RESUMEN
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
Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Toxinas Bacterianas/química , Células CACO-2 , Clostridioides difficile/enzimología , Cristalografía por Rayos X , Citosol/metabolismo , Enterotoxinas/química , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Rubidio/química , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Pathways of human epidermal growth factor (EGF) receptors are activated upon ligand-dependent or -independent homo- or heterodimerization and their subsequent transphosphorylation. Overexpression of these receptors positively correlates with transphosphorylation rates and increased tumor growth rates. MEDI4276, an anti-human epidermal growth factor receptor 2 (HER2) biparatopic antibody-drug conjugate, has two paratopes within each antibody arm. One, 39S, is aiming at the HER2 site involved in receptor dimerization and the second, single chain fragment (scFv), mimicking trastuzumab. Here we present the cocrystal structure of the 39S Fab-HER2 complex and, along with biophysical and functional assays, determine the corresponding epitope of MEDI4276 and its underlying mechanism of action. Our results reveal that MEDI4276's uniqueness is based first on the ability of its 39S paratope to block HER2 homo- or heterodimerization and second on its ability to cluster the receptors on the surface of receptor-overexpressing cells.
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Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Multimerización de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Trastuzumab/farmacología , Secuencia de Aminoácidos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cristalografía por Rayos X , Femenino , Humanos , Modelos Moleculares , Fosforilación , Conformación Proteica , Homología de Secuencia , Células Tumorales CultivadasRESUMEN
Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.
Asunto(s)
Antibacterianos/metabolismo , Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimología , Enterocitos/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferasas/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Antibacterianos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Sitios de Unión de Anticuerpos , Células CACO-2 , Secuencia Conservada , Cristalografía por Rayos X , Enterocitos/efectos de los fármacos , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidad , Mapeo Epitopo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/toxicidad , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Secuencias Repetitivas de AminoácidoRESUMEN
Monoclonal antibodies (mAbs) are complex molecular structures. They are often prone to development challenges particularly at high concentrations due to undesired solution properties such as reversible self-association, high viscosity, and liquid-liquid phase separation. In addition to formulation optimization, applying protein engineering can provide an alternative mitigation strategy. Protein engineering during the discovery phase can provide great benefits to optimize molecular properties, resulting in improved developability profiles. Here, we present a case study utilizing complementary analytical and predictive in silico methods. We have systematically identified and reengineered problematic residues responsible for the self-association of a model mAb, driven by a complex combination of hydrophobic and electrostatic interactions. Noteworthy findings include a more dominant contribution of hydrophobic interactions to self-association and potential feasibility of mutations in the CDR regions to mitigate self-association. The engineered mutation panel enabled us to assess potential correlations among commonly utilized developability screening assays, including affinity capture self-interaction nanospectroscopy (AC-SINS), dynamic light scattering (DLS), and apparent solubility by PEG-precipitation. In addition, we evaluated the correlations between experimental measurements and computational predictions. CamSol, an in silico computational tool that accounts for complex molecular interactions and neighboring hotspots, was found to be an effective screening tool. Our work led to reengineered mAb variants, better suited for high-concentration liquid formulation development. The engineered mAbs exhibited enhanced in vitro and simulated in vivo solubility and reduced self-association propensity, while maintaining binding affinity and thermal stability.
