RESUMEN
Bicycle toxin conjugates (BTCs) are a promising new class of molecules for targeted delivery of toxin payloads into tumors. Herein we describe the discovery of BT8009, a Nectin-4 targeting BTC currently under clinical evaluation. Nectin-4 is overexpressed in multiple tumor types and is a clinically validated target for selective delivery of cytotoxic payloads. A Nectin-4 targeting bicyclic peptide was identified by phage display, which showed highly selective binding for Nectin-4 but suffered from low plasma stability and poor physicochemical properties. Multiparameter chemical optimization involving introduction of non-natural amino acids resulted in a lead Bicycle that demonstrated high affinity for Nectin-4, good stability in biological matrices, and a much-improved physicochemical profile. The optimized Bicycle was conjugated to the cytotoxin Monomethyl auristatin E via a cleavable linker to give the targeted drug conjugate BT8009, which demonstrates potent anticancer activity in in vivo rodent models.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Inmunotoxinas , Neoplasias , Humanos , Nectinas , Ciclismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Moléculas de Adhesión Celular , Línea Celular TumoralRESUMEN
Multiple tumor types overexpress Nectin-4 and the antibody-drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a "Bicycle Toxin Conjugate" (BTC) consisting of a Nectin-4-binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1-2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.
Asunto(s)
Carcinoma de Células Transicionales , Inmunoconjugados , Inmunotoxinas , Ratas , Animales , Nectinas , Ciclismo , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Moléculas de Adhesión Celular/metabolismo , Carcinoma de Células Transicionales/tratamiento farmacológicoRESUMEN
After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.
Asunto(s)
Inmunoconjugados/uso terapéutico , Oligopéptidos/uso terapéutico , Polímeros/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunoconjugados/farmacología , Ratones , Ratones SCID , Oligopéptidos/farmacología , Polímeros/farmacologíaRESUMEN
Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Polímeros/uso terapéutico , Animales , Femenino , Humanos , Inmunoconjugados/farmacología , Ratones , Ratones SCID , Oligopéptidos/farmacología , Polímeros/farmacologíaRESUMEN
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
CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antígenos de Neoplasias/inmunología , Linfocitos B/efectos de los fármacos , Inmunotoxinas/uso terapéutico , Maitansina/análogos & derivados , Terapia Molecular Dirigida , Tetraspaninas/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Anticuerpos Monoclonales de Origen Murino/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Clorhidrato de Bendamustina , Línea Celular Tumoral/efectos de los fármacos , Ciclofosfamida/administración & dosificación , Citotoxicidad Inmunológica/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Maitansina/administración & dosificación , Maitansina/farmacología , Maitansina/uso terapéutico , Ratones , Ratones SCID , Compuestos de Mostaza Nitrogenada/uso terapéutico , Prednisona/administración & dosificación , Rituximab , Vincristina/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Depletion of CD4(+)CD25(+)FoxP3(+) Treg using PC61 mAb (anti-murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated approximately 70% of CD4(+)FoxP3(+) cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcgamma receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcgamma receptor (FcgammaR)III(-/-) mice were resistant to Treg depletion. FcgammaRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab-dependent-cellular-cytotoxicity and Ab-dependent-cellular-phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab-dependent-cellular-phagocytosis of CD25(+) cells by WT or FcgammaRIIB(-/-), but not FcgammaRIII(-/-), macrophages. Altogether these data demonstrate the critical role of FcgammaRIII(+) phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Depleción Linfocítica , Macrófagos/inmunología , Receptores de IgG/inmunología , Linfocitos T Reguladores , Animales , Anticuerpos Monoclonales/farmacología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Ratones Endogámicos C57BL , Fagocitos/inmunología , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Inmunotoxinas/farmacocinética , Maitansina/análogos & derivados , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacocinética , Reactivos de Enlaces Cruzados/farmacología , Disulfuros/química , Disulfuros/farmacocinética , Disulfuros/farmacología , Células HT29 , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Maitansina/química , Maitansina/farmacocinética , Ratones , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA-Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.