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Supplementary Materials and Methods, and Supplementary Figures

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells.Significance:The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. Significance: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy. See related commentary by Burton and Tawbi, p. 1008. This article is highlighted in the In This Issue feature, p. 995

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1

In the wake of the success of modern immunotherapy, oncolytic viruses (OVs) are currently seen as a potential therapeutic option for patients with cancer who do not respond or fail to achieve durable responses following treatment with immune checkpoint inhibitors. OVs offer a multifaceted therapeutic platform because they preferentially replicate in tumour cells, can be engineered to express transgenes that augment their cytotoxic and immunostimulatory activities, and modulate the tumour microenvironment to optimize immune-mediated tumour eradication, both at locoregional and systemic sites of disease. Lysis of tumour cells releases tumour-specific antigens that trigger both the innate and adaptive immune systems. OVs also represent attractive combination partners with other systemically delivered agents by virtue of their highly favourable safety profiles. Rational combinations of OVs with different immune modifiers and/or antitumour agents, based on mechanisms of tumour resistance to immune-mediated attack, may benefit the large, currently underserved, population of patients who respond poorly to immune checkpoint inhibition. Oncolytic viruses are currently seen as a potential therapeutic option for patients with cancer who do not respond to immune checkpoint inhibitors. This Review discusses the different therapeutic approaches to develop oncolytic viruses, delivery modalities and methods for assessing their biological activity.

Phosphoinositide 3-kinase (PI3K) is an important target in oncology due to the deregulation of the PI3K/Akt signaling pathway in a wide variety of tumors. A series of 4-amino-6-methyl-1,3,5-triazine sulfonamides were synthesized and evaluated as inhibitors of PI3K. The synthesis, in vitro biological activities, pharmacokinetic and in vivo pharmacodynamic profiling of these compounds are described. The most promising compound from this investigation (compound 3j) was found to be a pan class I PI3K inhibitor with a moderate (>10-fold) selectivity over the mammalian target of rapamycin (mTOR) in the enzyme assay. In a U87 MG cellular assay measuring phosphorylation of Akt, compound 3j displayed low double digit nanomolar IC(50) and exhibited good oral bioavailability in rats (F(oral)=63%). Compound 3j also showed a dose dependent reduction in the phosphorylation of Akt in a U87 tumor pharmacodynamic model with a plasma EC(50)=193 nM (91 ng/mL).

The phosphoinositide 3-kinase family catalyzes the phosphorylation of phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-triphosphate, a secondary messenger which plays a critical role in important cellular functions such as metabolism, cell growth, and cell survival. Our efforts to identify potent, efficacious, and orally available phosphatidylinositol 3-kinase (PI3K) inhibitors as potential cancer therapeutics have resulted in the discovery of 4-(2-((6-methoxypyridin-3-yl)amino)-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (1). In this paper, we describe the optimization of compound 1, which led to the design and synthesis of pyridyltriazine 31, a potent pan inhibitor of class I PI3Ks with a superior pharmacokinetic profile. Compound 31 was shown to potently block the targeted PI3K pathway in a mouse liver pharmacodynamic model and inhibit tumor growth in a U87 malignant glioma glioblastoma xenograft model. On the basis of its excellent in vivo efficacy and pharmacokinetic profile, compound 31 was selected for further evaluation as a clinical candidate and was designated AMG 511.

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (1) is a potent and efficacious inhibitor of PI3Kα and mTOR in vitro and in vivo. However, in hepatocyte and in vivo metabolism studies, 1 was found to undergo deacetylation on the 2-amino substituent of the benzothiazole. As an approach to reduce or eliminate this metabolic deacetylation, a variety of 6,5-heterocyclic analogues were examined as an alternative to the benzothiazole ring. Imidazopyridazine 10 was found to have similar in vitro potency and in vivo efficacy relative to 1, while only minimal amounts of the corresponding deacetylated metabolite of 10 were observed in hepatocytes.

The phosphoinositide 3-kinase (PI3K) family catalyzes the ATP-dependent phosphorylation of the 3'-hydroxyl group of phosphatidylinositols and plays an important role in cell growth and survival. There is abundant evidence demonstrating that PI3K signaling is dysregulated in many human cancers, suggesting that therapeutics targeting the PI3K pathway may have utility for the treatment of cancer. Our efforts to identify potent, efficacious, and orally available PI3K/mammalian target of rapamycin (mTOR) dual inhibitors resulted in the discovery of a series of substituted quinolines and quinoxalines derivatives. In this report, we describe the structure-activity relationships, selectivity, and pharmacokinetic data of this series and illustrate the in vivo pharmacodynamic and efficacy data for a representative compound.

A series of 2-aminothiadiazole of inhibitors of AKT1 is described. SAR relationships are discussed, along with selectivity for protein kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). Moderate selectivity observed in several compounds for AKT1 versus PKA is rationalized by X-ray crystallographic analysis. Key compounds showed activity in cellular assays measuring phosphorylation of two AKT substrates, PRAS40 and FKHRL1. Compound 30 was advanced to a mouse liver PD assay, where it showed dose-dependent inhibition of AKT activity, as measured by the inhibition of phospho-PRAS40.