Asunto(s)
Anticuerpos Monoclonales/genética , Desarrollo de Medicamentos , Mutagénesis Sitio-Dirigida , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Disponibilidad Biológica , Química Farmacéutica , Clonación Molecular , Simulación por Computador , Estabilidad de Medicamentos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Modelos Químicos , Mutación , Solubilidad , Electricidad Estática , ViscosidadRESUMEN
The mechanism of the new Radical Ring-opening Redox Polymerization (R3P) of 3,6-dioxa-1,8-octanedithiol (DODT) by triethylamine (TEA) and dilute H2O2 was investigated. Scouting studies showed that the formation of high molecular weight polymers required a 1:2 molar ratio of DODT to TEA and of DODT to H2O2. Further investigation into the chemical composition of the organic and aqueous phases by 1H-NMR spectroscopy and mass spectrometry demonstrated that DODT is ionized by two TEA molecules (one for each thiol group) and thus transferred into the aqueous phase. The organic phase was found to have cyclic disulfide dimers, trimers and tetramers. Dissolving DODT and TEA in water before the addition of H2O2 yielded a polymer with Mn = 55,000 g/mol, in comparison with Mn = 92,000 g/mol when aqueous H2O2 was added to a DODT/TEA mixture. After polymer removal, MALDI-ToF MS analysis of the residual reaction mixtures showed only cyclic oligomers remaining. Below the LCST for TEA in water, 18.7 °C, the system yielded a stable emulsion, and only cyclic oligomers were found. Below DODT/TEA and H2O2 1:2 molar ratio mostly linear oligomers were formed, with <20% cyclic oligomers. The findings support the proposed mechanism of R3P.
Asunto(s)
Polimerizacion , Polímeros/química , Éteres de Etila/química , Espectrometría de Masas , Oxidación-Reducción , Espectroscopía de Protones por Resonancia Magnética , Compuestos de Sulfhidrilo/químicaRESUMEN
COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.
Asunto(s)
COVID-19 , Profilaxis Pre-Exposición , Animales , Macaca mulatta , COVID-19/prevención & control , SARS-CoV-2/genética , Anticuerpos Antivirales , Anticuerpos Monoclonales , Macaca fascicularis , ADN , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.
Asunto(s)
Anticuerpos Neutralizantes , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , SARS-CoV-2/efectos de los fármacos , Humanos , COVID-19/virología , Anticuerpos Neutralizantes/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Cricetinae , Tratamiento Farmacológico de COVID-19 , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacocinética , Mesocricetus , Femenino , Masculino , Adulto , Anticuerpos Antivirales/inmunología , Mutación/genética , Anticuerpos Monoclonales , Enzima Convertidora de Angiotensina 2/metabolismo , Carga Viral/efectos de los fármacosRESUMEN
To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to FcγR and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Ratones , Humanos , SARS-CoV-2/genética , Pandemias , Anticuerpos NeutralizantesRESUMEN
AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/genética , Reproducibilidad de los Resultados , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Interferometría , Fragmentos de Inmunoglobulinas , Proteínas RecombinantesRESUMEN
Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2) in prophylactic and therapeutic settings. Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human FcγR transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes , Anticuerpos Antivirales/uso terapéutico , Combinación de Medicamentos , Humanos , Glicoproteínas de Membrana , Ratones , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio ViralRESUMEN
Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/terapia , Combinación de Medicamentos , Semivida , Humanos , Inmunización Pasiva , Primates , Glicoproteína de la Espiga del Coronavirus , Sueroterapia para COVID-19RESUMEN
Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.
Asunto(s)
Productos Biológicos , COVID-19 , Ácidos Nucleicos , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/prevención & control , ADN , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
In a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Receptores Fc/metabolismo , Animales , Semivida , Antígenos de Histocompatibilidad Clase I/farmacología , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Ratones , Ratones Transgénicos , Ingeniería de ProteínasRESUMEN
Neuregulin protein 1 (NRG1) is a large (> 60-amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.
