RESUMEN
New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeting in November 2013 in Berlin: (i)Approaches to refine use of non-human primates in non-clinical safety testing of biologics and current experience on the use of minipigs as alternative non-rodent species.(ii)Tissue distribution studies as a useful tool to support pharmacokinetic/pharmacodynamic (PKPD) assessment of biologics, in that they provide valuable mechanistic insights at drug levels at the site of action.(iii)Mechanisms of nonspecific toxicity of antibody drug conjugates (ADC) and ways to increase the safety margins.(iv)Although biologics toxicity typically manifests as exaggerated pharmacology there are some reported case studies on unexpected toxicity.(v)Specifics of non-clinical development approaches of noncanonical monoclonal antibodies (mAbs), like bispecifics and nanobodies.
Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Productos Biológicos/efectos adversos , Evaluación Preclínica de Medicamentos/métodos , Seguridad , Pruebas de Toxicidad , Animales , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Productos Biológicos/inmunología , Productos Biológicos/farmacocinética , Humanos , Modelos Animales , Primates , Anticuerpos de Dominio Único/efectos adversos , Porcinos , Porcinos Enanos , Distribución TisularRESUMEN
Trastuzumab emtansine (T-DM1) is the first antibody-drug conjugate (ADC) approved for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. The therapeutic premise of ADCs is based on the hypothesis that targeted delivery of potent cytotoxic drugs to tumors will provide better tolerability and efficacy compared with non-targeted delivery, where poor tolerability can limit efficacious doses. Here, we present results from preclinical studies characterizing the toxicity profile of T-DM1, including limited assessment of unconjugated DM1. T-DM1 binds primate ErbB2 and human HER2 but not the rodent homolog c-neu. Therefore, antigen-dependent and non-antigen-dependent toxicity was evaluated in monkeys and rats, respectively, in both single- and repeat-dose studies; toxicity of DM1 was assessed in rats only. T-DM1 was well tolerated at doses up to 40 mg/kg (~4400 µg DM1/m(2)) and 30 mg/kg (~ 6000 µg DM1/m(2)) in rats and monkeys, respectively. In contrast, DM1 was only tolerated up to 0.2mg/kg (1600 µg DM1/m(2)). This suggests that at least two-fold higher doses of the cytotoxic agent are tolerated in T-DM1, supporting the premise of ADCs to improve the therapeutic index. In addition, T-DM1 and DM1 safety profiles were similar and consistent with the mechanism of action of DM1 (i.e., microtubule disruption). Findings included hepatic, bone marrow/hematologic (primarily platelet), lymphoid organ, and neuronal toxicities, and increased numbers of cells of epithelial and phagocytic origin in metaphase arrest. These adverse effects did not worsen with chronic dosing in monkeys and are consistent with those reported in T-DM1-treated patients to date.
Asunto(s)
Anticuerpos Monoclonales Humanizados/toxicidad , Antineoplásicos/toxicidad , Plaquetas/efectos de los fármacos , Citotoxinas/toxicidad , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/efectos adversos , Antineoplásicos/efectos adversos , Plaquetas/metabolismo , Plaquetas/patología , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Citotoxinas/efectos adversos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Macaca fascicularis , Masculino , Maitansina/efectos adversos , Maitansina/toxicidad , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , TrastuzumabRESUMEN
Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.
Asunto(s)
Anticuerpos Monoclonales/toxicidad , Antineoplásicos/toxicidad , Inmunoconjugados/toxicidad , Pruebas de Toxicidad , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Antineoplásicos/química , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Guías como Asunto , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoconjugados/farmacocinética , Proyectos de Investigación , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/normasRESUMEN
In order to determine a threshold for nongenotoxic carcinogens, the traditional risk assessment approach has been to identify a mode of action (MOA) with a nonlinear dose-response. The dose-response for one or more key event(s) linked to the MOA for carcinogenicity allows a point of departure (POD) to be selected from the most sensitive effect dose or no-effect dose. However, this can be challenging because multiple MOAs and key events may exist for carcinogenicity and oftentimes extensive research is required to elucidate the MOA. In the present study, a microarray analysis was conducted to determine if a POD could be identified following short-term oral rat exposure with two nongenotoxic rodent carcinogens, fenofibrate and methapyrilene, using a benchmark dose analysis of genes aggregated in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes, which likely encompass key event(s) for carcinogenicity. The gene expression response for fenofibrate given to rats for 2days was consistent with its MOA and known key events linked to PPARα activation. The temporal response from daily dosing with methapyrilene demonstrated biological complexity with waves of pathways/biological processes occurring over 1, 3, and 7days; nonetheless, the benchmark dose values were consistent over time. When comparing the dose-response of toxicogenomic data to tumorigenesis or precursor events, the toxicogenomics POD was slightly below any effect level. Our results suggest that toxicogenomic analysis using short-term studies can be used to identify a threshold for nongenotoxic carcinogens based on evaluation of potential key event(s) which then can be used within a risk assessment framework.
