RESUMEN
The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.
Asunto(s)
Inmunoconjugados/uso terapéutico , Subunidad alfa del Receptor de Interleucina-3/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Xenoinjertos , Humanos , Inmunoconjugados/inmunología , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The promise of tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. ©2016 AACR.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Inmunoconjugados/farmacología , Animales , Antineoplásicos Alquilantes/química , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/química , ADN/metabolismo , Aductos de ADN , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inmunoconjugados/química , Ratones , Estructura Molecular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody-drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non-small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochemistry. IMGN853 displayed cytotoxic activity against FRα-negative cells situated near FRα-positive cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clinical development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptor 1 de Folato/antagonistas & inhibidores , Inmunoconjugados/farmacología , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Efecto Espectador/efectos de los fármacos , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Receptor 1 de Folato/inmunología , Humanos , Inmunoconjugados/inmunología , Maitansina/análogos & derivados , Maitansina/inmunología , Maitansina/farmacología , Ratones Desnudos , Ratones SCID , Terapia Molecular Dirigida/métodos , Neoplasias/inmunología , Neoplasias/metabolismo , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.
Asunto(s)
Maitansina/metabolismo , Tubulina (Proteína)/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Liposomas , Maitansina/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Unión ProteicaRESUMEN
The maytansinoid antibody-drug conjugates (ADCs) in clinical development for cancer therapy each contain a derivative of the microtubule-targeting agent, maytansine, covalently attached to the antibody via an engineered linker. A sample of any of these conjugates contains molecules with different numbers of maytansinoid molecules, or "drug" loads, the relative abundance of which can be determined by mass spectrometry. We examined the accuracy of the Poisson distribution and the binomial distribution in predicting the relative abundance of species with different drug loads for three antibody-maytansinoid conjugates with different antibodies and linker-maytansinoid pairings. We used variance, calculated from the experimental mass distribution data, as the parameter to determine the optimal value n of the binomial distribution number of trials. The accuracy of the Poisson distribution in predicting distribution of the species abundance in these conjugates varied among the conjugates. In contrast, the accuracy of the binomial distribution was similar for all three conjugates and comparable to the best accuracy of the Poisson distribution, as supported by a paired t-test.
Asunto(s)
Inmunoconjugados/farmacocinética , Maitansina/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacocinética , Humanos , Inmunoconjugados/química , Espectrometría de Masas , Modelos EstadísticosRESUMEN
PURPOSE: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984. EXPERIMENTAL DESIGN: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice. RESULTS: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells. CONCLUSION: These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies.
Asunto(s)
ADP-Ribosil Ciclasa 1/genética , Anticuerpos Monoclonales Humanizados/administración & dosificación , Neoplasias Hematológicas/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Glicoproteínas de Membrana/genética , Mieloma Múltiple/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Neoplasias Hematológicas/patología , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Mieloma Múltiple/patología , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated-NCAM1 expressing-"blastema" phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets.
Asunto(s)
Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Células Madre Neoplásicas/patología , Tumor de Wilms/metabolismo , Tumor de Wilms/patología , Antígeno AC133 , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/metabolismo , Antígeno CD56/metabolismo , Diferenciación Celular , Proliferación Celular , Separación Celular/métodos , Expresión Génica , Glicoproteínas/metabolismo , Humanos , Neoplasias Renales/genética , Neoplasias Renales/terapia , Maitansina/análogos & derivados , Maitansina/uso terapéutico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Retinal-Deshidrogenasa , Células Tumorales Cultivadas , Ensayo de Tumor de Célula Madre , Tumor de Wilms/genética , Tumor de Wilms/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Antineoplásicos Alquilantes/síntesis química , Indoles/síntesis química , Compuestos Organofosforados/síntesis química , Profármacos/síntesis química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Duocarmicinas , Femenino , Humanos , Indoles/química , Indoles/farmacología , Ratones , Ratones SCID , Trasplante de Neoplasias , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Profármacos/química , Profármacos/farmacología , Solubilidad , Estereoisomerismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. Antibody-maytansinoid conjugates (AMCs) bearing these sulfonate- or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.
