RESUMEN
PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01-0.3 mg/kg/dose) and GLP toxicity study (0.1-3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 µg/mL and 37.9 µg*h/mL, respectively.
Asunto(s)
Anticuerpos Monoclonales , Receptor de Muerte Celular Programada 1 , Animales , Macaca fascicularis , Interleucina-15 , Administración Intravenosa , Citocinas , Inhibidores de Puntos de Control InmunológicoRESUMEN
The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/terapia , Inmunoterapia , Interleucina-15/uso terapéutico , Melanoma Experimental/terapia , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Humanos , Interleucina-15/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Development of antagonistic mAbs that specifically target the immune checkpoint receptor, programmed cell death protein-1 (PD-1), is of great interest for cancer immunotherapy. Here, we report the biophysical characteristics and nonclinical antagonistic activities of sasanlimab (PF-06801591), a humanized anti-PD-1 antibody of IgG4 isotype. We show that sasanlimab binds selectively and with similar high potency to human and cynomolgus monkey PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, with no detectable Fc-dependent effector function. The binding of sasanlimab to human and cynomolgus PD-1 is associated with the formation of a stable complex, which is likely to be the main driver of this high-affinity interaction. In vitro, sasanlimab significantly augmented T-cell proliferation and cytokine production in mixed lymphocyte reaction and superantigen stimulation assays. In vivo, sasanlimab accelerated the incidence of GvHD by enhancing T-cell proliferation and cytokine secretion in a xenogeneic model of acute GvHD and halted the growth of MC-38 colon adenocarcinoma tumors in human PD-1 knock-in mice. Pharmacokinetic and toxicokinetic findings from cynomolgus monkey showed that sasanlimab was active and well-tolerated. Taken together, the data presented here support the clinical development of sasanlimab for the treatment of patients with advanced cancers as a single agent or in combination with other immunotherapies.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Animales , Línea Celular Tumoral , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , RatonesRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Complejo CD3/inmunología , Claudinas/inmunología , Inmunoconjugados/uso terapéutico , Neoplasias Pancreáticas/terapia , Neoplasias Gástricas/terapia , Adenocarcinoma/terapia , Animales , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Humanos , Inmunoconjugados/sangre , Ratones , Neoplasias Pancreáticas/sangre , Ratas , Neoplasias Gástricas/sangreRESUMEN
The increasing use of multiple immunomodulatory (IMD) agents for cancer therapies (e.g. antibodies targeting immune checkpoints, bispecific antibodies, and chimeric antigen receptor [CAR]-T cells), is raising questions on their potential immunogenicity and effects on treatment. In this review, we outline the mechanisms of action (MOA) of approved, antibody-based IMD agents, potentially related to their immunogenicity, and discuss the reported incidence of anti-drug antibodies (ADA) as well as their clinical relevance in patients with cancer. In addition, we discuss the impact of the administration route and potential strategies to reduce the incidence of ADA and manage treated patients. Analysis of published reports indicated that the risk of immunogenicity did not appear to correlate with the MOA of anti-programmed death 1 (PD-1)/PD-ligand 1 monoclonal antibodies nor to substantially affect treatment with most of these agents in the majority of patients evaluated to date. Treatment with B-cell depleting agents appears associated with a low risk of immunogenicity. No significant difference in ADA incidence was found between the intravenous and subcutaneous administration routes for a panel of non-oncology IMD antibodies. Additionally, while the data suggest a higher likelihood of immunogenicity for antibodies with T-cell or antigen-presenting cell (APC) targets versus B-cell targets, it is possible to have targets expressed on APCs or T cells and still have a low incidence of immunogenicity.
