RESUMEN
Patients with relapsed/refractory B-cell malignancies such as non-Hodgkin lymphoma (B-NHL) or acute lymphoblastic leukemia have a poor prognosis. Despite measurable clinical activity with new targeted therapies, many patients do not achieve a complete or durable response suggesting an opportunity to improve upon existing therapies. Here we describe SGN-CD19B, a pyrrolobenzodiazepine (PBD)-based anti-CD19 antibody drug conjugate (ADC) being investigated for treatment of B-cell malignancies, which has improved potency compared with other ADCs. CD19-expressing tumor cells rapidly internalize SGN-CD19B, and the released PBD drug induces DNA damage, resulting in G2/M cell cycle arrest and cell death. SGN-CD19B demonstrated activity against a broad panel of malignant B-cell lines and induced durable regressions in mice bearing xenografts derived from these B-cell malignancies. A single dose of SGN-CD19B induced durable regressions at 300 µg/kg (3 µg/kg drug equivalents); combination with rituximab decreased the curative dose to 100 µg/kg (1 µg/kg drug equivalents). These doses are significantly lower than the level of drug required with other ADC payloads. In cynomolgus monkeys, SGN-CD19B effectively depleted CD20+ B lymphocytes in peripheral blood and lymphoid tissues confirming that SGN-CD19B is pharmacodynamically active at well-tolerated doses. In summary, preclinical studies show SGN-CD19B is a highly active ADC, which releases a DNA cross-linking agent rather than a microtubule inhibitor. The distinct mechanism of action, broad potency, and potential to combine with rituximab suggest that SGN-CD19B may offer unique clinical opportunities in B-cell malignancies. A phase 1 clinical trial is in progress to investigate the therapeutic potential of SGN-CD19B in relapsed/refractory B-NHL. This trial was registered at www.clinicaltrials.gov as #NCT02702141.
Asunto(s)
Anticuerpos/uso terapéutico , Antígenos CD19/metabolismo , Benzodiazepinas/química , Linfoma de Células B/tratamiento farmacológico , Pirroles/química , Animales , Anticuerpos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Linfoma de Células B/patología , Macaca fascicularis , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A quaternary ammonium-based drug-linker has been developed to expand the scope of antibody-drug conjugate (ADC) payloads to include tertiary amines, a functional group commonly present in biologically active compounds. The linker strategy was exemplified with a ß-glucuronidase-cleavable auristatin E construct. The drug-linker was found to efficiently release free auristatin E (AE) in the presence of ß-glucuronidase and provide ADCs that were highly stable in plasma. Anti-CD30 conjugates comprised of the glucuronide-AE linker were potent and immunologically specific in vitro and in vivo, displaying pharmacologic properties comparable with a carbamate-linked glucuronide-monomethylauristatin E control. The quaternary ammonium linker was then applied to a tubulysin antimitotic drug that contained an N-terminal tertiary amine that was important for activity. A glucuronide-tubulysin quaternary ammonium linker was synthesized and evaluated as an ADC payload, in which the resulting conjugates were found to be potent and immunologically specific in vitro, and displayed a high level of activity in a Hodgkin lymphoma xenograft. Furthermore, the results were superior to those obtained with a related tubulysin derivative containing a secondary amine N-terminus for conjugation using previously known linker technology. The quaternary ammonium linker represents a significant advance in linker technology, enabling stable conjugation of payloads with tertiary amine residues. Mol Cancer Ther; 15(5); 938-45. ©2016 AACR.
