Erin Maloney

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.

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.

A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity.

The promise of tumor-selective delivery of cytotoxic agents in the form of antibody–drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870–8. ©2016 AACR.

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.

Abstract B126: Potent antigen‐specific anti‐tumor activity observed with antibody‐drug conjugates (ADCs) made using a new class of DNA‐crosslinking agents

The clinical support for ADC therapeutics has expanded as more highly‐engineered ADCs advance in human clinical testing. Most of the ADCs now in clinical testing contain a tubulin‐acting compound (a maytansine or dolastatin derivative) as the cytotoxic agent. While tubulin‐acting agents can be effective against many different types of cancers, some cancers are more responsive to DNA‐acting agents. To expand the therapeutic potential for ADCs, we sought to develop a new class of cytotoxic agents with a novel, DNA‐acting mechanism of action for use with tumor‐targeting antibodies. Herein, we report the development of our IGN family of cytotoxic agents. These IGN agents comprise indolino‐benzodiazepine dimers that are highly potent by virtue of their ability to alkylate and crosslink DNA. This novel class of compounds demonstrated sequence‐selective DNA adduct formation in vitro and cytotoxicity in the picomolar range towards cultured human cancer cells. The intense potency of these compounds, along with their desired aqueous solubility and stability, make them ideally suited for use in ADCs. A lead compound from this class was conjugated to an EpCAM‐binding antibody, B38.1, and to a CD33‐binding antibody, huMy9‐6, through amide bonds. The B38.1‐IGN conjugate was highly potent against three different EpCAM‐expressing cell lines ‐ COLO 205, LoVo and OVCAR‐3 ‐ with IC50 values of 1 pM, 5 pM and 18 pM, respectively. The addition of excess unconjugated B38.1 antibody abolished this cytotoxic effect, demonstrating that the activity of the conjugate is antigen specific. The B38.1‐IGN conjugate was considerably less potent towards the antigen‐negative Namalwa cell line, with an IC50 value of >1 nM, further demonstrating antigen specificity. Similar potent cytotoxicity was seen with a huMy9‐6‐IGN conjugate targeting the CD33‐positive human promyelocytic leukemia cell line, NB4 (IC50 ∼4pM), in spite of the low antigen expression level (∼10,000 molecules/cell) in this cell line. Of particular interest, the B38.1‐IGN conjugate also was potent towards multidrug resistant cancer cells. B38.1‐IGN had a IC50 value of 14 pM for COLO 205MDR, a COLO 205 clone engineered to overexpress MDR1 transporter, and 7 pM for HCT‐15, an EpCAM‐expressing cell line that naturally expresses MDR1. Antibody‐IGN conjugates demonstrated dose‐dependent activity in multiple human tumor xenograft models in mice, with anti‐tumor activity observed at non‐toxic doses. The unique mechanism of action of the IGN class of compounds, and the high antigen‐specific potency of antibody‐IGN conjugates seen in vitro and in vivo, provides a promising new cytotoxic agent for use in the development of new ADCs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B126.

Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing.

Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.

Abstract B120: Designing potent antibody‐drug conjugates: The impact of lysosomal processing efficiency and conjugate linker selection on anticancer activity

Six different ADCs are in clinical testing that contain the maytansinoid agent DM1 or DM4. The two most advanced of these, trastuzumab‐DM1 (T‐DM1) and IMGN901, have demonstrated encouraging activity and tolerability. These conjugates differ in the linker used to keep the maytansinoid agent (DMx) attached to the antibody until target‐cell entry and activation. T‐DM1 employs a non‐reducible thioether linker and IMGN901 employs a disulfide linker that is reducible intracellularly. Our findings support that the best linker choice can depend on the nature of the target cancer. We tested conjugates consisting of an anti‐EGF receptor antibody (anti‐EGFR Ab) attached to DMx using either a reducible disulfide linker or a non‐reducible thioether linker. In our initial cytotoxicity survey, we observed that anti‐EGFR‐DMx conjugates made with either type of linker were potent in vitro ( IC 50 = 0.07 nM) against MDA‐MB‐468 breast cancer cells, a cell line with high EGFR expression. In contrast, the reducible, disulfide‐linked conjugate was 15‐fold more potent ( IC 50 = 0.2 nM) than the non‐reducible thioether‐linked conjugate ( IC 50 = 3 nM) against the A431 epidermoid cell line, which also has high EGFR expression. The greater cytotoxicity of conjugate containing the disulfide linker over conjugate containing the non‐cleavable linker against A431 cells was observed using two different disulfide linkers and two different anti‐EGFR Abs. Use of fluorescently‐labeled anti‐EGFR Ab identified that trafficking to lysosomes was delayed in A431 cells, in contrast to the rapid lysosomal localization found in MDA‐MB‐468 cells. This observation of poor lysosomal processing in A431 cells was corroborated by a cellular metabolism study. Lower amounts of active metabolites were formed in A431 cells treated with the non‐reducible linker conjugate than with the disulfide linker design. The disulfide linker conjugate also showed early DMx metabolite release (8 h) due to disulfide cleavage before any lysine‐linker DMx metabolite (24 h) was produced by lysosomal degradation. The non‐reducible linker conjugate showed only the lysine‐linker DMx metabolite at 24 h. Making the disulfide linkage more prone to intracellular reduction (by chemical design) increased the amount of intracellular DMx thiol present and also improved the cytotoxicity of the conjugate. We previously have proposed a model for conjugate activation where lysosomal degradation precedes disulfide linker reduction. The data reported here support an additional pathway of conjugate activation for designs using disulfide linkers (intracellular disulfide cleavage prior to deficient lysosomal proteolysis), which can be beneficial for ADCs targeting cancer cells that have inefficient lysosomal processing or targeting tumor antigens that are, by their nature, inefficiently processed in lysosomes. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B120.

