RESUMO
Chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of hematologic malignancies and have potentials for solid tumor treatment. To overcome limited CAR T cell infiltration to solid tumors, local delivery of CAR T cells is a practical strategy that has shown promising therapeutic outcome and safety profile in the clinic. It is of great interest to understand the impact of dosing routes on CAR T cell distribution, subsequent proliferation and tumor killing in a quantitative manner to identify key factors that contribute to CAR T efficacy and safety. In this study, we established mouse minimal physiologically-based pharmacokinetic (mPBPK) models combined with pharmacodynamic (PD) components to delineate CAR T cell distribution, proliferation, tumor growth, and tumor cell killing in the cases of pleural and liver tumors. The pleural tumor model reasonably captured published CAR T cellular kinetic and tumor growth profiles in mice. The mPBPK-PD simulation of a liver tumor mouse model showed a substantial increase in initial tumor infiltration and earlier CAR T cell proliferation with local hepatic artery delivery compared to portal vein and intravenous (i.v.) injections whereas portal vein injection showed little difference from i.v. administration, suggesting the importance of having the injection site close to tumor for maximal effect of non-systemic administration. Blood flow rate in the liver tumor was found to be a sensitive parameter for cellular kinetics and efficacy, indicating a potential role of tumor vascularization in the efficacy of CAR T cell therapies.
Assuntos
Neoplasias Hepáticas , Receptores de Antígenos Quiméricos , Animais , Proliferação de Células , Modelos Animais de Doenças , Imunoterapia Adotiva , Camundongos , Linfócitos TRESUMO
The objective of this work was to develop a systems pharmacokinetics-pharmacodynamics (PK-PD) model that can characterize in vivo bystander effect of antibody-drug conjugate (ADC) in a heterogeneous tumor. To accomplish this goal, a coculture xenograft tumor with 50% GFP-MCF7 (HER2-low) and 50% N87 (HER2-high) cells was developed. The relative composition of a heterogeneous tumor for each cell type was experimentally determined by immunohistochemistry analysis. Trastuzumab-vc-MMAE (T-vc-MMAE) was used as a tool ADC. Plasma and tumor PK of T-vc-MMAE was analyzed in N87, GFP-MCF7, and coculture tumor-bearing mice. In addition, tumor growth inhibition (TGI) studies were conducted in all three xenografts at different T-vc-MMAE dose levels. To characterize the PK of ADC in coculture tumors, our previously published tumor distribution model was evolved to account for different cell populations. The evolved tumor PK model was able to a priori predict the PK of all ADC analytes in the coculture tumors reasonably well. The tumor PK model was subsequently integrated with a PD model that used intracellular tubulin occupancy to drive ADC efficacy in each cell type. The final systems PK-PD model was able to simultaneously characterize all the TGI data reasonably well, with a common set of parameters for MMAE-induced cytotoxicity. The model was later used to simulate the effect of different dosing regimens and tumor compositions on the bystander effect of ADC. The model simulations suggested that dose-fractionation regimen may further improve overall efficacy and bystander effect of ADCs by prolonging the tubulin occupancy in each cell type. SIGNIFICANCE STATEMENT: A PK-PD analysis is presented to understand bystander effect of Trastuzumab-vc-MMAE ADC in antigen (Ag)-low, Ag-high, and coculture (i.e., Ag-high + Ag-low) xenograft mice. This study also describes a novel single cell-level systems PK-PD model to characterize in vivo bystander effect of ADCs. The proposed model can serve as a platform to mathematically characterize multiple cell populations and their interactions in tumor tissues. Our analysis also suggests that fractionated dosing regimen may help improve the bystander effect of ADCs.