Background Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K- ras , BRAF , and PIK3CA gene mutations with tumor resistance to panitumumab alone. Patients and Methods From 3 phase II panitumumab metastatic colorectal cancer (mCRC) studies, 62 of 533 patient samples were available. Mutations were identified from genomic DNA by sequencing. Results Of the 62 samples, 24 (38.7%) harbored a K- ras mutation, and 38 (61.3%) were wild type. In the wild-type K- ras group, 11% of patients had a partial response (PR), 53% had stable disease (SD), and 37% had progressive disease (PD). In the mutant K- ras group, 21% of patients had SD, and 79% of patients had PD; there were no responses. The absence of a K- ras mutation was associated with response to panitumumab (PR vs. SD vs. PD; P = .0028). The hazard ratio for wild-type versus mutant K- ras was 0.4 (95% CI, 0.2-0.7) for progression-free survival and 0.5 (95% CI, 0.3-0.9) for overall survival. Four patients had a V600E BRAF mutation, and 2 patients had a PIK3CA mutation. Conclusion These data suggest that patients with mCRC with activating K- ras mutations are less likely to respond to panitumumab alone. The small sample size limits us from defining a predictive role of PIK3CA and BRAF mutations for panitumumab treatment.

We show in this study that PTEN regulates p53 protein levels and transcriptional activity through both phosphatase-dependent and -independent mechanisms. The onset of tumor development in p53+/−;Pten+/− mice is similar to p53−/− animals, and p53 protein levels are dramatically reduced in Pten−/− cells and tissues. Reintroducing wild-type or phosphatase-dead PTEN mutants leads to a significant increase in p53 stability. PTEN also physically associates with endogenous p53. Finally, PTEN regulates the transcriptional activity of p53 by modulating its DNA binding activity. This study provides a novel mechanism by which the loss of PTEN can functionally control “two” hits in the course of tumor development by concurrently modulating p53 activity.

Introduction: This two-part phase 2 study evaluated the efficacy and safety of panitumumab, a fully human anti–epidermal growth factor receptor monoclonal antibody, combined with carboplatin/paclitaxel in patients with previously untreated advanced non–small-cell lung cancer. Methods: In part 1, patients were sequentially enrolled to receive paclitaxel 200 mg/m 2 and carboplatin (area under the concentration-versus-time curve, 6 mg/min/ml) plus panitumumab (1.0, 2.0, or 2.5 mg/kg). In part 2, patients were randomized 2:1 to receive paclitaxel/carboplatin with (arm A) or without (arm B) the maximum tolerated dose of panitumumab identified in part 1. Primary endpoints in parts 1 and 2 were the incidence of dose-limiting toxicities and time to progression (TTP), respectively. Results: In part 1, four of 19 patients had dose-limiting toxicities: three at 2.0 mg/kg (fatigue, pain in extremity, dyspepsia) and one at 2.5 mg/kg (rash). The maximum tolerated dose was not reached; panitumumab 2.5 mg/kg was selected for part 2. In part 2, TTP was 18.1 weeks (95% confidence interval [CI], 13.6−23.3) in arm A and 23.0 weeks (95% CI, 15.9−24.1) in arm B (hazard ratio, 0.9; 90% CI, 0.66−1.21; p = 0.555). Progression-free survival in arms A and B was 17.6 weeks and 18.3 weeks, respectively, and the objective response rate was 15.2% and 11.1%. Adverse events occurring more frequently in arm A than in arm B included skin toxicity, diarrhea, stomatitis, vomiting, and dizziness. Exploratory analyses did not demonstrate associations between potential biomarkers and outcomes. Conclusion: Although toxicity was predictable and manageable, the addition of panitumumab to paclitaxel/carboplatin did not improve TTP in patients with previously untreated advanced non–small-cell lung cancer.

Purpose. A plasmid containing the luciferase ‘marker’ cDNA was constructed to test non viral gene delivery formulations in vivo.

Background Successful treatment of solid tumors relies on the ability of drugs to penetrate into the tumor tissue. Methods We examined the correlation of panitumumab (an anti-epidermal growth factor [EGFR] antibody) tumor penetration and EGFR saturation, a potential obstacle in large molecule drug delivery, using pharmacokinetics, pharmacodynamics, and tumor growth rate in an A431 epidermoid carcinoma xenograft model of human cancer. To determine receptor saturation, receptor occupancy, and levels of proliferation markers, immunohistochemical and flow cytometric methods were used. Pharmacokinetic data and modeling were used to calculate growth characteristics of panitumumab-treated tumors. Results Treatment with panitumumab in vivo inhibited pEGFR, Ki67 and pMAPK levels vs control. Tumor penetration and receptor saturation were dose- and time-dependent, reaching 100% and 78%, respectively. Significant tumor inhibition and eradication (p Conclusions These data demonstrate that the antitumor activity of panitumumab correlates with its ability to penetrate into tumor tissue, occupy and inhibit activation of EGFR, and inhibit markers of proliferation and MAPK signaling.

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

Through a combination of screening and structure-based rational design, we have discovered a series of N1-(5-(heterocyclyl)-thiazol-2-yl)-3-(4-trifluoromethylphenyl)-1,2-propanediamines that were developed into potent ATP competitive inhibitors of AKT. Studies of linker strand-binding adenine isosteres identified SAR trends in potency and selectivity that were consistent with binding interactions observed in structures of the inhibitors bound to AKT1 and to the counter-screening target PKA. One compound was shown to have acceptable pharmacokinetic properties and to be a potent inhibitor of AKT signaling and of in vivo xenograft tumor growth in a preclinical model of glioblastoma.