Abstract 2644: Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models

Most ADCs in the clinic utilize a tubulin-binding small molecule as the cytotoxic payload. Although many cancers are sensitive to tubulin agents, some are more responsive to DNA-interacting agents. We have developed a new class of highly potent cytotoxic agents, IGNs, for use in ADCs that consists of indolino-benzodiazepine dimers with a novel DNA-alkylating mechanism of action. We found that IGNs containing a di-imine moiety act via DNA-crosslinking and -alkylating, whereas IGNs with a mono-imine induce DNA alkylation only. Although ADCs with either form of IGN were highly active, ADCs containing di-imine IGNs caused delayed toxicity in mice, leading to mortality. The mono-imine IGN, DGN462, was selected as our lead IGN as, when conjugated to an antibody via a cleavable linkage, the resultant ADC had favorable tolerability in mice (maximally tolerated dose of 40 mg/kg; 700 µg/kg DGN462 dose) without delayed toxicity. The ability of DGN462 to alkylate DNA was demonstrated by measurement of DNA adduct formation with double-stranded DNA and in cells. DGN462 formed covalent DNA adducts through alkylation of the C2- amino group of guanine. Consistent with the activity of other DNA alkylating agents, DGN462-treated cells progressed slowly through S-phase and were arrested in G2/M phase of the cell cycle in a dose dependent manner. ADCs consisting of ∼3 molecules of DGN462 per antibody, attached via a cleavable linker, had desirable biochemical characteristics, including high monomer content (> 97%), low free drug ( A CD33-targeting DGN462 ADC was highly active against AML xenografts, with a minimal efficacious dose (MED) of 0.6 mg/kg (conjugate dose), while a non-targeting control was inactive. A DGN462 ADC targeting epidermal growth factor receptor was highly active and antigen-specific against a head and neck squamous cell carcinoma model, with a 1.6 mg/kg MED. The intact conjugate half-life was approximately 90 hours in mice, and bioactivity of conjugate was maintained at 72 hours post dosing, indicating that intact conjugate remains active in vivo. DGN462 ADCs have an acceptable safety profile and potent antigen-specific DNA-alkylating mechanism of action with the potential for activity in tumors with low sensitivity to tubulin agents, heterogeneous antigen expression and/or PgP-mediated drug resistance. Citation Format: Kathleen Whiteman, Charlene Audette, Andre Dandeneau, Megan Ellis, Nathan Fishkin, Lauren Harvey, Holly Johnson, Yelena Kovtun, Erin Maloney, Michael Miller, Alan Wilhelm, Ravi Chari. Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models. [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 2644. doi:10.1158/1538-7445.AM2014-2644

Abstract 4398: Maytansinoid release from thioether linked antibody maytansine conjugates (AMCs) under oxidative conditions: Implication for formulation and for ex vivo sample analysis in pharmacokinetic studies

