Katie Archer

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 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

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 2965:In vitroandin vivoactivity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating agent

Previously we have described the characterization of a proprietary class of indolino-benzodiazepine dimers, IGNs, with high potency against many cancer lines. Antibody-drug conjugates (ADCs) made with mono-imine containing IGNs were designed to only alkylate DNA and not cause DNA crosslinking. ADCs with the IGN linked via lysine residues of the antibody were shown to be highly potent and antigen specific. Here we apply our SeriMab site-specific technology platform, which employs N-terminal conjugatable aldehydes derived from oxidation of a serine residue, to link an IGN molecule to the antibody using a peptide linker. This ADC (SeriMab-IGN-P1) has exactly 2 IGN molecules per antibody conjugated to the N-termini of the heavy chains, as determined by MS analysis. Serimab-IGN-P1 was found to maintain binding to the target antigen with comparable affinity to the unconjugated antibody by FACS analysis (Kd = 300 pM). The conjugate was highly potent (IC50 = 4 pM) against a cancer cell line with high target expression (3 × 106 antibodies bound per cell) as well as a cell line with much lower target expression (2 × 104 antibodies bound per cell, IC50 = 22 pM). SeriMab-IGN-P1 also demonstrated strong bystander killing activity against proximal target-negative cancer cells, likely due to a cell permeable metabolite identified in cells expressing the target antigen. The unique oxime linkage in SeriMab-IGN-P1 was found to be stable in circulation in mice for 3 days, as determined by affinity capture LC-MS; the conjugate species with 2 IGNs per antibody remained predominant over this period with an estimated t1/2 of >10 days for payload release. In vivo, SeriMab-IGN-P1 was found to be effective against human lung cancer xenografts (NCI-H2110), with anti-tumor activity observed at single doses as low as 2 μg/kg (payload dose), and complete responses observed at 5 μg/kg. This conjugate was also well tolerated in CD-1 mice at doses significantly higher than the minimum effective dose that produced tumor regression. These data are highly encouraging for new ADC therapies using the SeriMab site-specific conjugation platform with potent DNA-acting payloads. Citation Format: Dilrukshi Vitharana, Alan Wilhelm, Luke Harris, Katie Archer, Manami Shizuka, Erin Maloney, Olga Ab, Rassol Laleau, Xiuxia Sun, Jan Pinkas, Michael Miller, Ravi Chari, Thomas Keating, Nathan Fishkin. In vitro and in vivo activity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating agent. [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 2965.

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.