Gordon Moody

Efficacy of Ganitumab (AMG 479), Alone and in Combination with Rapamycin, in Ewing's and Osteogenic Sarcoma Models

Ewings and osteogenic sarcoma are two of the leading causes of cancer deaths in children and adolescents. Recent data suggest that sarcomas may depend on the insulin-like growth factor type 1 (IGF-1) receptor (IGF1R) and/or the insulin receptor (INSR) to drive tumor growth, survival, and resistance to mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We evaluated the therapeutic value of ganitumab (AMG 479; C6472H10028N1728O2020S42), an anti-IGF1R, fully human monoclonal antibody, alone and in combination with rapamycin (mTORC1 inhibitor) in Ewings (SK-ES-1 and A673) and osteogenic (SJSA-1) sarcoma models. IGF1R was activated by IGF-1 but not by insulin in each sarcoma model. INSR was also activated by IGF-1 in the SJSA-1 and SK-ES-1 models, but not in the A673 model where insulin was the preferred INSR ligand. Ganitumab significantly inhibited the growth of SJSA-1 and SK-ES-1 xenografts; inhibition was associated with decreased IGF1R and Akt phosphorylation, reduced total IGF1R and bromodeoxyuridine detection, and increased caspase-3 expression. Ganitumab inhibited rapamycin-induced IGF1R, Akt, and glycogen synthase kinase-3β hyperphosphorylation in each sarcoma model. However, ganitumab in combination with rapamycin also resulted in a marked increase in INSR expression and activity in the SJSA-1 and A673 models. The in vivo efficacy of ganitumab in the two ganitumab-sensitive models (SJSA-1 and SK-ES-1) was significantly enhanced in combination with rapamycin. Our results support studying ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas and suggest that INSR signaling is an important mechanism of resistance to IGF1R blockade.

Ganitumab (AMG 479) Inhibits IGF-II–Dependent Ovarian Cancer Growth and Potentiates Platinum-Based Chemotherapy

Purpose: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer. Experimental Design: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I–, IGF-II–, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo. Results: In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho–IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I–, IGF-II–, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo. Conclusions: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho–IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947–58. ©2014 AACR.

IGF1R blockade with ganitumab results in systemic effects on the GH–IGF axis in mice

Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding assays showed that ganitumab recognized murine IGF1R with sub-nanomolar affinity (KD=0.22 nM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon carcinoma cells and tumors. Addition of ganitumab to 5-fluorouracil resulted in enhanced inhibition of tumor growth in the CT26 model. Pharmacological intervention with ganitumab in naïve nude mice resulted in a number of physiological changes described previously in animals with targeted deletions of Igf1 and Igf1r, including inhibition of weight gain, reduced glucose tolerance and significant increase in serum levels of GH, IGF1 and IGFBP3. Flow cytometric analysis identified GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These findings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly regulates serum IGF1 levels through Gh signaling.

Antibody-mediated neutralization of autocrine Gas6 inhibits the growth of pancreatic ductal adenocarcinoma tumors in vivo

Gas6 and its receptors Axl, Mer and Tyro‐3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor‐promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co‐expressed and their co‐expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6‐induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl‐Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6‐induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100–1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6‐induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.

Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing.

For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.

Abstract 2027: Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models

Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC Evasion of apoptosis represents an essential hallmark in the progression of many cancers. The Bcl-2 family of proteins plays a central role in regulating the apoptotic process. Targeting pro-survival Bcl-2 family members like Mcl-1 with small molecule inhibitors represents a viable therapeutic approach for the treatment of cancer. This study evaluated the in vitro and in vivo activity of AMG 176, a potent and selective Mcl-1 inhibitor currently in Phase I clinical development. AMG 176 binds with high affinity and selectivity to the BH3-binding groove of Mcl-1. In a cell based split-luciferase complementation assay, AMG 176 disrupted the interaction between Mcl-1 and Bak, leading to downstream activation of the intrinsic apoptosis pathway as measured by increased caspase activity and subsequent effects on viability. Oral administration of AMG 176 to mice bearing OPM-2 multiple myeloma xenografts resulted in a dose-dependent increase in activated Bak with a clear PK/PD relationship. Dosing regimens (20-60 mg/kg PO, QD) evaluating discontinuous and continuous administration of AMG 176 in established OPM2 xenografts demonstrated robust tumor growth inhibition with complete tumor regression at an elevated dose. Efficacy in this model was achieved at doses in agreement with those eliciting induction of apoptotic markers. Treatment of tumor cell lines with Compound A, a close structural analog of AMG 176, revealed a dose- and time-dependent increase in Mcl-1 protein levels that was reversible upon compound washout. Subsequent experiments performed with cycloheximide suggested that elevations in Mcl-1 protein levels were due to an increase in Mcl-1 protein half-life, likely driven by the compounds ability to disrupt proteasome-mediated degradation. Compound A was also used to characterize the kinetics of activating apoptosis. These studies revealed a rapid induction of apoptosis and loss of viability in Mcl-1 dependent multiple myeloma and AML cell lines. In cell lines highly dependent on Mcl-1, treatment with Compound A for as little as two hours was sufficient to achieve complete cell killing. Cell line profiling studies (>200 lines) revealed robust effects on cell viability in a subset of solid tumor cell lines and cell lines of hematological origin, including multiple myeloma, acute myeloid leukemia and non-Hodgkin lymphoma. Subsequent association analysis with molecular profiling endpoints identified an inverse correlation between BCLxL expression and sensitivity to Mcl-1 inhibition. Combination screens with Compound A revealed multiple highly synergistic combinations including compounds targeting the MAPK pathway, standard of care chemotherapeutics and agents targeting additional pro-survival members of the BCL-2 family. In conclusion, AMG 176 is a potent and selective Mcl-1 inhibitor, with significant in vitro and in vivo activity in Mcl-1 dependent cancer models. Citation Format: Sean R. Caenepeel, Brian Belmontes, Jan Sun, Angela Coxon, Gordon Moody, Paul E. Hughes. Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models [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 2027. doi:10.1158/1538-7445.AM2017-2027