Several antibody—maytansinoid conjugates (AMCs) are in various stages of clinical testing, with the most advanced being trastuzumab-DM1 (T-DM1) in Phase III testing. In T-DM1, the maytansinoid, DM1, is linked to the trastuzumab antibody via a non-reducible (“non-cleavable”) thioether bond using the SMCC (N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate) linker. It has been reported that T-DM1 shows evidence of maytansinoid loss in circulation over time based on ex vivo analysis (Liepold, AACR 2009 abstract #2914), and it has been assumed that a slow retro-Michael reaction is the cause of some linker cleavage. In the present study, however, we demonstrate that a more likely source of observed maytansinoid release is a reaction generated ex vivo. In our study, we used a model synthetic compound, DM1-MCC, which has the DM1 maytansinoid linked via a thioether bond like that in T-DM1, and also a thioether-linked AMC. When incubated in human plasma ex vivo, these were found to release maytansinoid sulfonate (DM1-SO3-) under physiological conditions (37°C, pH 7.4) at a slow rate. The formation of an initial sulphenic acid intermediate (RSOH) during β-elimination was confirmed with trapping experiments using dimedone (5,5-dimethyl-1,3-cyclohexanedione). Oxidatively-stressed AMCs and a model substrate with a more hindered thioether made with the maytansinoid DM4 showed a 3-fold increase in rate of sulfoxide formation, but an 8-fold lower rate of β-elimination compared to unhindered thioethers. DM1-MCC and thioether-linked AMCs were incubated at 37°C with thiol-trapping agents, such as 2,2′-dipyridyldisulfide. No release of DM1-thiol was detected under these conditions. In contrast, greater than 20% of conjugated maytansinoid was cleaved from a thioether-linked AMC under oxidative stress conditions (10−6 M H2O2 1 hr, 4°C dialysis, then incubation 18 h at pH 7.4, 37 °C). The present observations suggest that ex vivo thioether oxidation may lead to maytansinoid release from conjugates. This sulfoxide elimination is noted at 37 °C under aqueous conditions and is likely facilitated by the mildly acidic β-hydrogen on the maleimide (pKa ∼ 25). Oxidation of thioethers (such as methionine) is common during protein extraction and concentration, and the present studies underscore the importance of reducing autooxidation of thioether-linked AMCs with regard to sample handling during pharmacokinetic analyses and conjugate formulation. 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 4398.

Abstract 647: SeriMabs: N-terminal serine modification enables modular, site-specific payload incorporation into antibody-drug conjugates (ADCs)

Site-specific incorporation of cell-killing agents into cancer-targeting antibodies is an active area of innovation in the field of ADCs. We have developed a highly modular site-specific conjugation platform employing N-terminal serine engineered antibodies (SeriMabs), using ligation chemistry orthogonal to lysine and cysteine modification which typically employs maleimide containing linkers. Humanized IgG1 antibodies were engineered with N-terminal serine residues on either the light or heavy chain, with the precise position optimized for quantitative conversion to the corresponding glyoxyl aldehyde in the presence of sodium periodate. The aminooxy functional group of the heterobifunctional linker N-[(aminooxy)acetyl]-3-[(3-nitro-2-pyridinyl)dithio]-L-alanine was condensed with the aldehyde group on the antibody using 4-amino phenethyl alcohol catalyst, yielding a stable oxime bond under conditions that fully maintain antibody integrity. The dithiopyridine groups were then reacted with thiol-containing maytansinoid or IGN cell-killing agents, yielding disulfide-linked SeriMab ADCs with exactly 2 payload molecules per antibody, as determined by MS analysis, in > 90% yield and with a monomer content of > 98%. Conjugation of payload was found to be selective for the N-terminal serine based on MS/MS analysis. The site-specific ADCs were found to bind to their target antigens with affinity similar to the corresponding lysine-conjugated ADCs, and FcRn binding (pH 5.8) of the SeriMab ADCs were comparable to lysine-conjugated controls. DGN462, our proprietary DNA-acting IGN payload used in our preclinical candidate IMGN779, was conjugated to SeriMabs against two targets, and in both cases, high, antigen-specific in vitro potency was noted. The oxime linkage used in the serine site-specific conjugates was found to be stable at pH 5.5 and pH 7.4 in buffer, as little detectable payload was released over 4 days at 37°C. SeriMab antiFRα-DGN462 (2 DGN462 molecules per Ab) demonstrated potent, dose-dependent antitumor activity against NCI-H2110 non-small cell lung cancer xenografts in SCID mice. SeriMab antiFRα-DGN462 was highly active at a single injection dose of 50 and 25 μg/kg (DGN462 dose) with a minimal efficacious dose (MED) of 10 μg/kg. We have developed a modular method for preparing site-specific, disulfide-linked ADCs with good biochemical characteristics, through an optimized N-terminal serine engineered IgG, using the same thiol containing effector payloads employed in our lysine-conjugation platform. Citation Format: Luke Harris, Daniel Tavares, Lingyun Rui, Erin Maloney, Alan Wilhelm, Juliet Costoplus, Katie Archer, Megan Bogalhas, Lauren Harvey, Rui Wu, Xuan Chen, Xiangyang Xu, Sonia Connaughton, Lintao Wang, Kathleen Whiteman, Olga Ab, Erica Hong, Wayne Widdison, Manami Shizuka, Michael Miller, Jan Pinkas, Thomas Keating, Ravi Chari, Nathan Fishkin. SeriMabs: N-terminal serine modification enables modular, site-specific payload incorporation into antibody-drug conjugates (ADCs). [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 647. doi:10.1158/1538-7445.AM2015-647