RESUMEN
ABP 798 is a biosimilar candidate to rituximab reference product (RP). This comprehensive analytical similarity assessment was designed to assess the structural and functional similarity of ABP 798, rituximab (US), and rituximab (EU) using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. The similarity assessment was performed to evaluate product quality attributes associated with Fab, Fab/Fc, and Fc domains, including those known to affect the mechanisms of action. ABP 798 has the same amino acid sequence and exhibits similar secondary and tertiary structures, similar glycan and post-translational modification profiles, and biological activities as rituximab RP. There are minor differences in biochemical attributes, which are not considered clinically meaningful. The results of the analytical and functional similarity assessment demonstrate that ABP 798 is highly analytically similar to rituximab RP. These results support the totality of evidence and the scientific justification for extrapolation of ABP 798 to all therapeutic indications approved for rituximab.
Asunto(s)
Biosimilares Farmacéuticos/metabolismo , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Rituximab/metabolismo , Secuencia de Aminoácidos , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/metabolismo , Antineoplásicos Inmunológicos/farmacología , Apoptosis/efectos de los fármacos , Biosimilares Farmacéuticos/química , Biosimilares Farmacéuticos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Células Jurkat , Unión Proteica , Conformación Proteica , Estándares de Referencia , Rituximab/química , Rituximab/farmacologíaRESUMEN
PURPOSE: The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation. METHODS: This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters. RESULTS: The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action. CONCLUSIONS: These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.
Asunto(s)
Antineoplásicos/química , Biosimilares Farmacéuticos/química , Trastuzumab/química , Animales , Unión Competitiva , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Cinética , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales , Unión Proteica , Receptor ErbB-2/química , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.
Asunto(s)
Anticuerpos Antineoplásicos , Citotoxicidad Celular Dependiente de Anticuerpos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G , Ingeniería de Proteínas , Receptores de IgG/inmunología , Animales , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Línea Celular Tumoral , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Ratones , Ratones SCID , Mutación , Neoplasias , Receptores de IgG/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Non-small-cell lung cancer (NSCLC) is categorized into various histologic subtypes that play an important role in prognosis and treatment outcome. We investigated the antitumor activity of motesanib, a selective antagonist of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit, alone and combined with chemotherapy in five human NSCLC xenograft models (A549, Calu-6, NCI-H358, NCI-H1299, and NCI-H1650) containing diverse genetic mutations. RESULTS: Motesanib as a single agent dose-dependently inhibited tumor xenograft growth compared with vehicle in all five of the models (P < 0.05). When combined with cisplatin, motesanib significantly inhibited the growth of Calu-6, NCI-H358, and NCI-H1650 tumor xenografts compared with either single agent alone (P < 0.05). Similarly, the combination of motesanib plus docetaxel significantly inhibited the growth of A549 and Calu-6 tumor xenografts compared with either single agent alone (P < 0.05). In NCI-H358 and NCI-H1650 xenografts, motesanib with and without cisplatin significantly decreased tumor blood vessel area (P < 0.05 vs vehicle) as assessed by anti-CD31 staining. Motesanib alone or in combination with chemotherapy had no effect on tumor cell proliferation in vitro. CONCLUSIONS: These data demonstrate that motesanib had antitumor activity against five different human NSCLC xenograft models containing diverse genetic mutations, and that it had enhanced activity when combined with cisplatin or docetaxel. These effects appeared to be mediated primarily by antiangiogenic mechanisms.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas , Indoles/farmacología , Neoplasias Pulmonares , Niacinamida/análogos & derivados , Taxoides/farmacología , Animales , Antineoplásicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/farmacología , Docetaxel , Femenino , Expresión Génica , Humanos , Indoles/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Mutación , Niacinamida/administración & dosificación , Niacinamida/farmacología , Oligonucleótidos , Taxoides/administración & dosificación , Carga Tumoral/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.
Asunto(s)
Anticuerpos Biespecíficos/química , Inmunoglobulina G/química , Animales , Dimerización , Femenino , Humanos , Cinética , Leucocitos Mononucleares/citología , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Trasplante de Neoplasias , Conformación Proteica , Estructura Terciaria de Proteína , Factor de Necrosis Tumoral alfa/químicaRESUMEN
Despite the strong influence of pollination ecology on the evolution of selfing, we have little information on how distinct groups of insect pollinators influence outcrossing rate. However, differences in behavior between pollinator groups could easily influence how each group affects outcrossing rate. We examined the influence of distinct insect pollinator groups on outcrossing rate in the rocky mountain columbine, Aquilegia coerulea. The impact of population size, plant density, size of floral display, and herkogamy (spatial separation between anthers and stigmas) on outcrossing rate was also considered as these variables were previously found to affect outcrossing rate in some plant species. We quantified correlations between all independent variables and used simple and two-factor regressions to determine direct and indirect impact of each independent variable on outcrossing rate. Outcrossing rate increased significantly with hawkmoth abundance but not with the abundance of any of the other groups of floral visitors, which included bumblebees, solitary bees, syrphid flies, and muscidae. Outcrossing rate was also significantly affected by floral display size and together, hawkmoth abundance and floral display size explained 87% of the variation in outcrossing rate. None of the other independent variables directly affected the outcrossing rate. This is the first report of a significant impact of pollinator type on outcrossing rate. Hawkmoths did not visit fewer flowers per plant relative to other pollinator groups but preferred visiting female-phase flowers first on a plant. Both the behavior of pollinators and floral display size affected outcrossing rate via their impact on the level of geitonogamous (among flower) selfing. Given that geitonogamous selfing is never advantageous, the variation in outcrossing rate and maintenance of mixed mating systems in populations of A. coerulea may not require an adaptive explanation.
Asunto(s)
Aquilegia/anatomía & histología , Aquilegia/fisiología , Flores/anatomía & histología , Flores/fisiología , Insectos/fisiología , Polen/fisiología , Animales , Aquilegia/genética , Arizona , Cruzamientos Genéticos , Variación Genética , Reproducción/genética , Reproducción/fisiología , UtahRESUMEN
To quantitatively evaluate the extravasation, accumulation and selectivity to tumor tissues of liposomal vincristine (LV), dorsal skin-fold window chambers on athymic mice with or without LX-1, a human small cell lung cancer, xenograft implants and fluorescent intravital microscopy imaging were used. In vitro studies show that minimal loss of fluorescence marker DiI from liposomes occurs after 4 days of inoculation in murine plasma, and the release profiles of DiI-LV and LV were essentially the same with approximately 40% of the encapsulated vincristine sulfate (VCR) released after 26 h. Significantly faster extravasation of DiI-LV from tumor vessels was shown compared to non-tumor tissue after single dose i.v. administration. The relative interstitial amounts at 60 min (RIA(60)) for tumor and non-tumor tissues were 0.837+/-0.314 and 0.012+/-0.091, respectively (P=0.01). DiI-LV accumulation was significantly higher in tumor than in normal tissue, which continued beyond 48 h. Both DiI-LV and LV showed significant antitumor effects in window chambers and in flank tumors, compared with controls and VLS alone. The preferential extravasation of DiI-LV from tumor vasculature as well as its differential retention in tumor tissue provides the basis for the enhancement in antitumor activity of LV over VCR.