An IQ consortium analysis of starting dose selection for oncology small molecule first-in-patient trials suggests an alternative NOAEL-based method can be safe while reducing time to the recommended phase 2 dose.

The first-in-patient (FIP) starting dose for oncology agents should be reasonably safe and provide potential therapeutic benefit to the patient. For late-stage oncology patients, this dose is often based on the ICH S9 guidance, which was developed primarily based on experience with cytotoxic chemotherapeutic agents using the rodent STD10 or non-rodent HNSTD and an appropriate safety factor. With the increase in molecularly targeted chemotherapeutics, it is prudent to re-evaluate how the FIP dose is derived to ensure that the appropriate balance between risk and therapeutic benefit to the patient is achieved. Blinded data on 92 small molecule oncology compounds from 12 pharmaceutical companies who are members of the IQ DruSafe consortium were gathered to investigate if a NOAEL-based starting dose without a safety factor would have been tolerated in the FIP trial and if so, estimating how many dose escalation cohorts could have been reduced. Our analysis suggests that the NOAEL-based alternative starting dose would have been tolerated in most cases evaluated, with an anticipated mean reduction of 2.3 cohorts. Of the 12 cases where the alternative approach resulted in a starting dose that would have exceeded the MTD/RP2D, none of the nonclinical toxicities in these cases were considered irreversible and would be monitorable in all but one instance. Most non-tolerated cases were within two-threefold of the MTD/RP2D, with the clinical AEs considered manageable and mitigated by dose de-escalation. No one method of FIP dose calculation will likely be appropriate for all oncology small molecules and starting dose selection should be performed using a case-by-case approach. However, the NOAEL-based method that does not utilize a safety factor should be considered when appropriate to minimize the number of patients exposed to sub-therapeutic doses of an investigational oncology agent and accelerating development to RP2D.

Off-target immune cell toxicity caused by AG-012986, a pan-CDK inhibitor, is associated with inhibition of p38 MAPK phosphorylation.

AG-012986 is a pan-CDK (cyclin-dependent kinase) inhibitor that has in vitro and in vivo antitumor properties but was stopped in development due in part to rapid bone-marrow-independent white blood cell toxicity in preclinical studies and the potential for acute and delayed immunosuppression in humans. Because peripheral lymphocytes are largely nonproliferating, it was hypothesized the toxicity of AG-012986 was due to an off-target mechanism and not driven by the intended pharmacology. We show the toxicity mechanism in primary human immune cells is caspase driven. T-cells treated with AG-012986 and acutely stimulated through the T-cell receptor exhibited decreased toxicity while still maintaining cell division inhibition. This indicated that the pharmacology of AG-012986 functioned as expected but the toxicity had now been decoupled through activation. Induced phosphorylation of p38 and IL-2 production was impaired with AG-012986. Thus, AG-012986 could cause apoptosis of T-cells by targeting upstream kinases in the p38 Mitogen-activated protein kinase (MAPK) pathway and impairing cellular survival.

A tyrosine kinase inhibitor-induced myocardial degeneration in rats through off-target phosphodiesterase inhibition.

PF-04254644 is a selective kinase inhibitor of mesenchymal epithelial transition factor/hepatocyte growth factor receptor with known off-target inhibitory activity against the phosphodiesterase (PDE) family. Rats given repeated oral doses of PF-04254644 developed a mild to moderate myocardial degeneration accompanied by sustained increase in heart rate and contractility. Investigative studies were conducted to delineate the mechanisms of toxicity. Microarray analysis of Sprague-Dawley rat hearts in a 6 day repeat dose study with PF-04254644 or milrinone, a selective PDE3 inhibitor, revealed similar perturbation of the cyclic adenosine monophosphate (c-AMP) pathway. PDE inhibition and activation of c-AMP were further substantiated using PDE3B immunofluorescence staining and through a c-AMP response element reporter gene assay. The intracellular calcium and oxidative stress signaling pathways were more perturbed by treatment with PF-04254644 than milrinone. The rat cardiomyocytes calcium assay found a dose-dependent increase in intracellular calcium with PF-04254644 treatment. These data suggest that cardiotoxicity of PF-04254644 was probably due to activation of c-AMP signaling, and possibly subsequent disruption of intracellular calcium and oxidative stress signaling pathways. The greater response with PF-04254644 as compared with milrinone in gene expression and micro- and ultrastructural changes is probably due to the broader panel of PDEs inhibition.

