RESUMEN
Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.
Asunto(s)
Anticuerpos/química , Afinidad de Anticuerpos , Arginasa/inmunología , Regiones Determinantes de Complementariedad/química , Anticuerpos/genética , Anticuerpos/inmunología , Sitios de Unión de Anticuerpos , Regiones Determinantes de Complementariedad/inmunología , HumanosRESUMEN
Antibodies are an important class of therapeutics that have significant clinical impact for the treatment of severe diseases. Computational tools to support antibody drug discovery have been developing at an increasing rate over the last decade and typically rely upon a predetermined co-crystal structure of the antibody bound to the antigen for structural predictions. Here, we show an example of successful in silico affinity maturation of a hybridoma derived antibody, AB1, using just a homology model of the antibody fragment variable region and a protein-protein docking model of the AB1 antibody bound to the antigen, murine CCL20 (muCCL20). In silico affinity maturation, together with alanine scanning, has allowed us to fine-tune the protein-protein docking model to subsequently enable the identification of two single-point mutations that increase the affinity of AB1 for muCCL20. To our knowledge, this is one of the first examples of the use of homology modelling and protein docking for affinity maturation and represents an approach that can be widely deployed.
Asunto(s)
Afinidad de Anticuerpos/fisiología , Biología Computacional/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Quimiocina CCL20 , Simulación por Computador , Diseño de Fármacos , Región Variable de Inmunoglobulina , Ratones , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.
Asunto(s)
Abatacept/farmacología , Diseño de Fármacos , Inmunosupresores/farmacología , Abatacept/química , Animales , Antígeno B7-1/inmunología , Antígeno B7-2 , Estabilidad de Medicamentos , Humanos , Inmunosupresores/química , Unión Proteica/inmunologíaRESUMEN
Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.
Asunto(s)
Amidas/metabolismo , Anticuerpos Monoclonales/metabolismo , Asparagina/metabolismo , Espectrometría de Masas/métodos , Amidas/química , Anticuerpos Monoclonales/química , Asparagina/química , Catálisis , Medición de Intercambio de Deuterio , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismoRESUMEN
Several peptide dual agonists of the human glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R) are in development for the treatment of type 2 diabetes, obesity and their associated complications. Candidates must have high potency at both receptors, but it is unclear whether the limited experimental data available can be used to train models that accurately predict the activity at both receptors of new peptide variants. Here we use peptide sequence data labelled with in vitro potency at human GCGR and GLP-1R to train several models, including a deep multi-task neural-network model using multiple loss optimization. Model-guided sequence optimization was used to design three groups of peptide variants, with distinct ranges of predicted dual activity. We found that three of the model-designed sequences are potent dual agonists with superior biological activity. With our designs we were able to achieve up to sevenfold potency improvement at both receptors simultaneously compared to the best dual-agonist in the training set.
RESUMEN
Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33red) and oxidized IL-33 (IL-33ox) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products/epidermal growth factor receptor (RAGE/EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 107 M-1 s-1, to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33red and a fast association rate (8.5 × 107 M-1 s-1), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33red and IL-33ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.
Asunto(s)
Inflamación , Proteína 1 Similar al Receptor de Interleucina-1 , Ratones , Humanos , Animales , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Inflamación/metabolismo , Interleucina-33/metabolismo , Citocinas/metabolismo , Receptores ErbB/metabolismo , Transducción de SeñalRESUMEN
Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.
Asunto(s)
Seudópodos/metabolismo , Nexinas de Clasificación/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Femenino , Células HeLa , Humanos , Masculino , Nexinas de Clasificación/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.
Asunto(s)
Factor H de Complemento/metabolismo , Trypanosoma/fisiología , Moscas Tse-Tse/parasitología , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Bovinos , Membrana Celular/metabolismo , Complemento C3b/metabolismo , Factor H de Complemento/química , Cricetinae , Cricetulus , Ratones Endogámicos BALB C , Parasitemia/sangre , Unión Proteica , Dominios Proteicos , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/metabolismo , Regulación hacia ArribaRESUMEN
Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
Asunto(s)
Afinidad de Anticuerpos , Arginasa/química , Anticuerpos de Cadena Única/química , Regulación Alostérica , Cristalografía por Rayos X , HumanosRESUMEN
Doxorubicin is a chemotherapeutic agent that is commonly used to treat a broad range of cancers. However, significant cardiotoxicity, associated with prolonged exposure to doxorubicin, limits its continued therapeutic use. One strategy to prevent the uptake of doxorubicin into cardiac cells is the encapsulation of the drug to prevent non-specific uptake and also to improve the drugs' pharmacokinetic properties. Although encapsulated forms of doxorubicin limit the cardiotoxicity observed, they are not without their own liabilities as an increased amount of drug is deposited in the skin where liposomal doxorubicin can cause palmar-plantar erythrodysesthesia. Exosomes are small endogenous extracellular vesicles, that transfer bioactive material from one cell to another, and are considered attractive drug delivery vehicles due to their natural origin. In this study, we generated doxorubicin-loaded exosomes and demonstrate their rapid cellular uptake and re-distribution of doxorubicin from endosomes to the cytoplasm and nucleus resulting in enhanced potency in a number of cultured and primary cell lines when compared to free doxorubicin and liposomal formulations of doxorubicin. In contrast to other delivery methods for doxorubicin, exosomes do not accumulate in the heart, thereby providing potential for limiting the cardiac side effects and improved therapeutic index.
