Search

Figure S9 shows antitumor efficacy of combination therapy in the L55 NSCLC tumor xenograft model

Supplementary Table S1 - Therapeutic antibody induced EGFR ECR mutations; Supplementary Table S2 - Mean SPR KD values for binding of wild type (WT) and cetuximab resistance variants of sEGFR to immobilized Fab fragments from cetuximab (FabC225) and necitumumab (Fab11F8) and to EGF; Supplementary Table S3 - Crystallographic data collection and refinement statistics; Supplementary Table S4 - Changes in experimental binding and DSC4.1 computed energy terms for interaction of cetuximab (C225) and necitumumab (11F8) with wild type and epitope mutated EGFR variants; Supplementary Figure S1 - Binding of necitumumab and cetuximab to LK2 and HELA cells; Supplementary Figure S2 - Electron density near R468 in sEGFRd3-S468R/Fab11F8 structure; Supplementary Figure S3 - Comparison of the paratopes of necitumumab and panitumumab; Supplementary Figure S4 - Necitumumab exhibits structural plasticity in binding to sEGFRd3-S468R; Supplementary Figure S5 - Location of the epitope substitutions in the experimental test set and evaluation of the side chain conformations modeled with DSC4.1; Supplementary Figure S6 - CDR dynamics for necitumumab and cetuximab when bound to three cetuximab resistance variants, evaluated by MD simulations

Table S2 shows human immune effector ADCC and CDC activity EC50 (nM)

Supplemental Tables 1-2 & Figures 1-4. Supplemental Table 1. Total IR, IR-A, IR-B, IGF-IR, IGF-I and IGF-II mRNA expression in preclinical models of pediatric solid tumors. Supplemental Table 2. Patient-derived xenograft models of lung adenocarcinoma: clinical information and mutational status. Supplemental Figure 1. Effect of cixutumumab on circulating growth hormone (A), insulin (B) and IGF-I (C) levels in male C57BL/6 mice. Represented are mean values +/- SEM. p-values {less than or equal to} 0.05 indicate statistical significance (*) (Student's t-test). Supplemental Figure 2. IGF-I and IGF-II mRNA expression in patient-derived models of lung adenocarcinoma. Represented are mean values +/- SEM. Supplemental Figure 3. Overlay analysis of cell surface IR and IGF-IR expression by flow cytometry (A) and ligand-induced signal transduction by Western Blot (B) in A549 cell overexpressing empty vector (pLVX) or IR isoforms (IR-A and IR-B). Serum-starved cells (1h) were treated with rhIGF-I, rhIGF-II or recombinant human insulin (10 nM) for 5, 15 or 60 minutes. Proteins (10ug) extracted from cultured cells were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRbY1150/51/IGF-IRbY1135/36, anti-phospho-AktS473 and anti-phospho-p42/p44 MAPKT202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total Akt and p42/p44 MAPK. Supplemental Figure 4. Anchorage-dependent cell viability assay. MCF-7-Mock, MCF-7-IR-A and MCF-7-IRB cells were treated with increasing concentrations of cixutumumab (0.015-1,000 nM). Maximum inhibition for MCF-7-Mock, MCF-7-IR-A and MCF-7-IRB were 60.4%, 0% and 26.9% respectively, p-values < 0.01(Student's t-test). Represented are mean values +/- SEM.

Supplementary Materials and Methods

Supplemental Information. This file contains Supplemental Materials and Methods, Tables and Figure Legends

The distributions of mRNA gene expression levels IGF-1R, Total IR, IR-A, and IR-B

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.

S1 and S2

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.

Supplementary Figure Legends for Figures S1 and S2

Kaplan-Meier curves of PFS and OS in pooled group and arm A dichotomized by insulin receptor-A (IR-A) expression (high vs. low) using the 75th percentile of marker distribution as cutpoint.

Supplementary Table S1 and S2

The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy.

Background Modulation 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. Results Here 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. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.

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.

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.

