RESUMEN
Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC), combining a humanized monoclonal antibody (IgG1) targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and a potent cytotoxic maytansinoid derivative, DM4, inhibiting microtubule assembly. SAR408701 is currently in clinical development for the treatment of advanced solid tumors expressing CEACAM5. It is administered intravenously as a conjugated antibody with an average Drug Antibody Ratio (DAR) of 3.8. During SAR408701 clinical development, four entities were measured in plasma: conjugated antibody (SAR408701), naked antibody (NAB), DM4 and its methylated metabolite (MeDM4), both being active. Average DAR and proportions of individual DAR species were also assessed in a subset of patients. An integrated and semi-mechanistic population pharmacokinetic model describing the time-course of all entities in plasma and DAR measurements has been developed. All DAR moieties were assumed to share the same drug disposition parameters, excepted for clearance which differed for DAR0 (i.e. NAB entity). The conversion of higher DAR to lower DAR resulted in a DAR-dependent ADC deconjugation and was represented as an irreversible first-order process. Each conjugated antibody was assumed to contribute to DM4 formation. All data were fitted simultaneously and the model developed was successful in describing the pharmacokinetic profile of each entity. Such a structural model could be translated to other ADCs and gives insight of mechanistic processes governing ADC disposition. This framework will further be expanded to evaluate covariates impact on SAR408701 pharmacokinetics and its derivatives, and thus can help identifying sources of pharmacokinetic variability and potential efficacy and safety pharmacokinetic drivers.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Maitansina , Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/farmacocinética , Moléculas de Adhesión Celular , Humanos , Inmunoconjugados/farmacocinética , Maitansina/química , Maitansina/farmacocinéticaRESUMEN
Tusamitamab ravtansine is an anti-CEACAM5 antibody-drug conjugate indicated in patients with solid tumors. Based on a previous developed semimechanistic model describing simultaneously pharmacokinetic (PK) of SAR408701, two of its active metabolites: DM4 and methyl-DM4 and naked antibody, with integration of drug-to-antibody data, the main objective of the present analysis was to evaluate covariate's impact in patients from phase I/II study (n = 254). Demographic and pathophysiologic baseline covariates were explored to explain interindividual variability on each entity PK parameter. Model parameters were estimated with good precision. Five covariates were included in the final PK model: body surface area (BSA), tumor burden, albumin, circulating target, and gender. Comparison of BSA-adjusted dosing and flat dosing supported the current BSA-based dosing regimen, to limit under and over exposure in patients with extreme BSA. Overall, this model characterized accurately the PKs of all entities and highlighted sources of PK variability. By integrating mechanistic considerations, this model aimed to improve understanding of the SAR408701 complex disposition while supporting key steps of clinical development.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Maitansina , Neoplasias , Anticuerpos Monoclonales/farmacocinética , Antineoplásicos/farmacocinética , Humanos , Maitansina/farmacocinética , Neoplasias/tratamiento farmacológicoRESUMEN
Background We report a Phase 1 study of LY3076226, an antibody-drug conjugate composed of human IgG1 monoclonal antibody against the human FGFR3 attached with a cleavable linker to the maytansine derivative DM4 in patients with advanced or metastatic cancer. Methods This study was comprised of two parts: (A) dose escalation in patients with advanced or metastatic cancer and (B) dose expansion in patients with urothelial carcinoma with locally determined FGFR3 alterations. The dose range of LY3076226 tested was 0.2-5.0 mg/kg as an intravenous infusion on Day 1 of each 21-day cycle. The primary objective was to determine a recommended phase 2 dose (RP2D). Results Twenty-five patients were enrolled (Part A: 22, Part B: 3) and received ≥ 1 dose of LY3076226. No dose-limiting toxicities were reported. LY3076226 was generally well tolerated; most of the toxicities were Grade 1 or 2. Two patients experienced treatment-related Grade 3 toxicity (embolism and decreased platelet count). Four patients experienced serious adverse events (not treatment-related), all in Part A. Dose-proportional exposure was observed, with an estimated half-life of 2-7 days. No responses were seen with LY3076226 treatment. Stable disease persisting for > 6 months was observed in 1 patient receiving 3.2 mg/kg of LY3076226. Conclusion The study demonstrates acceptable safety and tolerability of LY3076226 up to the 5.0 mg/kg dose. Recruitment was stopped due to pipeline prioritization. Dose escalation of LY3076226 beyond 5.0 mg/kg in patients with advanced tumors may be possible. The trial was registered on August 19, 2015 under identifier NCT02529553 with ClinicalTrials.gov.