Antibody-maytansinoid Conjugates Research Articles

Sensitive LC-MS/MS quantification of unconjugated maytansinoid DM4 and its metabolite S-methyl-DM4 in human plasma.

Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.

Hepatotoxicity with antibody maytansinoid conjugates: A review of preclinical and clinical findings.

Maytansinoids, the potent cytotoxic derivatives of the alkaloid maytansine are used as payloads in antibody maytansinoid conjugates. This article reviews clinical and preclinical hepatotoxicity observed with antibody maytansinoid conjugates used to treat cancer. Specific aspects of drug distribution, metabolism and excretion that may impact hepatotoxicity are reviewed vis-à-vis the kind of maytansinoid in the conjugate, cleavable or non-cleavable linkers, linker-payload combinations, drug to antibody ratio, metabolite formation, hepatic enzyme induction in relation to drug-drug interactions and species, age and gender differences. The article also sheds light on factors that may protect the liver from toxic insults.

Effects of Drug–Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody–Maytansinoid Conjugates

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

αvβ3 Integrin-targeted reduction-sensitive micellar mertansine prodrug: Superb drug loading, enhanced stability, and effective inhibition of melanoma growth in vivo

Antibody-maytansinoid conjugates (AMCs) have emerged as one of the most promising active targeting cancer therapeutics. Their clinical use is, however, challenged by their low drug content, poor stability, high cost and potential immune response. Here, we designed and developed robust, cRGD-functionalized, reduction-sensitive polymeric micellar mertansine (DM1) prodrug (cRGD-MMP) that showed targeted treatment of B16F10 melanoma-bearing C57BL/6 mice. cRGD-MMP was obtained with a superb drug content of ~40wt.% and a small size of ~45nm from poly(ethylene glycol)-b-(poly(trimethylene carbonate)-graft-SSDM1) (PEG-P(TMC-g-SSDM1)) and cRGD-functionalized PEG-P(TMC-g-SSDM1) copolymers. cRGD-MMP exhibited excellent stability in 10% fetal bovine serum and cell culture medium while fast swelling and markedly accelerated drug release under a reductive environment. Confocal microscopy, flow cytometry and MTT assays indicated receptor-mediated uptake and high antitumor effect of cRGD-MMP in αvβ3 integrin over-expressing B16F10 melanoma cells. Notably, cRGD-MMP displayed a long elimination half-life of 5.25h and 4-fold better maximum-tolerated dose than free DM1. The in vivo studies demonstrated that cRGD-MMP effectively inhibited B16F10 melanoma growth and greatly improved mice survival rate as compared to free DM1 and non-targeted MMP control. cRGD-MMP with superior stability, drug loading, and αvβ3 targetability offers an attractive alternative to AMCs for malignant tumor therapy.

Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody–Maytansinoid Conjugates

Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.

New Insights in Tissue Distribution, Metabolism, and Excretion of [3H]-Labeled Antibody Maytansinoid Conjugates in Female Tumor-Bearing Nude Rats.

For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs to be well understood to mitigate toxicity risks and properly design the first in-patient studies. Therefore, a distribution, metabolism, and excretion (DME) study with a radiolabeled rat cross-reactive ADC ([(3)H]DM1-LNL897) targeting the P-cadherin receptor was conducted in female tumor-bearing nude rats. Although multiple components [total radioactivity, conjugated ADC, total ADC, emtansine (DM1) payload, and catabolites] needed to be monitored with different technologies (liquid scintillation counting, liquid chromatography/mass spectrometry, enzyme-linked immunosorbent assay, and size exclusion chromatography), the pharmacokinetic data were nearly superimposable with the various techniques. [(3)H]DM1-LNL897 was cleared with half-lives of 51-62 hours and LNL897-related radioactivity showed a minor extent of tissue distribution. The highest tissue concentrations of [(3)H]DM1-LNL897-related radioactivity were measured in tumor. Complimentary liquid extraction surface analysis coupled to micro-liquid chromatography-tandem mass spectrometry data proved that the lysine (LYS)-4(maleimidylmethyl) cyclohexane-1-carboxylate-DM1 (LYS-MCC-DM1) catabolite was the only detectable component distributed evenly in the tumor and liver tissue. The mass balance was complete with up to 13.8% ± 0.482% of the administered radioactivity remaining in carcass 168 hours postdose. LNL897-derived radioactivity was mainly excreted via feces (84.5% ± 3.12%) and through urine only to a minor extent (4.15% ± 0.462%). In serum, the major part of radioactivity could be attributed to ADC, while small molecule disposition products were the predominant species in excreta. We show that there is a difference in metabolite profiles depending on which derivatization methods for DM1 were applied. Besides previously published results on LYS-MCC-DM1 and MCC-DM1, maysine and a cysteine conjugate of DM1 could be identified in serum and excreta.