Asunto(s)
Anticuerpos Monoclonales/química , Neurregulina-1/química , Receptor ErbB-4/agonistas , Anticuerpos Monoclonales/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Ligandos , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Neurregulina-1/metabolismo , Unión Proteica , Receptor ErbB-4/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: We have used mass-spectrometry (MS) based proteomics platform to identify cell surface proteins over-expressed on a cisplatin resistant derivative of an ovarian cancer cell line A2780. METHODS: Membrane associated glycoproteins from A2780 and its cisplatin resistant derivative cell line, A2780cis, were processed for liquid chromatography (LC)-MS based analysis. The expression of proteins found at elevated levels in A2780cis cell line was confirmed using flow cytometry and Taqman analysis. The expression of these proteins was further evaluated in unrelated ovarian cancer cell lines using MS analysis and flow cytometry. Immunohistochemical (IHC) analysis was performed using ovarian tumor tissues to evaluate the protein density on the cell surface. Monoclonal antibodies were used in an alamar blue proliferation assay to examine the cytotoxic effects on cell proliferation in resistant cell lines. RESULTS: Six proteins were identified by LC-MS as being over-expressed on cell surface of A2780cis cell line. Mass spectrometry and flow cytometry confirmed the over-expression of CD49f, CD70 and Her-2/neu in other cisplatin resistant ovarian cancer cell lines. Immunohistochemical analysis revealed that only CD70 was expressed at moderate levels in ovarian tumors. When cisplatin resistant ovarian cell lines A2780cis and SKOV-3 were treated with antibody against CD70, there was a significant decrease in cell proliferation. CONCLUSION: Using a MS based proteomics approach we have shown that expression of CD70 is associated with cisplatin resistance in ovarian cancer cell lines. Follow-up examination of these tumor cell line findings in clinical tumor specimens with available pathology staging and cisplatin treatment history is warranted.
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Biomarcadores de Tumor/biosíntesis , Ligando CD27/biosíntesis , Cisplatino/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor/inmunología , Ligando CD27/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Resistencia a Antineoplásicos , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Espectrometría de Masas , Datos de Secuencia Molecular , Neoplasias Ováricas/inmunología , ProteómicaRESUMEN
Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R-mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system's potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform.
Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Neutralizantes/metabolismo , Interleucinas/inmunología , Macaca fascicularis/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Humanos , Hibridomas/inmunología , Inmunización , RatonesRESUMEN
Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.
Asunto(s)
Antineoplásicos , Benzodiazepinas/química , Inmunoconjugados , Pirroles/química , Receptor ErbB-2/antagonistas & inhibidores , Neoplasias Gástricas/tratamiento farmacológico , Trastuzumab , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Células MCF-7 , Ratones Desnudos , Ratas , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Trastuzumab/química , Trastuzumab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background Heart failure is one of the leading causes of death in Western countries, and there is a need for new therapeutic approaches. Relaxin-2 is a peptide hormone that mediates pleiotropic cardiovascular effects, including antifibrotic, angiogenic, vasodilatory, antiapoptotic, and anti-inflammatory effects in vitro and in vivo. Methods and Results We developed RELAX10, a fusion protein composed of human relaxin-2 hormone and the Fc of a human antibody, to test the hypothesis that extended exposure of the relaxin-2 peptide could reduce cardiac hypertrophy and fibrosis. RELAX10 demonstrated the same specificity and similar in vitro activity as the relaxin-2 peptide. The terminal half-life of RELAX10 was 7 days in mouse and 3.75 days in rat after subcutaneous administration. We evaluated whether treatment with RELAX10 could prevent and reverse isoproterenol-induced cardiac hypertrophy and fibrosis in mice. Isoproterenol administration in mice resulted in increased cardiac hypertrophy and fibrosis compared with vehicle. Coadministration with RELAX10 significantly attenuated the cardiac hypertrophy and fibrosis compared with untreated animals. Isoproterenol administration significantly increased transforming growth factor ß1 (TGF-ß1)-induced fibrotic signaling, which was attenuated by RELAX10. We found that RELAX10 also significantly increased protein kinase B/endothelial NO synthase signaling and protein S-nitrosylation. In the reversal study, RELAX10-treated animals showed significantly reduced cardiac hypertrophy and collagen levels. Conclusions These findings support a potential role for RELAX10 in the treatment of heart failure.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Miocardio/patología , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/prevención & control , Fibrosis/inducido químicamente , Fibrosis/tratamiento farmacológico , Fibrosis/prevención & control , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The accumulation, dissemination and clearance of monoclonal antibody-based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)-modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG-modified diabodies improved the PK of the parental diabody from a half-life of 40â¯min to over 40â¯h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody-PEG5K showing peak accumulation early on, but with the larger diabody-PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody-PEG20K-B with a hydrodynamic radius (Rh) of 6â¯nm had superior tumor uptake than the larger diabody-PEG40K-B with Rh of 12â¯nm, indicating that beyond 6â¯nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ~6â¯nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.