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Antineoplásicos , Inmunoconjugados , Maitansina , Neoplasias , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Área Bajo la Curva , Relación Dosis-Respuesta a Droga , Semivida , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Dosis Máxima Tolerada , Maitansina/administración & dosificación , Maitansina/efectos adversos , Maitansina/farmacocinética , Tasa de Depuración Metabólica , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/patología , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/farmacocinéticaRESUMEN
PURPOSE: This phase I study, which to our knowledge is the first-in-human study of this kind, investigates the safety, tolerability, pharmacokinetics, and clinical activity of anetumab ravtansine, an antibody-drug conjugate of anti-mesothelin antibody linked to maytansinoid DM4, in patients with advanced, metastatic, or recurrent solid tumors known to express the tumor-differentiation antigen mesothelin. PATIENTS AND METHODS: This phase I, open-label, multicenter, dose-escalation and dose-expansion study of anetumab ravtansine enrolled 148 adult patients with multiple solid tumor types. Ten dose-escalation cohorts of patients with advanced or metastatic solid tumors (0.15-7.5 mg/kg) received anetumab ravtansine once every 3 weeks, and 6 expansion cohorts of patients with advanced, recurrent ovarian cancer or malignant mesothelioma received anetumab ravtansine at the maximum tolerated dose once every 3 weeks, 1.8 mg/kg once per week, and 2.2 mg/kg once per week. RESULTS: Forty-five patients were enrolled across the 10 dose-escalation cohorts. The maximum tolerated dose of anetumab ravtansine was 6.5 mg/kg once every 3 weeks or 2.2 mg/kg once per week. Thirty-two patients were enrolled in the 6.5 mg/kg once-every-3-weeks, 35 in the 1.8 mg/kg once-per-week, and 36 in the 2.2 mg/kg once-per-week expansion cohorts. The most common drug-related adverse events were fatigue, nausea, diarrhea, anorexia, vomiting, peripheral sensory neuropathy, and keratitis/keratopathy. There were no drug-related deaths. Anetumab ravtansine pharmacokinetics were dose proportional; the average half-life was 5.5 days. Among 148 patients with mesothelioma or ovarian, pancreatic, non-small-cell lung, and breast cancers, 1 had a complete response, 11 had partial responses, and 66 had stable disease. High levels of tumor mesothelin expression were detected in patients with clinical activity. CONCLUSION: Anetumab ravtansine exhibited a manageable safety and favorable pharmacokinetic profile with encouraging preliminary antitumor activity in heavily pretreated patients with mesothelin-expressing solid tumors. The results allowed for the determination of recommended doses, schedules, and patient populations for anetumab ravtansine in phase II studies.
Asunto(s)
Antineoplásicos Inmunológicos/administración & dosificación , Proteínas Ligadas a GPI/antagonistas & inhibidores , Inmunoconjugados/administración & dosificación , Maitansina/análogos & derivados , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/farmacocinética , Femenino , Proteínas Ligadas a GPI/inmunología , Humanos , Inmunoconjugados/efectos adversos , Inmunoconjugados/farmacocinética , Masculino , Dosis Máxima Tolerada , Maitansina/administración & dosificación , Maitansina/efectos adversos , Maitansina/farmacocinética , Mesotelina , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Neoplasias/inmunología , Neoplasias/mortalidad , Neoplasias/patología , Supervivencia sin ProgresiónRESUMEN
Folate receptor α (FRα) is a well-studied tumor biomarker highly expressed in many epithelial tumors such as breast, ovarian, and lung cancers. Mirvetuximab soravtansine (IMGN853) is the antibody-drug conjugate of FRα-binding humanized monoclonal antibody M9346A and cytotoxic maytansinoid drug DM4. IMGN853 is currently being evaluated in multiple clinical trials, in which the immunohistochemical evaluation of an archival tumor or biopsy specimen is used for patient screening. However, limited tissue collection may lead to inaccurate diagnosis due to tumor heterogeneity. Herein, we developed a zirconium-89 (89Zr)-radiolabeled M9346A (89Zr-M9346A) as an immuno-positron emission tomography (immuno-PET) radiotracer to evaluate FRα expression in triple-negative breast cancer (TNBC) patients, providing a novel means to guide intervention with therapeutic IMGN853. In this study, we verified the binding specificity and immunoreactivity of 89Zr-M9346A by in vitro studies in FRαhigh cells (HeLa) and FRαlow cells (OVCAR-3). In vivo PET/computed tomography (PET/CT) imaging in HeLa xenografts and TNBC patient-derived xenograft (PDX) mouse models with various levels of FRα expression demonstrated its targeting specificity and sensitivity. Following PET imaging, the treatment efficiencies of IMGN853, pemetrexed, IMGN853 + pemetrexed, paclitaxel, and saline were assessed in FRαhigh and FRαlow TNBC PDX models. The correlation between 89Zr-M9346A tumor uptake and treatment response using IMGN853 in FRαhigh TNBC PDX model suggested the potential of 89Zr-M9346A PET as a noninvasive tool to prescreen patients based on the in vivo PET imaging for IMGN853-targeted treatment.