Pharmacodynamic analysis of an agonistic antibody to the costimulatory receptor GITR

GITR/TNFRSF18 is a member of the TNF-receptor superfamily preferentially expressed on regulatory T cells (Tregs) and activated T effector cells. Antibody agonists to GITR claim two distinct mechanisms to overcome the repressive tumor microenvironment and drive anti-tumor efficacy in vivo: receptor agonism (forward signaling) on T effector cells and FcγR-mediated Treg depletion. We sought to better understand the contribution of these two mechanisms using pharmacodynamic readouts relating target coverage, Treg depletion and efficacy using isotypic variants of a surrogate antibody against mouse GITR, DTA-1. First, target coverage was determined in spleen, tumor and draining lymph node following treatment with a single dose of mouse IgG2a DTA-1. In this study, efficacy correlated with doses that covered >90% GITR-expressing intratumoral leukocytes and depleted >90% intratumoral Tregs at 24 hours post-dose. Though displaying equivalent agonistic activity in vitro and achieving a similar level of target coverage, the mouse IgG1 N297A variant of DTA-1 neither depleted Tregs nor displayed anti-tumor activity in vivo, in confirmation of recent literature. To further explore the influence of Fc engagement, additional DTA-1 isotypic variants were generated and tested in vivo. In this study, we confirmed that preferential engagement of Fcγ receptors was necessary for optimal activity, as the mouse IgG1 DTA-1 variant failed to regress tumors. Additionally, we identified a variant with enhanced Treg depletion properties, however, the enhanced depletion did not translate to improved anti-tumor efficacy. Lastly, we sought to understand if mouse IgG2a DTA-1-would enhance the effect of PD-1 / PD-L1 blockade in vivo. Using the MC38 tumor model, we observed synergistic tumor regression in the combination group versus either monotherapy. Given the likely non-overlapping mechanism of the antibodies, the results suggest that an ADCC-enabled agonistic GITR antibody could provide benefit to human cancer patients in combination with, or refractory to, PD-1/PD-L1 inhibitors

Abstract C172: Efficacy of AMG 479, alone and in combination with cisplatin, in ovarian carcinoma xenograft models

AMG 479, a fully human monoclonal antibody raised to the type I insulin‐like growth factor receptor (IGF1R), is currently being studied in the treatment of patients with ovarian carcinoma. We have previously shown that AMG 479 inhibits the growth of pancreatic and sarcoma tumor models by inhibiting IGF1R signaling through ligand blockade and receptor internalization and degradation. In the present study, we tested the ability of AMG 479 to inhibit the growth of four human ovarian carcinoma xenografts (OV‐90, SK‐OV3, TOV‐21G, and OV‐CAR3) in nude mice. Recent evidence has shown that multiple players in the IGF‐axis are differentially regulated in human ovarian serous carcinomas, suggesting that the IGF1R pathway may play an important role in the growth of these tumors. The OV‐90 model showed exquisite sensitivity to single‐agent AMG 479, reaching significant (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C172.