Biological evaluation of antibody-maytansinoid conjugates as a strategy of RON targeted drug delivery for treatment of non-small cell lung cancer

BackgroundAberrant expression of the RON receptor tyrosine kinase, a member of the MET proto-oncogene family, in breast cancer and non-small cell lung cancer (NSCLC) has therapeutic implication. Here we evaluated the efficacy of a novel anti-RON antibody-drug maytansinoid conjugate Zt/g4-DM1 for treatment of breast and NSCLC xenograft tumors in mouse models and explored a treatment strategy by combination of Zt/g4-DM1 with chemotherapeutics to achieve the maximal therapeutic activity.MethodsMouse monoclonal antibody Zt/g4 (IgG1a/κ) specific to human RON was conjugated to DM1 via thioether linkage to form Zt/g4-DM1 with a drug-antibody ratio of 4:1. Several breast cancer and NSCLC cell lines, expressing different levels of RON, were used as the model. Immunofluorescence was used to determine Zt/g4-induced RON internalization. Flow cytometric analysis and cell viability assay were used to determine the effect of Zt-g4-DM1 on cell cycle and death. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of Zt/g4-DM1 alone or in combination with chemotherapeutics.ResultsIn vitro, Zt/g4 treatment of breast cancer and NSCLC cells rapidly induced cell surface RON internalization, which results in intracellular delivery of DM1 sufficient to arrest cell cycle at G2/M phase, reduce cell viability, and cause massive cell death. In mouse tumor xenograft models, Zt/g4-DM1 at 20 mg/kg in a Q12 × 2 regimen effectively blocked breast cancer and NSCLC cell- mediated tumor growth. More than 95 % inhibition of tumor growth among three tumor xenograft models tested was achieved according to the measured tumor volume. The minimal dose to balance the tumor growth and inhibition (tumoristatic concentration) was established at 2.02 mg/kg for H2228, 1.94 mg/kg for H358 cell, and 6.25 mg/kg for T-47D cell-mediated xenograft tumors.ConclusionZt/g4 is highly effective in RON-directed drug delivery for targeted inhibition of NSCLC cell-derived tumor growth in mouse xenograft models. This work provides the basis for clinical development of humanized Zt/g4-DM1 for potential cancer therapy in the future.

Constitutive overexpression of asm18 increases the production and diversity of maytansinoids in Actinosynnema pretiosum.

Ansamitocins isolated from Actinosynnema pretiosum, potent antitumor compounds, belong to the family of maytansinoids, and the antibody-maytansinoid conjugates are currently under different phases of clinical trials. The clinical applications of ansamitocins have stimulated extensive studies to improve their production yields. In this study, we investigated the function of a pathway-specific S treptomyces antibiotic regulatory protein (SARP) family regulator, Asm18, and observed that ectopic overexpression of the asm18 gene increased the production of N-demethyl-4,5-desepoxy-maytansinol (2) to 50 mg/L in the HGF052 + pJTU824-asm18 strain, an increase by 4.7-fold compared to that of the control strain HGF052 + pJTU824. Real-time PCR analysis showed that the overexpression of the asm18 gene selectively increased the transcription levels of the genes involved in the biosynthesis of the starter unit (asm43), polyketide assembly (asmA), post-PKS modification (asm21), as well as the transcription levels of the regulatory gene (asm8), which is a specific LAL-type activator in ansamitocin biosynthesis. With the increase of fermentation titre, seven ansamitocin analogs (1-7) including three new ones (1, 5, and 6) and maytansinol (7) were isolated from the HGF052 + pJTU824-asm18 strain. Our results not only pave the way for further improving the production of ansamitocin analogs but also indicate that the post-PKS modifications of ansamitocin biosynthesis are flexible, which brings a potential of producing maytansinol, the most fascinating intermediate for the synthesis of antibody-maytansinoid conjugates, by optimizing the HGF052 and/or HGF052 + pJTU824-asm18 strains.

