Emily E. Reid

Synthesis and Biological Evaluation of Antibody Conjugates of Phosphate Prodrugs of Cytotoxic DNA Alkylators for the Targeted Treatment of Cancer

The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.

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.

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.

Abstract C160: New class of DNA-alkylating agents with a suitable tolerability profile created for use in antibody-drug conjugates (ADCs).

The recent FDA approvals of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla) has spurred tremendous interest in ADCs for the treatment of cancer. Most clinical-stage ADCs, including the two approved ones, utilize a tubulin-acting small molecule as the cytotoxic payload. There is strong interest in preparing DNA-acting payload agents because some cancers are more responsive to DNA-acting agents than to tubulin-acting ones. The key challenge has been developing a DNA-acting payload agent with the necessary tolerability profile as well as potency. We have developed a new class of DNA-acting agents for use in ADCs, IGNs, which demonstrate the desired profile. Initially, our IGNs were indolino-benzodiazepine dimers that were highly potent in vitro (IC50 ∼10−12 M) by virtue of their ability to both alkylate and cross-link DNA. However, a resultant IGN-ADC did not have the desired in vivo tolerability, a known problem with highly potent DNA-acting agents. This IGN-ADC was found to be intolerable in mice (4.4 mg/kg, weekly x 3), resulting in delayed toxicity and mortality. To address this, we tested modifying the saturation level of the di-imine cross-linking groups by generating a mono-imine compound that could still alkylate DNA, but no longer act as a cross-linker. This new, mono-imine IGN, 2m (alkylator only), was highly potent, but with at least 2-fold better tolerability (9.1 mg/kg, weekly x 3) and, most importantly, not associated with delayed toxicity. To assess its potency in an ADC format, 2m and the lead IGN di-imine compound, 2d (crosslinker/alkylator) were conjugated to the folate receptor-α (FRα)-binding antibody, anti-FRα, and the CD33-binding antibody, anti-CD33, via amide bonds. In spite of their different imine saturation levels, anti-FRα-2d and anti-FRα-2m were both highly potent against the FRα-expressing KB cell line (IC50 values of 15 pM and 20 pM, respectively). The addition of excess unconjugated antibody abolished the cytotoxic effect (>3 nM), thus demonstrating that the activity of the conjugate is antigen specific. Similarly, anti-CD33-2d and anti-CD33-2m displayed comparable potency against a panel of CD33-expressing cell lines. This included the human promyelocytic leukemia cell line, NB4, where these two IGN-ADCs had IC50 values in the picomolar range despite the low level of antigen expression on the target cells (∼10,000 molecules/cell). In vivo, the anti-FRα-2m conjugate was found to be effective against KB tumors in mouse xenograft models at doses that were well below the MTD. The mechanism of action, potency, tolerability, and lack of delayed toxicity make IGNs a promising new class of cytotoxic small molecules for use in ADCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C160. Citation Format: Michael Miller, Nathan Fishkin, Wei Li, Emily Reid, Katie Archer, Erin Maloney, Yelena Kovtun, Gregory Jones, Megan Ellis, Rajeeva Singh, Kathleen Whiteman, Jan Pinkas, Ravi Chari. New class of DNA-alkylating agents with a suitable tolerability profile created for use in antibody-drug conjugates (ADCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C160.

A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody–Drug Conjugates (ADCs)

Tumor-selective delivery of cytotoxic agents in the form of antibody–drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA–cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650–60. ©2018 AACR.

Abstract 53: Antibody-drug conjugates (ADCs) of peptide-linked Indolino-Benzodiazepine (IGN) DNA-alkylator provides improved anti-tumor activity over that of a crosslinker

We recently disclosed highly active antibody-drug conjugates (ADCs) that incorporated the novel DNA alkylating indolino-benzodiazepine (termed IGN) dimer, DGN549 (IGN-P1). The stereochemistry of the alanyl moiety of the protease-cleavable alanine-alanine linker used was shown to impact ADC catabolism, bystander killing activity, and in vivo efficacy (Shizuka, et al., AACR 2016 #2959). Building upon these results, here we describe preclinical results from a head-to-head comparison of ADCs of the mono-imine containing DGN549 with its corresponding DNA cross-linking diimine version, IGN-P1 diimine. IGN-P1 diimine and DGN549 were conjugated to a folate receptor-α (FRα)-binding antibody and an EpCAM-binding antibody. The resulting ADCs demonstrated similar high in vitro potency (IC50 ~3-100 pM) and specificity towards several cancer cell lines. Further in vitro studies revealed that the DNA alkylating anti-FRα-DGN549 ADC demonstrated superior bystander cell-killing activity compared to its DNA crosslinking counterpart, anti-FRα-IGN-P1 diimine. In vivo, this improved bystander killing ability translated into better in vivo activity for the DNA alkylating ADC. In an endometrial tumor xenograft model established with Ishikawa cells, the anti-FRα-DGN549 induced complete regressions at a single dose of 140 µg/kg Ab dose (equivalent to 5 µg/kg linked IGN). The cross-linking anti-FRα-IGN-P1 diimine had to be used at twice the dose to achieve the same level of anti-tumor activity. The in vivo tolerability in CD-1 mice also displayed differences in the two ADCs. We found that the ADC of the DNA crosslinker was at least two-fold less tolerated than the corresponding ADC of the DNA alkylator. These results indicate that a ~4 fold greater therapeutic index can be achieved when using a DNA alkylating mono-imine DGN549 ADC as compared to the DNA crosslinking IGN-P1 diimine ADC. Citation Format: Michael L. Miller, Manami Shizuka, Jose F. Ponte, Leanne Lanieri, Dilrukshi Vitharana, Qifeng Qiu, Emily E. Reid, Katie E. Archer, Rui Wu, Erin K. Maloney, Olga Ab, Jan Pinkas, Ravi V. Chari. Antibody-drug conjugates (ADCs) of peptide-linked Indolino-Benzodiazepine (IGN) DNA-alkylator provides improved anti-tumor activity over that of a crosslinker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2017-53

Abstract 2960: Potentin vivoactivity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation

ADCs are widely studied for cancer therapy, with numerous agents in preclinical and clinical development embodying a wide array of targets, linker chemistries, and cytotoxic effector classes. A fourth element of ADC design that has received much attention recently is the site of conjugation of the cytotoxic molecule to the antibody. Historically, lysine- or interchain cysteine-directed conjugation has been used, but site-specific chemistries have become increasingly popular. Our previous evaluation of site-specific and lysine-linked ADCs utilizing a tubulin-acting maytansinoid effector molecule found the lysine-linked version was more active in vivo (Yoder et al., AACR 2015 #645). Here we present a comparison of engineered cysteine site-specific and lysine-linked ADCs utilizing the previously described indolino-benzodiazepine (henceforth referred to as IGN) effector IGN-P1 (Miller et al., AACR 2015 #652) which is designed to undergo proteolytic cleavage upon cell uptake to release a potently cytotoxic DNA alkylator. We show that HC-S442C mutants of human IgG1 can be conjugated via maleimide chemistry to IGN-P1 to give stable, potent, and homogeneous ADCs with drug to antibody ratio (DAR) of 2. The in vitro potency of engineered-cysteine IGN-P1 ADCs is largely dependent on the DAR of the ADC, although some difference is observed between HC-S442C and other cysteine mutants used for conjugation. Pharmacokinetic study of C442 maleimide conjugates suggests that the chemical linkage between effector and antibody is stable upon administration in mice. Further, and in contrast to our previous observations utilizing maytansinoid ADCs, the site-specific and Lys-linked IGN-P1 ADCs showed comparable efficacy in vivo on a molar drug basis. This effect was observed across two different antibodies targeting two different cell surface antigens. These results suggest that, in certain cases, site-specific conjugation chemistry can offer comparable activity to heterogeneous conjugation at well-tolerated doses. Citation Format: Nicholas C. Yoder, Chen Bai, Alan Wilhelm, Erin K. Maloney, Olga Ab, Emily E. Reid, Manami Shizuka, Daniel Tavares, Rassol Laleau, Xiuxia Sun, Megan E. Bogalhas, Lintao Wang, Jan Pinkas, Michael L. Miller, Ravi Chari, Thomas A. Keating. Potent in vivo activity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2960.

Abstract 652: Antibody-drug conjugates (ADCs) of indolino-benzodiazepine DNA-alkylating agents