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Receptor 1 de Folato/inmunología , Receptor 1 de Folato/metabolismo , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Maitansina/análogos & derivados , Radioisótopos/farmacocinética , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Circonio/farmacocinética , Animales , Anticuerpos Monoclonales Humanizados/química , Antineoplásicos Fitogénicos/química , Quimioterapia Combinada , Femenino , Células HeLa , Humanos , Inmunoconjugados/química , Masculino , Maitansina/química , Maitansina/farmacocinética , Maitansina/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Terapia Molecular Dirigida/métodos , Paclitaxel/uso terapéutico , Pemetrexed/uso terapéutico , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radioisótopos/química , Distribución Tisular , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Circonio/químicaRESUMEN
PURPOSE: Epidermal growth factor receptor variant III (EGFRvIII) is expressed in a significant percentage of primary and recurrent glioblastoma (GBM), a common malignant primary brain tumor in adults. AMG 595 is an antibody-drug conjugate comprising a fully human, anti-EGFRvIII monoclonal antibody linked to DM1. The study goals were to assess safety, tolerability, and pharmacokinetics of AMG 595 in GBM. METHODS: In this phase 1, first-in-human, open-label, sequential-dose, exploration study, adults with recurrent GBM received AMG 595 once every 3 weeks (Q3W) according to incremental dosing cohorts (0.5-3.0 mg/kg). Primary endpoints were to assess safety, the incidence of dose-limiting toxicities (DLTs), objective response (per Macdonald criteria), evaluate pharmacokinetics, and estimate the maximum tolerated dose (MTD). RESULTS: Of 382 patients screened, 32 were enrolled and received ≥ 1 dose of AMG 595. Ten patients experienced 18 DLTs (all grade 4 thrombocytopenia), and the MTD was 2.0 mg/kg. Twenty-eight patients (88%) experienced ≥ 1 treatment-related adverse event (AE); the most common AEs were thrombocytopenia (50%) and fatigue (25%). Grade ≥ 3 treatment-related AEs occurred in 17 patients (53%); 11 (34%) had serious treatment-emergent AEs, and none were considered treatment related. Pharmacokinetic profiles indicated low levels of circulating unconjugated antibody and cytotoxin, dose-proportional increases in plasma exposures for the conjugated antibody over the studied range, and less than twofold accumulation following multiple Q3W dosing. Two patients (6%) had partial responses; 15 (47%) had stable disease. CONCLUSIONS: AMG 595 exhibited favorable pharmacokinetics and is a unique therapy with possible benefit for some patients with EGFRvIII-mutated GBM with limited therapeutic options.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Inmunoconjugados/uso terapéutico , Maitansina/análogos & derivados , Adulto , Anciano , Neoplasias Encefálicas/patología , Femenino , Glioma/patología , Humanos , Inmunoconjugados/farmacocinética , Masculino , Maitansina/farmacocinética , Maitansina/uso terapéutico , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Somatostatin receptor 2 (SSTR2) is frequently overexpressed on several types of solid tumors, including neuroendocrine tumors and small-cell lung cancer. Peptide agonists of SSTR2 are rapidly internalized upon binding to the receptor and linking a toxic payload to an SSTR2 agonist is a potential method to kill SSTR2-expressing tumor cells. Herein, we describe our efforts towards an efficacious SSTR2-targeting cytotoxic conjugate; examination of different SSTR2-targeting ligands, conjugation sites, and payloads led to the discovery of 22 (PEN-221), a conjugate consisting of microtubule-targeting agent DM1 linked to the C-terminal side chain of Tyr3-octreotate. PEN-221 demonstrates in vitro activity which is both potent (IC50 = 10 nM) and receptor-dependent (IC50 shifts 90-fold upon receptor blockade). PEN-221 targets high levels of DM1 to SSTR2-expressing xenograft tumors, which has led to tumor regressions in several SSTR2-expressing xenograft mouse models. The safety and efficacy of PEN-221 is currently under evaluation in human clinical trials.