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Δ746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 μg/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation. [Mol Cancer Ther 2009;8(6):1536–46]

Therapies targeting epidermal growth factor receptor (EGFR) are a promising recent development in the treatment of metastatic colorectal cancer (mCRC). Panitumumab is a fully human anti-EGFR monoclonal antibody that competitively inhibits the binding of all known EGFR ligands, including epidermal growth factor and transforming growth factor alpha, to cells expressing EGFR. In patients with mCRC, panitumumab monotherapy has resulted in favorable clinical responses, including increases in objective response rate, stable disease rate, and progression-free survival. Panitumumab has also shown promising antitumor activity in combination with selected chemotherapy regimens. Responses and improvements in progression-free survival associated with panitumumab monotherapy in patients with mCRC appear to be confined to patients whose tumors express wild-type KRAS. Therapy with panitumumab is generally well tolerated; the most common adverse events observed include skin-related toxicities, gastrointestinal toxicities, and hypomagnesemia. Infusion reactions are rare, and the agent has low immunogenicity.

Purpose Panitumumab, a fully human antibody against the epidermal growth factor receptor (EGFR), has activity in a subset of patients with metastatic colorectal cancer (mCRC). Although activating mutations in KRAS, a small G-protein downstream of EGFR, correlate with poor response to anti-EGFR antibodies in mCRC, their role as a selection marker has not been established in randomized trials. Patients and Methods KRAS mutations were detected using polymerase chain reaction on DNA from tumor sections collected in a phase III mCRC trial comparing panitumumab monotherapy to best supportive care (BSC). We tested whether the effect of panitumumab on progression-free survival (PFS) differed by KRAS status. Results KRAS status was ascertained in 427 (92%) of 463 patients (208 panitumumab, 219 BSC). KRAS mutations were found in 43% of patients. The treatment effect on PFS in the wild-type (WT) KRAS group (hazard ratio [HR], 0.45; 95% CI: 0.34 to 0.59) was significantly greater (P < .0001) than in the mutant group (HR, 0.99; 95% CI, 0.73 to 1.36). Median PFS in the WT KRAS group was 12.3 weeks for panitumumab and 7.3 weeks for BSC. Response rates to panitumumab were 17% and 0%, for the WT and mutant groups, respectively. WT KRAS patients had longer overall survival (HR, 0.67; 95% CI, 0.55 to 0.82; treatment arms combined). Consistent with longer exposure, more grade III treatment-related toxicities occurred in the WT KRAS group. No significant differences in toxicity were observed between the WT KRAS group and the overall population. Conclusion Panitumumab monotherapy efficacy in mCRC is confined to patients with WT KRAS tumors. KRAS status should be considered in selecting patients with mCRC as candidates for panitumumab monotherapy.

Purpose To examine the interaction between panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, and radiation in head-and-neck squamous cell carcinoma and non–small-cell lung cancer cell lines and xenografts. Methods and Materials The head-and-neck squamous cell carcinoma lines UM-SCC1 and SCC-1483, as well as the non–small-cell lung cancer line H226, were studied. Tumor xenografts in athymic nude mice were used to assess the in vivo activity of panitumumab alone and combined with radiation. In vitro assays were performed to assess the effect of panitumumab on radiation-induced cell signaling, apoptosis, and DNA damage. Results Panitumumab increased the radiosensitivity as measured by the clonogenic survival assay. Radiation-induced epidermal growth factor receptor phosphorylation and downstream signaling through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was inhibited by panitumumab. Panitumumab augmented radiation-induced DNA damage by 1.2–1.6-fold in each of the cell lines studied as assessed by residual γ-H 2 AX foci after radiation. Radiation-induced apoptosis was increased 1.4–1.9-fold by panitumumab, as evidenced by Annexin V-fluorescein isothiocyanate staining and flow cytometry. In vivo, the combination therapy of panitumumab and radiation was superior to panitumumab or radiation alone in the H226 xenografts ( p = 0.01) and showed a similar trend in the SCC-1483 xenografts ( p = 0.08). In vivo, immunohistochemistry demonstrated the ability of panitumumab to augment the antiproliferative and antiangiogenic effects of radiation. Conclusion These studies have identified a favorable interaction in the combination of radiation and panitumumab in upper aerodigestive tract tumor models, both in vitro and in vivo . These data suggest that clinical investigations examining the combination of radiation and panitumumab in the treatment of epithelial tumors warrant additional pursuit.

MDM2 is an oncoprotein that controls tumorigenesis through both p53-dependent and -independent mechanisms. Mdm2 mRNA level is transcriptionally regulated by p53 in response to stress such as DNA damage, and its protein level and subcellular localization are post-translationally modulated by the AKT serine/threonine kinase. Previous studies showed that PTEN, a dual specificity phosphatase that antagonizes phosphatidylinositol 3-kinase/AKT signaling, is capable of blocking MDM2 nuclear translocation and destabilizing the MDM2 protein. Results from our current study demonstrate an additional role for PTEN in regulating MDM2 functions; PTEN modulates Mdm2 transcription and isoform selection by negatively regulating its P1 promoter. In Pten-null cell lines and prostate cancer tissues, Mdm2 P1 promoter activity is up-regulated, resulting in increased L-Mdm2 expression and enhanced p90(MDM2) isoform production. Furthermore, PTEN controls Mdm2 P1 promoter activity through its lipid phosphatase activity, independent of p53. Thus, our results provide a novel mechanism for PTEN in controlling MDM2 oncoprotein functions.

