplásicos/administração & dosagem , Imunotoxinas/administração & dosagem , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Animais , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Química Farmacêutica , Sistemas de Liberação de Medicamentos , Molécula de Adesão da Célula Epitelial , Humanos , Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at the C-prM junction. This system shows that processing at the C-terminus of protein C by the TBEV NS2B/3(pro) is highly membrane dependent and will allow the examination of how the membrane topology of protein C affects both SPaseI and NS2B/3(pro) processing.
Assuntos
Proteínas do Capsídeo/metabolismo , Doenças do Cão/virologia , Vírus da Encefalite Transmitidos por Carrapatos/enzimologia , Encefalite Transmitida por Carrapatos/veterinária , Membranas Intracelulares/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Doenças do Cão/metabolismo , Cães , Vírus da Encefalite Transmitidos por Carrapatos/química , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/virologia , Membranas Intracelulares/metabolismo , Microssomos/metabolismo , Microssomos/virologia , Dados de Sequência Molecular , Proteólise , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Designed ankyrin repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using "click chemistry" with nonnatural amino acids.
Assuntos
Repetição de Anquirina , Proteínas de Transporte/química , Desenho de Fármacos , Engenharia de Proteínas/métodos , Proteínas/uso terapêutico , Marcadores de Afinidade/química , Sequência de Aminoácidos , Animais , Pesquisa Biomédica , Química Click , Escherichia coli/química , Corantes Fluorescentes/química , Vetores Genéticos/química , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Polietilenoglicóis/química , Ligação Proteica , Estabilidade Proteica , Proteínas/química , Receptores de Superfície Celular/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Ribossomos/química , Termodinâmica , Titulometria , Vírus/químicaRESUMO
PURPOSE: Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)-specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A. EXPERIMENTAL DESIGN: DARPin Ec4 was genetically fused to a truncated form of Pseudomonas aeruginosa exotoxin A (ETAâ³) and expressed in Escherichia coli. The cytotoxicity of Ec4-ETAâ³ was measured against tumor cell lines of various histotypes in vitro. Tumor localization and antitumor activity were determined in mice bearing 2 different EpCAM-positive tumor xenografts. RESULTS: Ec4-ETAâ³ expressed very well in soluble form in the cytoplasm of E. coli and yielded up to 40 mg after purification per liter of culture. The protein was monomeric and the disulfides of ETAâ³ formed spontaneously. Ec4-ETAâ³ bound to EpCAM with low nanomolar affinity, similar to free Ec4. Furthermore, it was highly cytotoxic against various EpCAM-positive tumor cell lines in vitro with IC(50) values less than 0.005 pmol/L. This effect was competed by free Ec4, but not by unspecific DARPins. Upon systemic administration in athymic mice, Ec4-ETAâ³ efficiently localized to EpCAM-positive tumors to achieve maximum accumulation 48 to 72 hours after injection, whereas an irrelevant control fusion toxin did not accumulate. Tumor targeting with Ec4-ETAâ³ resulted in a strong antitumor response including complete regressions in some animals. CONCLUSIONS: Our data show for the first time the potential of DARPins for the generation of protein therapeutics for tumor targeting, and that Ec4-ETAâ³ deserves attention for clinical development.
Assuntos
ADP Ribose Transferases/farmacologia , Repetição de Anquirina , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Moléculas de Adesão Celular/farmacologia , Exotoxinas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Virulência/farmacologia , ADP Ribose Transferases/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/metabolismo , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Moléculas de Adesão Celular/administração & dosagem , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Exotoxinas/administração & dosagem , Exotoxinas/química , Exotoxinas/metabolismo , Feminino , Humanos , Injeções Intravenosas , Camundongos , Camundongos Nus , Engenharia de Proteínas , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Virulência/administração & dosagem , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Exotoxina A de Pseudomonas aeruginosaRESUMO
Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K(d)=68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.