Assuntos
Efeito Espectador/efeitos dos fármacos , Imunoconjugados/farmacologia , Imunoconjugados/farmacocinética , Modelos Biológicos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Humanos , Distribuição TecidualRESUMO
Antibiotics with novel bactericidal mechanisms of action are urgently needed. The antibiotic acyldepsipeptide 4 (ADEP4) activates the ClpP protease and causes cells to self-digest. The effects of ADEP4 and ClpP activation have not been characterized sufficiently for the enterococci, which are important pathogens known for high levels of acquired and intrinsic antibiotic resistance. In the present study, ADEP4 was found to be potently active against both Enterococcus faecalis and Enterococcus faecium, with MIC90s of 0.016 µg/ml and 0.031 µg/ml, respectively. ClpP purified from E. faecium was found to bind ADEP4 in a surface plasmon resonance analysis, and ClpP activation by ADEP4 was demonstrated biochemically with a ß-casein digestion assay. In addition, E. faecium ClpP was crystallized in the presence of ADEP4, revealing ADEP4 binding to ClpP in the activated state. These results confirm that the anti-enterococcal activity of ADEP4 occurs through ClpP activation. In killing curve assays, ADEP4 was found to be bactericidal against stationary-phase vancomycin-resistant E. faecalis (VRE) strain V583, and resistance development was prevented when ADEP4 was combined with multiple classes of approved antibiotics. ADEP4 in combination with partnering antibiotics also eradicated mature VRE biofilms within 72 h of treatment. Biofilm killing with ADEP4 antibiotic combinations was superior to that with the clinically used combinations ampicillin-gentamicin and ampicillin-daptomycin. In a murine peritoneal septicemia model, ADEP4 alone was as effective as ampicillin. ADEP4 coadministered with ampicillin was significantly more effective than either drug alone. These data suggest that ClpP-activating antibiotics may be useful for treating enterococcal infections.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Depsipeptídeos/farmacologia , Endopeptidase Clp/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Ampicilina/farmacologia , Animais , Antibacterianos/química , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cristalografia por Raios X , Depsipeptídeos/química , Modelos Animais de Doenças , Combinação de Medicamentos , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecium/enzimologia , Enterococcus faecium/genética , Enterococcus faecium/crescimento & desenvolvimento , Ativação Enzimática/efeitos dos fármacos , Feminino , Expressão Gênica , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Sepse/tratamento farmacológico , Sepse/microbiologia , Vancomicina/farmacologia , Enterococos Resistentes à Vancomicina/enzimologia , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/crescimento & desenvolvimentoRESUMO
The main objective of this work was to understand and mathematically characterize the cellular disposition of a tool antibody-drug conjugate (ADC), trastuzumab-valine-citrulline-monomethyl auristatin E (T-vc-MMAE). Toward this goal, three different analytical methods were developed to measure the concentrations of different ADC-related analytes in the media and cell lysate. A liquid chromatography-tandem mass spectrometry method was developed to quantify unconjugated drug (i.e., MMAE) concentrations, a forced deconjugation method was developed to quantify total drug concentrations, and an enzyme-linked immunosorbent assay method was developed to quantify total antibody (i.e., trastuzumab) concentrations. Cellular disposition studies were conducted in low-HER2-(GFP-MCF7) and high-HER2-expressing (N87) cell lines, following continuous or 2-hour exposure to MMAE and T-vc-MMAE. Similar intracellular accumulation of MMAE was observed between two cell lines following incubation with plain MMAE. However, when incubated with T-vc-MMAE, much higher intracellular exposures of unconjugated drug, total drug, and total antibody were observed in N87 cells compared with GFP-MCF7 cells. A novel single-cell disposition model was developed to simultaneously characterize in vitro pharmacokinetics of all three analytes of the ADC in the media and cellular space. The model was able to characterize all the data well and provided robust estimates of MMAE influx rate, MMAE efflux rate, and intracellular degradation rate for T-vc-MMAE. ADC internalization and degradation rates, HER2 expression, and MMAE efflux rate were found to be the key parameters responsible for intracellular exposure to MMAE, on the basis of a global sensitivity analysis. The single-cell pharmacokinetics model for ADCs presented here is expected to provide a better framework for characterizing bystander effect of ADCs.
Assuntos
Antineoplásicos Imunológicos/farmacocinética , Imunoconjugados/farmacocinética , Modelos Biológicos , Modelos Químicos , Oligopeptídeos/farmacocinética , Trastuzumab/farmacocinética , Efeito Espectador , Cromatografia Líquida de Alta Pressão/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Células MCF-7 , Modelos Estruturais , Espectrometria de Massas em Tandem/métodosRESUMO
Bacterial infections, caused by Mycobacterium tuberculosis and other problematic bacterial pathogens, continue to pose a significant threat to global public health. As such, new chemotype antibacterial agents are desperately needed to fuel and strengthen the antibacterial drug discovery and development pipeline. As part of our antibacterial research program to develop natural product-inspired new antibacterial agents, here we report synthesis, antibacterial evaluation, and structure-activity relationship studies of an extended chemical library of macrocyclic diarylheptanoids with diverse amine, amide, urea, and sulfonamide functionalities. Results of this study have produced macrocyclic geranylamine and 4-fluorophenethylamine substituted derivatives, exhibiting moderate to good activity against M. tuberculosis and selected Gram-positive bacterial pathogens.
