Kathryn Parsons

Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate.

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigen-expressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development.

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.

Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer

Trastuzumab (Herceptin®) is currently used as a treatment for patients whose breast tumors overexpress HER2/ErbB2. Trastuzumab-DM1 (T-DM1, trastuzumab emtansine) is designed to combine the clinical benefits of trastuzumab with a potent microtubule-disrupting drug, DM1 (a maytansine derivative). Currently T-DM1 is being tested in multiple clinical trials. The mechanisms of action for trastuzumab include inhibition of PI3K/AKT signaling pathway, inhibition of HER-2 shedding and Fcγ receptor mediated engagement of immune cells, which may result in antibody-dependent cellular cytotoxicity (ADCC). Here we report that T-DM1 retains the mechanisms of action of unconjugated trastuzumab and is active against lapatinib resistant cell lines and tumors.

Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Purpose: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate. Experimental Design and Results: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti–cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (μg/m2) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 μg DM1/m2) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 μg DM1/m2). Conclusions: The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies. Clin Cancer Res; 16(19); 4769–78. ©2010 AACR.

Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer

The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcgammaRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function-enhanced antibodies for solid tumor therapy.

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 P4-15-02: The PI3K inhibitor GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models

The phosphoinositide 3-kinases (PI3Ks) are lipid kinases that activate the PI3K signaling pathway and play an essential role in regulating breast tumor cell growth, migration, and survival. Activating and transforming mutations in the PIK3CA gene (PI3K alpha) are commonly found in HER2+ breast cancer and ER+ breast cancer. GDC-0032 is a selective, orally bioavailable inhibitor with a Ki of 0.29 nM for PI3K alpha with 30 fold less inhibition of PI3K beta. In addition, GDC-0032 has increased single agent activity against PI3K alpha mutant and HER2 amplified cancer models in vitro and in vivo. The aim of our study was to evaluate the efficacy of GDC-0032 in breast cancer models in combination with standard of care therapeutics including taxanes, endocrine therapies, and HER2 targeted therapies. We evaluated cell viability for a range of dose concentrations of GDC-0032, as single agent and in combinations, in eleven breast cancer cell lines that harbor mutations in PI3K alpha (K111N, E545K, H1047R) and/or over-expressed HER2. GDC-0032 was combined with taxanes (paclitaxel and docetaxel), endocrine therapies (fulvestrant and tamoxifen) or anti-HER2 agents (trastuzumab, pertuzumab or ado-trastuzumab-emtansine) and synergy quantified using the Chou and Talalay method of Combination Index (C.I.). The combination of GDC-0032 with taxanes, endocrine therapies or anti-HER2 therapies were synergistic in the breast cancer cell lines tested based on C.I. values of less than 1.0. The combination of GDC-0032 with docetaxel in vitro resulted in decreased viability as a result of increased cell death. The in vitro results translated in vivo as GDC-0032 enhanced the anti-tumor activity of docetaxel and paclitaxel in MCF7 (E545K) xenografts that resulted in increased tumor regressions when GDC-0032 was dosed daily or intermittently on the same schedule as the taxane. In addition, the combination of GDC-0032 and fulvestrant or tamoxifen resulted in greater efficacy in the MCF7 xenograft model when compared to either agent alone. Enhanced efficacy was also observed when GDC-0032 was combined with trastuzumab or ado-trastuzumab-emtansine in the HER2+ BT474M1 (K111N) xenograft model. Moreover, the triple combinations of GDC-0032/trastuzumab/pertuzumab or GDC-0032/trastuzumab/docetaxel resulted in durable tumor regressions that were sustained in the HER2+ KPL-4 (H1047R) and BT474M1 xenograft models, respectively. All drug combinations with GDC-0032 were well tolerated in vivo based on minimal changes in body weight. Collectively, our preclinical data demonstrate that GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models and provides a strong rationale for further evaluation in patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-02.

Abstract 4396: Conjugation site modulates the stability and biological activity of antibody drug conjugates

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Antibody drug conjugates (ADCs) are attractive targeted chemo-therapeutic molecules as they combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity. The successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency and mode of linker-drug conjugation to the antibody. Recently, we have developed antibodies with cysteine substitutions (THIOMABs) at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., Nature Biotech., 26, 925-932, 2008). These THIOMABs can then be derivatized through the engineered cysteine thiol group to obtain ADCs with uniform stoichiometry (∼2 drugs per antibody). Studies with multiple antibodies against different antigens have shown that THIOMAB drug conjugates (TDCs) are as efficacious as conventional conjugates in xenograft models and are tolerated at higher doses in relevant preclinical models. To further understand improved therapeutic activity and in vivo metabolism of TDCs, we have engineered TDCs with drug attachment at different parts of the antibody (light chain-Fab, heavy chain-Fab and heavy chain-Fc). TDCs produced by THIOMAB technology provided an unique advantage over conventional ADCs to answer several unresolved questions in ADC therapeutics due to their homogeneity and site-specific conjugation to cytotoxic drugs. We will discuss novel findings that were observed with these TDC varaints and the influence of conjugation site in modulating stability and biological activity of antibody drug conjugates. 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 4396.