Carter Fields

Ligand-Independent HER2/HER3/PI3K Complex Is Disrupted by Trastuzumab and Is Effectively Inhibited by the PI3K Inhibitor GDC-0941

Herceptin (trastuzumab) is the backbone of HER2-directed breast cancer therapy and benefits patients in both the adjuvant and metastatic settings. Here, we describe a mechanism of action for trastuzumab whereby antibody treatment disrupts ligand-independent HER2/HER3 interactions in HER2-amplified cells. The kinetics of dissociation parallels HER3 dephosphorylation and uncoupling from PI3K activity, leading to downregulation of proximal and distal AKT signaling, and correlates with the antiproliferative effects of trastuzumab. A selective and potent PI3K inhibitor, GDC-0941, is highly efficacious both in combination with trastuzumab and in the treatment of trastuzumab-resistant cells and tumors.

Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade in Antitumor Response to Combination Therapy

Purpose: Targeting HER2 with multiple HER2-directed therapies represents a promising area of treatment for HER2-positive cancers. We investigated combining the HER2-directed antibody–drug conjugate trastuzumab emtansine (T-DM1) with the HER2 dimerization inhibitor pertuzumab (Perjeta). Experimental Design: Drug combination studies with T-DM1 and pertuzumab were performed on cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-positive locally advanced or metastatic breast cancer (mBC), T-DM1 was dose-escalated with a fixed standard pertuzumab dose in a 3+3 phase Ib/II study design. Results: Treatment of HER2-overexpressing tumor cells in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation and induction of apoptotic cell death. The presence of the HER3 ligand, heregulin (NRG-1β), reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this effect was reversed by the addition of pertuzumab. Results from mouse xenograft models showed enhanced antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each agent. In patients with mBC previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1 could be dosed at the maximum tolerated dose (MTD; 3.6 mg/kg every 3 weeks) with standard dose pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity. Conclusions: Dual targeting of HER2 with the combination of T-DM1 and pertuzumab in cell culture and mouse xenograft models resulted in enhanced antitumor activity. In patients, this combination showed an encouraging safety and tolerability profile with preliminary evidence of efficacy. Clin Cancer Res; 20(2); 456–68. ©2013 AACR.

A class of 2,4-bisanilinopyrimidine Aurora A inhibitors with unusually high selectivity against Aurora B.

The two major Aurora kinases carry out critical functions at distinct mitotic stages. Selective inhibitors of these kinases, as well as pan-Aurora inhibitors, show antitumor efficacy and are now under clinical investigation. However, the ATP-binding sites of Aurora A and Aurora B are virtually identical, and the structural basis for selective inhibition has therefore not been clear. We report here a class of bisanilinopyrimidine Aurora A inhibitors with excellent selectivity for Aurora A over Aurora B, both in enzymatic assays and in cellular phenotypic assays. Crystal structures of two of the inhibitors in complex with Aurora A implicate a single amino acid difference in Aurora B as responsible for poor inhibitory activity against this enzyme. Mutation of this residue in Aurora B (E161T) or Aurora A (T217E) is sufficient to swap the inhibition profile, suggesting that this difference might be exploited more generally to achieve high selectivity for Aurora A.

Targeting LGR5 + cells with an antibody-drug conjugate for the treatment of colon cancer

An antibody-drug conjugate targeting LGR5 effectively treats intestinal cancer in preclinical models. Stemming the progression of cancer LGR5 is a well-known marker of intestinal cancer stem cells, which makes it an attractive target for anticancer treatments. Unfortunately, it is also found in healthy intestinal stem cells, giving rise to concerns about the potential toxicity of such treatments. Now, Junttila et al. used preclinical models of intestinal cancer to demonstrate that targeting LGR5 with an antibody-drug conjugate is effective for shrinking tumors without damaging the surrounding normal tissues. These observations of preclinical effectiveness as well as safety suggest that targeting LGR5-expressing cells may be a viable therapeutic strategy and a candidate for evaluation in human studies. Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat–containing G protein–coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.