Asunto(s)
Antineoplásicos Inmunológicos/efectos adversos , Enfermedades del Sistema Inmune/inmunología , Inmunoterapia Adoptiva/efectos adversos , Depleción Linfocítica/efectos adversos , Neoplasias/terapia , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/inmunología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Humanos , Enfermedades del Sistema Inmune/inducido químicamente , Enfermedades del Sistema Inmune/epidemiología , Enfermedades del Sistema Inmune/prevención & control , Inmunosupresores/uso terapéutico , Inmunoterapia Adoptiva/métodos , Incidencia , Depleción Linfocítica/métodos , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptores Quiméricos de Antígenos/inmunología , Resultado del TratamientoRESUMEN
Trop-2, also known as TACSTD2, EGP-1, GA733-1, and M1S1, is frequently expressed on a variety of human carcinomas, and its expression is often associated with poor prognosis of the diseases. However, it is also present on the epithelium of several normal tissues. A comprehensively designed Trop-2-targeting antibody-drug conjugate (ADC), balancing both efficacy and toxicity, is therefore necessary to achieve clinical utility. To this end, we developed a cleavable Trop-2 ADC (RN927C) using a site-specific transglutaminase-mediated conjugation method and a proprietary microtubule inhibitor (MTI) linker-payload, PF-06380101. Robust in vitro cytotoxicity of RN927C was observed on a panel of Trop-2-expressing tumor cell lines, with IC50 generally in the subnanomolar range. As expected for an MTI-containing ADC, RN927C readily induced mitotic arrest of treated cells in vitro and in vivo, followed by subsequent cell death. The in vivo efficacy of RN927C was tested in multiple cell line and patient-derived xenograft tumor models, including pancreatic, lung, ovarian, and triple-negative breast tumor types. Single-dose administration of RN927C at 0.75 to 3 mg/kg was generally sufficient to induce sustained regression of Trop-2-expressing tumors and showed superior efficacy over standard treatment with paclitaxel or gemcitabine. Administration of RN927C in nonhuman primate toxicity studies resulted in target-mediated effects in skin and oral mucosa, consistent with Trop-2 expression in these epithelial tissues with minimal, non-dose limiting off-target toxicities. On the basis of the combined efficacy and safety results, RN927C is postulated to have a favorable therapeutic index for treatment of solid tumors. Mol Cancer Ther; 15(11); 2698-708. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Inmunoconjugados/farmacología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Femenino , Expresión Génica , Humanos , Inmunoconjugados/química , Lisosomas , Ratones , Mitosis/efectos de los fármacos , Mitosis/genética , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Interleukin-7 receptor α (IL-7Rα) is associated with autoimmune disease. Blocking its activation by interleukin-7 (IL-7) with a therapeutic monoclonal antibody may reduce pathogenic T cells and effectively control the autoimmune response in these disorders. METHODS: Two flow cytometry-based assays were developed and implemented to evaluate the interaction between cell surface IL-7Rα and an anti-IL-7Rα monoclonal antibody (Ab1). The receptor occupancy assay utilized competing and noncompeting commercial detection antibodies for "free" and "total" IL-7Rα, respectively. STAT5 phosphorylation (pSTAT5) was measured as a proximal biomarker of IL-7Rα inhibition by Ab1. RESULTS: Monkeys administered Ab1 had no free IL-7Rα detectable on the CD3+ T cell surface at 0.25 hours postdose through day 4, in all treatment groups. Ab1 treatment resulted in a significant reduction in total IL-7Rα, dropping to 53%, 44%, and 55% on day 4 at 0.3, 3, and 30 mg/kg, respectively, compared to predose levels. There were treatment-related decreases in the ability of IL-7 to induce STAT5 phosphorylation in both CD4+ and CD8+ T cells in monkey blood samples from all treated animals from 0.25 hours through Day 4 postdose. CONCLUSIONS: The nonclinical receptor occupancy assay was developed and applied to detect free and total IL-7Rα on the surface of CD3+ T cells in cynomolgus monkeys treated with Ab1. The results showed good correlation with the phosphorylation of STAT5 and serum concentration of Ab1. The approach for IL-7Rα occupancy and pSTAT5 measurements established in monkeys can be utilized in clinical trials for pharmacokinetic/pharmacodynamic evaluation of Ab1 effect in humans.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Citometría de Flujo , Receptores de Interleucina-7/inmunología , Factor de Transcripción STAT5/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Enfermedades Autoinmunes/terapia , Humanos , Interleucina-7/inmunología , Macaca fascicularis/inmunología , Fosforilación , Receptores de Interleucina-7/antagonistas & inhibidores , Factor de Transcripción STAT5/antagonistas & inhibidoresRESUMEN
An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent drug molecule conjugated to a monoclonal antibody. As the number of ADCs in various stages of nonclinical and clinical development has been increasing, pharmaceutical companies have been exploring diverse approaches to understanding the disposition of ADCs. To identify the key absorption, distribution, metabolism, and excretion (ADME) issues worth examining when developing an ADC and to find optimal scientifically based approaches to evaluate ADC ADME, the International Consortium for Innovation and Quality in Pharmaceutical Development launched an ADC ADME working group in early 2014. This white paper contains observations from the working group and provides an initial framework on issues and approaches to consider when evaluating the ADME of ADCs.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Inmunoconjugados/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Industria Farmacéutica/métodos , HumanosRESUMEN
Bococizumab is a humanized monoclonal IgG2Δa antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9) for the treatment of hyperlipidemia. The evaluation of potential effects on embryo-fetal development was conducted in the rat. In a pharmacokinetic/pharmacodynamic study bococizumab was administered intravenously to pregnant Sprague-Dawley (SD) rats (n = 8/group) at 0, 10, 30, and 100 mg/kg during organogenesis. Maternal and fetal bococizumab, total cholesterol and HDL concentrations were determined. Bococizumab was well tolerated and there were no effects on ovarian or uterine parameters. Maternal and fetal bococizumab exposure increased with increasing dose, with a corresponding dose-dependent decrease in fetal cholesterol levels. Maternal cholesterol levels were decreased significantly, with reductions that were of a similar magnitude regardless of dose. In the definitive embryo-fetal development study bococizumab was administered to pregnant SD rats (n = 20/group) at 0, 10, 30, and 100 mg/kg and no adverse maternal or developmental effects were observed up to 100 mg/kg. These studies have provided an appropriate and relevant safety assessment of bococizumab in pregnant rats to inform human risk assessment, demonstrating no adverse effects on embryo-fetal development at magnitudes greater than anticipated clinical exposure and in the presence of maximal reductions in maternal cholesterol and dose-dependent reductions in fetal cholesterol.