Asunto(s)
Compuestos de Amonio/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Inmunoconjugados/química , Inmunoconjugados/farmacología , Animales , Anticuerpos Monoclonales/farmacocinética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Liberación de Fármacos , Estabilidad de Medicamentos , Humanos , Inmunoconjugados/farmacocinética , Cinética , Ratones , Estructura Molecular , Unión Proteica , Ratas , Tubulina (Proteína) , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: CD70 (CD27L) is a member of the tumor necrosis factor family aberrantly expressed on a number of hematologic malignancies and some carcinomas. CD70 expression on malignant cells coupled with its highly restricted expression on normal cells makes CD70 an attractive target for monoclonal antibody (mAb)-based therapies. We developed a humanized anti-CD70 antibody, SGN-70, and herein describe the antitumor activities of this mAb. EXPERIMENTAL DESIGN: CD70 expression on primary tumors was evaluated by immunohistochemical staining of Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and renal cell carcinoma tissue microarrays. The CD70-binding and cytotoxic activities of SGN-70 were tested in vitro using a number of cell-based assays. The in vivo antitumor properties of SGN-70 were tested in severe combined immunodeficient mice bearing disseminated lymphoma and multiple myeloma xenografts. Mechanism-of-action studies were conducted using SGN-70v, a variant mAb with equivalent target-binding activity but impaired Fcgamma receptor binding compared with SGN-70. RESULTS: Immunohistochemical analysis identified CD70 expression on approximately 40% of multiple myeloma isolates and confirmed CD70 expression on a high percentage of Hodgkin lymphoma Reed-Sternberg cells, non-Hodgkin lymphoma, and renal cell carcinoma tumors. SGN-70 lysed CD70+ tumor cells via Fc-dependent functions, including antibody-dependent cellular cytotoxicity and phagocytosis and complement fixation. In vivo, SGN-70 treatment significantly decreased tumor burden and prolonged survival of tumor-bearing mice. CONCLUSIONS: SGN-70 is a novel humanized IgG1 mAb undergoing clinical development for the treatment of CD70+ cancers. SGN-70 possesses Fc-dependent antibody effector functions and mediates antitumor activity in vivo.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Ligando CD27/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Antineoplásicos/farmacología , Ligando CD27/metabolismo , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Humanos , Inmunohistoquímica , Neoplasias Renales/inmunología , Neoplasias Renales/metabolismo , Linfoma/inmunología , Linfoma/metabolismo , Ratones , Ratones SCID , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increased expression of CD30 is associated with a variety of hematologic malignancies, including Hodgkin disease (HD) and anaplastic large cell lymphoma (ALCL). The anti-CD30 monoclonal antibody SGN-30 induces direct antitumor activity by promoting growth arrest and DNA fragmentation of CD30(+) tumor cells. In this study, we investigated the contributions of Fc-mediated effector cell functions to SGN-30 activity. We determined that antibody-dependent cellular phagocytosis, mediated by macrophages, to contribute significantly to antitumor activity in vitro. To delineate the identity of the host effector cells involved in mediating antitumor activity in vivo, we studied the effects of effector cell ablation in a disseminated model of HD (L540cy). Depletion of macrophages markedly reduced efficacy of SGN-30, demonstrating that macrophages contribute significantly to SGN-30 efficacy in this model. These findings may have implications for patient stratification or combination treatment strategies in clinical trials conducted with SGN-30 in HD and ALCL.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Enfermedad de Hodgkin/terapia , Macrófagos/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Humanos , Antígeno Ki-1/inmunología , Ratones , Ratones SCID , Trasplante de Neoplasias , Fagocitosis/efectos de los fármacos , Trasplante HeterólogoRESUMEN
Antigens expressed on malignant cells in the absence of significant expression on normal tissues are highly desirable targets for therapeutic antibodies. CD70 is a TNF superfamily member whose normal expression is highly restricted but is aberrantly expressed in hematologic malignancies including non-Hodgkin lymphoma (NHL), Hodgkin disease, and multiple myeloma. In addition, solid tumors such as renal cell carcinoma, nasopharyngeal carcinoma, thymic carcinoma, meduloblastoma, and glioblastoma express high levels of this antigen. To functionally target CD70-expressing cancers, a murine anti-CD70 monoclonal antibody was engineered to contain human IgG1 constant domains. The engineered antibody retained the binding specificity of the murine parent monoclonal antibody and was shown to induce Fc-mediated effector functions including antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis in vitro. Further, administration of this antibody significantly prolonged survival of severe combined immunodeficient (SCID) mice bearing CD70+ disseminated human NHL xenografts. Survival of these mice was dependent upon the activity of resident effector cells including neutrophils, macrophages, and natural killer (NK) cells. These data suggest that an anti-CD70 antibody, when engineered to contain human IgG1 constant domains, possesses effector cell-mediated antitumor activity and has potential utility for anticancer therapy.