Evaluation of the effects of a VEGFR-2 inhibitor compound on alanine aminotransferase gene expression and enzymatic activity in the rat liver.

BACKGROUND: Traditional assessment of drug-induced hepatotoxicity includes morphological examination of the liver and evaluation of liver enzyme activity in serum. The objective of the study was to determine the origin of drug-related elevation in serum alanine aminotransferase (ALT) activity in the absence of morphologic changes in the liver by utilizing molecular and immunohistochemical techniques. METHODS: Sixteen female Sprague-Dawley rats were divided into 2 groups (control and treated, n = 4 per group) and treated rats were dosed orally twice daily (400 mg/kg/day) for 7 days with a VEGFR-2 compound (AG28262), which in a previous study caused ALT elevation without morphological changes. Serum of both treated and control animals were evaluated on day 3 of treatment and at day 8. Three separate liver lobes (caudate, right medial, and left lateral) were examined for determination of ALT tissue activity, ALT gene expression and morphological changes. RESULTS: ALT activity was significantly (p < 0.01) elevated on day 3 and further increased on day 8. Histologic changes or increase in TUNEL and caspase3 positive cells were not observed in the liver lobes examined. ALT gene expression in the caudate lobe was significantly up-regulated by 63%. ALT expression in the left lateral lobe was not significantly affected. Statistically significant increased liver ALT enzymatic activity occurred in the caudate (96%) and right medial (41%) lobes but not in the left lateral lobe. CONCLUSIONS: AG28262, a VEFG-r2 inhibitor, causes an increase in serum ALT, due in part to both gene up-regulation. Differences between liver lobes may be attributable to differential distribution of blood from portal circulation. Incorporation of molecular data, such as gene and protein expression, and sampling multiple liver lobes may shed mechanistic insight to the evaluation of hepatotoxicity.

Microphysiological systems in early stage drug development: Perspectives on current applications and future impact.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

Phenotypic Characterization of Targeted Knockdown of Cyclin-Dependent Kinases in the Intestinal Epithelial Cells.

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition.

Pharmacologic Properties and Preclinical Activity of Sasanlimab, A High-affinity Engineered Anti-Human PD-1 Antibody.

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.

Corneal neovascularization and ocular irritancy responses in dogs following topical ocular administration of an EP4-prostaglandin E2 agonist.

Prostaglandin receptor agonists have intraocular pressure-lowering effects in humans and are of interest in the treatment of glaucoma. The prostanoid receptor agonist PF-04475270 is a potent and selective agonist of the prostaglandin E(2) receptor EP4. This paper characterizes the toxicity associated with topical ocular administration of PF-04475270 in beagles. Dogs were given PF-04475270 topically to the eye on a consecutive daily dosing schedule for one or four weeks followed by a one-or four-week reversal period, respectively. Clinical observations, ophthalmic, and laboratory parameters were recorded. Necropsies were conducted at the end of the dosing and recovery phases, and histologic examinations performed. Corneal neovascularization that was considered adverse was observed at doses of >or=1.0 microg/eye and was not reversed by the end of the recovery phase. Dogs dosed with >or=0.25 microg/eye developed a dose-related conjunctival hyperemia that persisted throughout the reversal period. Corneal neovascular cells stained positive with EP4 and the endothelial biomarker Factor VIII-vWF. Other histopathology findings observed at doses of >or=1.0 microg included single-cell necrosis and neutrophils in the cornea, inflammatory cell infiltrates in the iris/ciliary body, and iridal endothelial cell hypertrophy. A resolving acute to subacute inflammation in the iris/ciliary body was observed after the four-week recovery period.

Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model.

BACKGROUND: Benzalkonium chloride (BAC) is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D) corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. METHODS: The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC) and olopatadine (0.01% BAC) was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T) cell cultures, expression levels (mRNA and protein) of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 microL drops twice daily in 1 eye for 1 year) in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. RESULTS: In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC) and untreated eyes. CONCLUSION: The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC contained in ophthalmic solutions are not likely to cause significant direct toxicity to epithelium of otherwise normal corneas.

Pharmacologic properties of AG-012986, a pan-cyclin-dependent kinase inhibitor with antitumor efficacy.

