RESUMEN
Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.
Asunto(s)
Anticuerpos Monoclonales/química , Cisteína/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Lisina/química , Maitansina/inmunología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Animales , Supervivencia Celular/efectos de los fármacos , Femenino , Células HeLa , Humanos , Inmunoconjugados/administración & dosificación , Inyecciones Intravenosas , Ratones , Ratones SCID , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.
Asunto(s)
Alquilantes/química , Diseño de Fármacos , Inmunoconjugados/química , Animales , Anticuerpos Monoclonales/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Ratones , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two-hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-immunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, our results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Movimiento Celular/fisiología , Extensiones de la Superficie Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Sistema Nervioso/embriología , Análisis de Varianza , Animales , Proteínas de Caenorhabditis elegans/genética , Inmunoprecipitación , Proteínas de Microfilamentos/metabolismo , Mutación/genética , Interferencia de ARN , Técnicas del Sistema de Dos HíbridosRESUMEN
Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.
Asunto(s)
Antineoplásicos/uso terapéutico , Cadherinas/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Macaca fascicularis , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes â¼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.