Assuntos
Antibacterianos/síntese química , Antituberculosos/síntese química , Heptanos/química , Aminas/química , Antibacterianos/química , Antibacterianos/farmacologia , Antituberculosos/química , Antituberculosos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Heptanos/síntese química , Heptanos/farmacologia , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/química , Ureia/químicaRESUMO
Pretomanid (PA-824) is an important nitroimidazole antitubercular agent in late stage clinical trials. However, pretomanid is limited by poor solubility and high protein binding, which presents opportunities for improvement in its physiochemical properties. Conversely, the oxazolidinone linezolid has excellent physicochemical properties and has recently shown impressive activity for the treatment of drug resistant tuberculosis. In this study we explore if incorporation of the outer ring elements found in first and second generation oxazolidinones into the nitroimidazole core of pretomanid can be used to improve its physicochemical and antitubercular properties. The synthesis of pretomanid outer oxazolidinone ring hybrids was successfully performed producing hybrids that maintained antitubercular activity and had improved in vitro physicochemical properties. Three lead compounds were identified and evaluated in a chronic model of tuberculosis infection in mice. However, the compounds lacked efficacy suggesting that portions of PA-824 tail not found in the hybrid molecules are required for in vivo efficacy.
Assuntos
Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Nitroimidazóis/uso terapêutico , Oxazolidinonas/uso terapêutico , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/síntese química , Antituberculosos/química , Doença Crônica , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Nitroimidazóis/síntese química , Nitroimidazóis/química , Oxazolidinonas/síntese química , Oxazolidinonas/químicaRESUMO
Antibody-drug conjugates (ADCs) are designed to target antigen expressing (Ag+) cells in a tumor. Once processed by the Ag+ cells, ADCs can release cytotoxic drug molecules that can diffuse out of Ag+ cells into the neighboring antigen-negative (Ag-) cells to induce their cytotoxicity. This additional efficacy of ADCs on Ag- cells in the presence of Ag+ cells is known as the 'bystander effect'. Although the importance of this phenomena is widely acknowledged for effective killing of a heterogeneous tumor, the rate and extent of the bystander killing in a heterogeneous system is not quantitatively understood yet. Thus, the objectives of this manuscript were to: (1) synthesize and characterize a tool ADC Trastuzumab-vc-MMAE that is capable of exhibiting bystander effect, (2) quantify the time course of the bystander effect for the tool ADC using in vitro co-culture systems created using mixture of various HER2-expressing cell lines, and (3) develop a pharmacodynamic (PD) model that is capable of characterizing the bystander effect of ADCs. Co-culture studies conducted using GFP labelled MCF7 cells as Ag- cells and N87, BT474, and SKBR3 as Ag+ cells revealed that the bystander effect of ADC increases with increasing fraction of Ag+ cells in a co-culture system, and with increased expression level of target on Ag+ cells. A notable lag time after ADC incubation was also observed prior to significant bystander killing of Ag- cells. Based on our results we hypothesize that there may be other determinants apart from the antigen expression level that can also influence the ability of Ag+ cells to demonstrate the bystander effect in a co-culture system. The co-culture analysis also suggested that the bystander effect of the ADC can dissipate over the period of time as the population of Ag+ cells declines. A novel PD model was developed to mathematically characterize the bystander effect of ADCs by combining two different cell distribution models to represent the population of Ag+ and Ag- cells in a co-culture system. This PD model can be integrated with the systems PK model for ADCs in the future to generate a quantitative framework that is capable of supporting the discovery and development of novel ADCs with optimal bystander killing capabilities.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Efeito Espectador/efeitos dos fármacos , Imunoconjugados/farmacologia , Modelos Biológicos , Oligopeptídeos/farmacologia , Trastuzumab/farmacologia , Anticorpos Monoclonais/química , Antígenos de Neoplasias/genética , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Proteínas de Fluorescência Verde/genética , Humanos , Imunoconjugados/química , Células MCF-7 , Oligopeptídeos/síntese química , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Fatores de TempoRESUMO