Background: Non-uniform tumor penetration by antibodies may contribute to therapeutic failure. Competition experiments by radiolabeled antibody imaging can guide selection of the antibody dose that fully blocks target binding and thus corresponds to complete therapeutic coverage. However, because antibodies penetrate tumors slowly and non-uniformly, such a competition experiment may reflect only the most accessible sites. A novel approach, termed enhanced competition, has been proposed in which the radiolabeled moiety is smaller and more readily diffusible. In this study, simulations were conducted in order to examine the enhanced competition experiment. Materials and Methods: The Krogh cylinder distributed model was implemented to simulate the distribution of antibody, antibody fragment, affibody, or peptide in the tumor as a function of time and distance from the capillary wall. Simulations explored traditional and enhanced competition experiments, varying parameters including the nature of the small construct, time between administration of unlabeled and radiolabeled compounds, mass dose, affinity, antigen density, and internalization rate. Results were plotted as nanomolar concentration versus time and distance from the capillary wall. In addition, total tumor %ID/g is presented as a function of time and as a function of unlabeled antibody dose. Results: Simulation results showed that small constructs access sites in tumors at distances far from capillaries that are not readily accessible to intact antibodies. Under conditions of high affinity (1-10 nM), high antigen density (150,000 sites/cell), and compound internalization (t1/2 = 13 h), a mass of > 40 μg/kg of peptide or affibody construct required a higher dose of antibody to displace the radioactive signal compared to traditional competition with radiolabeled antibody. However, the difference in imaging signal between sub-saturating and fully displacing antibody doses is expected to be difficult to detect. Conclusions: The phenomenon predicted by the enhanced competition paradigm using small constructs is validated by modeling. Simulated imaging competition experiments indicate that affinity and mass dose levels are critical factors in this approach. High affinity small constructs and enhanced precision of imaging techniques will be required to fully evaluate target occupancy and saturation in vivo. Citation Format: Kelly D. Orcutt, Gregory P. Adams, Anna M. Wu, Matthew Silva, Jack Hoppin, Catey Harwell, Manabu Matsumura, Masakatsu Kotsuma, Daniel Freeman, Archie Tse, Jonathan Greenberg, Andrew Scott, Robert A. Beckman. Receptor occupancy and tumor penetration by antibodies, peptides, and antibody fragments: Molecular simulation of imaging assessment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4300. doi:10.1158/1538-7445.AM2014-4300

2618 Background: Activation of the receptor tyrosine kinase HER3 and its oncogenic downstream signaling pathways are thought to be critically involved in the development of various cancer types, in...

e19016 Background: HER3, an ErbB receptor with 6 docking sites for PI3K, is implicated in resistance to cancer treatment. P is a fully human mAb specific to HER3. Heregulin (HRG) is the ligand for ...

Introduction Platinum agents are a standard of care for the treatment of many cancers. Phase 3 trials have shown that patients (pts) with mutant (MT) KRAS tumors have shorter progression-free survival and overall survival when panitumumab or cetuximab is added to oxaliplatin (Ox)-containing chemotherapy (CT). This negative interaction has not been observed in pts with wild-type (WT) KRAS tumors receiving an epidermal growth factor receptor (EGFR) inhibitor and platinum-CT or with MT KRAS tumors receiving an EGFR inhibitor with irinotecan. Our goal was to understand the pharmacodynamic interaction between EGFR inhibitors and Ox in KRAS MT colorectal cancer (CRC) cells. Methods Isogenic MT and WT KRAS-expressing HCT116 CRC cells were treated with Ox, SN-38 (the active metabolite of irinotecan), panitumumab, gefitinib, or inhibitors of MEK, PI3K or Src as single agents or in combination. Viability was measured using an ATPlite assay. Cellular distribution of EGFR was visualized by a confocal microscopy. Downstream activation of MAPK and PI3K was detected by Western blotting. Results Treatment with gefitinib, but not panitumumab, reversed the anti-proliferative effects of Ox in the MT KRAS-expressing CRC cells vs Ox alone (p Conclusion Feedback though AKT may contribute to the increased resistance to Ox in KRAS MT cells. Inhibitors of MEK, PI3K, or Src reversed the negative interaction between the EGFR inhibitors and Ox in a MT-KRAS genetic background.

