Amelie Forest

Clinical and Translational Results of a Phase II, Randomized Trial of an AntiIGF-1R (Cixutumumab) in Women with Breast Cancer That Progressed on Endocrine Therapy

Purpose: This phase II trial evaluated the efficacy and safety of cixutumumab, a human anti–insulin-like growth factor receptor 1 (IGF-1R) monoclonal IgG1 antibody, and explored potential biomarkers in postmenopausal women with hormone receptor–positive breast cancer. Experimental Design: Patients with hormone receptor–positive breast cancer that progressed on antiestrogen therapy received (2:1 randomization) cixutumumab 10 mg/kg and the same antiestrogen (arm A) or cixutumumab alone (arm B) every 2 weeks (q2w). Primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS) and safety. Correlative analyses of IGF-1R, total insulin receptor (IR), and IR isoforms A (IR-A) and B (IR-B) expression in tumor tissue were explored. Results: Ninety-three patients were randomized (arm A, n = 62; arm B, n = 31). Median PFS was 2.0 and 3.1 months for arm A and arm B, respectively. Secondary efficacy measures were similar between the arms. Overall, cixutumumab was well tolerated. IGF-1R expression was not associated with clinical outcomes. Regardless of the treatment, lower IR-A, IR-B, and total IR mRNA expression in tumor tissue was significantly associated with longer PFS [IR-A: HR, 2.62 (P = 0.0062); IR-B: HR, 2.21 (P = 0.0202); and total IR: HR, 2.18 (P = 0.0230)] and OS [IR-A: HR, 2.94 (P = 0.0156); IR-B: HR, 2.69 (P = 0.0245); and total IR: HR, 2.72 (P = 0.0231)]. Conclusions: Cixutumumab (10 mg/kg) with or without antiestrogen q2w had an acceptable safety profile, but no significant clinical efficacy. Patients with low total IR, IR-A, and IR-B mRNA expression levels had significantly longer PFS and OS, independent of the treatment. The prognostic or predictive value of IR as a biomarker for IGF-1R–targeted therapies requires further validation. Clin Cancer Res; 22(2); 301–9. ©2015 AACR.

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

Intrinsic Resistance to Cixutumumab Is Conferred by Distinct Isoforms of the Insulin Receptor

Despite a recent shift away from anti–insulin-like growth factor I receptor (IGF-IR) therapy, this target has been identified as a key player in the resistance mechanisms to various conventional and targeted agents, emphasizing its value as a therapy, provided that it is used in the right patient population. Molecular markers predictive of antitumor activity of IGF-IR inhibitors remain largely unidentified. The aim of this study is to evaluate the impact of insulin receptor (IR) isoforms on the antitumor efficacy of cixutumumab, a humanized mAb against IGF-IR, and to correlate their expression with therapeutic outcome. The data demonstrate that expression of total IR rather than individual IR isoforms inversely correlates with single-agent cixutumumab efficacy in pediatric solid tumor models in vivo. Total IR, IR-A, and IR-B expression adversely affects the outcome of cixutumumab in combination with chemotherapy in patient-derived xenograft models of lung adenocarcinoma. IR-A overexpression in tumor cells confers complete resistance to cixutumumab in vitro and in vivo, whereas IR-B results in a partial resistance. Resistance in IR-B–overexpressing cells is fully reversed by anti–IGF-II antibodies, suggesting that IGF-II is a driver of cixutumumab resistance in this setting. The present study links IR isoforms, IGF-II, and cixutumumab efficacy mechanistically and identifies total IR as a biomarker predictive of intrinsic resistance to anti–IGF-IR antibody. Implications: This study identifies total IR as a biomarker predictive of primary resistance to IGF-IR antibodies and provides a rationale for new clinical trials enriched for patients whose tumors display low IR expression. Mol Cancer Res; 13(12); 1615–26. ©2015 AACR.