A pentacyclic aurora kinase inhibitor (AKI-001) with high in vivo potency and oral bioavailability.

Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC50 < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.

Abstract S3-6: Combination Therapy of the Novel PI3K Inhibitor GDC-0941 and Dual PI3K/mTOR Inhibitor GDC-0980 with Trastuzumab-DM1 Antibody Drug Conjugate Enhances Anti-Tumor Activity in Preclinical Breast Cancer Models In Vitro and In Vivo

The receptor tyrosine kinase, HER2/ErbB2, is a validated clinical target for HER2-amplified breast cancer, as evidenced by the U.S.F.D.A. approval of the humanized HER2 antibody, trastuzumab (Herceptin®), and the dual HER2/EGFR small molecule tyrosine kinase inhibitor lapatinib (Tykerb®). An alternative approach for targeting HER2 is the direct covalent coupling of a cytotoxic drug to trastuzumab. We have previously reported the potent in vitro and in vivo efficacy of T-DM1, trastuzumab (T) linked to the microtubule polymerization inhibitory drug maytansinoid (DM1), in trastuzumab-sensitive and-refractory breast tumor models (1). Inhibition of signaling through PI3K, which is hyperactivated in HER2-amplified breast cancer due to constitutive activity of overexpressed HER2 and/or through mutation of the p110-α subunit of PI3K, also offers an additional therapeutic approach. Therefore the specific aims of our study were to determine if the combination of a novel pan-PI3K inhibitor (GDC-0941) or a dual PI3K/mTOR inhibitor (GDC-0980) enhanced the anti-tumor activity of T-DM1 in HER2-amplified breast cancer lines in vitro and as xenografts in vivo. The breast cancer cell lines tested, MCF7 neo/HER2 and KPL4, harbor the E545K and H1047R PIK3CA mutations, respectively. Combination treatment of T-DM1 with either GDC-0941 or GDC-0980 in vitro resulted in a synergistic inhibition of cellular viability. Biochemical biomarker analyses revealed inhibition of phospho-Akt and phospho-ERK by both T-DM1 and GDC-0941, decreased phosphorylation of Rb and PRAS40 by GDC-0941, and increased levels of the mitotic markers phospho-histone H3 and cyclin B1 after treatment with T-DM1. In addition, T-DM1 treatment resulted in apoptosis as determined by appearance of the 23 kDa PARP-cleavage fragment, decreased levels of Bcl-XL, as well as activation of caspases 3 and 7. Addition of GDC-0941 to T-DM1 further enhanced apoptosis induction. In vivo, increased and sustained tumor regressions were observed when GDC-0941 was combined with T-DM1 as compared to single-agent activity in the MCF7 neo/HER2 and KPL4 sub-cutaneous xenograft models in a dose-dependent fashion. Moreover, an increased number of sustained complete regressions (CRs) were observed when GDC-0980 was combined with T-DM1 in the KPL4 xenograft model when compared to the combination treatment with GDC-0941 (% CRs = 88% for GDC-0980 + T-DM1 vs. 50% for GDC-0941 + T-DM1). The results of our pre-clinical studies provides evidence for the use of rational drug combinations of PI3K inhibitors such as GDC-0941 and GDC-0980 with T-DM1 in HER2-amplified breast cancer that harbor PIK3CA mutations and may offer additional treatment options for patients whose disease progresses on trastuzumab or lapatinib-based therapy. 1. Lewis Phillips, G. et al. Cancer Res 2008; 68: (22). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S3-6.