Asunto(s)
Descubrimiento de Drogas , Maitansina/farmacología , Receptores de Somatostatina/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/farmacología , Células CHO , Línea Celular , Cricetulus , Perros , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Maitansina/química , Maitansina/farmacocinética , Ratones , Receptores de Somatostatina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To investigate the safety, pharmacokinetics, and efficacy of trastuzumab emtansine (T-DM1) in combination with pertuzumab in Japanese patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. PATIENTS AND METHODS: Patients with HER2-positive advanced or recurrent breast cancer who had received trastuzumab and chemotherapy-containing therapies were eligible. Patients received T-DM1 (3.6 mg/kg) with full-dose pertuzumab (a loading dose of 840 mg and then 420 mg) intravenously every 3 weeks. This study was registered at the Japan Pharmaceutical Information Center-Clinical Trials Information (JapicCTI-101234). RESULTS: Six patients enrolled in this study. The median duration of treatment was 11 (range 1-32) cycles. The most common treatment-emergent adverse event (TEAE) for any grade was diarrhea. Grade 3 or greater TEAEs included aspartate aminotransferase increased, left ventricular ejection fraction (LVEF) decreased, and neutrophil count decreased. The dose-limiting toxicity of grade 3 LVEF decreased was observed in one patient during cycle 1; however, it resolved within 30 days. The pharmacokinetic parameters of T-DM1 and pertuzumab were not affected by co-administration of the drugs. The best overall response included a partial response (PR) in 3 patients (50%) and stable disease (SD) in 2 patients (33%). CONCLUSIONS: The combination of T-DM1 and pertuzumab was tolerated and showed exploratory efficacy in Japanese patients with HER2-positive metastatic breast cancer.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Diarrea/epidemiología , Maitansina/análogos & derivados , Trastuzumab/uso terapéutico , Ado-Trastuzumab Emtansina , Anciano , Anticuerpos Monoclonales Humanizados/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Mama/diagnóstico por imagen , Mama/patología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Diarrea/inducido químicamente , Esquema de Medicación , Femenino , Humanos , Japón/epidemiología , Imagen por Resonancia Magnética , Maitansina/farmacocinética , Maitansina/uso terapéutico , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Receptor ErbB-2/metabolismo , Criterios de Evaluación de Respuesta en Tumores Sólidos , Tomografía Computarizada por Rayos X , Trastuzumab/farmacocinéticaRESUMEN
INTRODUCTION: Our objective was to determine correlations between the tumor uptake and T/B ratios for 89Zr-labeled T-DM1 (89Zr-DFO-T-DM1) in mice with human BC xenografts by microPET/CT and biodistribution studies with HER2 expression and response to treatment with trastuzumab-DM1 (T-DM1). METHODS: The tumor and normal tissue uptake and T/B ratios for 89Zr-DFO-T-DM1 (10⯵g; 7.0â¯MBq) incorporated into a therapeutic dose (60⯵g) were determined by microPET/CT and biodistribution studies at 96â¯h p.i. in NOD/SCID mice with s.c. MDA-MB-231 (5â¯×â¯104 HER2/cell), MDA-MB-361 (5â¯×â¯105 HER2/cell) and BT-474 (2â¯×â¯106 HER2/cell) human BC xenografts. Mice bearing these tumors were treated with T-DM1 (3.6â¯mg/kg every 3â¯weeks) and the tumor doubling time estimated by fitting of tumor volume vs. time curves. A tumor doubling time ratio (TDR) was calculated by dividing the doubling time for T-DM1 and normal saline treated control mice. The clonogenic survival (CS) of BC cells with increasing HER2 expression treated for 72â¯h in vitro with T-DM1 or trastuzumab (0-100⯵g/mL) was compared. Correlations were determined between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression, TDR and CS, and between CS and TDR. RESULTS: Uptake of 89Zr-DFO-T-DM1 in MDA-MB-231, MDA-MB-361 and BT-474 tumors was 2.4⯱â¯0.4%ID/g, 6.9⯱â¯2.2%ID/g and 9.8⯱â¯1.1%ID/g, respectively. There was a non-linear but direct correlation between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression with the T/B ratio ranging from 4.5⯱â¯0.7 for MDA-MB-231 to 18.2⯱â¯1.8 for MDA-MB-361 and 35.9⯱â¯5.1 for BT-474 xenografts. Tumor intensity on microPET/CT images was proportional to HER2 expression. The standard uptake value (SUV) for the tumors on the images was strongly correlated with the T/B ratio in biodistribution studies. There was a direct linear correlation between the T/B ratio for 89Zr-DFO-T-DM1 and TDR, with TDR ranging from 0.9 for MDA-MB-231 to 1.6 for MDA-MB-361 and 2.1 for BT-474 tumors. The cytotoxicity of T-DM1 in vitro on BC cells was dependent on HER2 expression but T-DM1 was more potent than trastuzumab. There was an inverse correlation between the TDR for mice treated with T-DM1 and CS of BC cells exposed in vitro to T-DM1. CONCLUSIONS: Based on the direct correlations between the T/B ratio for 89Zr-DFO-T-DM1 by PET and HER2 expression and response to T-DM1, our results suggest that PET with 89Zr-DFO-T-DM1 may predict response of HER2-positive BC to treatment with T-DM1. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our results suggest that PET with 89Zr-DFO-T-DM1 may predict response to treatment with T-DM1 in HER-positive BC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Maitansina/análogos & derivados , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radioisótopos , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Trastuzumab/uso terapéutico , Circonio , Ado-Trastuzumab Emtansina , Animales , Transporte Biológico , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/terapia , Maitansina/sangre , Maitansina/metabolismo , Maitansina/farmacocinética , Maitansina/uso terapéutico , Ratones , Distribución Tisular , Trastuzumab/sangre , Trastuzumab/farmacocinética , Resultado del TratamientoRESUMEN