Abstract 4531: Effects of drug load on therapeutic index for antibody-maytansinoid conjugates

Abstract Antibody-drug conjugates (ADCs) are being actively pursued as a new modality to treat cancer following the regulatory approval of Adcetris@ and Kadcyla@. ADCs consist of a cytotoxic agent, or drug, conjugated to a targeting antibody (Ab) through a linker. The two approved ADCs (and most ADCs now in the clinic) are heterogeneous conjugates with an average molar drug to Ab ratio (DAR) of 3-4 (potentially ranging from 0-8 for individual molecules). To understand the effects of different DAR ranges on the preclinical properties of ADCs using a maytansinoid cytotoxic agent, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor alpha) or an uncleavable linker (J2898A-SMCC-DM1 targeting epidermal growth factor receptor) with varying DAR, and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. Both M9346A-sulfo-SPDB-DM4 and J2898A-SMCC-DM1 conjugates with low (average ∼2, range 0-4) to very high DAR (average 10, range 7-14) were prepared in good yield, high monomer content and low free drug levels. At constant Ab concentration, the in vitro potency consistently increased with increasing DAR. We then characterized the in vivo disposition of these ADCs. First, pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but at an average DAR around 9-10 rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR conjugates rapidly distribute to the liver, with the maximum%ID/g for this organ at 24-28% compared with 7-10% for lower DAR conjugates (all at 2-6 h post-injection). We further studied the efficacy of these ADCs in mouse xenograft models at both constant antibody and constant maytansinoid doses. At constant Ab dose, conjugates with average DAR ∼6 are more efficacious than conjugates with DAR ∼3. However, DAR 9-10 conjugates are either less or similarly active compared to DAR 6 conjugates, consistent with the observed rapid clearance. At constant maytansinoid dose, conjugates with 2 - 6 DAR show similar efficacy, while those with 9-10 DAR were again less active. Tolerability studies monitoring body weight loss show that conjugates with different DAR, even as high as 9-10, have comparable tolerability based on total administered maytansinoid dose. In summary, our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR in the range between 2 - 6 have better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs, but suggest the exploration of lower or higher DAR depending on the biology of the target antigen. Citation Format: Xiuxia Sun, Jose F. Ponte, Nicholas C. Yoder, Jennifer Coccia, Leanne Lanieri, Rassol Laleau, Qifeng Qiu, Rui Wu, Erica Hong, Megan Bogalhas, Lintao Wang, Erin K. Maloney, Olga Ab, Hans K. Erickson, Thomas A. Keating, Ravi Chari, John M. Lambert. Effects of drug load on therapeutic index for antibody-maytansinoid conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2015-4531

Metabolites of antibody-maytansinoid conjugates: characteristics and in vitro potencies.

Several antibody-maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical studies in mice, the cytotoxic metabolites initially formed in vivo are further processed in the mouse liver to give several oxidized metabolic species. In this work, the primary AMC metabolites were synthesized and incubated with human liver microsomes (HLMs) to determine if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how these impurities would be metabolized by HLM.

Statistics of the distribution of the abundance of molecules with various drug loads in maytansinoid antibody-drug conjugates.

The maytansinoid antibody-drug conjugates (ADCs) in clinical development for cancer therapy each contain a derivative of the microtubule-targeting agent, maytansine, covalently attached to the antibody via an engineered linker. A sample of any of these conjugates contains molecules with different numbers of maytansinoid molecules, or "drug" loads, the relative abundance of which can be determined by mass spectrometry. We examined the accuracy of the Poisson distribution and the binomial distribution in predicting the relative abundance of species with different drug loads for three antibody-maytansinoid conjugates with different antibodies and linker-maytansinoid pairings. We used variance, calculated from the experimental mass distribution data, as the parameter to determine the optimal value n of the binomial distribution number of trials. The accuracy of the Poisson distribution in predicting distribution of the species abundance in these conjugates varied among the conjugates. In contrast, the accuracy of the binomial distribution was similar for all three conjugates and comparable to the best accuracy of the Poisson distribution, as supported by a paired t-test.

