Computational Analysis of Cytokine Release Following Bispecific T-Cell Engager Therapy: Applications of a Logic-Based Model.

Bispecific T-cell engaging therapies harness the immune system to elicit an effective anticancer response. Modulating the immune activation avoiding potential adverse effects such as cytokine release syndrome (CRS) is a critical aspect to realizing the full potential of this therapy. The use of suitable exogenous intervention strategies to mitigate the CRS risk without compromising the antitumoral capability of bispecific antibody treatment is crucial. To this end, computational approaches can be instrumental to systematically exploring the effects of combining bispecific antibodies with CRS intervention strategies. Here, we employ a logical model to describe the action of bispecific antibodies and the complex interplay of various immune system components and use it to perform simulation experiments to improve the understanding of the factors affecting CRS. We performed a sensitivity analysis to identify the comedications that could ameliorate CRS without impairing tumor clearance. Our results agree with publicly available experimental data suggesting anti-TNF and anti-IL6 as possible co-treatments. Furthermore, we suggest anti-IFNγ as a suitable candidate for clinical studies.

Preclinical Assessment of a MUC12-Targeted BiTE (Bispecific T-cell Engager) Molecule.

MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo In vitro, the MUC12 HLE BiTE molecule mediated T-cell-redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 µg/kg with a step dose to 1,000 µg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.

GIPR antagonist antibodies conjugated to GLP-1 peptide are bispecific molecules that decrease weight in obese mice and monkeys.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) regulate glucose and energy homeostasis. Targeting both pathways with GIP receptor (GIPR) antagonist antibody (GIPR-Ab) and GLP-1 receptor (GLP-1R) agonist, by generating GIPR-Ab/GLP-1 bispecific molecules, is an approach for treating obesity and its comorbidities. In mice and monkeys, these molecules reduce body weight (BW) and improve many metabolic parameters. BW loss is greater with GIPR-Ab/GLP-1 than with GIPR-Ab or a control antibody conjugate, suggesting synergistic effects. GIPR-Ab/GLP-1 also reduces the respiratory exchange ratio in DIO mice. Simultaneous receptor binding and rapid receptor internalization by GIPR-Ab/GLP-1 amplify endosomal cAMP production in recombinant cells expressing both receptors. This may explain the efficacy of the bispecific molecules. Overall, our GIPR-Ab/GLP-1 molecules promote BW loss, and they may be used for treating obesity.

Quantification of Radiation Injury on Neutropenia and the Link between Absolute Neutrophil Count Time Course and Overall Survival in Nonhuman Primates Treated with G-CSF.

PURPOSE: To model absolute neutrophil count (ANC) suppression in response to acute radiation (AR) exposure and evaluate ANC time course as a predictor of overall survival (OS) in response to AR exposure with or without treatment with granulocyte colony-stimulating factor in nonhuman primates. METHODS: Source data were obtained from two pivotal studies conducted in rhesus macaques exposed to 750 cGy of whole body irradiation on day 0 that received either placebo, daily filgrastim, or pegfilgrastim (days 1 and 8 after irradiation). Animals were observed for 60 days with ANC measured every 1 to 2 days. The population model of ANC response to AR and the link between observed ANC time course and OS consisted of three submodels characterizing injury due to radiation, granulopoiesis, and a time-to-event model of OS. RESULTS: The ANC response model accurately described the effects of AR exposure on the duration of neutropenia. ANC was a valid surrogate for survival because it explained 76% (95% CI, 41%-97%) and 73.2% (95% CI, 38.7%-99.9%) of the treatment effect for filgrastim and pegfilgrastim, respectively. CONCLUSION: The current model linking radiation injury to neutropenia and ANC time course to OS can be used as a basis for translating these effects to humans.

Prediction of Survival Benefit of Filgrastim in Adult and Pediatric Patients With Acute Radiation Syndrome.

Acute exposure to high doses of radiation leads to severe myelosuppression, but few treatments are currently available to treat hematopoietic syndrome of acute radiation syndrome. Granulocyte colony stimulating factors (e.g., filgrastim) stimulate proliferation of neutrophil precursors and enhance mature neutrophil function. Owing to ethical constraints on conducting clinical research in lethally irradiated humans, we developed a model-based strategy to integrate preclinical experience in irradiated nonhuman primates (NHPs) and other clinical myelosuppressive conditions to inform filgrastim dosing to treat hematopoietic syndrome of acute radiation syndrome. Models predicting neutrophil counts and overall survival based on drug exposures were calibrated and scaled from NHPs to adult and pediatric human subjects. Several scenarios were examined investigating variations in filgrastim doses, dose frequency, treatment initiation, and duration, as well as the effect of age and radiation dose rate. Model-based simulations and established safety profiles supported that a subcutaneous filgrastim dose of 10 µg/kg once daily provides a significant survival benefit (50%) over placebo in both adults and children, provided that the treatment is initiated within 1-14 days after radiation exposure and lasts 2-3 weeks. For treatment durations of longer than 3 weeks, filgrastim treatment is not expected to provide significantly greater benefit. This survival benefit is expected to hold for the wide range of radiation doses and dose rates (0.01-1,000 Gy/hours) examined.