OBJECTIVES: Metronidazole, a mainstay treatment for Clostridium difficile infection (CDI), is often ineffective for severe CDI. Whilst this is thought to arise from suboptimal levels of metronidazole in the colon due to rapid absorption, empirical validation is lacking. In contrast, reutericyclin, an antibacterial tetramic acid from Lactobacillus reuteri, concentrates in the gastrointestinal tract. In this study, we modified metronidazole with reutericyclin's tetramic acid motif to obtain non-absorbed compounds, enabling assessment of the impact of pharmacokinetics on treatment outcomes. METHODS: A series of metronidazole-bearing tetramic acid substituents were synthesized and evaluated in terms of anti-C. difficile activities, gastric permeability, in vivo pharmacokinetics, efficacy in the hamster model of CDI and mode of action. RESULTS: Most compounds were absorbed less than metronidazole in cell-based Caco-2 permeability assays. In hamsters, lead compounds compartmentalized in the colon rather than the bloodstream with negligible levels detected in the blood, in direct contrast with metronidazole, which was rapidly absorbed into the blood and was undetectable in caecum. Accordingly, four leads were more efficacious (Pâ<â0.05) than metronidazole in C. difficile-infected animals. Improved efficacy was not due to an alternative mode of action, as the leads retained the mode of action of metronidazole. CONCLUSIONS: This study provides the clearest empirical evidence that the high absorption of metronidazole lowers treatment outcomes for CDI and suggests a role for the tetramic acid motif for colon-specific drug delivery. This approach also has the potential to lower systemic toxicity and drug interactions of nitroheterocyclic drugs for treating gastrointestine-specific diseases.
Assuntos
Antibacterianos/farmacocinética , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/tratamento farmacológico , Colo/química , Metronidazol/farmacocinética , Pirrolidinonas/farmacocinética , Ácido Tenuazônico/análogos & derivados , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Modelos Animais de Doenças , Masculino , Mesocricetus , Metronidazol/administração & dosagem , Metronidazol/química , Pirrolidinonas/química , Ácido Tenuazônico/química , Ácido Tenuazônico/farmacocinética , Resultado do TratamentoRESUMO
Characterization and prediction of the pharmacokinetics (PK) and pharmacodynamics (PD) of Antibody-Drug Conjugates (ADCs) is challenging, since it requires simultaneous quantitative understanding about the PK-PD properties of three different molecular species i.e., the monoclonal antibody, the drug, and the conjugate. Mathematical modeling and simulation provides an excellent tool to overcome these challenges, as it can simultaneously integrate the PK-PD of ADCs and their components in a quantitative manner. Additionally, the computational PK-PD models can also serve as a cornerstone for the model-based drug development and preclinical-to-clinical translation of ADCs. To provide an overview of this subject matter, this manuscript reviews the PK-PD models applicable to ADCs. Additionally, the usage of these models during different drug development stages (i.e., discovery, preclinical development, and clinical development) is also emphasized. The importance of PK-PD modeling and simulation in making rationale go/no-go decisions throughout the drug development process is also highlighted. There is an array of PK-PD models available, ranging from the systems models specifically developed for ADCs to the empirical models applicable to all chemotherapeutic agents, which one can employ for ADCs. The decision about which model to choose depends on the questions to be answered, time at hand, and resources available.
