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Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models.Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized.Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

XLS file, 33K, Summary of RG7212 monotherapy and combination antitumor efficacy.

PDF file, 306K, RG7212 antitumor efficacy and characterization of tumor models used to assess RG7212 antitumor efficacy.

PDF file, 771K, TWEAK promotes proliferation and survival signaling with minimal effect on cell viability.

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XLS file, 193K, Sorted Affymetrix gene array data.

PDF file, 29K, Lead antibodies have identical binding properties and neutralizing activities.

PDF file, 176K, Assessment of baseline gene expression in RG7212 responders and non-responders in qPCR array.

The BRAFV600E mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAFV600E kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAFV600E or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAFV600E-expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAFV600E gene. Cancer Res; 70(13); 5518–27. ©2010 AACR.

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Supplementary Methods, Tables 1-3, Figure Legend from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

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Background: BRAF mutations are a validated target for cancer therapy. A second-generation BRAF inhibitor with an improved preclinical safety profile (RG7256) was evaluated in a first-in-man study in order to determine the safety, efficacy, pharmacokinetics and pharmacodynamics in patients with BRAF V600-mutated advanced solid tumors. Patients and Methods: Patients received RG7256 orally over 8 dose levels from 200 mg once a day (QD) to 2400 mg twice a day (BID) (50-, 100- and 150-mg tablets) using a classic 3 + 3 dose escalation design. Results: In total, 45 patients were enrolled; most (87 %) had advanced melanoma (94 % BRAF V600E). RG7256 was rapidly absorbed, with limited accumulation and dose-proportional increase in exposure up to 1950 mg BID. The maximal tolerated dose (MTD) was not reached. The most common drug-related adverse events (AEs) were dyspepsia (20 %), dry skin (18 %), rash (18 %), fatigue (16 %) and nausea (13 %), mainly grade 1. Three patients (7 %) developed cutaneous squamous cell carcinoma. Photosensitivity, arthralgia and increased liver enzyme levels were each observed in only one patient each. Of 44 evaluable patients, 14 (32 %) had a partial response (melanoma and thyroid cancer). At high dose levels (>1200 mg BID), 10 of 16 (63 %) patients had a partial response. A decrease in maximum standardized uptake value (SUVmax) on FDG-PET of ≥25 % was observed in 19 of 37 patients. On-treatment reductions in pERK were documented in eight of ten paired tumor samples. Conclusions: RG7256 has a favorable safety profile compared to other BRAF inhibitors while maintaining clinical activity, and MTD was not reached. The excessive pill burden needed to provide the desired exposure, and thus concerns about patient compliance, limited further development of this agent. Study Identifier: ClinicalTrials.gov (NCT01143753)[MediaObject not available: see fulltext.] Refereed/Peer-reviewed

Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

A high percentage of patients with BRAFV600E mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAFV600E melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAFV600E inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. Cancer Res; 72(4); 969–78. ©2011 AACR.

The protein kinase BRAF is a key component of the RAS–RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAFV600E in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAFV600-expressing cell lines and inhibiting tumor growth in BRAFV600E bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAFV600E-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAFV600E mutation. Cancer Res; 72(3); 779–89. ©2011 AACR.

This chapter describes analytical documentation needs during the life cycle of a pharmaceutical product—from initial candidate screening and selection, through entry into humans/IND, to NDA and postapproval marketed product support. Analytical data are the foundation and backbone for pharmaceutical development, leading to approval and production of new drugs for the treatment of many diseases. Analytical documentation provides the critical links for the development team during the evolution and life cycle of a new pharmaceutical product—beginning from earliest studies, enabling entry into humans, through product launch and postapproval changes. The emerging concept of quality by design (QbD) emphasizes that quality should be built into a product by means of a thorough understanding of the product and process. The product is developed and manufactured with a knowledge of the risks involved in manufacturing the product together with an understanding of how best to mitigate those risks through continuous product improvement. Analytical data generated in these efforts play a pivotal role in describing the drug substance and drug product, and in establishing critical quality and process attributes. Regulatory, compliance, and research documents are the three major areas of analytical documentation essential to provide a complete history of a product.

Window factor analysis (WFA), a self-modeling chemometric method for obtaining the concentration profiles of individual components from evolutionary processes, is applied to unresolved liquid chromatograms. WFA makes use of the fact that each component lies in a specific region along the evolutionary axis, called the ‘window’. The high-performance liquid chromatograms of various mixtures of toluene, ethylbenzene, m -xylene and naphthalene, in methanol solvent, were recorded with an ultraviolet diode-array detector. Although the quantitative results obtained by WFA did not agree exactly with the known concentrations, the results were in agreement with those obtained by rank annihilation factor analysis and matrix regression analysis. Derivation of matrix regression analysis, developed during this investigation, is presented.

