RESUMEN
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
[This corrects the article DOI: 10.1371/journal.pone.0234268.].
RESUMEN
The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma de Células Claras/tratamiento farmacológico , Antígeno CTLA-4/metabolismo , Humanos , Inmunoterapia , Neoplasias Renales/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Linfocitos T/inmunologíaRESUMEN
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
There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of â¼200 attomoles and a limit of quantitation of â¼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.
Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/diagnóstico , Espectrometría de Masas/métodos , Proteínas de la Nucleocápside/análisis , Neumonía Viral/diagnóstico , Glicoproteína de la Espiga del Coronavirus/análisis , Secuencia de Aminoácidos , Betacoronavirus/aislamiento & purificación , COVID-19 , Cromatografía Líquida de Alta Presión/métodos , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus , Humanos , Límite de Detección , Nanotecnología , Proteínas de la Nucleocápside/química , Pandemias , Fosfoproteínas , Neumonía Viral/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Monoclonal antibodies from human sources are being increasingly recognized as valuable options in many therapeutic areas. These antibodies can show exquisite specificity and high potency while maintaining a desirable safety profile, having been matured and tolerized within human patients. However, the discovery of these antibodies presents important challenges, since the B cells encoding therapeutic antibodies can be rare in a typical blood draw and are short-lived ex vivo. Furthermore, the unique pairing of VH and VL domains in each B cell contributes to specificity and function; therefore, maintaining antibody chain pairing presents a throughput limitation. This work will review the various approaches aimed at addressing these challenges with an eye to next-generation methods for high-throughput discovery from the human B-cell repertoire.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Linfocitos B/inmunología , Descubrimiento de Drogas , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , HumanosRESUMEN
IgG4s are dynamic molecules that undergo a process called Fab-arm exchange. Disulfide bonds between heavy chains are transiently reduced, resulting in half antibodies that reform intact antibodies with other IgG4 half antibodies. In vivo, therapeutic IgG4s can recombine with endogenous IgG4s, resulting in a heterogeneous mixture of bispecific antibodies. A related issue that can occur for any therapeutic protein during manufacturing is interchain disulfide bond reduction. For IgG4s, this primarily results in high levels of half-mAb that persist through purification processes. The S228P mutation has been used to prevent half-mAb formation. However, we demonstrated that IgG4s with the S228P mutation are subject to half-mAb formation and Fab-arm exchange in reducing environments. We identified two novel mutations that stabilize the heavy-heavy chain interaction via incorporation of additional disulfide bonds in the hinge region. Individually, these mutations increase stability toward reduction and lessen Fab-arm exchange. Combination of all three mutations, Y219C, G220C, and S228P, has an additive benefit resulting in an IgG4 with Ë7-fold increase in stability toward reduction while preventing Fab-arm exchange. Importantly, the mutations do not affect antigen binding or Fc effector function. These mutations hold great promise for solving mAb reduction during manufacturing and preventing Fab-arm exchange in vivo.
Asunto(s)
Anticuerpos Monoclonales , Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G , Simulación de Dinámica Molecular , Sustitución de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Mutación MissenseRESUMEN
Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.
Asunto(s)
Anexina A1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Regulación hacia Arriba , Animales , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/irrigación sanguínea , RatonesRESUMEN
Efficacious use of therapeutic gene delivery via nanoparticles is hampered by the challenges associated with targeted delivery to tissues of interest. Systemic administration of lipid nanoparticle (LNP)-encapsulated mRNA leads to a protein expressed predominantly in the liver and spleen. Here, LNP encapsulating mRNA was covalently conjugated to an antibody, specifically binding plasmalemma vesicle-associated protein (PV1) as a means to target lung tissue. Systemic administration of PV1-targeted LNPs demonstrated significantly increased delivery of mRNA to the lungs and a 40-fold improvement in protein expression in the lungs, compared with control LNPs. We also investigated the effect of LNP size to determine optimal tissue distribution and transfection. Larger-size PV1-targeted LNPs not only have the elasticity to target the PV1 expressed in the caveolae but also enable robust mRNA expression in the lungs. Targeted delivery of mRNA to the lungs is a promising approach in the treatment of lung diseases.
Asunto(s)
Portadores de Fármacos/química , Lípidos/química , Pulmón/metabolismo , Nanopartículas/química , ARN Mensajero/farmacología , Animales , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Caveolas/inmunología , Femenino , Técnicas de Transferencia de Gen , Proteínas de la Membrana/inmunología , Ratones Endogámicos BALB CRESUMEN
Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Caveolas/metabolismo , Portadores de Fármacos/farmacocinética , Fragmentos Fab de Inmunoglobulinas/inmunología , Riñón/efectos de los fármacos , Proteínas de la Membrana/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Afinidad de Anticuerpos , Portadores de Fármacos/administración & dosificación , Femenino , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Riñón/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Distribución TisularRESUMEN
Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Inmunoglobulina G/metabolismo , Anticuerpos Biespecíficos/genética , Afinidad de Anticuerpos/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Reacciones Antígeno-Anticuerpo , Línea Celular Tumoral , Activación de Complemento , Complemento C1q/metabolismo , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Interferometría , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismoRESUMEN
The ability to genetically encode non-natural amino acids (nnAAs) into proteins offers an expanded tool set for protein engineering. nnAAs containing unique functional moieties have enabled the study of post-translational modifications, protein interactions, and protein folding. In addition, nnAAs have been developed that enable a variety of biorthogonal conjugation chemistries that allow precise and efficient protein conjugations. These are being studied to create the next generation of antibody-drug conjugates with improved efficacy, potency, and stability for the treatment of cancer. However, the efficiency of nnAA incorporation, and the productive yields of cell-based expression systems, have limited the utility and widespread use of this technology. We developed a process to isolate stable cell lines expressing a pyrrolysyl-tRNA synthetase/tRNApyl pair capable of efficient nnAA incorporation. Two different platform cell lines generated by these methods were used to produce IgG-expressing cell lines with normalized antibody titers of 3 g/L using continuous perfusion. We show that the antibodies produced by these platform cells contain the nnAA functionality that enables facile conjugations. Characterization of these highly active and robust platform hosts identified key parameters that affect nnAA incorporation efficiency. These highly efficient host platforms may help overcome the expression challenges that have impeded the developability of this technology for manufacturing proteins with nnAAs and represents an important step in expanding its utility.