Asunto(s)
Carcinógenos/toxicidad , Fenofibrato/toxicidad , Metapirileno/análisis , Metapirileno/toxicidad , Neoplasias/inducido químicamente , Toxicogenética/métodos , Animales , Carcinógenos/administración & dosificación , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Relación Dosis-Respuesta a Droga , Femenino , Fenofibrato/administración & dosificación , Expresión Génica , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Metapirileno/administración & dosificación , Neoplasias/genética , Nivel sin Efectos Adversos Observados , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Medición de RiesgoRESUMEN
Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inmunotoxinas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Receptores Acoplados a Proteínas G/inmunología , Animales , Antineoplásicos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Estudios de Factibilidad , Femenino , Regulación Neoplásica de la Expresión Génica , Genes APC , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Concentración 50 Inhibidora , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
Asunto(s)
Anticuerpos/sangre , Anticuerpos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoglobulina G/química , Ingeniería de Proteínas , Aminobenzoatos/química , Aminobenzoatos/inmunología , Animales , Anticuerpos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/inmunología , Línea Celular Tumoral , Supervivencia Celular , Cisteína/química , Humanos , Inmunoconjugados/administración & dosificación , Inmunoglobulina G/inmunología , Macaca fascicularis , Maleimidas/química , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/inmunología , Maitansina/química , Maitansina/inmunología , Ratones , Ratones Desnudos , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/inmunología , Conformación Proteica , Ratas , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , TrastuzumabRESUMEN
PURPOSE: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate. EXPERIMENTAL DESIGN AND RESULTS: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti-cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (µg/m(2)) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 µg DM1/m(2)) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 µg DM1/m(2)). CONCLUSIONS: The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Maitansina/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Compuestos de Sulfhidrilo/farmacología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Humanos , Macaca fascicularis , Maitansina/química , Ratones , Ratas , Ratas Sprague-Dawley , Receptor ErbB-2/química , Compuestos de Sulfhidrilo/química , TrastuzumabRESUMEN
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
Asunto(s)
Anticuerpos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Citotoxinas/farmacología , Inmunotoxinas/farmacocinética , Neoplasias Ováricas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/genética , Especificidad de Anticuerpos , Sitios de Unión , Antígeno Ca-125/inmunología , Línea Celular Tumoral , Química Farmacéutica/métodos , Cisteína/genética , Femenino , Humanos , Macaca fascicularis , Proteínas de la Membrana/inmunología , Ratones , Mutagénesis Sitio-Dirigida , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Dimerization is essential for activity of human epidermal growth factor receptors (HER1/EGFR, HER2/ErbB2, HER3/ErbB3, and ErbB4) and mediates intracellular signaling events leading to cancer cell proliferation, survival, and resistance to therapy. HER2 is the preferred dimerization partner. Activation of HER signaling pathways may be blocked by inhibition of dimer formation using a monoclonal antibody (MAb) directed against the dimerization domain of HER2. The murine MAb 2C4 that specifically binds the HER2 dimerization domain was cloned as a chimeric antibody, humanized using a computer-generated model to guide framework substitutions, and variants were tested as Fabs. Pharmacokinetics and toxicology were evaluated in rodents and cynomolgus monkeys. Cloning the variable domains of MAb 2C4 into a vector containing human kappa and CH1 domains allowed construction of a mouse-human chimeric Fab. DNA sequencing of the chimeric clone permitted identification of CDR residues. The full-length IgG1 of variant F-10 was equivalent in binding to chimeric IgG1 and was designated pertuzumab (rhuMAb 2C4; Omnitarg). Pertuzumab pharmacokinetics was best described by a two-compartment model with a distribution phase of <1 day, terminal half-life of approximately 10 days, and volume of distribution of approximately 40 mL/kg that approximates serum volume. With the exception of diarrhea, pertuzumab was generally well tolerated in cynomolgus monkeys. Pertuzumab, a recombinant humanized IgG1 MAb, is the first of a new class of agents known as HER dimerization inhibitors. Inhibition of HER dimerization may be an effective anticancer strategy in tumors with either normal or elevated expression of HER2.