An Open-Label, Multicenter, Randomized, Phase II Study of Cisplatin and Pemetrexed With or Without Cixutumumab (IMC-A12) as a First-Line Therapy in Patients With Advanced Nonsquamous Non-Small Cell Lung Cancer

Introduction: Type 1 insulin‐like growth factor receptor is deregulated in solid tumors. Cixutumumab, a monoclonal antibody that inhibits the activity of type 1 insulin‐like growth factor receptor, was investigated in combination with pemetrexed/cisplatin in the frontline setting. Methods: In this open‐label, phase II study, patients with stage IV nonsquamous NSCLC and a performance status of 0 to 1 were randomized (1:1) to receive 20 mg/kg cixutumumab, 500 mg/m2 pemetrexed, and 75 mg/m2 cisplatin (cixutumumab [n = 87]) or pemetrexed and cisplatin (control [n = 85]). Eligible patients received pemetrexed‐based maintenance therapy with cixutumumab (cixutumumab arm) or without it (control arm). The primary end point was progression‐free survival. Secondary end points assessed overall survival, objective response rate, and safety. Survival was analyzed by the Kaplan‐Meier method and Cox proportional hazard model. Exploratory correlative analyses were also performed. Results: The mean age of the intent‐to‐treat population (n = 172) was 59 years (range 32–83). Median progression‐free survival was 5.45 months with cixutumumab versus 5.22 months in the control (hazard ratio = 1.15, 95% confidence interval: 0.81–1.61; p = 0.44). Median overall survival was 11.33 months with cixutumumab versus 10.38 months in the control (hazard ratio = 0.93, 95% confidence interval: 0.64–1.36). Objective response rate did not differ between treatments (p = 0.338). Grade 3 or 4 hyperglycemia occurred at a higher rate with cixutumumab than in the control (9.4% versus 1.2%). One death possibly related to cixutumumab occurred. Conclusions: Efficacy was not improved in patients with nonsquamous NSCLC when cixutumumab was added to pemetrexed/cisplatin. Combination therapy was well tolerated and no new safety concerns were reported.

Mouse PDX Trial Suggests Synergy of concurrent Inhibition of RAF and EGFR in Colorectal Cancer with BRAF or KRAS mutations

Purpose: To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models. Experimental Design: Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group (n = 1) trial. Results: Consistent with clinical results, cetuximab was efficacious in wild-type KRAS and BRAF PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored KRAS or BRAF mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that BRAF and KRAS mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a P value of 0.01 compared with cetuximab alone. In 12 models with BRAF mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44 KRAS mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition. Conclusions: MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a KRAS or BRAF mutation. Clin Cancer Res; 23(18); 5547–60. ©2017 AACR.

Molecular Basis for Necitumumab Inhibition of EGFR Variants Associated with Acquired Cetuximab Resistance

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521–31. ©2017 AACR.

Discovery and preclinical characterization of the antagonist anti-PD-L1 monoclonal antibody LY3300054

BackgroundModulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types.ResultsHere we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways.ConclusionsLY3300054 is currently being evaluated in phase I clinical trials for oncology indications.

Abstract 583: The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade

Targeting cyclin dependent kinases 4 and 6 (CDK4/6) with inhibitors such as abemaciclib has shown promise in early and late phase clinical trials in both breast cancer and NSCLC. While there is evidence that patients benefit from single-agent abemaciclib, combination strategies leveraging this compound together with immunotherapy are of interest for the treatment of these and other cancers. Consequently, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. However, because most preclinical studies have been performed using xenograft tumors in immune-compromised mice, the potential immunomodulatory effects of abemaciclib have not been adequately ascertained. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with checkpoint immunotherapy in a murine syngeneic tumor model sensitive to abemaciclib using immuno-competent mice. Abemaciclib monotherapy of established murine CT26 tumors, which harbor KRAS G12C mutation and CDKN2A deletion, caused a dose-dependent delay in tumor growth. Surprisingly, gene expression analysis showed that treatment was associated with an increase in intra-tumor immune inflammation without major alteration in immune subset frequencies. Testing of various dosing regimens in this preclinical model found that monotherapy abemaciclib pretreatment followed by combination with anti-PD-L1 antibody therapy, induced an enhanced anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy exhibited superior anti-tumor efficacy, resulting in complete tumor regression (CR) in 50-60% of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice which maintained CRs after cessation of combination therapy were able to resist later CT26 rechallenge, demonstrating that abemaciclib in combination with anti-PD-L1 enabled the generation of an immunologic memory. Examination of intra-tumor gene expression during treatment found that combination therapy further amplified the immune/T cell activation signature compared to both monotherapies. Intra-tumoral suppression of cell cycle genes, which are indicative of inhibition of CDK4/6, was also greater during the combination therapy, suggesting that the effects anti-PD-L1 therapy may augment the cell cycle arrest induced by abemaciclib. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results demonstrate that it is possible to combine CDK4/6 inhibition by abemaciclib with checkpoint immunotherapy to improve tumor efficacy. The synergistic responses observed in terms of tumor efficacy, immune activation, and cell cycle control provides support for the clinical investigation of this combination. Citation Format: Jack Dempsey, Lysiane Huber, Amelie Forest, Jennifer R. Stephens, Thompson N. Doman, Jason Manro, Andrew Capen, Robert S. Flack, Gregory P. Donoho, Sean Buchanan, Alfonso De Dios, Kyla Driscoll, Michael Kalos, Ruslan Novosiadly, Richard P. Beckmann, David A. Schaer. The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2017-583