Asunto(s)
Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales Humanizados/administración & dosificación , Colesterol/sangre , Desarrollo Fetal/fisiología , Intercambio Materno-Fetal/fisiología , Serina Endopeptidasas/sangre , Animales , Anticuerpos Monoclonales Humanizados/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Desarrollo Fetal/efectos de los fármacos , Intercambio Materno-Fetal/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/sangre , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Proproteína Convertasa 9 , Ratas , Ratas Sprague-DawleyRESUMEN
The design of potent Pin1 inhibitors has been challenging because its active site specifically recognizes a phospho-protein epitope. The de novo design of phosphate-based Pin1 inhibitors focusing on the phosphate recognition pocket and the successful replacement of the phosphate group with a carboxylate have been previously reported. The potency of the carboxylate series is now further improved through structure-based optimization of ligand-protein interactions in the proline binding site which exploits the H-bond interactions necessary for Pin1 catalytic function. Further optimization using a focused library approach led to the discovery of low nanomolar non-phosphate small molecular Pin1 inhibitors. Structural modifications designed to improve cell permeability resulted in Pin1 inhibitors with low micromolar anti-proliferative activities against cancer cells.
Asunto(s)
Bencimidazoles/farmacología , Ácidos Carboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Fosfatos/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/metabolismo , Relación Estructura-ActividadRESUMEN
A mathematical pharmacokinetic/anti-drug-antibody (PK/ADA) model was constructed for quantitatively assessing immunogenicity for therapeutic proteins. The model is inspired by traditional pharmacokinetic/pharmacodynamic (PK/PD) models, and is based on the observed impact of ADA on protein drug clearance. The hypothesis for this work is that altered drug PK contains information about the extent and timing of ADA generation. By fitting drug PK profiles while accounting for ADA-mediated drug clearance, the model provides an approach to characterize ADA generation during the study, including the maximum ADA response, sensitivity of ADA response to drug dose level, affinity maturation rate, time lag to observe an ADA response, and the elimination rate for ADA-drug complex. The model also provides a mean to estimate putative concentration-time profiles for ADA, ADA-drug complex, and ADA binding affinity-time profile. When simulating ADA responses to various drug dose levels, bell-shaped dose-response curves were generated. The model contains simultaneous quantitative modeling and provides estimation of the characteristics of therapeutic protein drug PK and ADA responses in vivo. With further experimental validation, the model may be applied to the simulation of ADA response to therapeutic protein drugs in silico, or be applied in subsequent PK/PD models.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Fenómenos Inmunogenéticos/efectos de los fármacos , Fenómenos Inmunogenéticos/fisiología , Modelos Teóricos , Animales , Anticuerpos Monoclonales/inmunología , Relación Dosis-Respuesta Inmunológica , Macaca fascicularis , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs are mainly caused by proinflammatory cytokine or cytokine modulator-mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a "Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches" held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Proteínas/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Evaluación Preclínica de Medicamentos , Industria Farmacéutica , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Proteínas/farmacología , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The industry-wide biopharmaceutical (i.e., biologic, biotherapeutic) pipeline has been growing at an astonishing rate over the last decade with the proportion of approved new biological entities to new chemical entities on the rise. As biopharmaceuticals appear to be growing in complexity in terms of their structure and mechanism of action, so are interpretation, analysis, and prediction of their quantitative pharmacology. We present here a modeling and simulation (M&S) framework for the successful preclinical development of monoclonal antibodies (as an illustrative example of biopharmaceuticals) and discuss M&S strategies for its implementation. Critical activities during early discovery, lead optimization, and the selection of starting doses for the first-in-human study are discussed in the context of pharmacokinetic-pharmacodynamic (PKPD) and M&S. It was shown that these stages of preclinical development are and should be reliant on M&S activities including systems biology (SB), systems pharmacology (SP), and translational pharmacology (TP). SB, SP, and TP provide an integrated and rationalized framework for decision making during the preclinical development phase. In addition, they provide increased target and systems understanding, describe and interpret data generated in vitro and in vivo, predict human PKPD, and provide a rationalized approach to designing the first-in-human study.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Modelos Biológicos , Investigación Biomédica Traslacional/métodos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Técnicas de Apoyo para la Decisión , Modelos Animales de Enfermedad , Cálculo de Dosificación de Drogas , Humanos , Dinámicas no Lineales , Biología de SistemasRESUMEN
Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones Desnudos , Recurrencia , Esferoides Celulares/efectos de los fármacos , Taxoides/farmacología , Carga Tumoral , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The P21-activated kinases (PAK) are emerging antitumor therapeutic targets. In this paper, we describe the discovery of potent PAK inhibitors guided by structure-based drug design. In addition, the efflux of the pyrrolopyrazole series was effectively reduced by applying multiple medicinal chemistry strategies, leading to a series of PAK inhibitors that are orally active in inhibiting tumor growth in vivo.