Background Modulation 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. Results Here 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. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications. Electronic supplementary material The online version of this article (10.1186/s40425-018-0329-7) contains supplementary material, which is available to authorized users.

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.

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.

Summary: Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity. : Schaer, Beckmann et al. describe unique immune-modulating properties of abemaciclib that include upregulation of antigen presentation on tumor cells and increased T cell activation. These activities synergize with anti-PD-L1 therapy to further enhance immune activation, including macrophage and DC antigen presentation, and also lead to a reciprocal increase in abemaciclib-dependent cell cycle gene regulation. Keywords: CDK4/6, abemaciclib, PD-1, PD-L1, combination immunotherapy, 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. This work was supported by Eli Lilly and Company.

Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting. Citation Format: Veena Kandaswamy, Amelie Forest, Marianne Deroose, David A. Schaer, Ting Chen, Shengwu Liu, David Surguladze, Yung-mae Yao, Thompson Doman, Gerald Hall, Kwok-Kin Wong, Michael Kalos, Ruslan D. Novosiadly. Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3632.

The approval of abemaciclib and additional cyclin dependent kinases 4 & 6 (CDK4/6) inhibitors for the treatment of HR+ breast cancer has provided new therapeutic options to patients. As CDK4/6 inhibitors become part of the standard of care, combination strategies leveraging abemaciclib together with immunotherapy may represent an opportunity to extend benefit to more patients and additional cancers. Accordingly, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with PD-L1 blockade in immunocompetent murine syngeneic tumor models, and directly evaluated the tumor cell and immune cell intrinsic immunologic effect of abemaciclib in vitro. In vivo abemaciclib treatment of murine tumors (CT26, EMT6 and MC38) caused a dose-dependent delay in tumor growth and demonstrated the potential to induce complete tumor regression (CR ~10%). Combination with an anti-PD-L1 antibody after abemaciclib pretreatment greatly enhanced the anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy resulted in 50-60% CRs of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice maintaining CRs after cessation of combination therapy or abemaciclib monotherapy resisted later CT26 rechallenge, demonstrating the ability to generate immunological memory during abemaciclib therapy. Analysis of intra-tumor gene expression showed that abemaciclib monotherapy induced T cell activation and inflammation signatures. Combination therapy substantially enhanced this effect and was additionally associated with DC maturation, antigen presentation, cytokine signaling and helper T cell phenotype. Suppression of cell cycle genes indicative of inhibition of CDK4/6 was also more prominent during the combination therapy. In Jurkat and primary human T cells, treatment with abemaciclib in vitro resulted in a dose-dependent increase in NFAT activity upon TCR stimulation. This correlated with upregulation of both cell surface markers and genes associated with an enhanced T cell activation phenotype, while only modestly affecting T cell expansion. Abemaciclib also amplified expression of antigen presentation and other immune-related genes in human breast cancer cells. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results provide a strong rationale for combining abemaciclib with checkpoint immunotherapy to improve the anti-tumor efficacy. The T cell and tumor cell intrinsic effects, synergistic anti-tumor responses and intra-tumor immune activation, justify clinical investigation of this combination. Citation Format: David Schaer, Richard Beckmann, Jack Dempsey, Lysiane Huber, Amelie Forest, Nelusha Amaladas, Ying Cindy Wang, Erik Rasmussen, Darin Chin, Yanxia Li, Andrew Capen, Marianne Deroose, Carmine Carpenito, Xueqian Gong, Kirk Staschke, Linda Chung, Farhana Merzoug, Trent Stewart, Lacey Litchfield, Philip Iversen, Sean Buchanan, Alfonso de Dios, Ruslan Novosiadly, Michael Kalos. The CDK4/6 inhibitor abemaciclib synergizes with PD-L1 blockade to induce an immune inflamed tumor microenvironment through T cell and tumor cell intrinsic effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4569.

Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2730.