Abstract C129: Antitumor efficacy and non-clinical safety of the high affinity anti-FGFR4 antibody H4: Implications for targeting the FGF19-FGFR4 axis in oncology

While FGFR4 and its ligand FGF19 represent a promising target for cancers in which FGF19 is overexpressed or gene amplified, inhibition of this pathway carries a potential toxicity risk due to the role of FGFR4 in bile acid homeostasis. Treatment with an anti-FGF19 antibody caused significant toxicity in cynomolgus monkeys that was attributed to perturbations in the enterohepatic circulation of bile acids. Given that FGF19 signals through other FGFRs besides FGFR4, we hypothesized that the toxicity profile of an anti-FGFR4 antibody might be different than that of an anti-FGF19 antibody. To test this we set out to identify an anti-FGFR4 antibody that inhibits FGF19 binding and displays anti-tumor activity in models overexpressing FGF19, and then determine its toxicity profile. A phage screening effort yielded several anti-FGFR4 antibodies including H4, a high affinity fully human IgG1. H4 binds to the extracellular domain of FGFR4 (Kd = 12 pM) but does not cross react with FGFR1, FGFR2, or FGFR3. Furthermore, H4 efficiently blocks FGF19 binding to FGFR4 (IC50 = 589 pM) and inhibits FGF19-mediated downstream signaling in Hep3B2 cells. In human liver cancer cells that overexpress FGF19, H4 inhibits proliferation (IC50 250-1,100 nM). Anti-tumor activity was evaluated using cell line-derived xenograft tumors established from the FGF19 overexpressing and gene amplified cell lines HuH-7 and Hep3B2. H4 inhibited tumor growth in these models (T/C of 50% and 8%, respectively), and the antitumor effect was accompanied by alterations in several biomarkers associated with FGFR4 pathway inhibition including increased expression of CYP7A, the gene encoding the rate-limiting enzyme of bile acid synthesis. Finally, to assess the potential toxicity of H4 treatment, single dose intravenous toxicology studies were conducted in rats (20, 60, and 200 mg/kg) and cynomolgus monkeys (5, 20, and 100 mg/kg). Although H4 binds with similar affinity to monkey and rat FGFR4, the toxicology profiles were distinct. H4 was well tolerated in rats with no significant findings up to 200 mg/kg. Effects in monkeys included sporadic malformed feces, reduced food intake, elevated serum ALT (up to 44 fold over baseline) and AST activities, and elevated fecal bile acid concentrations at all doses. Hyperplasia of the gall bladder epithelium occurred at 100 mg/kg. There were no microscopic findings in the liver. Qualitatively this toxicity profile is similar to that of the anti-FGF19 antibody, although the maximum tolerated dose (MTD) of H4 in the monkey after a single dose was considered to be >100 mg/kg. These data are relevant for drug development in Oncology in light of the efforts aimed at developing anti-FGFR4 antibodies and small molecule inhibitors specific for FGFR4. Citation Format: Timothy R. Mack, Colleen Burns Burns, Xuemei Guo, William John Feaver, Marie Prewett, Jennifer Gruber, Amelie Forest, Armando R. Irizarry, Ruslan Novosiadly, Nick Loizos, Andrew Dropsey. Antitumor efficacy and non-clinical safety of the high affinity anti-FGFR4 antibody H4: Implications for targeting the FGF19-FGFR4 axis in oncology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C129.