3580 Background: Phase 3 trials have shown that pts with MT KRAS tumors have shorter progression-free survival and overall survival when an EGFR inhibitor is added to Ox-containing chemotherapy (CT) (Bokemeyer et al 2011, Douillard et al 2010). However, KRAS status alone is not predictive of pt outcomes in this setting. The negative interaction has not been observed in pts with wild-type (WT) KRAS tumors receiving an EGFR inhibitor and platinum-CT or with MT KRAS tumors receiving an EGFR inhibitor with irinotecan (Peeters et al 2010, Van Cutsem et al 2009). Our goal was to understand the pharmacodynamic interaction between EGFR inhibitors and Ox in KRAS MT CRC cells. Methods: Isogenic MT and WT KRAS-expressing HCT116 CRC cells were treated with Ox, SN-38 (the active metabolite of irinotecan), panitumumab, gefitinib, or inhibitors of MEK, PI3K or Src as single agents or in combination. Viability was measured using an ATPlite assay. Cellular distribution of EGFR was visualized by a confocal microscopy. Downstream activation of MAPK and PI3K was detected by Western blotting. Results: Treatment (tx) with gefitinib, but not panitumumab, reversed the anti-proliferative effects of Ox in the MT KRAS-expressing CRC cells vs Ox alone (p

Background: Somatic activating mutations of KRAS are the most frequent genetic event in lung cancer, occurring in approximately 20% of NSCLC. KRAS activates two major oncogenic signaling pathways, the PI3K pathway and the MAPK pathway. Studies have suggested that blockade of both pathways is required to inhibit the growth of tumors driven by KRAS mutation. In this study, we investigated the effect of inhibiting solely the PI3K pathway on the growth of Kras G12D lung tumors in mice using AMG 511, a potent and selective class I PI3K inhibitor. To track tumor growth in this genetically engineered mouse model of lung carcinoma, we performed longitudinal monitoring of lung tumor burden during treatment using magnetic resonance imaging (MRI). Methods: Lox-stop-lox (LSL) Kras G12D mice were infected with Ad-Cre by intra-tracheal injection. Eight weeks after administration of virus, mice were randomized into two treatment groups and treated daily with 10 mg/kg AMG 511 or vehicle (N=11 per group). At 8, 11, and 14 weeks post-infection, mice were imaged using ultra-short echo time (UTE) MRI (N=6 vehicle, N=5 AMG 511). UTE MRI was performed with 30, 75, 150, 500, and 1000 αs echo times to visualize lung and tumor tissue. Lung borders were traced manually on MR images, and lung tumor volume was determined within the identified lung region using an intensity threshold to segment denser tumor tissue from normal lung tissue. At 14 weeks, lungs were harvested, weighed, and processed for histological analysis. Lung tissue area fraction was determined histomorphometrically from multiple step sections. UTE MRI images were correlated with histological sections from the same animal. Body weights were recorded. Results: Once daily treatment with AMG 511 at 10 mg/kg significantly inhibited the development of Kras G12D mutant lung tumors. Lung tumor volume, as assessed by UTE MRI, was reduced by 64% at 11 weeks (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 980. doi:1538-7445.AM2012-980

The PI3K signaling pathway is frequently activated in cancer and has been implicated in many aspects of tumor growth and survival. Inhibition of this pathway represents a potential therapeutic path for the treatment of cancer. This study evaluated the in vitro characteristics of AMG 511, a potent and selective pan class I PI3K inhibitor exhibiting IC50 values of 8, 11, 2, and 6 nM against the PI3K β, α, β, and ≤ isoforms respectively. AMG 511 was shown to be inactive against members of the closely related phosphoinositide 3 kinase related kinases (PIKK) family of kinases and did not inhibit mTOR, hVPS34, PI4Kα or PI4Kα in-vitro (IC50 values > 1 μM). In addition, AMG 511 was inactive against a majority of protein kinases (372) in the human kinome as measured by in-vitro binding assays. AMG 511 inhibited PI3K pathway signaling in U87 MG glioblastoma cells as determined by dose-dependent reduction in AKT S473 phosphorylation (IC50 = 4 nM). AKT inhibition resulted in a concomitant reduction in PRAS40 phosphorylation (IC50 = 23 nM), a downstream effector of AKT. Reduced phosphorylation of mTORC1 substrates p70S6K (IC50 = 30 nM) and S6 (IC50 = 70 nM) but not 4EBP1 (T37/46), was also detected in U87 MG cells, suggesting that upstream blockade of PI3K pathway signaling with AMG 511 treatment leads to a selective reduction in downstream mTORC1 activity. Given the well documented role of mTORC1 in cap-dependent translation we profiled AMG 511 in a methionine-analog incorporation assay in U87 MG cells. However, no significant inhibition of bulk translation was observed following treatment with AMG 511 in U87 MG cells. Treatment of U87 MG cells with AMG 511 revealed a pronounced G1 arrest with a concurrent reduction in BrdU+ cells, detectable within 8 hours of treatment. This anti-proliferative effect was fully reversible by 18 hours following washout. In line with these anti-proliferative effects, reduced cyclin D1 levels and elevated p27 levels were detected within 4 hours of treatment. Minimal cell killing effects were detected with AMG 511 treatment in U87 MG cells as measured by induction of cleaved caspase-3 and DNA content < 2N. AMG 511 was profiled across a large panel of tumor cell lines encompassing several tumor types including breast and lung. A majority of the cell lines tested exhibited sensitivity to AMG 511 with a subset exhibiting evidence for cell death upon treatment with AMG 511. Breast cancer cell lines harboring activating mutations in PI3Kα, loss of PTEN, or amplification of Her2 tended to show greater sensitivity to AMG 511 treatment. In conclusion, AMG 511 is a potent and selective pan class I PI3K inhibitor, capable of inhibiting PI3K signaling and inducing robust anti-proliferative effects via a G1 arrest in many tumor cell lines, with evidence of cell killing in a subset of lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2805. doi:1538-7445.AM2012-2805