Asunto(s)
Aminoácidos/genética , Aminoacil-ARNt Sintetasas/genética , Antineoplásicos/química , Inmunoconjugados/genética , Inmunoglobulina G/genética , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Células CHO , Cricetulus , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoconjugados/química , Inmunoglobulina G/química , Lisina/análogos & derivados , Lisina/química , Procesamiento Proteico-PostraduccionalRESUMEN
Despite some clinical success with antibody-drug conjugates (ADCs) in patients with solid tumors and hematological malignancies, improvements in ADC design are still desirable due to the narrow therapeutic window of these compounds. Tumor-targeting antibody fragments have distinct advantages over monoclonal antibodies, including more rapid tumor accumulation and enhanced penetration, but are subject to rapid clearance. Half-life extension technologies such as PEGylation and albumin-binding domains (ABDs) have been widely used to improve the pharmacokinetics of many different types of biologics. PEGylation improves pharmacokinetics by increasing hydrodynamic size to reduce renal clearance, whereas ABDs extend half-life via FcRn-mediated recycling. In this study, we used an anti-oncofetal antigen 5T4 diabody conjugated with a highly potent cytotoxic pyrrolobenzodiazepine (PBD) warhead to assess and compare the effects of PEGylation and albumin binding on the in vivo efficacy of antibody fragment drug conjugates. Conjugation of 2× PEG20K to a diabody improved half-life from 40 min to 33 h, and an ABD-diabody fusion protein exhibited a half-life of 45 h in mice. In a xenograft model of breast cancer MDA-MB-436, the ABD-diabody-PBD showed greater tumor growth suppression and better tolerability than either PEG-diabody-PBD or diabody-PBD. These results suggest that the mechanism of half-life extension is an important consideration for designing cytotoxic antitumor agents.
Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Unión Competitiva , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Ensayo de Inmunoadsorción Enzimática , Femenino , Semivida , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Ratones , Ratones Desnudos , Polietilenglicoles/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASMCXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASMCXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASMCXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.
Asunto(s)
Biomimética/métodos , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Anticuerpos/genética , Quimiocina CXCL12/metabolismo , Células HEK293 , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Ligandos , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Receptores CXCR4/genética , Receptores Acoplados a Proteínas G/genética , Transducción de SeñalRESUMEN
Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.
Asunto(s)
Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Animales , Biomarcadores , Bleomicina/efectos adversos , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/etiología , Fibrosis Pulmonar Idiopática/patología , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , RatonesRESUMEN
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
Cell line development (CLD) for biotherapeutics is a time- and resource-intensive process requiring the isolation and screening of large numbers of clones to identify high producers. Novel methods aimed at enhancing cell line screening efficiency using markers predictive of productivity early in the CLD process are needed to reliably generate high-yielding cell lines. To enable efficient and selective isolation of antibody expressing Chinese hamster ovary cells by fluorescence-activated cell sorting, we developed a strategy for the expression of antibodies containing a switchable membrane-associated domain to anchor an antibody to the membrane of the expressing cell. The switchable nature of the membrane domain is governed by the function of an orthogonal aminoacyl transfer RNA synthetase/tRNApyl pair, which directs a nonnatural amino acid (nnAA) to an amber codon encoded between the antibody and the membrane anchor. The process is "switchable" in response to nnAA in the medium, enabling a rapid transition between the surface display and secretion. We demonstrate that the level of cell surface display correlates with productivity and provides a method for enriching phenotypically stable high-producer cells. The strategy provides a means for selecting high-producing cells with potential applications to multiple biotherapeutic protein formats.
Asunto(s)
Codón de Terminación , Vectores Genéticos/genética , Inmunoglobulina G/genética , Proteínas Recombinantes/genética , Animales , Técnicas de Cultivo Celular por Lotes/métodos , Células CHO , Cricetulus , Humanos , Transfección/métodosRESUMEN
The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. However, methods for mining primary antibody-expressing B cells are limited in their ability to rapidly isolate rare and antigen-specific binders. Here we show the encapsulation of two million primary B cells into picoliter-sized droplets, where their cognate V genes are fused in-frame to form a library of scFv cassettes. We used this approach to construct natively paired phage-display libraries from healthy donors and drove selection towards cross-reactive antibodies targeting influenza hemagglutinin. Within 4 weeks we progressed from B cell isolation to a panel of unique monoclonal antibodies, including seven that displayed broad reactivity to different clinically relevant influenza hemagglutinin subtypes. Most isolated antibody sequences were not detected by next-generation sequencing of the paired repertoire, illustrating how this method can isolate extremely rare leads not likely found by existing technologies.
RESUMEN
IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti-Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.
Asunto(s)
Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Membrana Mucosa/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/efectos de los fármacos , Animales , Transporte Biológico/inmunología , Células CHO , Cricetulus , Perros , Inmunoglobulina A/inmunología , Inmunoglobulina M/inmunología , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Membrana Mucosa/microbiología , Receptores de Inmunoglobulina Polimérica , Componente Secretorio , Transcitosis/inmunologíaRESUMEN
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.