Asunto(s)
Antineoplásicos/síntesis química , Pirazoles/síntesis química , Pirroles/síntesis química , Quinasas p21 Activadas/antagonistas & inhibidores , Administración Oral , Amidas/síntesis química , Amidas/farmacocinética , Amidas/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Carbamatos/química , Carbamatos/farmacocinética , Carbamatos/farmacología , Cristalografía por Rayos X , Perros , Humanos , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Conformación Molecular , Permeabilidad , Pirazoles/farmacocinética , Pirazoles/farmacología , Pirroles/farmacocinética , Pirroles/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (k(on) and k(off)), internalization of the drug-target complex (k(int)), target degradation rate (k(deg)), and target abundance (R(0)), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Simulación por Computador , Modelos Biológicos , Receptores de Activinas Tipo I/inmunología , Animales , Anticuerpos Monoclonales Humanizados , Ensayos Clínicos Fase I como Asunto , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Haplorrinos , Humanos , Inmunoglobulina G/inmunología , Ratones , Resonancia por Plasmón de Superficie , Venas UmbilicalesRESUMEN
Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvß3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvß3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvß3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a Kras(G12D-LSL)p53(fl/fl) subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/secundario , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Osteopontina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Factores Quimiotácticos/química , Factores Quimiotácticos/inmunología , Factores Quimiotácticos/metabolismo , Modelos Animales de Enfermedad , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Integrinas/metabolismo , Ratones , Ratones SCID , Datos de Secuencia Molecular , Osteopontina/química , Osteopontina/inmunología , Unión Proteica , Estabilidad Proteica , Trombina/metabolismoRESUMEN
The expression patterns and functional roles of three osteopontin splice variants (OPNa, b, and c) in cancer metastasis and progression are not well understood due to the lack of reliable assays to differentiate the isoforms. We have developed a mass spectrometric method to quantify OPN isoforms in human plasma. The method is based on the immunocapture of all OPN isoforms, followed by MRM-MS analysis of isoform-specific tryptic peptides. We were able to simultaneously identify and quantify all three isoforms in the plasma of 10 healthy individuals and 10 non-small cell lung cancer (NSCLC) patients. Our results show that none of the OPN splice variants is cancer specific. However, OPNa, the major isoform in healthy and NSCLC plasma, is substantially elevated in NSCLC patients, whereas OPNb and OPNc are at equivalent levels in two populations.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Osteopontina/sangre , ARN Neoplásico/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Estudios de Casos y Controles , Línea Celular Tumoral , Exones , Humanos , Inmunoprecipitación , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Osteopontina/genética , Fragmentos de Péptidos/análisis , Isoformas de Proteínas , Estructura Terciaria de Proteína , Estados UnidosRESUMEN
Increasing use of therapeutic proteins (TPs) in polypharmacy settings calls for more in-depth understanding of the biological interactions that can lead to increased toxicity or loss of pharmacological effect. Factors such as patient population, medications that are likely to be coadministered in that population, clearance mechanisms of a TP, and concomitant drugs have to be taken into account to determine the potential for drug-drug interactions (DDIs). The most well documented TP DDI mechanism involves cytokine-mediated changes in drug-metabolizing enzymes. Because of the limitations of the current preclinical models for addressing this type of DDI, clinical evaluation is currently the most reliable approach. Other DDI mechanisms need to be addressed on a case-by-case basis. These include altered clearance of TPs resulting from the changes in the target protein levels by the concomitant medication, displacement of TPs from binding proteins, modulation of Fcγ receptor expression, and others. The purpose of this review is to introduce the approach used by Pfizer scientists for evaluation of the DDI potential of novel TP products during drug discovery and development.