Background: Platinum agents are a standard of care for treatment many cancers. Clinical data has shown that patients (pts) with CRC with mutant (MT) KRAS do not respond to EGFR inhibitors (Amado et al 2009). Furthermore, results from phase 3 clinical trials with erlotinib, cetuximab, or panitumumab have shown that pts whose tumors express MT KRAS have shorter progression-free survival and overall survival when an EGFR inhibitor is added to a platinum-containing regimen vs chemotherapy alone (Eberhard et al 2005, Bokemeyer et al 2011, Douillard et al 2010). However, KRAS status is not predictive of outcomes in pts receiving Ox- or irinotecan-containing regimens without an EGFR inhibitor (Richman et al 2009). This negative interaction has not been observed in pts whose tumors express either wild type (WT) KRAS receiving an EGFR inhibitor and chemotherapy or whose tumors express MT KRAS receiving an EGFR inhibitor with irinotecan (Peeters et al 2010, Van Cutsem et al 2009). Our goal was to gain a molecular mechanistic understanding of the negative interaction between EGFR inhibitors and Ox in KRAS MT CRC cells. Methods: To investigate the negative interaction between Ox and EGFR inhibitors, isogenic MT and WT KRAS-expressing HCT116 CRC cells were treated with Ox, SN-38 (the active metabolite of irinotecan), panitumumab, or gefitinib as single agents or in combination for 72 hrs. Viability was measured using an ATPlite assay. To determine the cellular distribution of EGFR, cells were serum-starved, labeled with a fluorescent anti-EGFR mAb, and visualized by a confocal microscopy. To analyze the effects of single agent or combination treatment on downstream phosphorylation of the PI3K or MAPK pathways, cells were treated for 24 hrs and phospho-proteins were detected by Western blotting. To investigate whether negative interaction between Ox and EGFR inhibitors could be reversed, cells were treated with Ox and gefitinib in combination with inhibitors to MEK, PI3K or Src for 72 hrs. Results: Treatment with gefitinib, but not panitumumab, reversed the anti-proliferative effects of Ox in the MT KRAS-expressing CRC cells vs Ox alone (p Conclusion: We developed a preclinical model to further understand the negative interaction between MT KRAS-expressing cancer cells, platinum agents, and EGFR inhibitors. Feedback though AKT may contribute to the increased resistance to Ox in KRAS MT cells. Inhibitors of MEK, PI3K, or Src reversed the negative interaction between the EGFR inhibitors and Ox in a mutant-KRAS genetic background. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A150.

Background: Anti-EGFR mAbs have demonstrated clinical utility in SCCHN; however, most patients are resistant or acquire resistance to therapy. In preclinical models, activation of HER3 is a resistance mechanism to current HER inhibitors. Recent data (Wilson et al 2011) demonstrated that SCCHN cancer cell lines and primary tumors express both HER3 and heregulin, the ligand for HER3. This autocrine loop is one potential mechanism for the lack of sensitivity to anti-EGFR mAbs observed in SCCNH patients. Herein, we report in vitro and in vivo activity of U3–1287 (AMG 888) as a single agent and in combination with panitumumab, an anti-EGFR mAb, in the FaDu preclinical model of human SCCHN. Methods: FaDu (SCCHN) cells were treated with U3–1287 (AMG 888), panitumumab, the combination or control mAbs for 4 hours. To determine in vitro efficacy on cell proliferation, FaDu cells were treated with 1 μg/mL U3–1287 (AMG 888), 1 μg/ml panitumumab, the combination or control mAbs in serum containing medium. After 72 hours, the growth of treated cells was measured by ATPLite assay. Levels of pHER3, pEGFR, pERK1/2 and pAKT were measured by Western blot analyzes to determine the inhibition of HER3 oncogenic signaling. To determine in vivo efficacy, mice bearing ∼200 mm3 FaDu xenografts were treated 2×/week with U3–1287 (AMG 888), panitumumab, the combination or control mAbs. Results: Treatment with either U3–1287 (AMG 888) or panitumumab resulted in inhibition of pHER3 and pAKT or pEGFR and pERK1/2, respectively. Treatment with the combination resulted in inhibition of pHER3, pEGFR, pAKT and pERK1/2. Although treatment with panitumumab or U3–1287 (AMG 888) alone significantly inhibited in vitro cell proliferation versus control mAb treated cells (57% (p Conclusions: U3–1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling in FaDu SCCHN cells resulting in in vitro and in vivo growth inhibition. SCCHN xenografts were sensitive to U3–1287 (AMG 888) treatment as single agent or in combination with panitumumab. Combination treatment with U3–1287 (AMG 888) and panitumumab resulted durable regressions which was not observed with either single agent. These data together with the HER3/heregulin autocrine loop detected in patient samples (Wilson et al 2011) provides evidence for the potential clinical investigation of U3–1287 (AMG 888) in SCCHN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A182.

