Quantitative Systems Pharmacology Approaches for Immuno-Oncology: Adding Virtual Patients to the Development Paradigm.

Drug development in oncology commonly exploits the tools of molecular biology to gain therapeutic benefit through reprograming of cellular responses. In immuno-oncology (IO) the aim is to direct the patient's own immune system to fight cancer. After remarkable successes of antibodies targeting PD1/PD-L1 and CTLA4 receptors in targeted patient populations, the focus of further development has shifted toward combination therapies. However, the current drug-development approach of exploiting a vast number of possible combination targets and dosing regimens has proven to be challenging and is arguably inefficient. In particular, the unprecedented number of clinical trials testing different combinations may no longer be sustainable by the population of available patients. Further development in IO requires a step change in selection and validation of candidate therapies to decrease development attrition rate and limit the number of clinical trials. Quantitative systems pharmacology (QSP) proposes to tackle this challenge through mechanistic modeling and simulation. Compounds' pharmacokinetics, target binding, and mechanisms of action as well as existing knowledge on the underlying tumor and immune system biology are described by quantitative, dynamic models aiming to predict clinical results for novel combinations. Here, we review the current QSP approaches, the legacy of mathematical models available to quantitative clinical pharmacologists describing interaction between tumor and immune system, and the recent development of IO QSP platform models. We argue that QSP and virtual patients can be integrated as a new tool in existing IO drug development approaches to increase the efficiency and effectiveness of the search for novel combination therapies.

Phase 1 Dose Escalation Study of MEDI-565, a Bispecific T-Cell Engager that Targets Human Carcinoembryonic Antigen, in Patients With Advanced Gastrointestinal Adenocarcinomas.

INTRODUCTION: MEDI-565, a bispecific, single-chain antibody targeting human carcinoembryonic antigen on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex, showed antitumor activity in carcinoembryonic antigen-expressing tumors in murine models. PATIENTS AND METHODS: This phase I, multicenter, open-label dose escalation study enrolled adults with gastrointestinal adenocarcinomas. MEDI-565 was given intravenously over 3 hours on days 1 through 5 in 28-day cycles, with 4 single-patient (0.75-20 µg) and 5 standard 3 + 3 escalation (60 µg-3 mg; 1.5-7.5 mg with dexamethasone) cohorts. Primary objective was determining maximum tolerated dose; secondary objectives were evaluating pharmacokinetics, antidrug antibodies, and antitumor activity. RESULTS: Thirty-nine patients were enrolled (mean age, 59 years; 56% male; 72% colorectal cancer). Four patients experienced dose-limiting toxicities (2 at 3 mg; 2 at 7.5 mg + dexamethasone): hypoxia (n = 2), diarrhea, and cytokine release syndrome (CRS). Five patients reported grade 3 treatment-related adverse events: diarrhea, CRS, increased alanine aminotransferase, hypertension (all, n = 1), and hypoxia (n = 2); 6 experienced treatment-related serious adverse events: diarrhea, vomiting, pyrexia, CRS (all, n = 1), and hypoxia (n = 2). MEDI-565 pharmacokinetics was linear and dose-proportional, with fast clearance and short half-life. Nineteen patients (48.7%) had antidrug antibodies; 5 (12.8%) had high titers, 2 with decreased MEDI-565 concentrations. No objective responses occurred; 11 (28%) had stable disease as best response. CONCLUSIONS: The maximum tolerated dose of MEDI-565 in this patient population was 5 mg administered over 3 hours on days 1 through 5 every 28 days, with dexamethasone. Pharmacokinetics were linear. No objective responses were observed.

MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.

Functional identification of novel activities: activity-based selection of proteins from complete proteomes.

The identification of proteins with desired activities, especially from complex samples such as plasma and whole blood, is a continual challenge. We have developed a technology platform called Functional Identification of Novel Activities (FIoNA) to discover desired protein activities from complex biological samples. FIoNA uses immobilized libraries of combinatorial peptide ligands to purify and concentrate essentially all of the components of a complex mixture on ligands synthesized on individual beads. No depletion or prefractionation of the starting material is performed before it is incubated with the library, and no a priori knowledge of the active protein or of the ligand to which it binds is required. Instead, the protein-loaded beads are individually evaluated en masse in disease- relevant assays to identify proteins possessing a desired function. Beads associated with the activity are selected, and the ligand is sequenced and resynthesized in bulk on the original backbone for purification and characterization of the active component. Here we illustrate the use of FIoNA in a cell proliferation assay to detect a growth factor present in conditioned cell medium at nanogram/milliliter concentrations. We also have selected beads associated with hydrolysis of nerve agent analogs in assays performed in 100,000-well microtiter plates.

A fluorogenic substrate for detection of organophosphatase activity.

A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.

Failure of immunocompetitive capillary electrophoresis assay to detect disease-specific prion protein in buffy coat from humans and chimpanzees with Creutzfeldt-Jakob disease.