The BRAFV600E mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAFV600E kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAFV600E or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAFV600E-expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAFV600E gene. Cancer Res; 70(13); 5518–27. ©2010 AACR.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: In a retrospective analysis performed in a phase II study evaluating efficacy and safety of the MEK-inhibitor refametinib plus sorafenib in Asian patients with HCC (Lim et al., Clin Cancer Res, in press), patients with mutant RAS appeared to exhibit a particularly robust clinical response to refametinib plus sorafenib compared to patients with wild-type RAS. To further investigate clinical activity of refametinib as monotherapy or in combination with sorafenib in HCC patients with mutant RAS, two Ph II studies for patients with unresectable HCC have been initiated; refametinib monotherapy ([NCT01915589][1]) and refametinib plus sorafenib combination therapy ([NCT01915602][2]). Since RAS mutations are very rare events in HCC and biopsies are not routinely taken in patients with underlying liver cirrhosis, only liquid biopsies seemed feasible for prospective patient selection. Therefore, Sysmex Inostics’ BEAMing (Beads, Emulsions, Amplification, and Magnetics) technology based on cell-free circulating DNA in plasma is used to select patients for KRAS/NRAS Mutation status prior to enrollment. Patients are eligible if they exhibit any one or multiple of 13 KRAS/NRAS mutations. Results: Between Sep 2013 and Nov 2014 more than 1000 plasma samples from patients with unresectable HCC enrolled in the refametinib monotherapy study or the refametinib plus sorafenib combination study, have been tested at a CLIA certified laboratory in Baltimore, MD. The turnaround time from shipping the plasma sample from the clinical trial site to the results reporting was 6-10 days. Out of 1004 samples from 116 trial sites in 18 countries, 47 (4.7%) have been reported to harbor a KRAS and / or a NRAS mut. The following mutations have been reported: KRAS\_G12A, KRAS\_G12C, KRAS\_G12D, KRAS\_G12R, KRAS_ G12S, KRAS\_G12V, KRAS\_G13D, NRAS\_Q61K, NRAS\_Q61L, NRAS\_Q61H, NRAS\_Q61R. The frequency of the mutant allele ranged from 0.02% to 9.75%. No obvious differences in the incidence of any of the mutations with regards to geography, demographic factors are other baseline characteristics have been observed. Conclusion: To our knowledge this is the largest dataset of KRAS and NRAS mutations in HCC. The observed prevalence of RAS mutations of approximately 5% across both trials is well in line with published data including the COSMIC database. Our data support that prospective selection of patients with HCC for KRAS or NRAS mutation is feasible with a turn-around time of 1-2 weeks depending on geographical region. These data further support the broad utility of such approaches for large randomized prospective studies in patients with specific tumor genetic profiles. The clinical outcome of the 2 clinical studies will help understand whether KRAS or NRAS can potentially be a predictive marker for response to refametinib in patients with HCC. Citation Format: Heiko Krissel, Andrea Hennig, Danny Zhang, Rodrigo Ito, Christine Gonschorek, Fabricio Souza, Martina Poethig, Kathleen Schostack, Joachim Reischl, Philipp Angenendt, Barrett H. Childs, Michael Teufel. Liquid biopsies to prospectively select patients with KRAS or NRAS mutant hepatocellular carcinoma (HCC) in two phase II studies with Refametinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2015-5252 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01915589&atom=%2Fcanres%2F75%2F15_Supplement%2F5252.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01915602&atom=%2Fcanres%2F75%2F15_Supplement%2F5252.atom

Few drugs are currently approved for the treatment of melanoma, including dacarbazine (chemotherapy) and interferon alpha (immunotherapy). However, only a small fraction of patients with metastatic melanoma respond to dacarbazine and a survival benefit has not been shown with this or any other chemotherapy regimen. Taxanes have demonstrated some activity in melanoma, including modest response rates in combination with carboplatin or bevacizumab. These results contrast with the extraordinary Phase I clinical data reported with the BRAFV600E kinase inhibitor RG7204 (PLX4032), which has demonstrated a remarkable response rate (N Engl J Med 363(9):809-819, 2010). Here we report our preclinical investigation of RG7204 in combination with the biologics interferon alpha-2a (Pegasys) or B20-4.1 (B20) (mouse/ human cross-reactive anti-VEGF MAb), and the chemotherapies paclitaxel and carboplatin in the BRAFV600E mutant LOX-IMVI melanoma model in nude mice.The combination of RG7204 and paclitaxel provided superior survival as compared to either monotherapy. Conversely, the combination of RG7204 plus carboplatin or RG7204 plus B20 did not extend survival as compared to RG7204 monotherapy. When RG7204, paclitaxel, and B20 were given in triplet combination, no improvement in survival was observed over any doublet. Similarly, the triplet combination with RG7204, carboplatin and B20 failed to demonstrate improved survival over any doublets or RG7204 monotherapy. Although complete suppression of tumor growth was readily achieved with either RG7204 or Pegasys monotherapy, the combination provided a remarkable survival benefit with median survival extended by nearly a year as compared to either monotherapy. Taken together, these data support clinical testing of RG7204 with interferon alpha in melanoma patients harboring BRAFV600E mutation in order to extend survival, and may allow for use of lower dose interferon. Clinical investigation of RG7204 and paclitaxel combination may also be warranted. While our data do not support combined therapy with RG7204 plus anti-VEGF therapy, it should be noted that the LOX model was resistant to B20 monotherapy, and therefore these findings might not apply to tumors with intrinsic sensitivity to anti-VEGF therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2562. doi:10.1158/1538-7445.AM2011-2562