Abstract 2978: The utilization of a human MCL1 knockin mouse suggests that reductions in B-cells and monocytes may serve as clinically relevant pharmacodynamic markers of MCL1 inhibition

The development of small-molecule inhibitors targeting pro-survival BCL-2 family members, such as MCL1 and BCL-2, is an attractive approach for the treatment of cancer. AMG 176 is a potent and selective first-in-class MCL1 inhibitor currently in phase I clinical trials in multiple myeloma and AML. AMG 176 and its analogue AM-8621 exhibit picomolar affinity for human MCL1. However, binding affinity for murine MCL1 is reduced by ~200-fold. Examination of the AMG 176 binding pocket in human MCL1 suggests Leu248 contributes to the high affinity interaction with AMG 176. In mouse MCL1, the corresponding amino acid is a phenylalanine, reducing the affinity of AMG 176 for the mouse protein. This reduced affinity makes it challenging to assess the pharmacodynamic effect of AMG 176 on normal hematopoietic cell types at efficacious doses in human tumor xenograft models. To overcome this challenge we generated a human MCL1 knockin mouse in which the mouse genomic Mcl1 locus was replaced with its human counterpart. Human MCL1 knockin (KI) mice were created by targeting embryonic stem (ES) cells with a vector containing the human MCL1 genomic locus flanked by the homologous mouse sequences up- and downstream of mouse Mcl1. Targeted ES cells were confirmed by Southern blot and used to generate chimeric mice. Germline transmission of the human MCL1 KI allele and the removal of selectable markers was accomplished by breeding with cre-expressing transgenic mice. Homozygous human MCL1 KI mice were created by breeding heterozygous human MCL1 KI mice lacking the Cre transgene and selectable markers. We used a splenocyte viability assay to confirm the functional replacement of murine Mcl1 with its human ortholog. The in vitro treatment of splenocytes with AM-8621 for 6 hours resulted in dose-dependent Caspase 3 activation and reduced viability in B and T cells derived from human MCL1 knockin mice. Oral administration of AMG 176 at doses of 30 and 60 mg/kg resulted in the induction of apoptosis and the subsequent inhibition and regression of OPM-2 multiple myeloma tumor xenografts (54% tumor growth inhibition and 21% regression, respectively). The human MCL1 knockin mice were used to test the effect of AMG 176 at an equivalent dose and schedule on B cells, T cells and monocytes derived from peripheral blood, spleen and bone marrow. Samples treated with 30 and 60 mg/kg of AMG 176 were harvested 24 hours post-second dose of the first and second cycles. We observed a dose-dependent decrease in monocytes and B cells at both time points, with the most significant reductions observed in monocytes (70.1% and 94.5%, respectively) and B cells (81.5% and 94.0%, respectively) derived from peripheral blood after the first cycle. In conclusion, these data suggest that reductions in B cells and monocytes may serve as clinically relevant pharmacodynamic endpoints of MCL1 inhibition. Citation Format: Brian Belmontes, Sean R. Caenepeel, Jan Sun, Danny Chui, Angela Coxon, Gordon Moody, Jude Canon, Paul Hughes. The utilization of a human MCL1 knockin mouse suggests that reductions in B-cells and monocytes may serve as clinically relevant pharmacodynamic markers of MCL1 inhibition [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 2978.

Abstract 4038: Developmentof a murine tumor immunophenotyping platform to support drug discovery anddevelopment in immuno-oncology

Recent clinical data highlights the importance of immune cell localization, phenotype, and gene signature as it correlates to a productive anti-tumor response. Preclinically, multiple syngeneic mouse models have been used to study the effects of immunomodulation and define anti-tumor responses to transplanted “self” tumors. However, while the literature describes distinct aspects of many of these models, there is no comprehensive dataset comparing and contrasting their tumor-immune microenvironments across models. These data are critical for better understanding the role that various immune populations play in the anti-tumor response and interpreting observed changes in tumor clearance following treatment with immunomodulatory agents. We have therefore established a platform that 1) quantitates the types of immune cells within murine tumor models, and 2) describes the location of these cells within the tumor. In parallel to the immunophenotyping efforts we have benchmarked tumor models based on their response to antibodies against T cell checkpoint pathways. We sought to use this immunophenotyping platform to identify specific immune modulation that occurs in syngeneic tumors post depletion of macrophages via CSF1R blockade. Tumor-associated macrophages (TAMs) are believed to help promote tumor survival through suppression of the adaptive immune response and the secretion of growth factors that promote tumor growth and angiogenesis. Therefore depletion of TAMs should lead to T-cell recruitment and bolster the antitumor T-cell response. Here we show that treatment of CT-26 and RENCA syngeneic tumors with a CSF1R antagonist leads to depletion of MHCII+ and F4/80+ expressing cells. Future experiments will seek to understand if CSF1R blockade improves the response to T-cell checkpoint immunotherapies. In summary, the development of a murine tumor immunophenotyping platform has allowed insight and evaluation of immune cells in the tumor microenvironment that can ultimately be leveraged to understand the synergistic effects of immunotherapeutics. Citation Format: Brian Belmontes, Stephanie Matyas, Sarah O’Brien, Hong Tan, Kenneth Ganley, Kimberly Merriam, Jim Rottman, Jackson Egen, Pedro Beltran, Gordon Moody. Developmentof a murine tumor immunophenotyping platform to support drug discovery anddevelopment in immuno-oncology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4038.