Background: Understanding the mechanism of action (MOA) of novel therapeutic agents is instrumental for their recommended use in the clinic. U3-1287 (AMG 888) is a novel, fully human mAb that targets HER3, a member of the EGFR family involved in tumor growth and resistance to anti-HER inhibitors. To understand its MOA, we evaluated HER3 internalization, degradation, and downstream signaling in tumor cell lines in response to U3-1287 (AMG 888) treatment. Methods: Confocal microscopy was used to locate antibody/receptor complexes. MDA-MB-453 and MDA-MB-175 breast cancer cells were serum starved overnight and treated on ice with 10 ug/mL of quantum dot 585 (Invitrogen) conjugated U3-1287 (AMG 888) and 2 ug/mL of quantum dot 655 (Invitrogen) conjugated HER2 Ab5 (Labvision). Images were taken at time 0 and after 1 hour incubation at 37°C. In addition, cell surface reduction of HER3 was measured by flow cytometry. T47D breast cancer cells were incubated at 37°C for 0, 1, 4 and 24 hours with either U3-1287 (AMG 888), c225, trastuzumab or c2C4 (10 µg/ml). The cells were re-stained with the same primary antibodies (10 µg/ml) at 4°C, and analyzed. To evaluate the consequence of longer term U3-1287 (AMG 888) treatment, BxPC3 pancreatic cells were treated from 0-96 hours in vitro and A549 NSCLC xenografts were treated for 2 weeks in vivo. Total HER3, pHER3 and pAKT were measured by Western blot and HER3 mRNA was measured by RT-PCR. Results: U3-1287 (AMG 888) treatment of MDA-MB-453 and MDA-MB-175 cells resulted in the internalization of HER3 but not HER2. Treatment of T47D cells with trastuzumab, c2C4 or c225, U3-1287 (AMG 888) resulted in a 8, 4, 0 and 73% reduction of cell surface HER2, HER2, HER1 and HER3, respectively at the 1 hr time point. A ∼75% reduction of HER3 was maintained during the 24 hr treatment with U3-1287 (AMG 888). Although a 60% reduction in total HER3 was not observed until 4 hours post treatment of BxPC3 cells with U3-1287 (AMG 888), pHER3 and pAKT were reduced >90% within 15 minutes. Neither U3-1287 (AMG 888) nor heregulin (the ligand for HER3) treatment resulted in a significant change in HER3 mRNA levels over the 96 hr treatment period. Analyses of A549 tumor xenograft tissue demonstrated a dose dependent loss of total HER3 and pHER3 in response to U3-1287 (AMG 888) administration versus IgG control treatment. Conclusions: We demonstrate that U3-1287 (AMG 888)-induced inhibition of HER3 signaling and HER3 degradation are separate events, whereby loss of HER3 receptor in response to U3-1287 (AMG 888) treatment is preceded by U3-1287 (AMG 888)-mediated inhibition of HER3 activation. HER3 transcription is not affected by U3-1287 (AMG 888) or heregulin in BxPC3 cells. These findings suggest that U3-1287 (AMG 888) mediates both inhibition of HER3 downstream signaling and HER3 degradation and that these mechanisms may contribute to the activity of U3-1287 (AMG 888) in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-306.

Background: EGFR TKIs have demonstrated activity in NSCLC, however most patients are resistant or acquire resistance to therapy. HER3 is a member of the EGFR family of receptors and is considered a target for NSCLC treatment. Though HER3 lacks kinase activity, it is a scaffold for PI3K signaling for the HER family via heterodimeric interactions. In preclinical models, activation of HER3 is a resistance mechanism (de novo and acquired) to current HER inhibitors. Herein, we report the activity of U3-1287 (AMG 888) in preclinical models of human NSCLC. We also evaluate human NSCLC tumor specimens for detectable pHER3, pAKT and HRG, the ligand for HER3. Methods: To determine the inhibition of HER3 oncogenic signaling, A549 and Calu-3 NSCLC cells were treated with 100 nM of U3-1287 (AMG 888), panitumumab, lapatinib or control for 1 hour. To determine in vitro efficacy on anchorage independent growth, A549 cells were treated with 10 g/mL U3-1287 (AMG 888), other HER family Abs or control mAb in serum containing medium. Tumor cell colonies formed in the absence of exogenous ligand for 10 days and were stained with MTT and quantified using a Scanalyzer HTS camera imaging system. To determine in vivo efficacy, mice bearing ∼200 mm3 A549 or Calu-3 NSCLC xenografts were treated 2x/week with anti-HER family inhibitors or control. A549 xenograft tumors were analyzed for the inhibition of pHER3 byWestern blotting. The anti-tumor effect of U3-1287 (AMG 888) with EGFR inhibitors was tested in the Calu-3 NSCLC xenograft model. Levels of pHER3, pAKT and HRG in 20 NSCLC patient specimens were determined by Western blotting. Results: Treatment with U3-1287 (AMG 888) resulted in inhibition of pHER3 in both NSCLC cell lines. pAKT was inhibited in the A549 cell line with U3-1287 (AMG 888) as a single agent and in A549 and Calu-3 cells to a greater extent when combined with another HER family inhibitor. U3-1287 (AMG 888) inhibited colony growth by 50% (p Conclusions: U3-1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling and cell growth in NSCLC cell lines in vitro and in vivo. NSCLC xenografts are sensitive to U3-1287 (AMG 888) treatment as single agent or in combination with the anti-EGFR agents. The preclinical data together with the detectable levels of activated HER3 in patient samples provide evidence for the potential clinical application of U3-1287 (AMG 888) in NSCLC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B171.