Assuntos
Anticorpos Monoclonais , Descoberta de Drogas/métodos , Imunoconjugados , Modelos Biológicos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Simulação por Computador , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Distribuição TecidualRESUMO
Chimeric antigen receptor (CAR) T-cell subsets and immunophenotypic composition of the pre-infusion product, as well as their longitudinal changes following infusion, are expected to affect CAR T-cell expansion, persistence, and clinical outcomes. Herein, we sequentially evolved our previously described cellular kinetic-pharmacodynamic (CK-PD) model to incorporate CAR T-cell product-associated attributes by utilizing published preclinical and clinical datasets from two affinity variants (FMC63 and CAT19 scFv) anti-CD19 CAR T-cells. In step 1, a unified cell-level PD model was used to simultaneously characterize the in vitro killing datasets of two CAR T-cells against CD19+ cell lines at varying effector:target ratios. In step 2, an augmented CK-PD model for anti-CD19 CAR T-cells was developed, by integrating CK dataset(s) from two bioanalytical measurements (quantitative polymerase chain reaction and flow cytometry) in patients with cancer. The model described the differential in vivo expansion properties of CAR T-cell subsets. The estimated expansion rate constant was ~1.12-fold higher for CAR+CD8+ cells in comparison to CAR+CD4+ T-cells. In step 3, the model was extended to characterize the disposition of four immunophenotypic populations of CAR T-cells, including stem-cell memory, central memory, effector memory, and effector cells. The model adequately characterized the longitudinal changes in immunophenotypes post anti-CD19 CAR T-cell infusion in pediatric patients with acute lymphocytic leukemia. Polyclonality in the pre-infusion product was identified as a categorical covariate influencing differentiation of immunophenotypes. In the future, this model could be leveraged a priori toward optimizing the composition of CAR T-cell infusion product, and further understand the CK-PD relationship in patients.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Criança , Receptores de Antígenos Quiméricos/metabolismo , Cinética , Subpopulações de Linfócitos T/metabolismo , Imunoterapia Adotiva , Antígenos CD19/genética , Antígenos CD19/metabolismo , Receptores de Antígenos de Linfócitos TRESUMO
Immuno-oncology (IO) is a fast-expanding field due to recent success using IO therapies in treating cancer. As IO therapies do not directly kill tumor cells but rather act upon the patients' own immune cells either systemically or in the tumor microenvironment, new and innovative approaches are required to inform IO therapy research and development. Quantitative systems pharmacology (QSP) modeling describes the biological mechanisms of disease and the mode of action of drugs with mathematical equations, which has significant potential to address the big challenges in the IO field, from identifying patient populations that respond to different therapies to guiding the selection, dosing, and scheduling of combination therapy. To assess the perspectives of the community on the impact of QSP modeling in IO drug development and to understand current applications and challenges, the IO QSP working group-under the QSP Special Interest Group (SIG) of the International Society of Pharmacometrics (ISoP)-conducted a survey among QSP modelers, non-QSP modelers, and non-modeling IO program stakeholders. The survey results are presented here with discussions on how to address some of the findings. One of the findings is the differences in perception among these groups. To help bridge this perception gap, we present several case studies demonstrating the impact of QSP modeling in IO and suggest actions that can be taken in the future to increase the real and perceived impact of QSP modeling in IO drug research and development.
Assuntos
Neoplasias , Farmacologia , Humanos , Farmacologia em Rede , Desenvolvimento de Medicamentos , Neoplasias/tratamento farmacológico , Imunoterapia , Oncologia , Modelos Biológicos , Microambiente TumoralRESUMO
Despite tremendous success of chimeric antigen receptor (CAR) T cell therapy in clinical oncology, the dose-exposure-response relationship of CAR-T cells in patients is poorly understood. Moreover, the key drug-specific and system-specific determinants leading to favorable clinical outcomes are also unknown. Here we have developed a multiscale mechanistic pharmacokinetic (PK)-pharmacodynamic (PD) model for anti-B-cell maturation antigen (BCMA) CAR-T cell therapy (bb2121) to characterize (i) in vitro target cell killing in multiple BCMA expressing tumor cell lines at varying effector to target cell ratios, (ii) preclinical in vivo tumor growth inhibition and blood CAR-T cell expansion in xenograft mice, and (iii) clinical PK and PD biomarkers in patients with multiple myeloma. Our translational PK-PD relationship was able to effectively describe the commonly observed multiphasic CAR-T cell PK profile in the clinic, consisting of the rapid distribution, expansion, contraction, and persistent phases, and accounted for the categorical individual responses in multiple myeloma to effectively calculate progression-free survival rates. Preclinical and clinical data analysis revealed comparable parameter estimates pertaining to CAR-T cell functionality and suggested that patient baseline tumor burden could be more sensitive than dose levels toward overall extent of exposure after CAR-T cell infusion. Virtual patient simulations also suggested a very steep dose-exposure-response relationship with CAR-T cell therapy and indicated the presence of a "threshold" dose, beyond which a flat dose-response curve could be observed. Our simulations were concordant with multiple clinical observations discussed in this article. Moving forward, this framework could be leveraged a priori to explore multiple infusions and support the preclinical/clinical development of future CAR-T cell therapies.