RESUMEN
INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.
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Animales Modificados Genéticamente , Trasplante de Corazón , Inflamación , Papio , Trasplante Heterólogo , Animales , Trasplante Heterólogo/métodos , Trasplante de Corazón/métodos , Porcinos , Inflamación/inmunología , Coagulación Sanguínea/efectos de los fármacos , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Humanos , Xenoinjertos/inmunología , Galactosiltransferasas/genética , Inmunosupresores/farmacología , Citocinas/metabolismoRESUMEN
Cumulative evidence from several pre-clinical studies suggests that restoration of plasma DNase activity in a thrombo-inflammatory state may improve clinical outcomes. Following injury, hyperactivated immune cells release large amounts of granular proteins together with DNA, which often accumulate in the surrounding environment in so-called neutrophil extracellular traps (NETs). Degradation of excess NETs by systemic DNase administration offers a promising therapeutic approach to ameliorate inflammation and dissolve intravascular clots. In order to expand the therapeutic utility of human DNase I, a variant of the enzyme was developed that has both a prolonged systemic half-life and a higher catalytic activity compared to Dornase alfa (Pulmozyme®), the recombinant form of DNase I approved for inhaled therapy of cystic fibrosis. The hyperactive enzyme was "PASylated" by genetic fusion with a strongly hydrophilic and biodegradable PAS-polypeptide to increase its hydrodynamic volume and retard kidney filtration. A stable TurboCell™ CHO-K1-based cell line was generated which is suitable for the future production of PASylated DNase I according to good manufacturing practice (GMP). Furthermore, a robust bioprocess strategy was devised and an effective downstream process was developed. The final protein product is characterized by excellent purity, favorable physicochemical properties, a 14-fold higher DNA-degrading activity than Dornase alfa and a sustained pharmacokinetic profile, with a 22-fold slower clearance in rats.
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Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.
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Alanina/química , Cristalografía por Rayos X/métodos , Péptidos/química , Polietilenglicoles/química , Prolina/química , Cristalización/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Muramidasa/química , Proteínas de Plantas/química , Proteína O-Metiltransferasa/química , SolubilidadRESUMEN
The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.
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Descubrimiento de Drogas/métodos , Ingeniería de Proteínas/métodos , Proteínas/administración & dosificación , Animales , Repetición de Anquirina , Humanos , Unión Proteica , Proteínas/metabolismo , Proteínas/farmacologíaRESUMEN
Initially emerging as a highly innovative concept in the late 1990s, the concept of creating novel binding reagents based on stable protein scaffolds from outside the immunoglobulin (Ig) superfamily has become a well-developed area of research and discovery today. Numerous scaffolds based on extracellular, membrane-bound or intracellular proteins (or their domains) have been recruited, yielding versatile research reagents and even biological drug candidates to serve as a viable alternative to antibodies. This minireview discusses both established and novel concepts in this field and summarizes the current state of clinical development of the more advanced protein scaffolds, in particular Affibody, Adnectin, Anticalin and DARPin drug candidates.
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Ingeniería de Proteínas , ProteínasRESUMEN
We describe the comparative X-ray structural analysis of three Anticalin proteins directed against the extra-domain B (ED-B) of oncofetal fibronectin (Fn), a validated marker of tumor neoangiogenesis. The Anticalins were engineered from the human lipocalin 2 (Lcn2) scaffold via targeted randomization of the structurally variable loop region and selection by phage display, resulting in 15-19 exchanged residues. While the four reshaped loops exhibit diverse conformations (with shifts in Cα positions up to 20.4 Å), the ß-barrel core of the lipocalin remains strongly conserved, thus confirming the extraordinary robustness of this scaffold. All three Anticalins bind the cc' hairpin loop of ED-B, the most exposed motif in the context of its neighboring Fn domains, but reveal entirely different binding modes, with orientations differing by up to 180°. Hence, each Anticalin recognizes its molecular target in an individual manner, in line with the distinct epitope specificities previously seen in binding experiments.