Abstract 1207: Preclinical development of 2nd generation HER2-directed antibody-drug conjugates

The HER2 receptor tyrosine kinase is amplified in approximately 20% of human breast cancer and is associated with poor clinical outcome. The humanized antibodies trastuzumab and pertuzumab are approved for use in both early and metastatic HER2-positive breast cancer, and are most often given with chemotherapy. Antibody-drug conjugates (ADCs) are anti-tumor agents designed to deliver potent cytotoxic drugs selectively to target-expressing tumor cells. Trastuzumab emtansine is a HER2-directed ADC comprised of trastuzumab covalently linked to the microtubule inhibitor DM1, through the stable MCC linker. Trastuzumab emtansine is approved for use in HER2-positive metastatic breast cancer as a single agent in patients who have received prior trastuzumab and a taxane. We are now exploring new HER2-directed ADCs (‘2nd generation ADCs’) with different mechanisms of action (MOA) than trastuzumab emtansine by investigating ADCs utilizing DNA-damaging agents, such as pyrrolobenzodiazepine (PBD) dimers and cyclopropylbenzindole (CBI) dimers, as the cytotoxic drug components. These agents have been conjugated to either trastuzumab or the humanized anti-HER2 antibody 7C2 (hu7C2) using both uncleavable and cleavable linkers. As free drugs and ADCs, the PBDs and CBIs show similar or greater potency in cell proliferation assays in vitro compared to DM1 and trastuzumab emtansine. However, unlike DM1, these agents are not strong substrates of Pgp/MDR1. Moreover, the PBDs and CBIs are active on non-dividing cells, whereas microtubule inhibitors such as DM1 do not affect non-dividing cells. Robust anti-tumor activity was observed in vivo in the fo5 HER2 transgenic tumor transplant model with the 2nd generation ADCs. Efficacious doses resulting in tumor stasis or regression ranged from 0.25-3 mg/kg administered as a single injection. In contrast, doses of trastuzumab emtansine required for stasis/regression in this model are 10 and 15 mg/kg, respectively. Efficacious doses were well-tolerated in the mouse xenograft models. Further tolerability studies of the 2nd generation ADCs were performed in rats. As rats are a non-binding species for trastuzumab and hu7C2, these studies assessed antigen-independent toxicities. Maximum tolerated doses for the different ADCs ranged from 2.5-15 mg/kg administered as a single injection, compared to 46 mg/kg for trastuzumab emtansine (Poon et al., 2013), likely reflecting both the different MOA and greater potency of the cytotoxic agents utilized in the 2nd generation HER2-directed ADCs. Overall, our findings demonstrate robust in vitro and in vivo activity of HER2 ADCs comprised of DNA-active agents, allowing for further development of a HER2-directed ADC distinct from trastuzumab emtansine. Citation Format: Gail D. Lewis Phillips, Guangmin Li, Jun Guo, Jeffrey Lau, Shang-Fan Yu, Thomas Pillow, Byoung-Chul Lee, Jack Sadowsky, Melissa Schutten, Carter Fields, Mark X. Sliwkowski. Preclinical development of 2nd generation HER2-directed antibody-drug conjugates. [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 1207.