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Mieloma Múltiplo/terapia , Receptores de Antígenos Quiméricos/uso terapêutico , Animais , Antineoplásicos Imunológicos/administração & dosagem , Biomarcadores Farmacológicos/análise , Linhagem Celular Tumoral/efeitos dos fármacos , Simulação por Computador , Relação Dose-Resposta a Droga , Humanos , Imunoterapia Adotiva/métodos , Infusões Intravenosas , Camundongos , Camundongos Endogâmicos NOD , Modelos Teóricos , Farmacocinética , Intervalo Livre de Progressão , Receptores de Antígenos Quiméricos/administração & dosagem , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Chimeric antigen receptor (CAR)-T cell therapy has achieved considerable success in treating B-cell hematologic malignancies. However, the challenges of extending CAR-T therapy to other tumor types, particularly solid tumors, remain appreciable. There are substantial variabilities in CAR-T cellular kinetics across CAR-designs, CAR-T products, dosing regimens, patient responses, disease types, tumor burdens, and lymphodepletion conditions. As a "living drug," CAR-T cellular kinetics typically exhibit four distinct phases: distribution, expansion, contraction, and persistence. The cellular kinetics of CAR-T may correlate with patient responses, but which factors determine CAR-T cellular kinetics remain poorly defined. Herein, we developed a cellular kinetic model to retrospectively characterize CAR-T kinetics in 217 patients from 7 trials and compared CAR-T kinetics across response status, patient populations, and tumor types. Based on our analysis results, CAR-T cells exhibited a significantly higher cell proliferation rate and capacity but a lower contraction rate in patients who responded to treatment. CAR-T cells proliferate to a higher degree in hematologic malignancies than in solid tumors. Within the assessed dose ranges (107 -109 cells), CAR-T doses were weakly correlated with CAR-T cellular kinetics and patient response status. In conclusion, the developed CAR-T cellular kinetic model adequately characterized the multiphasic CAR-T cellular kinetics and supported systematic evaluations of the potential influencing factors, which can have significant implications for the development of more effective CAR-T therapies.
Assuntos
Proliferação de Células , Imunoterapia Adotiva , Ativação Linfocitária , Modelos Imunológicos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Morte Celular , Ensaios Clínicos como Assunto , Simulação por Computador , Humanos , Memória Imunológica , Cinética , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Estudos Retrospectivos , Linfócitos T/metabolismoRESUMO
The development of mechanism-based, multiscale pharmacokinetic-pharmacodynamic (PK-PD) models for chimeric antigen receptor (CAR)-T cells is needed to enable investigation of in vitro and in vivo correlation of CAR-T cell responses and to facilitate preclinical-to-clinical translation. Toward this goal, we first developed a cell-level in vitro PD model that quantitatively characterized CAR-T cell-induced target cell depletion, CAR-T cell expansion and cytokine release. The model accounted for key drug-specific (CAR-affinity, CAR-densities) and system-specific (antigen densities, E:T ratios) variables and was able to characterize comprehensive in vitro datasets from multiple affinity variants of anti-EGFR and anti-HER2 CAR-T cells. Next, a physiologically based PK (PBPK) model was developed to simultaneously characterize the biodistribution of untransduced T-cells, anti-EGFR CAR-T and anti-CD19 CAR-T cells in xenograft -mouse models. The proposed model accounted for the engagement of CAR-T cells with tumor cells at the site of action. Finally, an integrated PBPK-PD relationship was established to simultaneously characterize expansion of CAR-T cells and tumor growth inhibition (TGI) in xenograft mouse model, using datasets from anti-BCMA, anti-HER2, anti-CD19 and anti-EGFR CAR-T cells. Model simulations provided potential mechanistic insights toward the commonly observed multiphasic PK profile (i.e., rapid distribution, expansion, contraction and persistence) of CAR-T cells in the clinic. Model simulations suggested that CAR-T cells may have a steep dose-exposure relationship, and the apparent Cmax upon CAR-T cell expansion in blood may be more sensitive to patient tumor-burden than CAR-T dose levels. Global sensitivity analysis described the effect of other drug-specific parameters toward CAR-T cell expansion and TGI. The proposed modeling framework will be further examined with the clinical PK and PD data, and the learnings can be used to inform design and development of future CAR-T therapies.
Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Animais , Movimento Celular , Proliferação de Células , Simulação por Computador , Receptores ErbB/imunologia , Xenoenxertos , Humanos , Camundongos , Modelos Teóricos , Neoplasias/terapia , Ligação Proteica , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologiaRESUMO
It has been proposed that the binding-site barrier (BSB) for antibody-drug conjugates (ADCs) can be overcome with the help of antibody coadministration. However, broad utility of this strategy remains in question. Consequently, here, we have conducted in vivo experiments and pharmacokinetics-pharmacodynamics (PK-PD) modeling and simulation (M&S) to further evaluate the antibody coadministration hypothesis in a quantitative manner. Two different Trastuzumab-based ADCs, T-DM1 (no bystander effect) and T-vc-MMAE (with a bystander effect), were evaluated in high-HER2 (N87) and low-HER2 (MDA-MB-453) expressing tumors, with or without the coadministration of 1, 3, or 8-fold higher Trastuzumab. The tumor growth inhibition (TGI) data was quantitatively characterized using a semi-mechanistic PK-PD model to determine the nature of drug interaction for each coadministration regimen, by estimating the interaction parameter ψ. It was found that the coadministration strategy improved ADC efficacy under certain conditions and had no impact on ADC efficacy in others. The benefit was more pronounced for N87 tumors with very high antigen expression levels where the effect on treatment was synergistic (a synergistic drug interaction, ψ = 2.86 [2.6-3.12]). The benefit was diminished in tumor with lower antigen expression (MDA-MB-453) and payload with bystander effect. Under these conditions, the coadministration regimens resulted in an additive or even less than additive benefit (ψ ≤ 1). As such, our results suggest that while antibody coadministration may be helpful for ADCs in certain circumstances, one should not broadly apply this strategy to all the scenarios without first identifying the costs and benefits of this approach.
Assuntos
Ado-Trastuzumab Emtansina/farmacocinética , Antineoplásicos Imunológicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/farmacocinética , Modelos Biológicos , Oligopeptídeos/farmacocinética , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Trastuzumab/farmacocinética , Ado-Trastuzumab Emtansina/administração & dosagem , Animais , Antineoplásicos Imunológicos/administração & dosagem , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Efeito Espectador , Linhagem Celular Tumoral , Simulação por Computador , Composição de Medicamentos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Oligopeptídeos/administração & dosagem , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Distribuição Tecidual , Trastuzumab/administração & dosagem , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Here, we have developed a cell-level systems PK-PD model to characterize the bystander effect of antibody-drug conjugates (ADCs). Cytotoxicity data generated following incubation of Trastuzumab-vc-MMAE in cocultures of high HER2-expressing N87 and low HER2-expressing GFP-MCF7 cells were used to build the model. Single-cell PK model for ADC was used to characterize the PK of trastuzumab-vc-MMAE and released MMAE in N87 and GFP-MCF7 cells. The 2 cell-level PK models were mechanistically integrated to mimic the coculture condition. MMAE-induced intracellular occupancy of tubulin was used to drive the efficacy of ADC, and improvement in the tubulin occupancy of GFP-MCF7 cells in the presence of N87 cells was used to drive the bystander effect of trastuzumab-vc-MMAE. The "dual" cell-level PK-PD model was able to capture the observed data reasonably well. It was found that similar and high occupancy of tubulin by MMAE was required to achieve the cytotoxic effect in each cell line. In addition, estimated model parameters suggested that â¼60% improvement in the tubulin occupancy was required to attain half of the maximum bystander killing effect by the ADC. The presented model provides foundation for in vivo systems PK-PD model to characterize and predict the bystander effect of ADCs.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/farmacocinética , Efeito Espectador/efeitos dos fármacos , Imunoconjugados/farmacologia , Imunoconjugados/farmacocinética , Oligopeptídeos/farmacologia , Oligopeptídeos/farmacocinética , Trastuzumab/farmacologia , Trastuzumab/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Humanos , Células MCF-7RESUMO
Here, we have presented the development of a systems pharmacokinetics-pharmacodynamics (PK-PD) model for antibody-drug conjugates (ADCs), which uses intracellular target occupancy to drive in-vivo efficacy. The model is built based on PK and efficacy data generated using Trastuzumab-Valine-Citrulline-Monomethyl Auristatin E (T-vc-MMAE) ADC in N87 (high-HER2) and GFP-MCF7 (low-HER2) tumor bearing mice. It was observed that plasma PK of all ADC analytes was similar between the two tumor models; however, total trastuzumab, unconjugated MMAE, and total MMAE exposures were >10-fold, ~1.6-fold, and ~1.8-fold higher in N87 tumors. In addition, a prolonged retention of MMAE was observed within the tumors of both the mouse models, suggesting intracellular binding of MMAE to tubulin. A systems PK model, developed by integrating single-cell PK model with tumor distribution model, was able to capture all in vivo PK data reasonably well. Intracellular occupancy of tubulin predicted by the PK model was used to drive the efficacy of ADC using a novel PK-PD model. It was found that the same set of PD parameters was able to capture MMAE induced killing of GFP-MCF7 and N87 cells in vivo. These observations highlight the benefit of adopting a systems approach for ADC and provide a robust and predictive framework for successful clinical translation of ADCs.
