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Developing Second Generation Antibody−Drug Conjugates: The Quest for New Technologies Carlos Garcia-Echeverria* Lead Generation to Compound Realization, Sanofi-Aventis, 94400 Vitry sur Seine, France ABSTRACT: The field of antibody−drug conjugates (ADCs) has gained significant momentum after the recent regulatory approval of two ADCs, and significant research efforts are directed to identify more effective payloads and simplify current manufacturing challenges.
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Several aspects (the target, the antigen, the antibody, the linker, and the cytotoxic payload) must be thoroughly investigated and balanced during the design and synthesis of an ADC. In this context, the importance of linker−drug stability and conjugate homogeneity has recently been further realized because subtle improvements could have profound effects on increase efficacy and tolerability, particularly for the second generation of clinically validated ADC targets. To this end, two linkers based on 6-maleimidohexanoic acid (MC) and maleimidopolyethylenene glycol adipic acid (MPA) are proposed and investigated by Pillow et al.3 The corresponding linker−maytansinoid derivatives are readily available from the natural product analog maytansinol. This molecule can be obtained by control reduction of ansamitocins obtained by fermentation of the microorganism Actinosynnema pretiosum. Modifications of maytansinol are tolerated and synthetically amenable, providing a practical and scalable approach to prepare the proposed MC- and MPA-maytansinoid payloads.5 These molecules were conjugated at the LC-V205C site of trastuzumab to produce homogeneous (DAR = 1.8) ADC molecules: ThioTmab-MC-May and ThioTmab-MPA-May. A set of experiments against a panel of human tumor cell lines that express various levels of HER2 at the cellular membrane demonstrate that this change in the linker−drugs has no impact on the antigen specificity or the cytotoxic effect. The reduce cell killing activity observed for the ThioTmab ADCs containing the stable linker−maytansinoid payloads was interpreted by the fact that these molecules have half the cytotoxic load of Tmab-MCC-DM1. Although this explanation is certainly plausible, no information is provided on the intracellular fate and the role of MC and MPA-based linkers on the amount, nature, and cytotoxic activity of the catabolites formed upon internalization and processing of the ThioTmab ADCs. To gauge the relative stabilities of the linkers, in vitro plasma studies in mouse and human serum were performed. Overall, less deconjugation and improved stability were observed with the MC and MPA-based ThioTmab ADCs. These results confirm the original working hypothesis and validate the synthetic design strategy, but no in vivo pharmacokinetic
irst generation antibody−drug conjugate (ADC) technologies have matured as evidence by the recent regulatory approval of two ADCs, brentuximab vedotin (SGN-35, Seattle Genetics/Takeda)1 and trastuzumab emtansine (T-DM1/ Tmab-MCC-DM1, Roche/Genentech in partnership with ImmunoGen)2 and the number of molecules that have reached the clinic. Most of the ADCs currently undergoing clinical trials utilize tubulin polymerization inhibitors (e.g., maytansine or auristatin derivatives) or DNA-acting payloads (calicheamicin or pyrrolobenziodiazepines derivatives) conjugated via stable (e.g., thioether) or cleavable (e.g., hydrazine or disulfide) linkers to the side chains of natural lysine or cysteine residues in the targeting antibody. As a result of this nonselective conjugation technology, the drug−antibody ratio (also known as DAR) is variable and the synthetic ADC product is heterogeneous, which may potentially impact clinical efficacy and safety. In this issue, Pillow et al.3 report a new approach to prepare homogeneous trastuzumab based-ADCs with increased preclinical therapeutic activity compared to trastuzumab emtansine and other recently reported derivatives thereof. The proposed technology relies upon the use of a trastuzumab THIOMAB,4 which allows uniform and site-specific conjugation by the placement of four engineered cysteines and a new maytansine linker−payload that lacks a reversible functional group between the linker and the cytotoxic drug. According to the data reported in the literature,2,3 TmabMCC-DM1 shows an appreciable level of deconjugation and instability in plasma. This developmental flag, which could contribute to its narrow clinical therapeutic index (doselimiting toxicity is 4.8 mg/kg, while the maximum tolerated dose is 3.6 mg/kg/intravenous infusion once every 21 days), may be attributed to the reversibility of the thiol-containing maytansine derivative and the maleimide-based linker. It is worthy to mention that the first step in the synthesis of this ADC involves the reaction of succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SMCC) with the surface-accessible amino side chain of lysine residues on trastuzumab to form an amide bond. Subsequently, the maleimido moiety undergoes a Michael type addition with the thiol-containing maytansinoid, DM1, to form a thioether bond with the cytotoxic agent. On the basis of in vitro and in vivo results, a DAR value of 3.5 was found to be optimal and the heterogeneous Tmab-MCC-DM1 was selected as clinical candidate.2 © XXXX American Chemical Society
