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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

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: 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.