Patients with HER2-positive tumors often suffer resistance to therapy, warranting development of novel treatment modalities. Affibody molecules are small affinity proteins which can be engineered to bind to desired targets. They have in recent years been found to allow precise targeting of cancer specific molecular signatures such as the HER2 receptor. In this study, we have investigated the potential of an affibody molecule targeting HER2, ZHER2:2891, conjugated with the cytotoxic maytansine derivate MC-DM1, for targeted cancer therapy. ZHER2:2891 was expressed as a monomer (ZHER2:2891), dimer ((ZHER2:2891)2) and dimer with an albumin binding domain (ABD) for half-life extension ((ZHER2:2891)2-ABD). All proteins had a unique C-terminal cysteine that could be used for efficient and site-specific conjugation with MC-DM1. The resulting affibody drug conjugates were potent cytotoxic molecules for human cells over-expressing HER2, with sub-nanomolar IC50-values similar to trastuzumab emtansine, and did not affect cells with low HER2 expression. A biodistribution study of a radiolabeled version of (ZHER2:2891)2-ABD-MC-DM1, showed that it was taken up by the tumor. The major site of off-target uptake was the kidneys and to some extent the liver. (ZHER2:2891)2-ABD-MC-DM1 was found to have a half-life in circulation of 14â¯h. The compound was tolerated well by mice at 8.5â¯mg/kg and was shown to extend survival of mice bearing HER2 over-expressing tumors. The findings in this study show that affibody molecules are a promising class of engineered affinity proteins to specifically deliver small molecular drugs to cancer cells and that such conjugates are potential candidates for clinical evaluation on HER2-overexpressing cancers.
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Antineoplásicos Fitogénicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Maitansina/administración & dosificación , Neoplasias/tratamiento farmacológico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Antineoplásicos Fitogénicos/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Maitansina/farmacocinética , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
Blood-brain/blood-tumor barriers (BBB and BTB) and interstitial transport may constitute major obstacles to the transport of therapeutics in brain tumors. In this study, we examined the impact of focused ultrasound (FUS) in combination with microbubbles on the transport of two relevant chemotherapy-based anticancer agents in breast cancer brain metastases at cellular resolution: doxorubicin, a nontargeted chemotherapeutic, and ado-trastuzumab emtansine (T-DM1), an antibody-drug conjugate. Using an orthotopic xenograft model of HER2-positive breast cancer brain metastasis and quantitative microscopy, we demonstrate significant increases in the extravasation of both agents (sevenfold and twofold for doxorubicin and T-DM1, respectively), and we provide evidence of increased drug penetration (>100 vs. <20 µm and 42 ± 7 vs. 12 ± 4 µm for doxorubicin and T-DM1, respectively) after the application of FUS compared with control (non-FUS). Integration of experimental data with physiologically based pharmacokinetic (PBPK) modeling of drug transport reveals that FUS in combination with microbubbles alleviates vascular barriers and enhances interstitial convective transport via an increase in hydraulic conductivity. Experimental data demonstrate that FUS in combination with microbubbles enhances significantly the endothelial cell uptake of the small chemotherapeutic agent. Quantification with PBPK modeling reveals an increase in transmembrane transport by more than two orders of magnitude. PBPK modeling indicates a selective increase in transvascular transport of doxorubicin through small vessel wall pores with a narrow range of sizes (diameter, 10-50 nm). Our work provides a quantitative framework for the optimization of FUS-drug combinations to maximize intratumoral drug delivery and facilitate the development of strategies to treat brain metastases.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Ado-Trastuzumab Emtansina , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Humanos , Maitansina/administración & dosificación , Maitansina/análogos & derivados , Maitansina/farmacocinética , Ratones , Microburbujas , Trastuzumab/administración & dosificación , Trastuzumab/farmacocinética , Ultrasonografía/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Our aim was to synthesize 89Zr-labeled trastuzumab-emtansine (89Zr-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of 89Zr-DFO-T-DM1 with 89Zr-DFO-trastuzumab. T-DM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing 89Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl3. SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with 89Zr at a specific activity of 0.55 MBq/µg in a 2 M Na2CO3/0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. 89Zr-DFO-T-DM1 exhibited high affinity HER2 binding ( Kd = 3.7 ± 0.4 nM) that was not significantly different than 89Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P = 0.06). The optimal time for tumor imaging with 89Zr-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with 89Zr-DFO-T-DM1 (10 µg, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake of 89Zr-DFO-T-DM1 was 4.1-fold significantly higher than 89Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ± 4.3%ID/g vs 10.6 ± 5.4%ID/g, respectively; P < 0.001). Tumor uptake of 89Zr-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher than 89Zr-DFO-trastuzumab (10.1 ± 3.6%ID/g vs 2.7 ± 0.5%ID/g; P < 0.001). The higher tumor uptake of 89Zr-DFO-T-DM1 compared to 89Zr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that 89Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET with 89Zr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that 89Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.
