RESUMEN
Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.
Asunto(s)
Compuestos de Anilina/química , Antineoplásicos Fitogénicos/química , Inmunoconjugados/química , Maitansina/química , Compuestos de Anilina/farmacocinética , Compuestos de Anilina/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/uso terapéutico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Maitansina/farmacocinética , Maitansina/uso terapéutico , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
Conjugates of the anti-CanAg humanized monoclonal antibody huC242 with the microtubule-formation inhibitor DM1 (a maytansinoid), or with the DNA alkylator DC1 (a CC1065 analogue), have been evaluated for their ability to eradicate mixed cell populations formed from CanAg-positive and CanAg-negative cells in culture and in xenograft tumors in mice. We found that in culture, conjugates of either drug killed not only the target antigen-positive cells but also the neighboring antigen-negative cells. Furthermore, we showed that, in vivo, these conjugates were effective in eradicating tumors containing both antigen-positive and antigen-negative cells. The presence of antigen-positive cells was required for this killing of bystander cells. This target cell-activated killing of bystander cells was dependent on the nature of the linker between the antibody and the drug. Conjugates linked via a reducible disulfide bond were capable of exerting the bystander effect whereas equally potent conjugates linked via a nonreducible thioether bond were not. Our data offer a rationale for developing optimally constructed antibody-drug conjugates for treating tumors that express the target antigen either in a homogeneous or heterogeneous manner.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Inmunoconjugados/farmacología , Maitansina/análogos & derivados , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Reactivos de Enlaces Cruzados/química , Femenino , Células HT29 , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Maitansina/química , Maitansina/farmacología , Ratones , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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
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
Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5-10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno CD56/inmunología , Inmunoterapia/métodos , Neoplasias Pulmonares/terapia , Maitansina/análogos & derivados , Maitansina/metabolismo , Carcinoma Pulmonar de Células Pequeñas/terapia , Moduladores de Tubulina/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Maitansina/química , Maitansina/inmunología , Ratones , Ratones SCID , Carcinoma Pulmonar de Células Pequeñas/inmunología , Moduladores de Tubulina/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N(ε)-MCC-DM1, and lysine-N(ε)-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Maitansina/química , Maitansina/metabolismo , Maitansina/farmacocinética , Maitansina/farmacología , Ratones , Ratones Desnudos , Receptor ErbB-2/metabolismo , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.