Pharmacokinetic-pharmacodynamic modelling of neutrophil response to G-CSF in healthy subjects and patients with chemotherapy-induced neutropenia.

AIM: The objective of the present study was to use pharmacokinetic-pharmacodynamic modelling to characterize the effects of chemotherapy on the granulopoietic system and to predict the absolute neutrophil counts (ANCs) for patients with chemotherapy-induced neutropenia treated with filgrastim and pegfilgrastim. METHODS: Data were extracted from 10 phase I-III studies conducted in 110 healthy adults, and 618 adult and 52 paediatric patients on chemotherapy following administration of filgrastim or pegfilgrastim. The structural model accounted for ANC dynamics and the effects of filgrastim and pegfilgrastim, chemotherapy and corticosteroids. The impact of neutrophils on drug disposition was based on a drug receptor-binding model that assumed quasi-equilibrium and stimulation of the production and maturation of neutrophils upon treatment. The chemotherapy and corticosteroid effects were represented by kinetic-pharmacodynamic-type models, where chemotherapy stimulated elimination of neutrophil precursors at the mitotic stage, and corticosteroids stimulated neutrophil production. RESULTS: The systemic half-lives of filgrastim (2.6 h) and pegfilgrastim (10.1 h) were as expected. The effective half-life of chemotherapy was 9.6 h, with a 2-day killing effect. The rate of receptor elimination from mitotic compartments exhibited extreme interindividual variability (% coefficient of variation >200), suggesting marked differences in sensitivity to chemotherapy effects on ANCs. The stimulatory effects of pegfilgrastim were significantly greater than those of filgrastim. Model qualification confirmed the predictive capability of this model. CONCLUSION: This qualified model simulates the time course of ANC in the absence or presence of chemotherapy and predicts nadir, time to nadir and time of recovery from different grades of neutropenia upon treatment with filgrastim and pegfilgrastim.

A cell-level model of pharmacodynamics-mediated drug disposition.

We aimed to develop a cell-level pharmacodynamics-mediated drug disposition (PDMDD) model to analyze in vivo systems where the PD response to a drug has an appreciable effect on the pharmacokinetics (PK). An existing cellular level model of PD stimulation was combined with the standard target-mediated drug disposition (TMDD) model and the resulting model structure was parametrically identifiable from typical in vivo PK and PD data. The PD model of the cell population was controlled by the production rate k in and elimination rate k out which could be stimulated or inhibited by the number of bound receptors on a single cell. Simulations were performed to assess the impact of single and repeated dosing on the total drug clearance. The clinical utility of the cell-level PDMDD model was demonstrated by fitting published data on the stimulatory effects of filgrastim on absolute neutrophil counts in healthy subjects. We postulated repeated dosing as a means of detecting and quantifying PDMDD as a single dose might not be sufficient to elicit the cellular response capable of altering the receptor pool to visibly affect drug disposition. In the absence of any PD effect, the model reduces down to the standard TMDD model. The applications of this model can be readily extended to include chemotherapy-induced cytopenias affecting clearance of endogenous hematopoietic growth factors, different monoclonal antibodies and immunogenicity effects on PK.

Pharmacodynamic model for chemoradiotherapy-induced thrombocytopenia in mice.

A mechanistic model describing the effects of chemotherapy and radiation on platelet counts and endogenous thrombopoietin (eTPO) in mice was developed. Thrombocytopenia was induced in mice by injection of carboplatin followed by the whole body irradiation on days 0, 28, and 56, with platelet and eTPO samples collected over 84 days. The pharmacodynamic model consisted of a series of aging compartments representing proliferating megakaryocyte precursors, megakaryocytes, and platelets with possible eTPO clearance through internalization. The cytotoxic effects of treatment were described by the kinetics of the effect (K-PD) model, and stimulation of platelet production by eTPO was considered to be driven by receptor occupancy. The proposed PD model adequately described the platelet counts and eTPO concentrations in mice by accounting for nadirs and peaks of platelet count, and rebounds in eTPO time course profiles. The estimates of model parameters were in good agreement with their physiological values reported in literature for mice with platelet lifespan of 4.3 days and 185 cMpl receptors per platelet. The predicted duration of the treatment effect was 0.82 h (approximately 5 carboplatin half-lives in mice). The data was not informative about the eTPO stimulatory effect as the nominal precursor production rate was sufficient to account for platelet response to treatment. The model quantified the inverse relationship between eTPO levels and platelet counts and offered an explanation of the tolerance effect observed in the eTPO data. The simulated rebound in free receptors levels correlated with rebounds in eTPO levels. The model suggests that the duration of the toxic effects is determined by the turnover of the proliferating cells in the bone marrow. This indicates that the lifespan of the target cells (megakaryocyte precursors, megakaryocytes and platelets) is a key determinant in the duration of both drug exposure and toxicity due to treatment. The model can be extended to account for pharmacokinetics of exogenous drugs and be applied to analysis of human data.