Asunto(s)
Antineoplásicos Inmunológicos/farmacocinética , Neoplasias de la Mama/diagnóstico por imagen , Inmunoconjugados/farmacocinética , Maitansina/análogos & derivados , Receptor ErbB-2/metabolismo , Trastuzumab/farmacocinética , Ado-Trastuzumab Emtansina , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Maitansina/administración & dosificación , Maitansina/química , Maitansina/farmacocinética , Ratones , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Radioisótopos , Receptor ErbB-2/antagonistas & inhibidores , Distribución Tisular , Trastuzumab/administración & dosificación , Trastuzumab/química , Microtomografía por Rayos X/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , CirconioRESUMEN
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) designed for the treatment of HER2-positive cancers. T-DM1 is composed of the humanized monoclonal antibody trastuzumab connected to a maytansine derivative cytotoxic drug, via a nonreducible thioether linker at random lysine residues, and therefore has a very complex molecular structure. It was anticipated that T-DM1 undergoes biotransformations in circulation. However, there was limited knowledge on these structural changes due to bioanalytical challenges. Here, we have investigated the in vivo biotransformations of T-DM1 using a high-resolution accurate-mass (HR/AM) mass spectrometry approach. Three types of biotransformations were characterized for T-DM1 in circulation in tumor-bearing mice, including cysteine or glutathione adduct formation via maleimide exchange, loss of maytansinol via ester hydrolysis, as well as addition of H2O via linker-drug hydrolysis. These results provide new insights into in vivo catabolism of T-DM1.
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Antineoplásicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Espectrometría de Masas/métodos , Maitansina/análogos & derivados , Neoplasias Gástricas/tratamiento farmacológico , Trastuzumab/farmacocinética , Ado-Trastuzumab Emtansina , Animales , Antineoplásicos/uso terapéutico , Biotransformación , Femenino , Humanos , Maitansina/farmacocinética , Maitansina/uso terapéutico , Ratones , Ratones SCID , Estructura Molecular , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugates are an emerging class of biopharmaceuticals changing the landscape of targeted chemotherapy. These conjugates combine the target specificity of monoclonal antibodies with the anti-cancer activity of small-molecule therapeutics. Several antibody-drug conjugates have received approval for the treatment of various types of cancer including gemtuzumab ozogamicin (Mylotarg®), brentuximab vedotin (Adcetris®), trastuzumab emtansine (Kadcyla®), and inotuzumab ozogamicin, which recently received approval (Besponsa®). In addition to these approved therapies, there are many antibody-drug conjugates in the drug development pipeline and in clinical trials, although these fall outside the scope of this article. Understanding the pharmacokinetics and pharmacodynamics of antibody-drug conjugates and the development of pharmacokinetic/pharmacodynamic models is indispensable, albeit challenging as there are many parameters to incorporate including the disposition of the intact antibody-drug conjugate complex, the antibody, and the drug agents following their dissociation in the body. In this review, we discuss how antibody-drug conjugates progressed over time, the challenges in their development, and how our understanding of their pharmacokinetics/pharmacodynamics led to greater strides towards successful targeted therapy programs.
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Inmunoconjugados , Modelos Biológicos , Ado-Trastuzumab Emtansina , Aminoglicósidos/farmacocinética , Aminoglicósidos/farmacología , Animales , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/farmacología , Gemtuzumab , Humanos , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inotuzumab Ozogamicina , Maitansina/análogos & derivados , Maitansina/farmacocinética , Maitansina/farmacología , Neoplasias/metabolismo , Trastuzumab/farmacocinética , Trastuzumab/farmacologíaRESUMEN
Antibody-mertansine (DM1) conjugates (AMCs) are among the very few active targeting therapeutics that are approved or clinically investigated for treating various cancers including metastatic breast cancer. However, none of the AMCs are effective for the treatment of triple-negative breast cancers (TNBCs). Here, we show that cRGD-decorated, redox-activatable micellar mertansine prodrug (cRGD-MMP) can effectively target and deliver DM1 to αvß3 integrin overexpressing MDA-MB-231 TNBC xenografts in nude mice, resulting in potent tumor growth inhibition. