RESUMEN
The receptor tyrosine kinase HER2 acts as oncogenic driver in numerous cancers. Usually, the gene is amplified, resulting in receptor overexpression, massively increased signaling and unchecked proliferation. However, tumors become frequently addicted to oncogenes and hence are druggable by targeted interventions. Here, we design an anti-HER2 biparatopic and tetravalent IgG fusion with a multimodal mechanism of action. The molecule first induces HER2 clustering into inactive complexes, evidenced by reduced mobility of surface HER2. However, in contrast to our earlier binders based on DARPins, clusters of HER2 are thereafter robustly internalized and quantitatively degraded. This multimodal mechanism of action is found only in few of the tetravalent constructs investigated, which must target specific epitopes on HER2 in a defined geometric arrangement. The inhibitory effect of our antibody as single agent surpasses the combination of trastuzumab and pertuzumab as well as its parental mAbs in vitro and it is effective in a xenograft model.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias de la Mama/terapia , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Femenino , Células HeLa , Humanos , Inmunoglobulina G/inmunología , Inmunoterapia/métodos , Células MCF-7 , Ratones , Ratones SCID , Trastuzumab/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.
RESUMEN
Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.
Asunto(s)
Proteínas del Dominio Armadillo/química , Proteínas Portadoras/química , Repetición de Anquirina , Proteínas del Dominio Armadillo/genética , Proteínas Portadoras/genética , Cristalografía por Rayos X , Humanos , Ingeniería de ProteínasRESUMEN
A number of agents designed for immunotherapy of Acute Myeloid Leukemia (AML) are in preclinical and early clinical development. Most of them target a single antigen on the surface of AML cells. Here we describe the development and key biological properties of a tri-specific agent, the dual-targeting triplebody SPM-2, with binding sites for target antigens CD33 and CD123, and for CD16 to engage NK cells as cytolytic effectors. Primary blasts of nearly all AML patients carry at least one of these target antigens and the pair is particularly promising for the elimination of blasts and leukemia stem cells (LSCs) from a majority of AML patients by dual-targeting agents. The cytolytic activity of NK cells mediated by SPM-2 was analyzed in vitro for primary leukemic cells from 29 patients with a broad range of AML-subtypes. Blasts from all 29 patients, including patients with genomic alterations associated with an unfavorable genetic subtype, were lysed at nanomolar concentrations of SPM-2. Maximum susceptibility was observed for cells with a combined density of CD33 and CD123 above 10,000 copies/cell. Cell populations enriched for AML-LSCs (CD34pos and CD34pos CD38neg cells) from 2 AML patients carried an increased combined antigen density and were lysed at correspondingly lower concentrations of SPM-2 than unsorted blasts. These initial findings raise the expectation that SPM-2 may also be capable of eliminating AML-LSCs and thus of prolonging survival. In the future, patients with a broad range of AML subtypes may benefit from treatment with SPM-2.
RESUMEN
Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.
Asunto(s)
Adenovirus Humanos/genética , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Hígado/fisiología , Adenovirus Humanos/patogenicidad , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Humanos , Hígado/virología , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Bazo/virología , Virión/química , Virión/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.
Asunto(s)
Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/química , Ingeniería de Proteínas/métodos , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Repetición de Anquirina , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.
RESUMEN
The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.
Asunto(s)
Repetición de Anquirina/genética , Ingeniería de Proteínas/métodos , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/química , Línea Celular , Espacio Extracelular/química , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Dominios Proteicos , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells.SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC50-values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors.These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies.
Asunto(s)
Antígenos CD19/inmunología , Antineoplásicos Inmunológicos/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Linfocitos Intraepiteliales/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfoma de Células B/tratamiento farmacológico , Antineoplásicos Inmunológicos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Linfocitos Intraepiteliales/inmunología , Células Asesinas Naturales/inmunología , Cinética , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Rituximab/farmacología , Células Tumorales CultivadasRESUMEN
Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.
Asunto(s)
Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas del Dominio Armadillo/química , Cristalografía por Rayos X , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS-p110α interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerization-incompetent state, which entail pan-ErbB inhibition and a permanent OFF state in the oncogenic signalling, thereby triggering extensive apoptosis in ErbB2-addicted tumours. Thus, these novel insights into mechanisms underlying network robustness provide a guide for overcoming adaptation response to ErbB2/ErbB3-targeted therapy.
Asunto(s)
Neoplasias de la Mama/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Proteínas ras/metabolismo , Repetición de Anquirina , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Mapas de Interacción de Proteínas , Interferencia de ARN , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/antagonistas & inhibidores , Transducción de Señal , Trastuzumab/farmacología , Proteínas ras/antagonistas & inhibidoresRESUMEN
DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 ß-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.
