Ravi V. J. Chari

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.

Antibody–Drug Conjugates: An Emerging Concept in Cancer Therapy

Traditional cancer chemotherapy is often accompanied by systemic toxicity to the patient. Monoclonal antibodies against antigens on cancer cells offer an alternative tumor-selective treatment approach. However, most monoclonal antibodies are not sufficiently potent to be therapeutically active on their own. Antibody-drug conjugates (ADCs) use antibodies to deliver a potent cytotoxic compound selectively to tumor cells, thus improving the therapeutic index of chemotherapeutic agents. The recent approval of two ADCs, brentuximab vedotin and ado-trastuzumab emtansine, for cancer treatment has spurred tremendous research interest in this field. This Review touches upon the early efforts in the field, and describes how the lessons learned from the first-generation ADCs have led to improvements in every aspect of this technology, i.e., the antibody, the cytotoxic compound, and the linker connecting them, leading to the current successes. The design of ADCs currently in clinical development, and results from mechanistic studies and preclinical and clinical evaluation are discussed. Emerging technologies that seek to further advance this exciting area of research are also discussed.

Targeted delivery of chemotherapeutics: tumor-activated prodrug therapy.

The potential of targeted delivery of chemotherapeutic drugs for the treatment of cancer has not yet been realized owing to the difficulty of delivering therapeutic concentrations to the target site. While in vivo studies in animal tumor models have produced very encouraging results, clinical studies with antibody-drug conjugates have been less successful. This paper will review the current status of the targeted delivery approach and analyze some of the reasons for the lack of success so far. Starting with a historical perspective, this review will end with a description of newer, more potent and specific antibody-drug conjugates, which behave like tumor-activated prodrugs that may yet fulfil the promise of the targeted delivery approach for the treatment of cancer.

Cantuzumab Mertansine, a Maytansinoid Immunoconjugate Directed to the CanAg Antigen: A Phase I, Pharmacokinetic, and Biologic Correlative Study

PURPOSE To determine the maximum tolerated dose and pharmacokinetics of cantuzumab mertansine, an immunoconjugate of the potent maytansine derivative (DM1) and the humanized monoclonal antibody (huC242) directed to CanAg, intravenously (i.v.) once every 3 weeks and to seek evidence of antitumor activity. PATIENTS AND METHODS Patients with CanAg-expressing solid malignancies were treated with escalating doses of cantuzumab mertansine administered i.v. every 3 weeks. The pharmacokinetic parameters of cantuzumab mertansine, the presence of plasma-shed CanAg, and the development of both human antihuman and human anti-DM1 conjugate antibodies also were characterized. RESULTS Thirty-seven patients received 110 courses of cantuzumab mertansine at doses ranging from 22 to 295 mg/m2. Acute, transient, and reversible elevations of hepatic transaminases were the principal toxic effects. Nausea, vomiting, fatigue, and diarrhea were common but rarely severe at the highest dose levels. Dose, peak concentration, and area under the concentration-time curve correlated with the severity of transaminase elevation. The mean (+/- SD) clearance and terminal elimination half-life values for cantuzumab mertansine averaged 39.5 (+/-13.1) mL/h/m2 and 41.1 (+/-16.1) hours, respectively. Strong expression (3+) of CanAg was documented in 68% of patients. Two patients with chemotherapy-refractory colorectal carcinoma had minor regressions, and four patients had persistently stable disease for more than six courses. CONCLUSION The recommended dose for cantuzumab mertansine is 235 mg/m2 i.v. every 3 weeks. The absence of severe hematologic toxic effects, preliminary evidence of cantuzumab mertansine tumor localization, and encouraging biologic activity in chemotherapy-refractory patients warrant further broad clinical development of this immunoconjugate in CanAg-expressing tumors.

Ado-trastuzumab Emtansine (T-DM1): An Antibody–Drug Conjugate (ADC) for HER2-Positive Breast Cancer

Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that combines the antitumor properties of the humanized anti-human epidermal growth factor receptor 2 (HER2) antibody, trastuzumab, with the maytansinoid, DM1, a potent microtubule-disrupting agent, joined by a stable linker. Upon binding to HER2, the conjugate is internalized via receptor-mediated endocytosis, and an active derivative of DM1 is subsequently released by proteolytic degradation of the antibody moiety within the lysosome. Initial clinical evaluation led to a phase III trial in advanced HER2-positive breast cancer patients who had relapsed after prior treatment with trastuzumab and a taxane, which showed that T-DM1 significantly prolonged progression-free and overall survival with less toxicity than lapatinib plus capecitabine. In 2013, T-DM1 received FDA approval for the treatment of patients with HER2-positive metastatic breast cancer who had previously received trastuzumab and a taxane, separately or in combination, the first ADC to receive full approval based on a randomized study.

Antibody-Maytansinoid Conjugates Designed to Bypass Multidrug Resistance

Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.

Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

Synthesis and Evaluation of Hydrophilic Linkers for Antibody–Maytansinoid Conjugates

The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. Antibody-maytansinoid conjugates (AMCs) bearing these sulfonate- or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.

Maytansine and Cellular Metabolites of Antibody-Maytansinoid Conjugates Strongly Suppress Microtubule Dynamics by Binding to Microtubules

Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar KD values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (KD, 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule. Mol Cancer Ther; 9(10); 2689–99. ©2010 AACR.

Maytansinoid-Antibody Conjugates Induce Mitotic Arrest by Suppressing Microtubule Dynamic Instability

Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability. Mol Cancer Ther; 9(10); 2700–13. ©2010 AACR.