Elaine Mai

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.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

MetMAb, the One-Armed 5D5 Anti-c-Met Antibody, Inhibits Orthotopic Pancreatic Tumor Growth and Improves Survival

The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.

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.

The Effect of Different Linkers on Target Cell Catabolism and Pharmacokinetics/Pharmacodynamics of Trastuzumab Maytansinoid Conjugates

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody–drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-Nϵ-MCC-DM1, and lysine-Nϵ-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor. Mol Cancer Ther; 11(5); 1133–42. ©2012 AACR.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent

Significance Therapeutic antibodies have revolutionized the treatment of human disease. Despite these advances, antibody bivalency limits their utility against some targets. Here, we describe the development of a one-armed (monovalent) antibody, onartuzumab, targeting the receptor tyrosine kinase MET. While initial screening of bivalent antibodies produced agonists of MET, engineering them into monovalent antibodies produced antagonists instead. We explain the structural basis of the mechanism of action with the crystal structure of onartuzumab antigen-binding fragment in complex with MET and HGF-β. These discoveries have led to an additional antibody-based therapeutic option and shed light on the underpinnings of HGF/MET signaling. Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

HER2-Targeted Therapy Reduces Incidence and Progression of Midlife Mammary Tumors in Female Murine Mammary Tumor Virus huHER2-Transgenic Mice

Purpose: This study examined the effectiveness of early and prolonged mu4D5 (the murine form of trastuzumab/Herceptin) treatment in transgenic mice that overexpress human HER2 (huHER2), under the murine mammary tumor virus promoter, as a model of huHER2-overexpressing breast cancer. Experimental Design: Mice were randomly assigned to one of three treatment groups and received i.p. injections from 17 weeks of age until either 52 weeks of age or morbidity. Fourteen mice received 100 mg/kg mu4D5, 14 mice received 100 mg/kg antiherpes simplex virus glycoprotein D control antibody, and 11 mice received a diluent control. Results: High levels of huHER2 expression were detectable in mammary glands of young virgin founder mice. Mammary adenocarcinomas were frequently found in female founders and progeny at an average age of 28 weeks, with some progressing to metastatic disease. The incidence of mammary tumors was significantly reduced, and tumor growth inhibition was observed in mice receiving mu4D5 compared with control mice. In addition, Harderian gland neoplasms, highly associated with overexpression of huHER2 in this transgenic line, were entirely absent in the mu4D5 treatment group, indicating down-regulation of huHER2 in vivo activity. Conclusions: Early intervention with mu4D5 was of benefit in our transgenic mice at high risk for developing huHER2-overexpressing breast cancer. This study suggests a potential benefit of early treatment with Herceptin in HER2-positive primary breast cancer.

Modeling the efficacy of trastuzumab-DM1, an antibody drug conjugate, in mice

Trastuzumab-DM1 (T-DM1) is a novel antibody–drug conjugate under investigation for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. One challenge in oncologic drug development is determining the optimal dose and treatment schedule. A novel dose regimen-finding strategy was developed for T-DM1 using experimental data and pharmacokinetic/pharmacodynamic modeling. To characterize the disposition of T-DM1, pharmacokinetic studies were conducted in athymic nude and beige nude mice. The pharmacokinetics of T-DM1 were described well by a two-compartment model. Tumor response data were obtained from single-dose, multiple-dose and time–dose-fractionation studies of T-DM1 in animal models of HER2-positive breast cancer, specifically engineered to be insensitive to trastuzumab. A sequential population-based pharmacokinetic/pharmacodynamic modeling approach was developed to describe the anti-tumor activity of T-DM1. A cell-cycle-phase nonspecific tumor cell kill model incorporating transit compartments captured well the features of tumor growth and the activity of T-DM1. Key findings of the model were that tumor cell growth rate played a significant role in the sensitivity of tumors to T-DM1; anti-tumor activity was schedule independent; and tumor response was linked to the ratio of exposure to a concentration required for tumor stasis.

Characterization of the drug-to-antibody ratio distribution for antibody-drug conjugates in plasma/serum.

BACKGROUND Antibody-drug conjugates (ADCs) are a new class of cancer therapeutics that deliver potent cytotoxins specifically to tumors to minimize systemic toxicity. However, undesirable release of covalently linked drugs in circulation can affect safety and efficacy. The objective of this manuscript was to propose and assess the assays that allow for the characterization of the drug deconjugation in plasma/serum. RESULTS ADCs of three main drug conjugation platforms, linked via lysine, site-specific engineered cysteine or reduced interchain disulfide cysteine residues, were analyzed using affinity capture for sample enrichment coupled with LC-MS or hydrophobic interaction chromatography-UV for detection. These novel approaches enabled measurement of the relative abundance of individual ADC species with different drug-to-antibody ratios, while maintaining their structural integrity. CONCLUSION The characterization data generated by affinity capture LC-MS or hydrophobic interaction chromatography-UV provided critical mechanistic insights into understanding the stability and bioactivity of ADCs in vivo, and also helped the development of appropriate quantitative ELISAs.

Anti-CD20/CD3 T cell–dependent bispecific antibody for the treatment of B cell malignancies

Anti-CD20/CD3 T cell–dependent bispecific antibodies may be useful for the treatment of B cell malignancies. Two-headed cancer therapy Immunotherapeutic approaches harness either humoral (antibody-mediated) or cellular (T cell–mediated) immunity to fight cancer. Sun et al. combine these approaches by designing a CD3/CD20 TDB (T cell–dependent bispecific), a dual-targeted antibody that recruits T cells to CD20-expressing cells. Their humanized TDB induces T cells to kill primary patient leukemia and lymphoma cells both in vitro and in a mouse model and can deplete CD20-expressing B cells in a macaque model with similar properties as conventional antibodies. If these data hold true in clinical studies, this CD20/CD3 TDB could add to our expanding arsenal of cancer immunotherapeutics. Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell–recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell–targeting anti-CD20/CD3 T cell–dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using “knobs-into-holes” technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.