Asunto(s)
Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Exosomas/metabolismo , Apoptosis/efectos de los fármacos , Transporte Biológico , Línea Celular , Exosomas/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , CinéticaRESUMEN
Both fibroblast growth factors (FGFs), by binding to FGF receptors (FGFRs), and activation of the gravitostat, by artificial loading, decrease the body weight (BW). Previous studies demonstrate that both the FGF system and loading have the capacity to regulate BW independently of leptin. The aim of the current study was to determine the possible interactions between the effect of increased loading and the FGF system for the regulation of BW. We observed that the BW-reducing effect of increased loading was abolished in mice treated with a monoclonal antibody directed against FGFR1c, suggesting interactions between the two systems. As serum levels of endocrine FGF21 and hepatic FGF21 mRNA were increased in the loaded mice compared with the control mice, we first evaluated the loading response in FGF21 over expressing mice with constant high FGF21 levels. Leptin treatment, but not increased loading, decreased the BW in the FGF21-overexpressing mice, demonstrating that specifically the loading effect is attenuated in the presence of high activity in the FGF system. However, as FGF21 knockout mice displayed a normal loading response on BW, FGF21 is neither mediating nor essential for the loading response. In conclusion, the BW-reducing effect of increased loading but not of leptin treatment is blocked by high activity in the FGF system. We propose that both the gravitostat and the FGF system regulate BW independently of leptin and that pharmacologically enhanced activity in the FGF system reduces the sensitivity of the gravitostat.
Asunto(s)
Peso Corporal/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Hígado/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica/efectos de los fármacos , Leptina/farmacología , Hígado/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Obesidad/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/inmunología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
To maintain prolonged infection of mammals, African trypanosomes have evolved remarkable surface coats and a system of antigenic variation1. Within these coats are receptors for macromolecular nutrients such as transferrin2,3. These must be accessible to their ligands but must not confer susceptibility to immunoglobulin-mediated attack. Trypanosomes have a wide host range and their receptors must also bind ligands from diverse species. To understand how these requirements are achieved, in the context of transferrin uptake, we determined the structure of a Trypanosoma brucei transferrin receptor in complex with human transferrin, showing how this heterodimeric receptor presents a large asymmetric ligand-binding platform. The trypanosome genome contains a family of around 14 transferrin receptors4, which has been proposed to allow binding to transferrin from different mammalian hosts5,6. However, we find that a single receptor can bind transferrin from a broad range of mammals, indicating that receptor variation is unlikely to be necessary for promiscuity of host infection. In contrast, polymorphic sites and N-linked glycans are preferentially found in exposed positions on the receptor surface, not contacting transferrin, suggesting that transferrin receptor diversification is driven by a need for antigenic variation in the receptor to prolong survival in a host.
Asunto(s)
Interacciones Huésped-Parásitos/inmunología , Evasión Inmune , Receptores de Transferrina/química , Receptores de Transferrina/inmunología , Transferrina/metabolismo , Trypanosoma brucei brucei/inmunología , Variación Antigénica , Variación Genética , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Tripanosomiasis Africana/inmunologíaRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Inhibidor 1 de Activador Plasminogénico/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Especificidad de Anticuerpos , Humanos , Ratones , Modelos Moleculares , Inhibidor 1 de Activador Plasminogénico/química , Conformación Proteica , RatasRESUMEN
P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Neuralgia/metabolismo , Neuralgia/prevención & control , Receptores Purinérgicos P2X4/metabolismo , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Inyecciones Espinales , Ratones , Ratones Endogámicos C57BL , Unión Proteica/fisiología , Antagonistas del Receptor Purinérgico P2X/administración & dosificación , Antagonistas del Receptor Purinérgico P2X/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antiprotozoarios/administración & dosificación , Benzodiazepinas/administración & dosificación , Pirroles/administración & dosificación , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/química , Antiprotozoarios/química , Benzodiazepinas/química , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Pirroles/química , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/parasitologíaRESUMEN
The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Biología Computacional/métodos , Epítopos/química , Algoritmos , Secuencia de Aminoácidos , Anticuerpos/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Modelos MolecularesRESUMEN
C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a-C5aR1 receptor are well defined, whereas C5a-C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement-mediated bacterial cell killing. Unlike other anti-C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a-C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia-reperfusion injury.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Complemento C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptores de Quimiocina/antagonistas & inhibidores , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Complemento C5a/química , Complemento C5a/inmunología , Complemento C5a/metabolismo , Mapeo Epitopo/métodos , Epítopos , Células HEK293 , Humanos , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Quimiocina/química , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Relación Estructura-ActividadRESUMEN
The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.
Asunto(s)
Aminoácidos/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/metabolismo , Fosfatasa Alcalina/metabolismo , Transporte Biológico , Medios de Cultivo Condicionados/química , Vectores Genéticos , Células HEK293 , Humanos , Interferón-alfa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.