Asunto(s)
Anticuerpos/uso terapéutico , Antineoplásicos , Ligando CD27/inmunología , Linfoma no Hodgkin/terapia , Ingeniería de Proteínas/métodos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular Tumoral , Proteínas del Sistema Complemento , Humanos , Sistema Inmunológico/citología , Inmunoglobulina G , Ratones , Ratones SCID , Fagocitosis , Tasa de Supervivencia , Trasplante HeterólogoRESUMEN
The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.
Asunto(s)
Antígeno Ki-1/química , Lisosomas/metabolismo , Oligopéptidos/química , Anticuerpos/química , Antígenos CD20/química , Antineoplásicos/farmacología , Western Blotting , Catepsina B/química , Línea Celular , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Endocitosis , Endopeptidasas/química , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Microscopía Fluorescente , Modelos Químicos , Péptido Hidrolasas/química , Péptidos/química , Unión Proteica , Fracciones Subcelulares/metabolismo , Temperatura , Factores de Tiempo , beta-Galactosidasa/metabolismoRESUMEN
Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.
Asunto(s)
Antígenos CD/biosíntesis , Antineoplásicos/administración & dosificación , Carcinoma de Células Renales/inmunología , Inmunoconjugados/inmunología , Inmunoconjugados/farmacología , Neoplasias Renales/inmunología , Proteínas de la Membrana/biosíntesis , Factores de Necrosis Tumoral/biosíntesis , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Apoptosis/efectos de los fármacos , Ligando CD27 , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Ciclo Celular/efectos de los fármacos , Humanos , Inmunohistoquímica , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Activación de Linfocitos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Desnudos , Microtúbulos/efectos de los fármacos , Oligopéptidos/administración & dosificación , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SGN-40 is a humanized IgG1 antihuman CD40 that is currently in a phase I clinical trial for the treatment of multiple myeloma. As surface CD40 expression on B-lineage cells is maintained from pro-B cells to plasma cells, SGN-40 may be applicable to treatment of other B-cell neoplasias, including non-Hodgkin's lymphoma. In this study, we examined potential in vitro and in vivo anti-B-lineage lymphoma activity of SGN-40. Recombinant SGN-40 was expressed and purified from Chinese hamster ovary cells and characterized based on binding affinity, specificity, and normal B-cell stimulation. The ability of SGN-40 to target neoplastic B cells was examined in vitro by proliferation inhibition, cytotoxicity, and antibody-dependent cell cytotoxicity assays and in vivo by human lymphoma xenograft models. Recombinant SGN-40 showed high affinity, Kd of approximately 1 nmol/L, and specific binding to CD40. Whereas SGN-40 was a weak agonist in stimulating normal B-cell proliferation in the absence of IL-4 and CD40L, it delivered potent proliferation inhibitory and apoptotic signals to, and mediated antibody-dependent cytotoxicity against, a panel of high-grade B-lymphoma lines. These in vitro antilymphoma effects were extended to disseminated and s.c. xenograft CD40 tumor models. In these xenograft models, the antitumor activity of SGN-40 was comparable with that of rituximab. The preclinical in vitro and in vivo antilymphoma activity of SGN-40 observed in this study provides a rationale for the clinical testing of SGN-40 in the treatment of CD40+ B-lineage lymphomas.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD40/inmunología , Inmunoglobulina G/farmacología , Linfoma de Células B/terapia , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Caspasa 3 , Caspasas/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/inmunología , Línea Celular Tumoral , Activación Enzimática , Humanos , Inmunoglobulina G/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyleneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos Alquilantes/síntesis química , ADN/química , Dipéptidos/química , Inmunoconjugados/química , Indoles/síntesis química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Compuestos Aza/síntesis química , Compuestos Aza/química , Compuestos Aza/farmacología , Catepsina B/química , Línea Celular Tumoral , Ciclopropanos/síntesis química , Ciclopropanos/química , Ciclopropanos/farmacología , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indoles/química , Indoles/farmacología , Ratones , Relación Estructura-ActividadRESUMEN