Abstract 1618: New peptide-linked anilino-maytansinoid antibody-drug conjugates (ADCs) for the treatment of cancer

Abstract A number of ADCs consisting of a maytansinoid cytotoxic agent linked to an antibody via a cleavable disulfide linker or a non-cleavable thioether linker are in the clinic. In preclinical models, antibody-maytansinoid conjugates (AMCs) utilizing a cleavable disulfide linker have been shown to be able to kill both the targeted antigen-positive (Ag+) cancer cells and also adjacent Ag- “bystander” cancer cells because of intra-cellular generation of maytansinoid catabolites that can travel out of the Ag+ cells, into adjacent Ag- cells, and kill these cells when they divide. Such bystander killing of Ag- cancer cells can be advantageous for some tumor types, such as those with heterogeneous target expression on the cancer cells. We thus developed a new dipeptide linker designed to promote bystander killing more efficiently than current disulfide linkers. It is used to attach an aniline-bearing maytansinoid to the targeting antibody. To determine the effect of amino acid stereochemistry on the therapeutic index, dipeptide-anilino AMCs (dpa-AMCs) were prepared with the four possible L- and D-alanine configurations using an anti-EGFR antibody. These were compared to a dpa-AMC containing the L-Val-L-Cit dipeptide, which is a commonly utilized dipeptide for ADC linkage. Dipeptide stereochemistry had a significant effect on the in vitro potency and bystander killing activity of the AMCs. Based on the results, the three most potent designs - those bearing the L-Ala-L-Ala, D-Ala-L-Ala, and L-Val-L-Cit peptides - were further investigated. In tolerability experiments in mice, with AMC dosing based on linked maytansinoid, the MTDs of the D-Ala-L-Ala dpa-AMCs and a disulfide linked control AMC were both approximately 1.4 mg/kg, while the L-Val-L-Cit and L-Ala-L-Ala dpa-AMCs were not as well tolerated. The AMC with the disulfide linker and the three most potent dpa-AMCs had similar in vivo efficacy in tumor xenograft models in mice where the antigen was expressed homogeneously. The D-Ala-L-Ala dpa-AMC however was selected as the lead based on its favorable MTD and because its anti-CanAg conjugate had 2-3 fold greater efficacy in vivo than a disulfide linked anti-CanAg AMC in a HT-29 tumor model, where the antigen is expressed heterogeneously. Cellular processing studies revealed that the anti-CanAg D-Ala-L-Ala dpa-AMC produced a unique, highly potent anilino-maytansinoid catabolite that can diffuse into, and kill, bystander cells. In conclusion, we have developed a promising new type of maytansinoid ADC, one that provides a high degree of bystander killing, improved activity in a heterogeneous tumor model in vivo, and a different mechanism of cytotoxic catabolite release than current AMC designs. Citation Format: Wayne C. Widdison, Joe Ponte, Jennifer Coccia, Yulius Setiady, Ling Dong, Anja Skaletskaya, Nathan Fishkin, Yelena Kovtun, Rui Wu, Rajeeva Singh, Luke Harris, Greg Jones, Leanne Lanieri, Erin Maloney, Charlene Audette, Andre Dandeneau, Ravi Chari, Juliet Costoplus, Karen Veale, Sharon Wilhelm. New peptide-linked anilino-maytansinoid antibody-drug conjugates (ADCs) for the treatment of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1618. doi:10.1158/1538-7445.AM2014-1618

ADME of antibody-maytansinoid conjugates.

The concept of treating cancer with antibody-drug conjugates (ADCs) has gained momentum with the favorable activity and safety of trastuzumab emtansine (T-DM1), SAR3419, and lorvotuzumab mertansine (IMGN901). All three ADCs utilize maytansinoid cell-killing agents which target tubulin and suppress microtubule dynamics. Each ADC utilizes a different optimized chemical linker to attach the maytansinoid to the antibody. Characterizing the absorption, distribution, metabolism, and excretion (ADME) of these ADCs in preclinical animal models is important to understanding their efficacy and safety profiles. The ADME properties of these ADCs in rodents were inferred from studies with radio-labeled ADCs prepared with nonbinding antibodies since T-DM1, SAR3419, IMGN901 all lack cross-reactivity with rodent antigens. For studies exploring tumor localization and activation in tumor-bearing mice, tritium-labeled T-DM1, SAR3419, and IMGN901 were utilized. The chemical nature of the linker was found to have a significant impact on the ADME properties of these ADCs-particularly on the plasma pharmacokinetics and observed catabolites in tumor and liver tissues. Despite these differences, T-DM1, SAR3419, and IMGN901 were all found to facilitate efficient deliveries of active maytansinoid catabolites to the tumor tissue in mouse xenograft models. In addition, all three ADCs were effectively detoxified during hepatobiliary elimination in rodents.