Simulations of site-specific target-mediated pharmacokinetic models for guiding the development of bispecific antibodies.

Bispecific antibodies (BAbs) are novel constructs that are under development and show promise as new therapeutic modalities for cancer and autoimmune disorders. The aim of this study is to develop a semi-mechanistic modeling approach to elucidate the disposition of BAbs in plasma and possible sites of action in humans. Here we present two case studies that showcase the use of modeling to guide BAb development. In case one, a BAb is directed against a soluble and a membrane-bound ligand for treating systemic lupus erythematosus, and in case two, a BAb targets two soluble ligands as a potential treatment for ulcerative colitis and asthma. Model simulations revealed important differences between plasma and tissues, when evaluated for drug disposition and target suppression. Target concentrations at tissue sites and type (soluble vs membrane-bound), tissue-site binding, and binding affinity are all major determinants of BAb disposition and subsequently target suppression. For the presented case studies, higher doses and/or frequent dosing regimens are required to achieve 80 % target suppression in site specific tissue (the more relevant matrix) as compared to plasma. Site-specific target-mediated models may serve to guide the selection of first-in-human doses for new BAbs.

Structural and functional proteomics of intracytoplasmic membrane assembly in Rhodobacter sphaeroides.

The results of a detailed structural and functional proteomic analysis of intracytoplasmic membrane (ICM) assembly in the model purple phototrophic bacterium Rhodobacter sphaeroides are reviewed in this report. Proteomics approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized within the internal cell membranes, together with their structural and functional correlates. For the examination of temporal protein expression, procedures were established for the induction of ICM formation at low oxygen tension and for ICM remodeling in cells adapting to low intensity illumination, which permitted isolation by rate-zone sedimentation of ICM growth initiation sites (CM invaginations) in an upper-pigmented band (UPB), together with more mature ICM vesicles (chromatophores) as the main band. Nondenaturing clear native gel electrophoresis of the chromatophore fraction gave rise to four pigmented bands: the top and bottom bands contained the reaction center-light-harvesting 1 (RC-LH1) core complex and the LH2 peripheral antenna, respectively, while two bands of intermediate migration exhibited distinct associations of LH2 and core complexes. Proteomic analysis of the gel bands revealed developmental changes including increasing levels of LH2 polypeptides relative to those of core complexes as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits, and the cytochrome bc1 complex. High counts were also observed for RSP6124, a protein of unknown function, that were correlated with increasing LH2 levels. RC-LH1-containing clear native electrophoresis gel bands from the UPB were enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane assembly factors (viz., preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg translocation subunit TatA), thereby confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination in which preferential assembly of the RC-LH1 complex occurs. Functional aspects of the photosynthetic unit assembly process were monitored by fluorescence induction/relaxation measurements of the variable fluorescence arising from LH-bacteriochlorophyll a. Slowing of the rate of RC electron transfer turnover (τQA), as assessed from the relaxation phase, was correlated with the growth of the functional absorption cross section (σ) and LH2/LH1 molar ratios. This is thought to arise from the imposition of constraints upon free diffusion of ubiquinone (UQ) redox species between the RC and cytochrome bc1 complex as the ICM bilayer becomes densely packed with LH2 rings. Such LH2 packing was confirmed in a comparison by high-resolution atomic force microscopy of ICM patches from cells grown at high and low light intensity [Adams and Hunter: Biochim Biophys Acta 2012;1817:1616-1627], in which the increasing LH2 levels form densely packed LH2-only domains, representing the light-responsive antenna complement arising under low illumination. In contrast, LH2 is initially dispersed in rows and small cluster-separating linear arrays of largely dimeric RC-LH1 core complexes, which become filled with LH2 during acclimation to reduced light intensity. In phototrophically grown cells that were transferred to oxic conditions in the dark, fluorescence induction/relaxation measurements showed that despite a growth burst independent of photosynthetic pathways, functional photosynthetic units were maintained for up to 24 h after the transition. The τQA was accelerated from ∼1 to 0.5 ms by 8 h, reflecting the decrease in LH2 levels, facilitating more rapid UQ redox species diffusion in the membrane bilayer as crowding by LH2 is overcome. Under these circumstances, UPB levels were elevated with significant increases in LH1/LH2 molar ratio. These changes indicate that vesiculation of CM growth initiation sites to form vesicular ICM was arrested under oxic conditions.