RESUMO
Acyldepsipeptides are a unique class of antibiotics that act via allosterically dysregulated activation of the bacterial caseinolytic protease (ClpP). The ability of ClpP activators to kill nongrowing bacteria represents a new opportunity to combat deep-seated biofilm infections. However, the acyldepsipeptide scaffold is subject to rapid metabolism. Herein, we explore alteration of the potentially metabolically reactive α,ß unsaturated acyl chain. Through targeted synthesis, a new class of phenyl urea substituted depsipeptide ClpP activators with improved metabolic stability is described. The ureadepsipeptides are potent activators of Staphylococcus aureus ClpP and show activity against Gram-positive bacteria, including S. aureus biofilms. These studies demonstrate that a phenyl urea motif can successfully mimic the double bond, maintaining potency equivalent to acyldepsipeptides but with decreased metabolic liability. Although removal of the double bond from acyldepsipeptides generally has a significant negative impact on potency, structural studies revealed that the phenyl ureadepsipeptides can retain potency through the formation of a third hydrogen bond between the urea and the key Tyr63 residue in the ClpP activation domain. Ureadepsipeptides represent a new class of ClpP activators with improved drug-like properties, potent antibacterial activity, and the tractability to be further optimized.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Depsipeptídeos/química , Endopeptidase Clp/metabolismo , Ativadores de Enzimas/química , Staphylococcus aureus/enzimologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Depsipeptídeos/metabolismo , Depsipeptídeos/farmacologia , Endopeptidase Clp/química , Endopeptidase Clp/genética , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Domínios Proteicos , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Ureia/química , Ureia/metabolismoRESUMO
Recombinant human erythropoietin (rHuEPO) is used effectively in the treatment of various anemic disorders. Belgrade rat is a useful animal model of anemia caused by defect in iron utilization. The objective of the present study was to investigate the dynamics of erythropoietic biomarkers in Belgrade rats receiving rHuEPO. Pharmacokinetics of rHuEPO was evaluated in Belgrade rats and normal rats after intravenous administration of single doses of the drug (100 and 1350 IU/kg). Pharmacodynamic biomarkers included levels of red blood cells, hemoglobin, and reticulocytes following administration of a single intravenous dose of rHuEPO (100 IU/kg). Red blood cell survival was assessed after treatment with rHuEPO (450 IU/kg), three times a week for 2 weeks. It was found that rHuEPO exhibited non-linear pharmacokinetics in both Belgrade and control rats. At the low dose, plasma concentrations and AUC (area under the curve) were significantly lower while clearance and volume of distribution were higher in Belgrade rats (p < 0.05). At the higher dose, there was no difference in pharmacokinetics between the two groups. Erythropoietic effect of rHuEPO was negligible in Belgrade rats at the dose of 100 IU/kg whereas all studied erythropoietic biomarkers were increased in normal rats. The levels of red blood cells, hemoglobin were significantly lower whereas the percentage of reticulocytes was higher in Belgrade rats compared to that in normal rats (p < 0.05). RHuEPO increased red blood cell survival in both animal groups. In conclusion, rHuEPO effect on erythropoietic biomarkers was stronger in normal rats than Belgrade rats at the studied doses. The findings from this study may provide further insights into understanding of anemic disorders resulting from mutations in the divalent metal transporter.
RESUMO
REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.