AG-012986 is a multitargeted cyclin-dependent kinase (CDK) inhibitor active against CDK1, CDK2, CDK4/6, CDK5, and CDK9, with selectivity over a diverse panel of non-CDK kinases. Here, we report the potent antitumor efficacies of AG-012986 against multiple tumor lines in vitro and in vivo. AG-012986 showed antiproliferative activities in vitro with IC(50)s of <100 nmol/L in 14 of 18 tumor cell lines. In vivo, significant antitumor efficacy induced by AG-012986 was seen (tumor growth inhibition, >83.1%) in 10 of 11 human xenograft tumor models when administered at or near the maximum tolerated dose for 8 or 12 days. AG-012986 caused dose-dependent hypophosphorylation at Ser(795) of the retinoblastoma protein, cell cycle arrest, and apoptosis in vitro. Colony-forming assays indicated that the potency of AG-012986 substantially decreased with treatment time of <24 h. In vivo, AG-012986 also showed dose-dependent retinoblastoma Ser(795) hypophosphorylation, cell cycle arrest, decreased Ki-67 tumor staining, and apoptosis in conjunction with antitumor activity. Studies comparing i.p. bolus with s.c. implanted minipump dosing regimens revealed that in vivo efficacy correlated with the duration of minimally effective plasma levels rather than maximal drug plasma levels. Dosing optimization of AG-012986 provided guidance for selecting a treatment schedule to achieve the best antitumor efficacy while minimizing the risk of adverse side effects.

Intestinal Toxicity in Rats Following Administration of CDK4/6 Inhibitors Is Independent of Primary Pharmacology.

Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib, and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles, however, are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however, it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. To understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib, and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathologic findings, and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli, and mucosal inflammation. In the jejunum of abemaciclib-treated rats, downregulation of enterocyte membrane transporters and upregulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacologic targets.

Antisense inhibition of 11betahydroxysteroid dehydrogenase type 1 improves diabetes in a novel cortisone-induced diabetic KK mouse model.

The inhibition of 11betahydroxysteroid dehydrogenase 1 (11betaHSD1), an enzyme that catalyzes the conversion of inactive cortisone to active cortisol, is an attractive target to treat diabetes by suppressing hepatic gluconeogenesis. To test this hypothesis, we developed a novel glucocorticoid-induced diabetic KK mouse model and used 11betaHSD1 antisense oligonucleotide (ASO) as an inhibitory tool. KK mice were treated with 25 or 50mg/kg/day of 11betaHSD1 ASO for 28 days. On day 25, cortisone pellets were surgically implanted to induce diabetes. In the ASO-treated mice, plasma blood glucose levels were significantly reduced by up to 54%. In parallel, cortisol and other diabetes endpoints were also significantly reduced. Hepatic 11betaHSD1 mRNA was suppressed by up to 84% with a concomitant respective decrease of up to 49% in the expression of PEPCK. The results suggest that inhibition of 11betaHSD1 activity reduces the availability of cortisol to activate the glucocorticoid receptor, down regulates gluconeogenesis and thus reduces plasma glucose levels in cortisone-induced diabetic KK mice.

Lysosomal adaptation: How cells respond to lysosomotropic compounds.

Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.

Validation of Cross-Species Reactivity of the VEGF-A/PDGFRß Bifunctional Antibody PF-06653157.

PURPOSE: PF-06653157 is a bifunctional antagonist monoclonal antibody (mAb) that targets human VEGF-A ligand and PDGF-Rß. With the advent of PF-06653157 as an angiogenesis inhibitor and potential treatment for angiogenesis deregulation diseases, a relevant toxicology species is needed for toxicity and efficacy studies. Investigative studies were conducted to validate the mAb dual antagonist properties in a human system and determine its cross-reactive pharmacology in nonhuman cells. METHODS: Sequence alignment was used to determine percent sequence identity of VEGF and PDGF receptors and ligands; qualitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the presence of PDGF-Rß on cells of interest. The functional activity of PF-06653157 antibody was assessed in human, dog, porcine, rabbit, rat, mouse, and cynomolgus monkey cells treated with VEGF and PDGF ligands through cell proliferation assays and western blot analysis of AKT and p44/p42 (ERK1/2) protein phosphorylation and enzyme-linked immunosorbent assay. RESULTS: PF-06653157 attenuated phosphorylation of AKT and p44/p42 proteins in human and cynomolgus monkey cells. The antibody did not attenuate AKT nor p44/p42 phosphorylation in any other species tested. PDGFR signaling could not be activated with human PDGF ligand in the porcine cells, so PF-06653157 activity in porcine remains inconclusive. CONCLUSION: The PF-06653157 mAb cross-reacts with cynomolgus monkey cells in a similar manner to human cells. Therefore, cynomolgus monkeys are considered the appropriate species for efficacy and regulatory toxicology studies in PF-06653157 development.