Combinatorial chemotherapeutic efficacy in non-Hodgkin lymphoma can be predicted by a signaling model of CD20 pharmacodynamics.

Combination chemotherapy represents the standard-of-care for non-Hodgkin lymphoma. However, the development of new therapeutic regimens is empirical and this approach cannot be used prospectively to identify novel or optimal drug combinations. Quantitative system pharmacodynamic models could promote the discovery and development of combination regimens based upon first principles. In this study, we developed a mathematical model that integrates temporal patterns of drug exposure, receptor occupancy, and signal transduction to predict the effects of the CD20 agonist rituximab in combination with rhApo2L/TNF-related apoptosis inducing ligand or fenretinide, a cytotoxic retinoid, upon growth kinetics in non-Hodgkin lymphoma xenografts. The model recapitulated major regulatory mechanisms, including target-mediated disposition of rituximab, modulation of proapoptotic intracellular signaling induced by CD20 occupancy, and the relative efficacy of death receptor isoforms. The multiscale model coupled tumor responses to individual anticancer agents with their mechanisms of action in vivo, and the changes in Bcl-xL and Fas induced by CD20 occupancy were linked to explain the synergy of these drugs. Tumor growth profiles predicted by the model agreed with cell and xenograft data, capturing the apparent pharmacologic synergy of these agents with fidelity. Together, our findings provide a mechanism-based platform for exploring new regimens with CD20 agonists.

Differential pharmacodynamic effects of paclitaxel formulations in an intracranial rat brain tumor model.

Nano- and microparticulate carriers can exert a beneficial impact on the pharmacodynamics of anticancer agents. To investigate the relationships between carrier and antitumor pharmacodynamics, paclitaxel incorporated in liposomes (L-pac) was compared with the clinical standard formulated in Cremophor-EL/ethanol (Cre-pac) in a rat model of advanced primary brain cancer. Three maximum-tolerated-dose regimens given by intravenous administration were investigated: 50 mg/kg on day 8 (d8) after implantation of 9L gliosarcoma tumors; 40 mg/kg on d8 and d15; 20 mg/kg on d8, d11, and d15. Body weight change and neutropenia were assessed as pharmacodynamic markers of toxicity. The pharmacodynamic markers of antitumor efficacy were increase in lifespan (ILS) and tumor volume progression, measured noninvasively by magnetic resonance imaging. At equivalent doses, neutropenia was similar for both formulations, but weight loss was more severe for Cre-pac. No regimen of Cre-pac extended survival, whereas L-pac at 40 mg/kg x2 doses was well tolerated and mediated 26% ILS (p < 0.0002) compared with controls. L-pac at a lower cumulative dose (20 mg/kg x3) was even more effective (40% ILS; p < 0.0001). In striking contrast, the identical regimen of Cre-pac was lethal. Development of a novel semimechanistic pharmacodynamic model permitted quantitative hypothesis testing with the tumor volume progression data, and suggested the existence of a transient treatment effect that was consistent with sensitization or "priming" of tumors by more frequent L-pac dosing schedules. Therefore, improved antitumor responses of carrier-based paclitaxel formulations can arise both from dose escalation, because of reduced toxicity, and from novel carrier-mediated alterations of antitumor pharmacodynamic effects.

Control-relevant modeling of the antitumor effects of 9-nitrocamptothecin in SCID mice bearing HT29 human colon xenografts.

The mathematical model structure selected to describe system behavior is at least partially dependent on the proposed use of the model. In this paper, a pharmacokinetic(PK)/pharmacodynamic (PD) model for use in drug delivery algorithm synthesis is developed. The antitumor agent 9-nitrocamptothecin (9NC) was administered orally to severe combined immunodeficient (SCID) mice bearing subcutaneously implanted HT29 human colon xenografts, and the effect of 9NC on those xenografts was characterized. Different PK model structures were considered in characterizing the dynamics of the drug concentration in the plasma. Akaike's Information Criterion (AIC) was used to select the model structure maximizing fit accuracy while simultaneously minimizing the number of model parameters. The resulting PK model was a set of coupled linear ordinary differential equations able to describe the nonlinear dynamic behavior (e.g. plateauing, etc.) of the drug concentrations observed in the plasma. Pharmacodynamics were modeled by characterizing tumor growth in both the untreated and drug-treated animals. The resulting PK/PD model related drug administration to effect, and this model has a structure that facilitates future control algorithm synthesis. Control algorithms in this context would directly utilize PK/PD model predictions. These predictions would be used to determine the amount and frequency of drug administration in order to reduce the tumor burden without violating clinically relevant constraints. This methodology could then be used to aid the clinician in selecting dose levels and schedules, and extension to patient tailored treatment may eventually be feasible with this approach.