Background: HER3 is required for proliferation in HER2 amplified (HER2+) breast cancer cell lines, but may also be important in the HER2 negative (HER2−) setting. Though HER3 lacks kinase activity, it is a scaffold for PI3K signaling for the HER family and other receptor kinases via heterodimeric interactions. In preclinical models, activation of HER3 is a resistance mechanism to current HER family inhibitors. We report the efficacy of U3-1287 (AMG 888) in HER2+ and HER2− preclinical breast cancer models. We also evaluate human breast tumor samples for detectable levels of pHER3 and pAKT. Methods: Levels of pHER3, pERK and pAKT were determined by western blotting. To determine the inhibition of HER3 oncogenic signaling, HER2+ (SKBR3, MDA-MB-453 and HCC1569) and HER2− (MDA-MB-175 VII) breast cancer cells were treated with 10 µg/ml of U3-1287 (AMG 888), cetuximab, c2C4, trastuzumab, lapatinib (250 to 1500 nM) or controls prior to heregulin (HRG) stimulation. To determine the activity on cell proliferation in the presence of 0.4% FBS, breast cancer cell lines were incubated with 10 µg/ml of U3-1287 (AMG 888), cetuximab, c2C4, trastuzumab, lapatinib (50 to 1500 nM) or control for 1 hour prior to HRG stimulation. After 4 days, the growth of treated cells was measured with alamarBlue™. To determine the activity on anchorage-independent growth, breast cancer cells were treated with 2 to 5 g/ml U3-1287 (AMG 888), anti-HER antibodies, 500 nM lapatinib, or control. Tumor cell colonies formed in the absence or presence of HRG for 6 to 10 days and were stained with MTT for 4 to 6 hours and quantified. Results: Treatment of breast cancer cell lines with U3-1287 (AMG 888) resulted in an inhibition of pHER3 and pAKT. In cell proliferation assays, U3- 1287 (AMG 888) reduced heregulin-stimulated SkBR-3 proliferation up to 40% (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B161.

Background: HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. Although HER3 lacks intrinsic kinase activity, it serves as a scaffold for PI3K/AKT signaling for the HER family via heterodimeric interactions. The goal of this study was to use preclinical modeling to predict a minimal effective dose regimen for objective response using preclinical PK and BxPC3 xenograft mice anti-tumor efficacy and PD data. Materials and Methods: U3-1287 (AMG 888) concentration-time data obtained from mice (0.025 to 2.5 mg/mouse), rats (1 to 100 mg/kg) and monkeys (1 to 200 mg/kg) were combined and analyzed using a target-mediated drug disposition model. Animal PK parameters were scaled based on body weight to predict human PK characteristics. An Emax model was used to relate drug concentration and inhibition of pHER (measure by ELISA) in BxPC3 xenograft tumors. A PK/PD/efficacy model (based on Simeoni et al 2004) was used to analyze tumor growth data from mice bearing BxPC3 pancreatic xenografts (∼200 mm3) treated twice per week at 25, 100, 200, or 500 g for a 1 month. The model was validated with tumor growth data following additional doses of 400 g biweekly and 200 g biweekly, weekly and twice a week. The relationship between drug concentration, the inhibition of pHER3 in animals and interspecies PK scaling was used to select the minimal effective dose for the first in human study. Results: U3-1287 (AMG 888) treatment of BxPC3 xenografts resulted in a statistically significant inhibition of tumor growth and pHER3 levels in a dose and schedule dependent manner (p Conclusion: The anti-tumor efficacy in the BxPC3 pancreatic xenograft model was correlated with an increased serum concentration of U3-1287 (AMG 888) and a decrease in pHER3 levels, allowing for the development of a PK/PD/Efficacy relationship. This relationship combined with interspecies PK scaling was used to determine a dose and schedule for U3-1287 (AMG 888) to investigate in a FIH study. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B167.

14536 Background: Panitumumab, a fully human monoclonal antibody against epidermal growth factor receptor (EGFR), has anti- tumor activity as monotherapy in both preclinical models and clinical trials. The objective of this study was to identify the epitope on EGFR for panitumumab and compare it to that of cetuximab, a chimeric anti-EGFR Ab. Methods: The extracellular domain of EGFR (amino acids 1–618) and domains I, II, III, IV individually were expressed to determine the domain on EGFR that was necessary for panitumumab binding. Chromatography was used to compare the binding affinity of panitumumab vs cetuximab. By flow cytometry and amino acid replacement scanning, the critical residues involved in panitumumab and cetuximab binding for EGFR were determined. Using diffraction data from the panitumumab Fab’2 and published data for cetuximab, crystal structure models of EGFR and the residues determined to be critical for panitumumab or cetuximab binding were built. To evaluate in vitro activity, A549 NSCL...

War powers hang in a delicate balance, with conflicting statutes overlying contrasting constitutional prerogatives. As Congress has filled nearly every shadowy corner of Justice Jackson's "zone of twilight" with its own imprimatur, war powers debates now hinge on traditional statutory interpretation, albeit in a unique context. This Note establishes context-specific canons for interpreting war powers legislation and effectuating its underlying values, aiming to provide principled resolution to seemingly intractable conflicts. In so doing, it draws upon the complete set of judicial opinions assessing authorizations of the use of military force and analyzes the institutional framework beneath them.