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that cRGD-MMP had obvious targetability to MDA-MB-231 cells with a low half-maximal inhibitory concentration (IC50) of 0.18 µM, which was close to that of free DM1 and 2.2-fold lower than that of micellar mertansine prodrug (MMP; nontargeting control). The confocal microscopy studies demonstrated that cRGD-MMP mediated a clearly more efficient cellular uptake and intracellular release of doxorubicin (used as a fluorescent anticancer drug model) in MDA-MB-231 cells. Notably, cRGD-MMP loaded with 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR; a hydrophobic near-infrared dye) was shown to quickly accumulate in the MDA-MB-231 tumor with strong DiR fluorescence from 2 to 24 h post injection. MMP loaded with DiR could also accumulate in the tumor, although significantly less than cRGD-MMP. The biodistribution studies revealed a high DM1 accumulation of 8.1%ID/g in the tumor for cRGD-MMP at 12 h post injection. The therapeutic results demonstrated that cRGD-MMP effectively suppressed MDA-MB-231 tumor growth at 1.6 mg DM1 equiv./kg without causing noticeable side effects, as shown by little body weight loss and histological analysis. This MMP has appeared as a promising platform for potent treatment of TNBCs.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Maitansina/farmacología , Terapia Molecular Dirigida/métodos , Profármacos/farmacocinética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Doxorrubicina/química , Femenino , Humanos , Maitansina/farmacocinética , Ratones Desnudos , Micelas , Oligopéptidos/química , Oligopéptidos/metabolismo , Profármacos/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate comprising trastuzumab conjugated via a stable thioether linker to DM1, a highly potent cytotoxic agent. A population pharmacokinetics (PK) analysis was performed to characterize T-DM1 PK and evaluate the impact of patient characteristics on T-DM1 PK in previously treated patients with HER2-positive advanced gastric cancer (AGC). METHODS: Following T-DM1 weekly or every three weeks dosing, T-DM1 concentration measurements (n = 780) were collected from 136 patients in the GATSBY (NCT01641939) study and analyzed using nonlinear mixed effects modeling. The influence of demographic, baseline laboratory, and disease characteristics on T-DM1 PK was examined. RESULTS: T-DM1 PK was best described by a two-compartment model with parallel linear and nonlinear (Michaelis-Menten) elimination from the central compartment. The final population model estimated linear clearance (CL) of 0.79 L/day, volume of distribution in the central compartment (V c) of 4.48 L, distribution clearance (Q) of 0.62 L/day, volume of distribution in the peripheral compartment (V p) of 1.49 L, nonlinear CL of 2.06 L/day, and KM of 1.63 µg/mL. Parameter uncertainty was low to moderate for fixed effects, except KM (estimated with poor precision). Patients with high body weight and low baseline trastuzumab concentrations had significantly faster linear CL; those with higher body weight had significantly larger V c. CONCLUSIONS: In a HER2-positive AGC population, T-DM1 PK was best described by a two-compartment model with parallel linear and nonlinear elimination. Baseline body weight and trastuzumab concentration were identified as significant covariates for T-DM1 PK in a HER2-positive AGC population.
Asunto(s)
Maitansina/análogos & derivados , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Trastuzumab/uso terapéutico , Ado-Trastuzumab Emtansina , Adulto , Anciano , Humanos , Maitansina/farmacocinética , Maitansina/uso terapéutico , Persona de Mediana Edad , Trastuzumab/farmacocinética , Adulto JovenRESUMEN
Systems pharmacokinetic (PK) models that can characterize and predict whole body disposition of antibody-drug conjugates (ADCs) are needed to support (i) development of reliable exposure-response relationships for ADCs and (ii) selection of ADC targets with optimal tumor and tissue expression profiles. Towards this goal, we have developed a translational physiologically based PK (PBPK) model for ADCs, using T-DM1 as a tool compound. The preclinical PBPK model was developed using rat data. Biodistribution of DM1 in rats was used to develop the small molecule PBPK model, and the PK of conjugated trastuzumab (i.e., T-DM1) in rats was characterized using platform PBPK model for antibody. Both the PBPK models were combined via degradation and deconjugation processes. The degradation of conjugated antibody was assumed to be similar to a normal antibody, and the deconjugation of DM1 from T-DM1 in rats was estimated using plasma PK data. The rat PBPK model was translated to humans to predict clinical PK of T-DM1. The translation involved the use of human antibody PBPK model to characterize the PK of conjugated trastuzumab, use of allometric scaling to predict human clearance of DM1 catabolites, and use of monkey PK data to predict deconjugation of DM1 in the clinic. PBPK model-predicted clinical PK profiles were compared with clinically observed data. The PK of total trastuzumab and T-DM1 were predicted reasonably well, and slight systemic deviations were observed for the PK of DM1-containing catabolites. The ADC PBPK model presented here can serve as a platform to develop models for other ADCs.