Abstract A24: Beta-klotho expression is associated with the antitumor activity of pan-FGFR inhibitor in human malignancies with FGF19 amplification

Fibroblast growth factor receptor 4 (FGFR4) is an attractive target in Oncology. A gene encoding fibroblast growth factor 19 (FGF19), the main ligand of FGFR4, is frequently amplified (A) and/or overexpressed (OE) in human malignancies according to The Cancer Genome Atlas (TCGA) (breast cancer 12.8% A, 0.6% OE; head and neck cancer 20.5% A, 7.1% OE; squamous cell carcinoma of the lung 11.6% A, 11.8% OE; hepatocellular carcinoma (HCC) 4.2% A, 21.1% OE; colorectal carcinoma 0% A, 15.4% OE; pancreatic carcinoma 0% A, 32.1% OE; endometrial carcinoma 0.8% A, 17.7% OE; bladder carcinoma 8.5% A, 1.9% OE). The objective of this study was to identify the hallmarks of FGFR4 pathway dependence in human tumors. LY2874455, a selective SMI with a potent activity against FGFR1, 2, 3 and 4, was tested in vitro using Cancer Cell Line Sensitivity Panel that included 539 histologically and genetically diverse tumor cell lines representing human malignancies. Cell lines that were most sensitive to LY2874455 were enriched for genetic FGFR pathway aberrations including FGF19 amplification. We therefore hypothesized that FGF19 amplification and/or overexpression might be molecular predictors of antitumor efficacy of LY2874455. We further tested LY2874455 in a panel of 14 HCC, 4 colorectal, 4 esophageal, 2 breast, 2 pancreatic and 1 head and neck carcinoma cell lines with FGF19 amplification and/or expression using a cell viability assay. Among all cell lines tested, LY2874455 activity was restricted to HCC cell lines with concurrent FGF19 amplification and expression (IC50 = 0.001-72 nM). It is known that endocrine effects of FGF19 require beta-klotho (KLB), a co-receptor whose expression is restricted to very few cell types including hepatocytes. We thus speculated that KLB expression could underlie FGFR4 pathway dependence in a subset of HCC with FGF19 amplification and/or overexpression. To corroborate this hypothesis, we employed doxycycline-inducible shRNA-mediated KLB knockdown in FGF19-amplified HCC cell lines, and demonstrated that ablated KLB expression decreased sensitivity to LY2874455 in JHH-7 (IC50 shift from 3.9 to 198 nM) and Hep3B (IC50 shift from 0.6 to 18 nM). Furthermore, in Hep3B cells, KLB knockdown was associated with a disrupted downstream signaling as exemplified by abrogated early growth response 1 (EGR1) and FBJ murine osteosarcoma viral oncogene homolog (FOS) expression upon FGF19 stimulation. These results suggest that FGF19 amplification or overexpression per se do not confer tumor cell sensitivity to FGFR blockade. Instead, FGFR4 inhibition seems to be most efficacious in FGF19-amplified tumor cells with concomitant FGF19, FGFR4 and KLB expression. This study established a mechanistic link between KLB expression and FGFR SMI efficacy in the setting of FGF19 amplification and/or overexpression and revealed a molecular profile of HCC patients that may benefit from FGFR inhibitors. Citation Format: Amelie Forest, Sandra Nakasone, Ying Wang, Xuemei Guo, Timothy R. Mack, Genshi Zhao, Yong G. Yue, Xueqian Gong, Trent Stewart, Sean Buchanan, Christoph Reinhard, Ruslan Novosiadly. Beta-klotho expression is associated with the antitumor activity of pan-FGFR inhibitor in human malignancies with FGF19 amplification. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A24.