Asunto(s)
Repetición de Anquirina , Cristalografía por Rayos X/métodos , Proteínas/química , Secuencia de Aminoácidos , Cristalización , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Unión a Maltosa/química , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , beta-Lactamasas/químicaRESUMEN
The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20µs for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Biología Computacional/métodos , Simulación de Dinámica Molecular , Neurotensina/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas del Dominio Armadillo/química , Humanos , Neurotensina/química , Conformación Proteica , Secuencias Repetitivas de AminoácidoRESUMEN
An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into CD30-positive cells. Fused to the C-terminus of the type-II transmembrane protein hemagglutinin (H) of measles virus and expressed in LV packaging cells, gene transfer mediated by the released LV particles was inefficient. A series of point mutations in the scFv framework regions addressing its biophysical properties, which substantially improved production and increased the melting temperature without impairing its kinetic binding behavior to CD30, also improved the performance of LV particles. Gene transfer into CD30-positive cells increased â¼100-fold due to improved transport of the H-scFv protein to the plasma membrane. Concomitantly, LV particle aggregation and syncytia formation in packaging cells were substantially reduced. The data suggest that syncytia formation can be triggered by trans-cellular dimerization of H-scFv proteins displayed on adjacent cells. Taken together, we show that the biophysical properties of the targeting ligand have a decisive role for the gene transfer efficiency of receptor-targeted LVs.
Asunto(s)
Técnicas de Transferencia de Gen , Fragmentos de Inmunoglobulinas/química , Inmunoterapia , Antígeno Ki-1/química , Anticuerpos de Cadena Única/química , Animales , Línea Celular , Vectores Genéticos , Humanos , Hibridomas , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Antígeno Ki-1/genética , Antígeno Ki-1/inmunología , Lentivirus/genética , Ligandos , Ratones , Pliegue de Proteína , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Repetición de Anquirina , Toxinas Bacterianas/farmacología , Ligasas de Carbono-Nitrógeno/farmacología , Proteínas Portadoras/farmacología , Proteínas de Escherichia coli/farmacología , Exotoxinas/farmacología , Proteínas Represoras/farmacología , Factores de Virulencia/farmacología , Transporte Biológico , Línea Celular , Citoplasma/metabolismo , Humanos , Células MCF-7 , Ingeniería de Proteínas , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins connected by a short linker, one targeting extracellular subdomain I and the other subdomain IV, causes much stronger cytotoxic effects on the HER2-addicted breast cancer cell line BT474, surpassing the therapeutic antibody trastuzumab. We determined crystal structures of these DARPins in complex with the respective subdomains. Detailed models of the full-length receptor, constrained by its rigid domain structures and its membrane anchoring, explain how the bispecific DARPins connect two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to a complete loss of signaling.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Animales , Repetición de Anquirina , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Hormonales/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Mapeo Epitopo , Humanos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/química , Proteínas Recombinantes de Fusión/química , Células Sf9 , Transducción de Señal , Spodoptera , TrastuzumabRESUMEN
Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.
Asunto(s)
Adenovirus Humanos/genética , Ingeniería de Proteínas/métodos , Adenovirus Humanos/metabolismo , Repetición de Anquirina/genética , Línea Celular Tumoral , Cristalografía por Rayos X , Escherichia coli/genética , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy. We describe here a novel vector (CD8-LV) derived from lentivirus, which delivers genes exclusively and specifically to CD8(+) cells. CD8-LV mediated stable in vitro and in vivo reporter gene transfer as well as efficient transfer of genes encoding TCRs recognizing the melanoma antigen tyrosinase. Strikingly, T cells genetically modified with CD8-LV killed melanoma cells reproducibly more efficiently than CD8(+) cells transduced with a conventional lentiviral vector. Neither TCR expression levels, nor the rate of activation-induced death of transduced cells differed between both vector types. Instead, CD8-LV transduced cells showed increased granzyme B and perforin levels as well as an up-regulation of CD8 surface expression in a small subpopulation of cells. Thus, a possible mechanism for CD8-LV enhanced tumor cell killing may be based on activation of the effector functions of CD8(+) T cells by the vector particle displaying OKT8-derived CD8-scFv and an increase of the surface density of CD8, which functions as coreceptor for tumor-cell recognition. CD8-LV represents a powerful novel vector for TCR gene therapy and other applications in immunotherapy and basic research requiring CD8(+) cell-specific gene delivery.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Citotoxicidad Inmunológica/genética , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/fisiología , Células Cultivadas , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Células HEK293 , Humanos , Inmunoterapia Adoptiva/métodos , Células Jurkat , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Biológicos , Neoplasias/genética , Especificidad de Órganos/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each amino acid position. By combining a previously developed FACS-based selection system for functional expression [Sarkar C, et al. (2009) Proc Natl Acad Sci USA 105:14808-14813] with ultradeep 454 sequencing, we determined the amino acid preference in every position and identified several positions in the natural sequence that restrict functional expression. A strong accumulation of shifts, i.e., a residue preference different from wild type, is detected for helix 1, suggesting a key role in receptor biosynthesis. Furthermore, under selective pressure we observe a shift of the most conserved residues of the N-terminal helices. This unique data set allows us to compare the in vitro evolution of a GPCR to the natural evolution of the GPCR family and to observe how selective pressure shapes the sequence space covered by functional molecules. Under the applied selective pressure, several positions shift away from the wild-type sequence, and these improve the biophysical properties. We discuss possible structural reasons for conserved and shifted residues.
Asunto(s)
Mutación , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Citometría de Flujo , Modelos Moleculares , Datos de Secuencia Molecular , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genéticaRESUMEN
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.