Asunto(s)
Anticuerpos/química , Inmunoconjugados/química , Maitansina/química , Animales , Química Farmacéutica/métodos , Diseño de Fármacos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Modelos Químicos , Trasplante de Neoplasias , Polietilenglicoles/química , Succinimidas/química , Sulfonas/químicaRESUMEN
One approach to improving activity of anticancer drugs is to conjugate them to antibodies that recognize tumor-associated, cell-surface antigens. The antibody-drug conjugate concept evolved following major advances, first, in the development of humanized and fully human antibodies; second, in the discoveries of highly cytotoxic compounds ('drugs) linkable to antibodies; and finally, in the optimization of linkers that couple the drug to the antibody and provide sufficient stability of the antibody-drug conjugate in the circulation, optimal activation of the drug in the tumor, and the ability of the activated drug to overcome multidrug resistance. In this article, we will review the considerations for selecting a target antigen, the design of the conjugate, and the pre-clinical and clinical experiences with the current generation of antibody-drug conjugates.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/administración & dosificación , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/efectos adversos , Humanos , Inmunoconjugados/efectos adversosRESUMEN
Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.
Asunto(s)
Inmunotoxinas/farmacología , Maitansina/análogos & derivados , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacocinética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Maleimidas/química , Maitansina/química , Maitansina/farmacocinética , Maitansina/farmacología , Ratones , Ratones SCID , Polietilenglicoles/químicaRESUMEN
Apoptosis is a major problem in animal cell cultures during production of biopharmaceuticals, such as recombinant proteins or viral vectors. A 293 cell line constitutively expressing vMIA (viral mitochondria-localized inhibitor of apoptosis) was constructed and examined on production of a model recombinant protein, green fluorescent protein (GFP) in the adenovirus-293 expression system, and on production of a model infectious adenoviral vector. vMIA-293 cells were more resistant than the parental 293 cells to apoptosis induced by either oxidative stress, or by adenovirus infection. The yield of GFP produced in vMIA-293 cell cultures was consistently higher (approximately 140%) compared to that in the parental cells. vMIA reduced production of adenovirus infectious particles, which was not due to a decline of adenovirus replication, since adenoviral DNA replication rate in vMIA-293 cells was higher than that in the parental cells. In conclusion, introduction of the vMIA gene into the 293 cell line is a promising strategy to improve recombinant protein production in the adenovirus-293 expression system.
Asunto(s)
Adenoviridae/genética , Proteínas Mitocondriales/genética , Proteínas Recombinantes/biosíntesis , Proteínas Virales/genética , Adenoviridae/metabolismo , Apoptosis , Muerte Celular , Células Cultivadas , Citometría de Flujo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Microscopía Fluorescente , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Proteínas Recombinantes/genética , Transfección , Proteínas Virales/metabolismoRESUMEN
Apoptosis is increasingly implicated as an early line of defense against viral infections. Viruses have devised numerous strategies to delay apoptosis of infected cells. Many viruses encode cell death suppressors that target mitochondrial apoptotic signaling pathway, indicating the importance of this pathway in the anti-viral response. Human and primate cytomegaloviruses encode the viral mitochondria-localized inhibitor of apoptosis vMIA, but no overt homologue of vMIA was identified in any non-primate cytomegalovirus. Here we report that m38.5 protein encoded by murine cytomegalovirus, which is unrelated to vMIA in its amino acid sequence, delays death receptor ligation-induced cell death, and that m38.5 associates with Bax, recruits it to mitochondria, and blocks Bax-mediated but not Bak-mediated mitochondrial outer membrane permeabilization. Thus, primate and murine cytomegaloviruses have evolved non-homologous but functionally similar cell death suppressors selectively targeting the Bax-mediated branch of the mitochondrial apoptotic signaling pathway, indicating the importance of this branch in the response of diverse host organisms against cytomegalovirus infections.