In Vitro Characterization of the Drug-Drug Interaction Potential of Catabolites of Antibody-Maytansinoid Conjugates

The in vitro characterization of the inhibition potential of four representative maytansinoid species observed upon hepatic and/or tumor in vivo processing of antibody-maytansine conjugates (AMCs) with cleavable and noncleavable linkers is reported. We investigated the free maytansinoid species N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1), (S)-methyl-DM1, and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) as representative cleavable linker catabolites and Lysine-N(ε)-N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate-DM1 (Lys-MCC-DM1) as the representative noncleavable linker catabolite. Studies with recombinant human cytochromes P450 (P450s) indicate CYP2D6, CYP3A4, and CYP3A5 are the primary isoforms responsible for the oxidative metabolism of DM1, (S)-methyl-DM1, and DM4. Lys-MCC-DM1 was not metabolized by any of the P450 isoforms studied. DM1 was shown to be a reversible inhibitor of CYP2C8 (K(i) = 11 ± 3 μM) and CYP2D6 (K(i) = 14 ± 2 μM). Lys-MCC-DM1 and (S)-methyl-DM1 showed no reversible or time-dependent inactivation of any of the P450s studied. DM1 and DM4 inactivated CYP3A from human liver microsomes with K(i)/k(inact) values of 4.8 ± 0.9 μM/0.035 ± 0.002 min(-1) and 3.3 ± 0.2 μM/0.114 ± 0.002 min(-1), respectively. DM1 and DM4 inactivated recombinant CYP3A4 with K(i)/k(inact) values of 3.4 ± 1.0 μM/0.058 ± 0.005 min(-1) and 1.4 ± 0.3 μM/0.117 ± 0.006 min(-1), respectively. Because of instability in plasma, further characterization of the DM1 and DM4 intramolecular and intermolecular disulfide conjugates observed in vivo is required before an accurate drug-drug interaction (DDI) prediction can be made. AMCs with noncleavable thioether-linked DM1 as the cytotoxic agent are predicted to have no potential for a DDI with any of the major human P450s studied.

Abstract 4628: Anti-tumor activity and pharmacokinetics of the anti-FOLR1-maytansinoid conjugate IMGN853 is maintained over a wide range of maytansinoid-to-antibody ratios

Abstract IMGN853 (M9346A-sulfo-SPDB-DM4) is an antibody-maytansinoid conjugate consisting of the cytotoxic maytansinoid, DM4, covalently linked to the humanized monoclonal antibody M9346A, which selectively binds to folate receptor 1 (FOLR1). Trastuzumab emtansine and other antibody-maytansinoid conjugates that have demonstrated favorable activity and safety in the clinic have maytansinoid-to-antibody ratios (MAR) around 3.5. We assessed the impact of varying MAR on conjugate activity and pharmacokinetics, to determine whether a similar maytansinoid load is optimal for IMGN853. Conjugates of M9346A were prepared with maytansinoid (DM4) loads per antibody ranging from 1.4 to 5.7. All conjugates had high (>90%) monomer content, low (<0.1 %) levels of free maytansinoid, and normal distribution profiles by LC/MS. Conjugates spanning the range of MARs demonstrated comparable binding to FOLR1-expressing cells as assessed by flow cytometry and had comparable in vitro cytotoxicity (based on DM4 concentration) against antigen-positive cells. The plasma pharmacokinetics of M9346A-sulfo-SPDB-[3H]DM4 conjugates with low (2.0), intermediate (3.6) and high (5.1) MAR levels were evaluated in CD-1 mice. There was no impact of varying maytansinoid load, and all conjugates had comparable pharmacokinetic parameters, with plasma circulation half-life of about 5.5 days compared with about 12 days for M9346A. The anti-tumor activity of conjugates with MARs from 1.4 to 5.7 was assessed in two FOLR1-positive xenograft models (KB cervical carcinoma and IGROV-1 ovarian cancer) in SCID mice. Mice bearing established subcutaneous KB or IGROV-1 xenografts were treated at doses known to be active without causing complete tumor regressions (25 or 50 µg/kg based on DM4 concentration, respectively) to allow detection of slight changes in activity based on MAR. Interestingly, conjugates with low MAR were found to have a therapeutic advantage over the higher MAR conjugates in the high FOLR1-expressing KB model, with strong activity of conjugates from 1.4 to 4.7 MAR and a slight reduction in activity observed with the highest MAR conjugates. Conjugate activity across the range of MAR was similar in IGROV-1 tumor-bearing mice. High-quality conjugates over a broad range of maytansinoid loads can be made with the sulfo-SPDB-DM4 linker format, with indistinguishable in vitro activities and without affecting pharmacokinetic properties. The activity of conjugates across a broad MAR range was comparable and not highly dependent on maytansinoid load in high FOLR1-expressing xenograft models. These results, along with the favorable safety and efficacy associated with antibody-maytansinoid conjugates currently under clinical evaluation, led to the selection of a MAR of 3.5 for IMGN853. 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 4628. doi:1538-7445.AM2012-4628