Mechanistic Investigation of Bone Marrow Suppression Associated with Palbociclib and its Differentiation from Cytotoxic Chemotherapies.

PURPOSE: Palbociclib (PD-0332991) is the first selective cyclin-dependent kinase (CDK) 4/6 inhibitor approved for metastatic breast cancer. Hematologic effects, especially neutropenia, are dose-limiting adverse events for palbociclib in humans. EXPERIMENTAL DESIGN: Reversible hematologic effects and bone marrow hypocellularity have been identified in toxicology studies in rats and dogs after palbociclib treatment. To understand the mechanism by which the hematologic toxicity occurs, and to further differentiate it from the myelotoxicity caused by cytotoxic chemotherapeutic agents, anin vitroassay using human bone marrow mononuclear cells (hBMNC) was utilized. RESULTS: This work demonstrated that palbociclib-induced bone marrow suppression occurred through cell-cycle arrest, with no apoptosis at clinically relevant concentrations, was not lineage-specific, and was reversible upon palbociclib withdrawal. In contrast, treatment with chemotherapeutic agents (paclitaxel and doxorubicin) resulted in DNA damage and apoptotic cell death in hBMNCs. In the presence or absence of the antiestrogen, palbociclib-treated hBMNCs did not become senescent and resumed proliferation following palbociclib withdrawal, consistent with pharmacologic quiescence. The breast cancer cells, MCF-7, conversely, became senescent following palbociclib or antiestrogen treatment with additive effects in combination and remained arrested in the presence of antiestrogen. CONCLUSIONS: Palbociclib causes reversible bone marrow suppression, clearly differentiating it from apoptotic cell death caused by cytotoxic chemotherapeutic agents. This study also distinguished the cell-cycle arresting action of palbociclib on normal bone marrow cells from the senescent effects observed in breast cancer cells. These results shed light on the mechanism and support risk management of palbociclib-induced bone marrow toxicity in the clinic.

Contribution of membrane trafficking perturbation to retinal toxicity.

The retina is a highly structured tissue that is formed by layers containing 7 different cell types. The photoreceptor cell is a specialized type of neuron in the retina that is capable of absorbing and converting light into electrophysiological signals. There is a constant renewal process for photoreceptors consisting of intermittent shedding of the distal tips of the photosensitive outer segment and subsequent phagocytosis (uptake, degradation and recycling) by retinal pigmented epithelial (RPE) cells. This rebuilding process is essential for vision and the survival of photoreceptors and RPE cells. Drugs with a basic moiety have the potential to accumulate in the lysosome and impair its functions including the phagocytosis process, which could hinder clearance of outer segments and ultimately induce retinopathy. To determine the prevalence of this cellular mechanism in retinal toxicity, a collection of proprietary compounds associated with retinal toxicity were subjected to a battery of in vitro tests using the human adult retinal pigmented epithelium cell line, ARPE-19. The tests included a phagocytosis assay, and lysosomal and autophagosomal staining. The compounds that induced retinopathy clustered in the basic and lipophilic region, which drives lysosomal sequestration. This accumulation coincided with phagocytosis inhibition and an increase in autophagosome staining, suggesting a blockage of the membrane trafficking process. A correlation between the physicochemical properties and in vitro lysosomal pathway effects was established. These data reveal the importance of physicochemical properties of compounds and lysosome accumulation as a potential mechanism for drug-induced retinopathy and demonstrate the usefulness of in vitro screening in predicting this liability.

Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 T cells in a poorly immunogenic tumor model.

Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti-4-1BB/anti-PD-1 combination with that of the anti-PD-1/anti-LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti-PD-1 and anti-4-1BB concomitantly, while combining anti-PD-1 with anti-LAG-3 led to a modest degree of tumor suppression. The activity of the anti-4-1BB/anti-PD-1 combination was dependent on IFNγ and CD8(+) T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8(+) T cells by anti-4-1BB/anti-PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8(+)/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8(+) CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti-4-1BB/anti-PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients.

Expression profiling during adipocyte differentiation of 3T3-L1 fibroblasts.

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.

Assessment of hepatocytes and liver slices as in vitro test systems to predict in vivo gene expression.

The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.