The anti-CD20 antibody rituximab is useful in the treatment of certain B-cell malignancies, most notably non-Hodgkin's lymphoma. Its efficacy has been increased when used in combination with chemotherapy, yet anti-CD20 monoclonal antibodies (mAbs) directly conjugated with drugs such as doxorubicin (Dox) have failed to deliver drug or to demonstrate antitumor activity. We have produced anti-CD20 antibody-drug conjugates that possess potent antitumor activity by using the anti-mitotic agent, monomethyl auristatin E (MMAE), linked via the lysosomally cleavable dipeptide, valine-citrulline (vc). Two anti-CD20 conjugates, rituximab-vcMMAE and 1F5-vcMMAE, were selectively cytotoxic against CD20(+) B-lymphoma cell lines, with IC(50) values ranging from 50 ng/mL to 1 microg/mL. Unlike rituximab, which showed diffuse surface localization, rituximab-vcMMAE capped and was internalized within 4 hours after binding to CD20(+) B cells. Internalization of rituximab-vcMMAE was followed by rapid G(2)-M phase arrest and onset of apoptosis. Anti-CD20 antibody-drug conjugates prepared with Dox were internalized and localized as with rituximab-vcMMAE, yet these were not effective for drug delivery (IC(50) > 50 microg/mL). Consistent with in vitro activity, rituximab-vcMMAE showed antitumor efficacy in xenograft models of CD20-positive lymphoma at doses where rituximab or rituximab-Dox conjugates were ineffective. These data indicate that anti-CD20-based antibody-drug conjugates are effective antitumor agents when prepared with a stable, enzyme-cleavable peptide linkage to highly potent cytotoxic agents such as MMAE.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD20/inmunología , Antineoplásicos/farmacología , Linfoma de Células B , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales de Origen Murino , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Fase G2/efectos de los fármacos , Humanos , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Ratones , Oligopéptidos/efectos adversos , Oligopéptidos/química , Rituximab , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The chimeric monoclonal antibody cAC10, directed against CD30, induces growth arrest of CD30+ cell lines in vitro and has pronounced antitumor activity in severe combined immunodeficiency (SCID) mouse xenograft models of Hodgkin disease. We have significantly enhanced these activities by conjugating to cAC10 the cytotoxic agent monomethyl auristatin E (MMAE) to create the antibody-drug conjugate cAC10-vcMMAE. MMAE, a derivative of the cytotoxic tubulin modifier auristatin E, was covalently coupled to cAC10 through a valine-citrulline peptide linker. The drug was stably attached to the antibody, showing only a 2% release of MMAE following 10-day incubation in human plasma, but it was readily cleaved by lysosomal proteases after receptor-mediated internalization. Release of MMAE into the cytosol induced G2/M-phase growth arrest and cell death through the induction of apoptosis. In vitro, cAC10-vcMMAE was highly potent and selective against CD30+ tumor lines (IC50 less than 10 ng/mL) but was more than 300-fold less active on antigen-negative cells. In SCID mouse xenograft models of anaplastic large cell lymphoma or Hodgkin disease, cAC10-vcMMAE was efficacious at doses as low as 1 mg/kg. Mice treated at 30 mg/kg cAC10-vcMMAE showed no signs of toxicity. These data indicate that cAC10-vcMMAE may be a highly effective and selective therapy for the treatment of CD30+ neoplasias.