Abstract 1212: The dual action antibody MEHD7945A targeting EGFR and HER3 enhances chemotherapy induced cytotoxicity in vitro and in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Dysregulation of the epidermal growth factor receptor family (EGFR, HER2, HER3, HER4) by mutation and/or overexpression plays an important role in tumorigenesis, and targeted agents directed against two members of the HER/ErbB family, epidermal growth factor receptor (EGFR/HER1), and HER2/ErbB2, are used in the treatment of cancer. Extensive crosstalk seen among these receptors implies that blocking signaling of more than one receptor may be more effective in inhibiting tumor growth and circumventing resistance mechanisms than targeting individual receptors. In particular, HER3 is considered a key mediator of resistance to many targeted agents. We generated a “two-in-one” antibody, MEHD7945A, that binds to EGFR and HER3 with high affinity, inhibits receptor function and is more broadly efficacious in various tumor types when compared to monospecific anti-EGFR or anti-HER3 antibodies. Given the ability of HER3 to potently activate the PI3K survival pathway, we investigated if antagonizing ligand-dependent HER signaling with MEHD7945A in the presence of chemotherapy augments cytotoxicity. We calculated combination index values the effects of combining MEHD7945A with commonly used chemotherapeutic agents in NSCLC and colorectal cell lines in vitro and in vivo. The NSCLC lines NCI-H292, NCI-H1666, NCI-H358 and HCC827 were treated with MEHD7945A plus gemcitabine over a wide range of drug concentrations. Cell proliferation data were analyzed using CalcuSyn software and all combination index values were <1, demonstrating that the combination of MEHD7945A and gemcitabine inhibited proliferation synergistically in these cells. To further explore the enhanced cytotoxic effect we evaluated the combination of MEHD7945A and gemcitabine, versus each single agent, in the NCI-H1975 NSCLC xenograft model, and observed enhanced tumor regression. Similar results were observed when MEHD7945A was combined with a range of chemotherapeutic agents, including pemetrexed, docetaxel and irinotecan in H1975, H441 or SW948 xenograft models. In summary, these in vitro and in vivo results demonstrate that MEHD7945A potentiates various cytotoxic agents in a variety of tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1212. doi:1538-7445.AM2012-1212

Abstract 5607: Dual targeting of HER2: Enhanced antitumor efficacy of trastuzumab-DM1 combined with pertuzumab

The EGFR family of transmembrane receptors consists of HER1 (ErbB1/EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These receptors are often dysregulated in human solid tumors. For example, amplification and overexpression of HER2 occur in approximately 20 percent of human breast cancers and are predictive of poor clinical outcome. A principal means by which these receptors exert their growth-stimulatory effect is through homo- and hetero-dimerization which can occur in either a ligand-dependent or -independent manner and subsequently promotes receptor tyrosine kinase activity, leading to autophosphorylation and activation of downstream signal transduction pathways. The humanized HER2 antibody, trastuzumab (Herceptin®), is approved for use in adjuvant and metastatic HER2-positive breast cancer and has shown significant clinical benefit. Trastuzumab-DM1 (T-DM1, TMAb-mcc-DM1, trastuzumab emtansine) is an antibody-cytotoxic drug conjugate composed of the maytansine derivative DM1 directly coupled, through a thioether SMCC linker, to trastuzumab. We previously reported the potent in vitro and in vivo efficacy of T-DM1 in trastuzumab-sensitive and -refractory breast tumor models. Both trastuzumab and T-DM1 bind domain IV of the HER2 extracellular domain (ECD) and inhibit HER2 function in a ligand-independent manner. T-DM1 provides additional efficacy through selective delivery of the cytotoxic agent DM1 to HER2-overexpressing cancer cells. Pertuzumab is a humanized HER2 antibody that binds domain II of the HER2 ECD and thereby inhibits ligand-induced activation of HER2 by blocking dimerization of HER2 with other HER family members. Pertuzumab in combination with trastuzumab has shown impressive preclinical as well as clinical activity in HER2-positive breast cancer. The purpose of our studies was to evaluate the in vitro and in vivo efficacy of T-DM1 combined with pertuzumab. Our in vitro results show that combination treatment resulted in synergistic inhibition of cell proliferation in human breast and lung cancer cells that over-express HER2. Drug combination effects were analyzed by the Chou and Talalay method for determining combination index values. Similarly, we observed enhanced in vivo efficacy when administering T-DM1 in combination with pertuzumab, as compared with either agent alone, in breast and lung cancer xenograft models. Treatment of cells with T-DM1 combined with pertuzumab also resulted in a synergistic increase in apoptosis as measured by increased activity of the apoptotic enzymes, caspases 3 and 7, and increased levels of the apoptotic population in cell cycle experiments. Together, these studies support the hypothesis that T-DM1 in combination with pertuzumab for HER2-amplified cancer may offer an additional therapeutic approach for patients whose disease progresses on trastuzumab and lapatinib-based therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5607.