The emergence of a new environmentally caused variant of Creutzfeldt-Jakob disease (vCJD), the result of food-born infection by the causative agent of bovine spongiform encephalopathy (BSE), has stimulated research on a practical diagnostic screening test. The immunocompetitive capillary electrophoresis (ICCE) assay has been reported to detect disease-specific, proteinase-resistant prion protein (PrPres) in the blood of scrapie-infected sheep. We have applied this method to blood from CJD-infected chimpanzees and humans. The threshold of detection achieved with our ICCE was 0.6 nM of synthetic peptide corresponding to the prion protein (PrP) C-terminus, and 2 nM of recombinant human PrP at the optimized conditions. However, the test was unable to distinguish between extracts of leucocytes from healthy and CJD-infected chimpanzees, and from healthy human donors and patients affected with various forms of CJD. Thus, the ICCE assay as presently performed is not suitable for use as a screening test in human transmissible spongiform encephalopathies (TSEs).

The use of the uromodulin promoter to target production of recombinant proteins into urine of transgenic animals.

A uromodulin promoter has been isolated, sequenced, and used to generate two sets of transgenic mice for expression of the lacZ marker gene and for production of the human recombinant erythropoietin (rhEPO) in urine. We demonstrated that the 5.6-kb fragment of the uromodulin gene containing the 3.7-kb promoter area and, both the first exon and part of the second exon, were sufficient to provide kidney-specific expression of the lacZ gene. Histological analysis of the lacZ expression pattern revealed beta-galactosidase activity specifically in the thick limb of Henle's loop. However, due to random integration of the transgene, ectopic expression was detected in some transgenic lines. Analysis of the EPO-transgenic mice showed that rhEPO was secreted into the urine of founder mice (up to 6 ng/ml). We were able to breed and analyze only two sublines with a very low expression level of rhEPO (up to 260 pg/ml). All of our transgenic mice expressing rhEPO in urine developed disease symptoms similar to polycythemia in humans. These included a considerable increase in red blood cell counts, hemoglobin concentration, and hematocrit concomitant with severe thrombocytopenia, all of which were detected in the rhEPO-expressing mice. Although our model did not prove to be beneficial for commercial production of rhEPO, we concluded that the uromodulin promoter could be useful for expression of other important therapeutic proteins into the urine of transgenic animals.

Expression of factor VIII in recombinant and transgenic systems.

Deficiency in a coagulation factor VIII (FVIII) causes a genetic disorder hemophilia A, which is treated by repeated infusions of expensive FVIII products. Recombinant FVIII (rFVIII), the culmination of years of extensive international research, is an important alternative to plasma-derived FVIII (pdFVIII) and is considered to have a higher margin of safety. Advances in biotechnology allowed production of rFVIII at industrial scale, which significantly improved treatment of hemophilia A patients. We review the contemporary methods used for FVIII expression in mammalian cell culture systems and discuss the factors responsible for insufficient recoveries of rFVIII, such as inefficient accumulation of FVIII mRNA in the cell, complexity of the mechanisms of FVIII secretion, and instability of secreted FVIII. The approaches to improve the yield of rFVIII in cell culture systems include genetic engineering of B-domain-deleted FVIII, introduction of introns into FVIII cDNA constructs for more efficient processing and accumulation of FVIII mRNA, and introduction of mutations into chaperone-binding sites of FVIII to improve its secretion. Design of FVIII with prolonged half-life in vivo is considered as another promising direction in improving rFVIII protein and efficiency of hemophilia A therapy. As an alternative to expression of rFVIII in cell culture systems, we discuss production of rFVIII in transgenic animals, where high levels of rFVIII have been successfully secreted into milk. We also pay attention to the major limitations of this approach, such as safety issues associated with potential transmission of animal pathogens. Finally, we present a brief characterization of commercial recombinant FVIII products currently available on the market for hemophilia A treatment.

Uromodulin promoter directs high-level expression of biologically active human alpha1-antitrypsin into mouse urine.

We have recently shown that the regulatory sequence of the uromodulin gene, containing the 3.7 kb promoter, exon 1 and a part of exon 2, provided for kidney-specific expression of the reporter lacZ gene in transgenic mice [Zbikowska, Soukhareva, Behnam, Chang, Drews, Lubon, Hammond and Soukharev (2002) Transgenic Res., in the press]. In the present study, we generated transgenic mice harbouring the regulatory sequence of the uromodulin gene to direct the expression of human alpha1-antitrypsin (alpha1AT) into urine. Of the 13 founder mice that tested positive by PCR, seven showed the presence of the human protein in their urine. The concentration of the recombinant human (rh) alpha1AT in the urine, estimated by using ELISA, ranged from 0.5 to 14 microg/ml in the F(0)-generation mice, and reached up to 65 microg/ml in the F1 generation. The transgenically produced rh alpha1AT was found to be N-glycosylated and biologically active. The N-terminal sequence analysis confirmed the identity of the human protein and revealed that the recombinant alpha1AT was correctly processed with the signal peptide cleaved off. Our results demonstrate for the first time that the uromodulin regulatory sequence provides a very attractive option for the potential large-scale production of functional therapeutic proteins in livestock.