Asunto(s)
Antineoplásicos Inmunológicos/farmacocinética , Maitansina/análogos & derivados , Modelos Biológicos , Trastuzumab/farmacocinética , Ado-Trastuzumab Emtansina , Animales , Humanos , Maitansina/farmacocinética , RatasRESUMEN
AIMS: We conducted population pharmacokinetic (PopPK) and exposure-response analyses for trastuzumab emtansine (T-DM1), to assess the need for T-DM1 dose optimization in patients with low exposure by using TH3RESA [A Study of Trastuzumab Emtansine in Comparison With Treatment of Physician's Choice in Patients With human epidermal growth factor receptor 2 (HER2)-positive Breast Cancer Who Have Received at Least Two Prior Regimens of HER2-directed Therapy] study data (NCT01419197). The randomized phase III TH3RESA study investigated T-DM1 vs. treatment of physician's choice (TPC) in patients with heavily pretreated HER2-positive advanced breast cancer. METHODS: We compared a historical T-DM1 PopPK model with T-DM1 pharmacokinetics in TH3RESA and performed exposure-response analyses using model-predicted cycle 1 maximum concentration (Cmax ), cycle 1 minimum concentration (Cmin ) and area under the concentration-time curve at steady state (AUCss ). Kaplan-Meier analyses [overall survival (OS), progression-free survival (PFS)] and logistic regression [overall response rate (ORR), safety] were stratified by T-DM1 exposure metrics. Survival hazard ratios (HRs) in the lowest exposure quartile (Q1) of cycle 1 Cmin were compared with matched TPC-treated patients. RESULTS: T-DM1 concentrations in TH3RESA were described well by the historical PopPK model. Patients with higher cycle 1 Cmin and AUCss exhibited numerically longer median OS and PFS and higher ORR than patients with lower exposure. Exposure-response relationships were less evident for cycle 1 Cmax . No relationship between exposure and safety was identified. HRs for the comparison of T-DM1-treated patients in the Q1 subgroup with matched TPC-treated patients were 0.96 [95% confidence interval (CI) 0.63, 1.47] for OS and 0.92 (95% CI 0.64, 1.32) for PFS. CONCLUSIONS: Exposure-response relationships for efficacy were inconsistent across exposure metrics. HRs for survival in patients in the lowest T-DM1 exposure quartile vs. matched TPC-treated patients suggest that, compared with TCP, the approved T-DM1 dose is unlikely to be detrimental to patients with low exposure.
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Antineoplásicos Inmunológicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Maitansina/análogos & derivados , Modelos Biológicos , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/farmacocinética , Moduladores de Tubulina/farmacocinética , Ado-Trastuzumab Emtansina , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/sangre , Área Bajo la Curva , Neoplasias de la Mama/sangre , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/mortalidad , Supervivencia sin Enfermedad , Femenino , Humanos , Infusiones Intravenosas , Estimación de Kaplan-Meier , Modelos Logísticos , Maitansina/administración & dosificación , Maitansina/efectos adversos , Maitansina/sangre , Maitansina/farmacocinética , Tasa de Depuración Metabólica , Dinámicas no Lineales , Modelos de Riesgos Proporcionales , Receptor ErbB-2/metabolismo , Medición de Riesgo , Trastuzumab/administración & dosificación , Trastuzumab/efectos adversos , Trastuzumab/sangre , Resultado del Tratamiento , Moduladores de Tubulina/administración & dosificación , Moduladores de Tubulina/efectos adversos , Moduladores de Tubulina/sangreRESUMEN
Successful clinical translation of antibody-drug conjugates (ADCs) can be challenging due to complex pharmacokinetics and differences between preclinical and clinical tumors. To facilitate this translation, we have developed a general pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulation (M&S)-based strategy for ADCs. Here we present the validation of this strategy using T-DM1 as a case study. A previously developed preclinical tumor disposition model for T-DM1 (Singh and Shah, AAPSJ. 2015; 18(4):861-875) was used to develop a PK-PD model that can characterize in vivo efficacy of T-DM1 in preclinical tumor models. The preclinical data was used to estimate the efficacy parameters for T-DM1. Human PK of T-DM1 was a priori predicted using allometric scaling of monkey PK parameters. The predicted human PK, preclinically estimated efficacy parameters, and clinically observed volume and growth parameters for breast cancer were combined to develop a translated clinical PK-PD model for T-DM1. Clinical trial simulations were performed using the translated PK-PD model to predict progression-free survival (PFS) and objective response rates (ORRs) for T-DM1. The model simulated PFS rates for HER2 1+ and 3+ populations were comparable to the rates observed in three different clinical trials. The model predicted only a modest improvement in ORR with an increase in clinically approved dose of T-DM1. However, the model suggested that a fractionated dosing regimen (e.g., front loading) may provide an improvement in the efficacy. In general, the PK-PD M&S-based strategy presented here is capable of a priori predicting the clinical efficacy of ADCs, and this strategy has been now retrospectively validated for all clinically approved ADCs.
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Inmunoconjugados/farmacología , Maitansina/análogos & derivados , Trastuzumab/farmacología , Ado-Trastuzumab Emtansina , Humanos , Inmunoconjugados/farmacocinética , Maitansina/farmacocinética , Maitansina/farmacología , Modelos Biológicos , Trastuzumab/farmacocinéticaRESUMEN
Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of â¼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below â¼6 had comparable clearance rates, but for those with an average DAR of â¼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (â¼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.