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Antígenos de Neoplasias/química , Epítopos/química , Fibronectinas/química , Lipocalina 2/química , Péptidos/química , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Epítopos/genética , Epítopos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Lipocalina 2/genética , Lipocalina 2/metabolismo , Modelos Moleculares , Biblioteca de Péptidos , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Pharmacokinetic (PK) extension is no longer just a means to create improved second generation biologics (so-called biobetters), but constitutes an accepted strategy in biopharmaceutical drug development today. Although PEGylation has become a widely applied methodology to furnish therapeutic proteins and peptides with prolonged plasma half-life, the immunogenicity and missing biodegradability of this synthetic polymer has prompted an evident need for alternatives. PASylation is based on biological polypeptides made of the small l-amino acids Pro, Ala and/or Ser (PAS), which adopt a random coil structure in aqueous buffers with surprisingly similar biophysical properties as PEG. In contrast, PAS sequences can be conjugated to pharmaceutically active proteins and peptides both via chemical coupling and at the genetic level, as so-called fusion proteins. PASylation has been successfully applied to numerous biologics, including cytokines, growth factors, antibody fragments, enzymes as well as various peptides, and validated in diverse animal models, from mice to monkeys. Here we compare PASylation with other current strategies for half-life extension and we discuss the utility of these approaches for the design of innovative peptide-based therapeutics.
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Descubrimiento de Drogas/métodos , Péptidos/química , Péptidos/farmacocinética , Alanina/química , Alanina/farmacocinética , Animales , Semivida , Humanos , Modelos Moleculares , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Prolina/química , Prolina/farmacocinética , Serina/química , Serina/farmacocinéticaRESUMEN
ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.
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Alineación de Secuencia/métodos , Programas Informáticos , Secuencia de Aminoácidos , Modelos Moleculares , Ingeniería de Proteínas/métodos , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína/métodosRESUMEN
The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the γ-emitter (123) I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.
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Anticuerpos/metabolismo , Neoplasias Encefálicas/diagnóstico , Fibronectinas/análisis , Glioblastoma/diagnóstico , Lipocalinas/inmunología , Imagen Molecular/métodos , Neoplasias Encefálicas/química , Línea Celular Tumoral , Fibronectinas/metabolismo , Glioblastoma/química , Humanos , Inmunohistoquímica , Lipocalinas/metabolismo , Biblioteca de Péptidos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6Å resolution and reveals binding to the gfcc' sheet and cc' loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved ß-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.
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Anticuerpos/metabolismo , Fibronectinas/metabolismo , Lipocalinas/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/genética , Sitios de Unión/genética , Unión Competitiva , Células CACO-2 , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Fibronectinas/química , Fibronectinas/genética , Humanos , Lipocalinas/genética , Lipocalinas/metabolismo , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.
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Fibronectinas/química , Multimerización de Proteína , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel ß-barrel, which is open to the solvent at one end. There, four loops connect the ß-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.
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Proteínas de Fase Aguda/química , Proteínas Portadoras/química , Sistemas de Liberación de Medicamentos/métodos , Lipocalinas/química , Biblioteca de Péptidos , Proteínas Proto-Oncogénicas/química , Proteínas de Fase Aguda/síntesis química , Proteínas de Fase Aguda/uso terapéutico , Animales , Proteínas Portadoras/síntesis química , Proteínas Portadoras/uso terapéutico , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/química , Vectores Genéticos/química , Humanos , Lipocalina 2 , Lipocalinas/síntesis química , Lipocalinas/uso terapéutico , Mutagénesis Sitio-Dirigida/métodos , Plásmidos/química , Ingeniería de Proteínas/métodos , Proteínas Proto-Oncogénicas/síntesis química , Proteínas Proto-Oncogénicas/uso terapéutico , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , Especificidad por SustratoRESUMEN
Antibodies have been the paradigm of binding proteins with desired specificities for more than one century and during the past decade their recombinant or humanized versions have entered clinical application with remarkable success. Meanwhile, a new generation of receptor proteins was born, which is derived from small and robust non-immunoglobulin "scaffolds" that can be equipped with prescribed binding functions using the methods of combinatorial protein design. Their ongoing development does not only provide valuable insights into the principles of molecular recognition and protein structure-function relationships but also yields novel reagents for medical use. This technology goes hand in hand with our expanding knowledge about the molecular pathologies of cancer, immunological, and infectious diseases. Currently, questions regarding the choice of suitable medically relevant targets with regard to a certain protein scaffold, the methodology for engineering high affinity, arming with effector functions, routes of administration, plasma half-life, and immunogenicity are in the focus. While many protein scaffolds have been proposed during the past years, the technology shows a trend toward consolidation with a smaller set of systems that are being applied against multiple targets and in different settings, with emphasis on the development of drug candidates for therapy or in vivo diagnostics: Adnectins, Affibodies, Anticalins, DARPins, and engineered Kunitz-type inhibitors, among others. Only few data from early clinical studies are available yet, but many more are likely to come in the near future, thus providing a growing basis for assessing the therapeutic potential--but possibly also some limitations--of this exciting new class of protein drugs.