First- , second- , and third-generation EGFR tyrosine kinase inhibitors (TKIs) demonstrate profound tumor responses in EGFR mutation-positive NSCLC. Nevertheless, most patients invariably develop acquired resistance. The objective of the present study was to evaluate the effects of dual EGFR blockade using a ligand-blocking antibody (necitumumab) and second- or third-generation TKIs (afatinib or osimertinib) in preclinical NSCLC models with EGFR mutations. Afatinib and osimertinib (AZD9291), second- and third-generation EGFR TKIs, respectively, are irreversible covalent inhibitors of the EGFR tyrosine kinase, with the latter specifically targeting a mutant form of the receptor (EGFR-T790M). We sought to investigate whether combination of necitumumab (LY3012211) with afatinib and osimertinib would provide any benefit over the monotherapies in preclinical mouse models of lung cancer. Using PC-9 and NCI-H1975 xenograft models of NSCLC that harbor EGFR mutations, we observed that dual EGFR blockade of ligand binding and receptor tyrosine kinase activity improved the antitumor efficacy and, more importantly, resulted in durable tumor remissions compared to either single-agent therapy. Additionally, we explored two dosing schedules of necitumumab/osimertinib combination treatment (concurrent versus phased). Concurrent administration of both agents for four weeks was significantly more efficacious compared to the phased schedule (e.g., administration of osimertinib for one week followed by necitumumab and osimertinib combination for additional three weeks). To provide mechanistic insights into the combinatorial activity observed, we employed high-content gene expression analysis using nCounter Pan-Cancer Pathways assay. Pathway analysis of the combination and single-agent treatment groups identified multiple shared and treatment-specific pathway alterations. Genes implicated in FGFR, PI3K pathways and extracellular remodeling appeared to be upregulated by combination and monotherapies and suggested potential common mechanisms of acquired resistance to EGFR inhibitors, whereas compromised DNA repair mechanisms were identified in PC-9 and NCI-H1975 tumors upon treatment with both combinations. In conclusion, these data demonstrate that combination of necitumumab with second- or third-generation EGFR TKIs results in an improved antitumor efficacy compared to the respective monotherapies; a more profound inhibition of EGFR pathway and compromised DNA repair mechanisms may underlie this effect. A phase I clinical trial of necitumumab and osimertinib in EGFR mutation-positive stage IV or recurrent NSCLC who have progressed on a previous EGFR TKI is currently ongoing (NCT02496663). Citation Format: Amelie Forest, Erik R. Rasmussen, Thompson N. Doman, Michael Amatulli, Rajiv Bassi, Gerald E. Hall, Jason R. Manro, Manisha Brahmachary, David Surguladze, Yung-mae M. Yao, Michael D. Kalos, Ruslan D. Novosiadly. Combination of necitumumab with second- or third-generation EGFR tyrosine kinase inhibitors downregulates DNA repair mechanisms and induces durable tumor remissions in xenograft NSCLC models with EGFR mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A160.

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.

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

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

In this study, we used electron tomography as well as immunogold labeling to analyze the morphology and distribution of proteins within postsynaptic densities (PSDs) isolated from rats before birth (embryonic day 19) and at postnatal days 2, 21, and 60. Our data provide direct evidence of distinct morphological and compositional differences in PSDs throughout development. Not all PSD components are present at the early stages of development, with a near lack of the scaffolding molecule PSD-95 at E19 and P2. The presence of NR1 and NR2b suggests that PSD-95 is not directly required for clustering of N-methyl-D-aspartic acid (NMDA) receptors in PSDs early in development. α-Actinin is abundant by E19, suggesting that it is a core structural component of the PSD. Both α and β isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) are present early on but then rise in labeling density by approximately fourfold by P21. Among all the molecules studied, only calmodulin (CaM) was found in higher abundance early in PSD development and then fell in amount over time. Spatial analysis of the immunogold label shows a nonrandom distribution for all the proteins studied, lending support to the idea that the PSD is systematically assembled in an organized fashion. Morphological data from electron tomography shows that the PSD undergoes major structural changes throughout development. J. Comp. Neurol. 518:4243–4260, 2010. © 2010 Wiley-Liss, Inc.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

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.

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.