Asunto(s)
Membranas Mitocondriales/metabolismo , Muromegalovirus/metabolismo , Proteínas Virales/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Muerte Celular , Células HCT116 , Células HeLa , Humanos , Ratones , Permeabilidad , Unión Proteica , Conformación Proteica , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Receptores de Muerte Celular/metabolismo , Proteína X Asociada a bcl-2/química , Receptor fas/metabolismoAsunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Antígeno CD56/inmunología , Linfoma Extranodal de Células NK-T/patología , Ado-Trastuzumab Emtansina , Anticuerpos Monoclonales Humanizados , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Maitansina/análogos & derivados , Trastuzumab , Células Tumorales CultivadasRESUMEN
EC131, a new folate receptor (FR)-targeted drug conjugate, was prepared by covalently attaching the vitamin folic acid (FA) to a potent microtubule-inhibiting agent, maytansinoid DM1, via an intramolecular disulfide bond. When tested on cells in culture, EC131 was found to retain high affinity for FR-positive cells and to provide FR-specific cytotoxicity with an IC(50) in the low nanomolar range. The activity of EC131 was completely blocked in the presence of an excess of free FA, and no activity was detected against FR-negative cells. When evaluated against s.c. FR-positive M109 tumors in BALB/c mice, EC131 showed marked antitumor efficacy. Furthermore, this therapeutic effect occurred in the apparent absence of weight loss or noticeable organ tissue degeneration. In contrast, no significant antitumor activity was observed in EC131-treated animals that were codosed with an excess of FA, thus demonstrating the targeted specificity of the in vivo activity. EC131 also showed marked antitumor activity against FR-positive human KB tumors, but not against FR-negative A549 tumors, in nude mice with no evidence of systemic toxicity during or after the therapy. In contrast, therapy with the free maytansinoid drug (in the form of DM1-S-Me) proved not to be effective against the KB model when administered at its maximum tolerated dose (MTD). Taken together, these results indicate that EC131 is a highly potent agent capable of producing therapeutic benefit in murine tumor models at sub-MTD levels.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Ácido Fólico/farmacología , Maitansina/farmacología , Extractos Vegetales/farmacología , Receptores de Superficie Celular/metabolismo , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Receptores de Folato Anclados a GPI , Ácido Fólico/química , Ácido Fólico/metabolismo , Humanos , Células KB , Dosis Máxima Tolerada , Maitansina/química , Maytenus/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
Antibody-drug conjugates (ADCs) are designed to specifically bind to and kill cells expressing their target antigens. In addition to the obvious requirement of the presence of the target antigen on the cell surface, several other factors contribute to the sensitivity of target cells to the action of ADCs. These include (i) the rate of internalization of the ADC, (ii) its proteolytic degradation in late endosomes and lysosomes and the subsequent release of cytotoxic drug, and (iii) the intracellular concentration of the released drug. In addition to killing antigen-expressing cells, some ADCs were found to kill bystander cells irrespective of their antigen expression. This review summarizes the current knowledge of the mechanisms of killing of antigen-expressing and bystander cells by antibody-drug conjugates.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/farmacología , Inmunoconjugados/farmacología , Neoplasias/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Humanos , Inmunoconjugados/metabolismoRESUMEN
Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.
Asunto(s)
Apoptosis , Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Proteínas Inmediatas-Precoces/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Virales/fisiología , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Citomegalovirus/genética , Efecto Citopatogénico Viral , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibroblastos/virología , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/toxicidad , Ratones , Proteínas Mitocondriales/genética , Células 3T3 NIH , Fosforilación Oxidativa/efectos de los fármacos , Polímeros/metabolismo , Proteínas Virales/genética , Proteínas Virales/toxicidad , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genéticaRESUMEN
Maytansine, a highly cytotoxic natural product, failed as an anticancer agent in human clinical trials because of unacceptable systemic toxicity. The potent cell killing ability of maytansine can be used in a targeted delivery approach for the selective destruction of cancer cells. A series of new maytansinoids, bearing a disulfide or thiol substituent were synthesized. The chain length of the ester side chain and the degree of steric hindrance on the carbon atom bearing the thiol substituent were varied. Several of these maytansinoids were found to be even more potent in vitro than maytansine. The targeted delivery of these maytansinoids, using monoclonal antibodies, resulted in a high, specific killing of the targeted cells in vitro and remarkable antitumor activity in vivo.
Asunto(s)
Antineoplásicos/síntesis química , Maitansina/análogos & derivados , Maitansina/síntesis química , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Disulfuros/síntesis química , Disulfuros/química , Disulfuros/farmacología , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Maitansina/química , Maitansina/farmacología , Ratones , Ratones SCID , Trasplante de Neoplasias , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Trasplante HeterólogoRESUMEN
The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.
Asunto(s)
Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Mitocondrias/metabolismo , Modificación Traduccional de las Proteínas , Eliminación de Secuencia , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Retículo Endoplásmico/genética , Glicosilación , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inmediatas-Precoces/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Proteínas de la Membrana/metabolismo , Pliegue de Proteína , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Modificación Traduccional de las Proteínas/genética , Señales de Clasificación de Proteína/genética , Estructura Secundaria de Proteína , Transporte de Proteínas/genética , Serina Endopeptidasas/metabolismo , Proteínas Virales/genéticaRESUMEN
Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen-specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, we used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, we examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibody-maytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates.