The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ∼2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.

Purpose and Methods Trop-2 is a glycoprotein over-expressed in many solid tumors but at low levels in normal human tissue, providing a potential therapeutic target. We conducted a phase 1 dose-finding study of PF-06664178, an antibody-drug conjugate that targets Trop-2 for the selective delivery of the cytotoxic payload Aur0101. The primary objective was to determine the maximum tolerated dose and recommended phase 2 dose. Secondary objectives included further characterization of the safety profile, pharmacokinetics and antitumor activity. Eligible patients were enrolled and received multiple escalating doses of PF-06664178 in an open-label and unblinded manner based on a modified continual reassessment method. Results Thirty-one patients with advanced or metastatic solid tumors were treated with escalating doses of PF-06664178 given intravenously every 21 days. Doses explored ranged from 0.15 mg/kg to 4.8 mg/kg. Seven patients experienced at least one dose limiting toxicity (DLT), either neutropenia or rash. Doses of 3.60 mg/kg, 4.2 mg/kg and 4.8 mg/kg were considered intolerable due to DLTs in skin rash, mucosa and neutropenia. Best overall response was stable disease in 11 patients (37.9%). None of the patients had a partial or complete response. Systemic exposure of PF-06664178 increased in a dose-related manner. Serum concentrations of free Aur0101 were substantially lower than those of PF-06664178 and total antibody. No correlation of Trop-2 expression and objective response was observed, although Trop-2 overexpression was not required for study entry. The intermediate dose of 2.4 mg/kg appeared to be the highest tolerated dose, but this was not fully explored as the study was terminated early due to excess toxicity. Conclusion PF-06664178 showed toxicity at high dose levels with modest antitumor activity. Neutropenia, skin rash and mucosal inflammation were dose limiting toxicities. Findings from this study may potentially aid in future antibody drug conjugate design and trials.

PF‐06647263, a novel antibody–drug conjugate consisting of an anti‐EFNA4 antibody linked to a calicheamicin payload, has shown potent antitumor activity in human xenograft tumor models, including triple‐negative breast cancer (TNBC). In the dose‐escalation part 1 of this multicenter, open‐label, phase I study (NCT02078752), successive cohorts of patients (n, 48) with advanced solid tumors and no available standard therapy received PF‐06647263 every 3 weeks (Q3W) or every week (QW), following a modified toxicity probability interval (mTPI) method (initial dosing: 0.015 mg/kg Q3W). Primary objective in part 1 was to estimate the maximum tolerated dose (MTD) and select the recommended phase 2 dose (RP2D). In part 2 (dose‐expansion cohort), 12 patients with pretreated, metastatic TNBC received PF‐06647263 at the RP2D to further evaluate tumor response and overall safety. PF‐06647263 QW administration (n, 23) was better tolerated than the Q3W regimen (n, 25) with only 1 DLT reported (thrombocytopenia). The most common AEs with the QW regimen (fatigue, nausea, vomiting, mucosal inflammation, thrombocytopenia, and diarrhea) were mostly mild to moderate in severity. The MTD was not estimated. PF‐06647263 exposures increased in a dose‐related manner across the doses evaluated. The RP2D was determined to be 0.015 mg/kg QW. Six (10%) patients achieved a confirmed partial response and 22 (36.7%) patients had stable disease. No correlations were observed between tumor responses and EFNA4 expression levels. Study findings showed manageable safety and favorable PK for PF‐06647263 administered QW at the RP2D, with preliminary evidence of limited antitumor activity in patients with TNBC and ovarian cancer.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3g for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3g levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3g (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3g in tissue, and reduction of body weight in obese mice was used to generate a pharmacokineticpharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

2511 Background: PF-06647263 is an anti-Ephrin-A4 (EFNA4) antibody drug conjugate (ADC) composed of a humanized mAb, a hydrazone cleavable linker, and calicheamicin, a potent DNA damaging agent. Higher levels of EFNA4 expression have been shown in tumor versus normal tissue, including in two thirds of triple negative breast cancers (TNBC). In vivo preclinical studies demonstrate PF-06647263 induced tumor regression in TNBC models. Methods: In Part 1 of a dose escalation, cohorts of 2-12 patient (pts) with solid tumors that were unselected for EFNA4 expression received escalating doses of PF-06647263 once every 3 weeks (Q3W, Cohort A) or weekly (QW, Cohort B). Escalations were based on mTPI design. An expansion cohort enrolled TNBC patients (n=12) unselected for EFNA4 expression. Efficacy, safety, EFNA4 RNA expression, pharmacokinetic (PK) and anti-drug antibody development were assessed. Results: Part 1 (dose escalation):A total of 48 pts (25 in A and 23 in B) wereenrolled. The most common treatment related adverse events (AE) were fatigue (65%), and nausea (60%), thrombocytopenia (40%), and decreased appetite (38%). DLTs were observed in 6 and 2 pts in the Q3W and QW regimens, respectively. One confirmed VOD and one suspected VOD were observed in two patients in the Q3W schedule. The maximum tolerated dose (and RP2D) was determined to be 0.015 mg/kg QW. Confirmed partial responses (PR) were observed in 5 pts (3 Ovarian Ca and 2 TNBC). Part 2 (TNBC dose expansion at RP2D)data are available on 10 of 12 pts treated (2 ongoing). The most common adverse events (AE) were nausea (40%), asthenia (30%), vomiting (30%) and mucosal inflammation (30%). No objective RECIST response was observed; there was no dependency with duration of treatment relative to EFNA4 expression. Conclusions: In Part 1 of this study, PF-06647263 was generally well-tolerated in the QW schedule and some anti-tumor activity was observed in heavily pretreated pts with EFNA4 unselected advanced malignancies. However, in the expansion cohort (Part 2) at RP2D in TNBC no objective responses were observed regardless of EFNA4 expression. Final safety, efficacy, expression and PK data will be reported at the meeting. Clinical trial information: NCT02078752.