Abstract 5677: A comparative study of the interaction of the maytansinoid, DM1-Me, vinblastine, and taxol with human cells

Abstract The current interest in antimitotic compounds that interact with microtubules is caused, in part, by their clinical importance in oncology. Several microtubule-binding compounds, including Vinca alkaloids, taxanes, maytansinoids, and auristatins, are in clinical use, some additionally being evaluated as antibody conjugates. To better understand the factors that determine the potency of antibody-maytansinoid conjugates in killing cells, we examined the interaction of the maytansinoid, DM1-Me, with MCF7 cells, in comparison with vinblastine and taxol. DM1-Me was more potent in arresting mitosis than vinblastine or taxol (5- and 13-fold, respectively). We compared the levels of accumulation of these three compounds by MCF7 cells following a 24 hour exposure at the mitotic IC50 concentration and found that, at this concentration, cells accumulated comparable amounts of vinblastine and DM1-Me, but required more than a 10-fold greater concentration of taxol to achieve a similar effect. Additionally, cells exposed to each compound for 2 hours prior to being washed into drug free medium, retained most of the accumulated DM1-Me or taxol for at least 24 h, while most vinblastine was released into the medium within the first 2 h. DM1-Me and taxol had similar affinities for MCF7 cells, while that of vinblastine was 4-6-fold lower. These properties of DM1-Me, vinblastine and taxol suggest that maytansinoids are a better choice for antibody conjugates than vinblastine or taxol, a conclusion which is consistent with the experimental data and clinical experience with antibody conjugates made with compounds of these three classes. 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 5677. doi:1538-7445.AM2012-5677

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.

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.

Design of Antibody−Maytansinoid Conjugates Allows for Efficient Detoxification via Liver Metabolism

Antibody-maytansinoid conjugates (AMCs) are targeted chemotherapeutic agents consisting of a potent microtubule-depolymerizing maytansinoid (DM1 or DM4) attached to lysine residues of a monoclonal antibody (mAb) using an uncleavable thioether linker or a stable disulfide linker. Most of the administered dose of an antibody-based therapeutic is slowly catabolized by the liver and other tissues of the reticuloendothelial system. Maytansinoids released from an AMC during this catabolic process could potentially be a source of toxicity. To investigate this, we isolated and identified liver metabolites in mice for three different [(3)H]AMCs with structures similar to those currently undergoing evaluation in the clinic. We then synthesized each metabolite to confirm the identification and assessed their cytotoxic potencies when added extracellularly. We found that the uncleavable mAb-SMCC-[(3)H]DM1 conjugate was degraded to a single major maytansinoid metabolite, lysine-SMCC-[(3)H]DM1, that was nearly 50-fold less cytotoxic than maytansine. The two disulfide-linked conjugates, mAb-SPP-[(3)H]DM1 and mAb-SPDB-[(3)H]DM4, were also found to be catabolized to the analogous lysine-linked maytansinoid metabolites. However, subsequent reduction, S-methylation, and NADPH-dependent oxidation steps in the liver yielded the corresponding S-methyl sulfoxide and S-methyl sulfone derivatives. The cytotoxic potencies of the oxidized maytansinoids toward several human carcinoma cell lines were found to be 5- to 50-fold less potent than maytansine. Our results suggest that liver plays an important role in the detoxification of both cleavable and uncleavable AMCs.

A novel pathway for maytansinoid release from thioether linked antibody–drug conjugates (ADCs) under oxidative conditions

A novel pathway for ex vivo maytansinoid release from thioether linked antibody maytansinoid conjugates (AMCs) upon incubation in human plasma has been identified. A thioether succinimide-linked AMC can undergo chemical oxidation followed by sulfoxide elimination under mild aqueous conditions (pH 5.5-7.5, 37 °C). Oxidized thioether-linked AMCs exhibit high, target-specific cytotoxicity toward cancer cells.