Received: August 25, 2014
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dx.doi.org/10.1021/jm501298k | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
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lymphoma following previous treatment failure. Drugs 2013, 73, 371− 381. (2) Lambert, J. M.; Chari, R. V. J. Ado-trastuzumab emtansine (TDM1): an antibody-drug conjugate (ADC) for HER2-positive breast cancer. J. Med. Chem. 2014, 57, 6949−6964. (3) Pillow, T. H.; Tien, J.; Parsons-Reponte, K. L.; Bhakta, S.; Li, H.; Staben, L. R.; Li, G.; Chuh, J.; Fourie-O’Donohue, A.; Darwish, M.; Yip, V.; Liu, L.; Leipold, D. D.; Su, D.; Wu, E.; Spencer, S. D.; Shen, B.-Q.; Xu, K.; Kozak, K. R.; Raab, H.; Vandlen, R.; Lewis Phillips, G. D.; Scheller, R. H.; Polakis, P.; Sliwkowski, M. X.; Flygare, J. A.; Junutula, J. R. Site-specific trastuzumab maytansinoid antibody−drug conjugates with improved therapeutic activity through linker and antibody engineering. J. Med. Chem. 2014, DOI: 10.1021/jm500552c. (4) Behrens, C. R.; Liu, B. Methods for site-specific drug conjugates to antibodies. mAbs 2014, 6, 46−53. (5) Kupchan, S. M.; Sneden, A. T.; Branfman, A. R.; Howie, G. A.; Rebhun, L. I.; McIvor, W. E.; Wang, R. W.; Schaitman, T. C. Structural requirements for antileukemic activity among the naturally occurring and semisynthetic maytansinoids. J. Med. Chem. 1978, 21, 31−37.
studies in mouse or other relevant preclinical species are reported, and therefore, it is difficult to determine if the in vitro data predict and match with in vivo half-life and exposure. The in vivo efficacy of the new trastuzumab ADCs was evaluated in the trastuzumab-resistant MMTV-HER2+ Fo5 mammary tumor model. This is a syngeneic tumor-transplant model where murine tumor cells express high levels (3+ by immunohistochemistry) of human HER2. It is a suitable cancer model to assess the value of armed trastuzumabs because it is not sensitive to antibody alone. Similar to the results obtained in cellular settings and despite improvement of the stability of the linker−drugs, ThioTmab-MC-May and ThioTmab-MPAMay (DAR = 1.8) are statistically less efficacious than TmabMCC-DM1 (DAR = 3.5). To address this, a ThioTmab containing four engineered cysteines in both the heavy (HCA114C) and light chain (LC-V205C) was prepared and conjugated with the new stable linker−maytansinoids to give a DAR of 3.9. Under the same experimental conditions, the 4DAR ThioTmab ADCs showed improved efficacy, particularly in the time to tumor progression parameter, vis-à-vis TmabMCC-DM1. As noted previously, the lack of pharmacokinetic and metabolism data from the in vivo efficacy experiments precludes a deeper understanding of the pharmacological properties of these new ThioTmab ADCs and the nature and contribution of catabolites to the observed antitumor activity. Moreover, a more thorough comparison to other relevant HER2+ models (e.g., KPL-4 or BT474-EEI breast tumors) is missing. One important aspect that is not covered by Pillow et al. is tolerability. Although it is reasonable to assume that increasing the preclinical efficacy could improve the clinical activity of second generation trastuzumab maytansinoid ADCs, it is unclear to what extent the new linkers and homogeneous products provide a better therapeutic index. On the basis of antigen recognition, tolerability studies in rats and cynomolgus monkeys would have allowed the assessment of antigenindependent and -dependent toxicity, respectively. However, while grade 4 thrombocytopenia defined the dose-limiting toxicity of Tmab-MCC-DM1 in cancer patients, the severity of this adverse event in the non-human primate toxicology studies was minimal. Liver and bone marrow/hematologic systems were the primary organs for toxicity in this species.2 This lack of predictability from preclinical toxicological studies is not necessarily an impediment to advance second generation trastuzumab maytansinoid ADCs but could represent a major challenge to demonstrate the superior safety of new formats in preclinical settings. In summary, the identification of more potent and safer cytotoxic platforms and improvements to simplify downstream purification processes in the manufacturing of homogeneous products will positively impact future developments in the ADC field. We can only hope that next-generation ADCs will further confirm the effectiveness of this modality to treat a wide array of solid and liquid tumors.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: carlos.garcia-echeverria@sanofi.com. Phone: +33 1 58 93 30 00.
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REFERENCES
(1) Garnock-Jones, K. P. Brentuximab vedotin: a review of its use in patients with hodgkin lymphoma and systemic anaplastic large cell B
dx.doi.org/10.1021/jm501298k | J. Med. Chem. XXXX, XXX, XXX−XXX