There are over thirty ADCs in the clinic today, reflecting the interest in ADCs for the treatment of cancer. Most of these ADCs utilize a tubulin-interacting small molecule as their cytotoxic payload. In order to continue to extend the utility of ADCs to more types of cancers, there is a strong interest in developing cytotoxic payloads with other mechanisms of action. Previously we have described the development of a class of indolino-benzodiazepine dimers (IGNs) with high potency and specificity. ADCs made with a mono-imine containing IGN, which only alkylate DNA, were found to display improved tolerability, along with the absence of delayed toxicity, compared to their DNA-cross-linking di-imine counterparts. Additional structure-activity relationship studies with mono-imine containing IGNs have resulted in two promising new leads, IGN-D1 and IGN-P1, which are conjugated to an antibody via either a disulfide or peptide linker, respectively. ADCs of IGN-D1 and IGN-P1 were prepared for evaluation using a folate receptor α (FRα)-binding antibody, anti-FRα, and EGFR-binding antibody, anti-EGFR. Anti-FRα-IGN-D1 and anti-FRα-IGN-P1 were found to be highly potent against the FRα-expressing KB cell line (IC50 values of 8 pM and 4 pM, respectively) and also demonstrated strong activity against nearby target-negative cancer cells. The addition of excess unconjugated antibody blocked the cytotoxic effect of these conjugates, thus demonstrating that their activity is antigen specific. In addition, both anti-FRα-IGN conjugates were found to be highly potent (IC50 values of 100 pM and 30 pM, respectively) against the breast ductal carcinoma cell line, T47D, despite only a moderate level of antigen expression on the target cells. Similarly, anti-EGFR-IGN-D1 and anti-EGFR-IGN-P1 were found to have IC50 values in the picomolar range against a panel of EGFR-expressing cell lines. In vivo, anti-FRα-IGN-D1 and anti-FRα-IGN-P1 were found to be effective against NCI-H2110 non-small cell lung cancer xenografts, with anti-tumor activity observed at single doses as low as 3 μg/kg (payload dose, equivalent to 0.15 mg/kg Ab dose). These data support evaluation of new ADC therapies with these two new IGNs for solid tumor indications as well as tumors with low antigen expression. Citation Format: Michael L. Miller, Manami Shizuka, Nathan Fishkin, Emily Reid, Katie Archer, Erin Maloney, Chen Bai, Olga Ab, Nick C. Yoder, Rui Wu, Erica Hong, Megan Bogalhas, Alan Wilhelm, Kathleen Whiteman, Ravi Chari. Antibody-drug conjugates (ADCs) of indolino-benzodiazepine DNA-alkylating agents. [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 652. doi:10.1158/1538-7445.AM2015-652

Abstract C162: Antibody-Drug Conjugates (ADCs) with novel IGN DNA-alkylating agents display potent antigen-specific activity against hematologic and solid tumor xenograft models.

The clinical benefit demonstrated by brentuximab vedotin and ado-trastuzumab emtansine has attracted considerable interest in ADCs. Most ADCs in the clinic, including these two approved ones, utilize a tubulin-acting cytotoxic agent. Although many cancers are sensitive to tubulin agents, some cancers are more responsive to DNA-acting agents. We have developed a new class of highly potent cytotoxic agents, IGNs, for use in ADCs which consist of indolino-benzodiazepine dimers with a novel DNA-alkylating mechanism of action. The intense in vitro potency (in the picomolar range) of these IGNs, along with desired characteristics such as aqueous solubility and stability, make them well-suited for use in ADCs. A series of IGN-ADCs was prepared using di-imine (DNA-crosslinking and -alkylating) and mono-imine (DNA-alkylating only) versions of the IGNs, attached using noncleavable and cleavable linkers. The tolerability of IGN-ADCs in mice was found to be dependent upon a) linker format: conjugates with cleavable linkers were better tolerated than those with noncleavable linkers; and b) mechanism of action: conjugates with IGNs with DNA-crosslinking ability (di-imine IGNs) had delayed toxicity that was absent with the DNA-alkylating only (mono-imine) IGN. We found that the cleavable linker format avoids liver toxicity, which was the dose-limiting toxicity of IGN-ADCs with non-cleavable linkers. This absence of liver toxicity resulted in a 4-fold increase in the maximally tolerated dose for the cleavable linker ADC compared with the noncleavable linker design (40 mg/kg and 10 mg/kg conjugate dose, respectively). Thus, IGN-ADCs utilizing a cleavable linker with a mono-imine IGN demonstrated greatly improved tolerability. The in vivo efficacy of ADCs with the selected IGN, 3m (mono-imine) attached using a cleavable linker was evaluated in xenograft models of both hematologic and solid tumor indications. A CD33-targeting IGN-ADC, anti-CD33-3m, was highly active against acute myeloid leukemia xenografts, with a minimal efficacious dose (MED) of 0.6 mg/kg (conjugate dose), while a non-targeting control was inactive. A folate receptor α-targeting IGN-ADC, anti-FRα-3m, and an epidermal growth factor receptor-targeting IGN-ADC, anti-EGFR-3m, were evaluated in non-small cell lung cancer models and found to be highly active and antigen-specific, with MEDs of 1.6 mg/kg. Anti-CD56-3m was highly active and antigen-specific against small-cell lung cancer xenografts, with a MED of 2.9 mg/kg. The desired safety profile (absence of delayed toxicity and liver toxicity) combined with the potent antigen-specific activity of these IGN 3m-ADCs (mono-imine IGN, cleavable linker) against various cancer types supports DNA-acting ADCs with the necessary therapeutic index can now be achieved. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C162. Citation Format: Kathleen Whiteman, Holly Johnson, Alan Wilhelm, Michael Miller, Wei Li, Emily Reid, Katie Archer, Nathan Fishkin, Andre Dandeneau, Erin Maloney, Jan Pinkas, Ravi Chari. Antibody-Drug Conjugates (ADCs) with novel IGN DNA-alkylating agents display potent antigen-specific activity against hematologic and solid tumor xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C162.