Thomas D. Chittenden

Abstract 5463: Development of a novel antibody-maytansinoid conjugate, IMGN289, for the treatment of EGFR-expressing solid tumors.

EGFR is an attractive target for the treatment of a variety of solid tumors because of its role as a driver oncogene and high level of expression. Four EGFR-targeting agents, including two antibodies (Abs), have been approved for clinical use. Despite anti-tumor benefits, inhibition of EGFR pathway is associated with significant dermatologic toxicities; resistance to EGFR antagonists also develops. To enhance potency with comparable or better tolerability, we developed IMGN289, an EGFR-targeting antibody-“drug” conjugate (ADC) that disrupts tumor growth both by inhibiting EGFR signaling and through direct anti-mitotic activity. To reduce potential dermatologic toxicities associated with EGFR pathway inhibition, a unique Ab discovery approach was employed. Hybridomas from mice immunized with EGFR-expressing tumor cells were screened for EGFR binding and selective inhibitory activity against EGFR-dependent tumor cells. This approach revealed a novel class of Ab with selective EGFR antagonistic activity. A humanized lead Ab was identified, J2898A, which was comparable in potency to cetuximab in vitro against a panel of EGFR-dependent tumor cell lines and in vivo against two head and neck tumor xenograft models. Notably, in cultures of human primary keratinocytes, this Ab was markedly less cytotoxic than cetuximab and did not affect TNFα-induced cytokine production, which has been implicated in chronic dermatologic toxicities induced by other anti-EGFR agents. To further enhance cytotoxic activity and to potentially overcome resistance to EGFR-targeting therapies, J2898A was conjugated to the maytansinoid DM1, a potent anti-tubulin agent, via a non-cleavable linker, SMCC. IMGN289 was not only more potent than J2898A against EGFR-dependent tumors, but also was effective against EGFR-positive tumor cells that grow independently of signaling via the EGFR pathway or have acquired resistance to EGFR inhibitors, including lung adenocarcinoma cell lines harboring the T790M EGFR mutation or MET gene amplification. Despite having potent activity against EGFR-expressing tumor cells, IMGN289 was less toxic to cultured keratinocytes than cetuximab. Moreover, a toxicology study in cynomolgus monkeys demonstrated that IMGN289 was well tolerated and exhibited a similar toxicity profile to that published for trastuzumab emtansine (T-DM1), another ADC which utilizes SMCC-DM1 as the selected linker-payload format. In summary, IMGN289 combines EGFR inhibition mediated by its J2898A Ab component with the potent cytotoxicity provided by its DM1 payload, and is highly active against EGFR-positive tumors regardless of their dependency on the EGFR pathway. IMGN289 thus represents a promising novel candidate for treatment of EGFR-expressing solid tumors. Citation Format: Yulius Y. Setiady, Peter U. Park, Jose F. Ponte, Ling Dong, Anna Skaletskaya, Jennifer A. Coccia, Erica Hong, Lauren Clancy, Lingyun Rui, Jan Pinkas, Robert J. Lutz, John M. Lambert, Thomas D. Chittenden. Development of a novel antibody-maytansinoid conjugate, IMGN289, for the treatment of EGFR-expressing solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5463. doi:10.1158/1538-7445.AM2013-5463

Abstract 2830: Antibody and linker selection for the anti-CD37 antibody-maytansinoid conjugate IMGN529 for the treatment of B-cell malignancies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD37 represents an attractive target for an antibody-maytansinoid conjugate (AMC) due to its prevalence in B-cell malignancies, such as non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and its restricted expression on normal tissue, where it is mainly found on B-cells in blood and lymphoid tissues. Additionally, since antibodies to CD37 have been described to have anti-tumor activity, this target has potential for the development of an AMC containing a functional antibody. To select the antibody for this AMC, a large panel of anti-CD37 antibodies was generated by immunizing mice with CD37+ cells. Anti-CD37 antibodies were selected based on their superior ability to induce apoptosis in Ramos and Raji cells in comparison to the anti-CD20 antibody, rituximab, and the anti-CD37 SMIP, TRU-016. Surprisingly, unlike TRU-016, these antibodies had potent apoptotic activity in the absence of cross-linking agent. After humanization by variable domain re-surfacing, the selected antibodies retained high affinity binding to CD37+ B-cells with an EC50 of < 1 nM. They had much stronger pro-apoptotic activity than rituximab against Ramos cells, with the K7153A antibody among those with the best EC50. They all had antibody-dependent cell-mediated cytotoxicity (ADCC) activity, with K7153A having the most potent activity against Daudi cells. When SMCC-DM1 conjugates of humanized antibodies were compared, the K7153A-SMCC-DM1 conjugate had the most potent specific cytotoxicity against Daudi and Granta-519 cells in vitro. Therefore, the K7153A anti-CD37 antibody provided the best overall anti-tumor activity in terms of its direct pro-apoptotic activity, effector function and potency when used in an AMC. To determine the most effective linker design, maytansinoid conjugates of K7153A were prepared with either hindered disulfide (SPP-DM1) or thioether (SMCC-DM1) linker chemistries. Both conjugates were highly active against lymphoma cells in vitro, with the SMCC-DM1 conjugate being somewhat more potent. In vivo, a single dose of either 10 mg/kg of K7153A-SMCC-DM1 or 5 mg/kg of K7153A-SPP-DM1 was highly active against established SU-DHL-4 sc xenograft tumors. Both treatments resulted in >50% tumor-free survivors at study end. Similarly, the same treatment dose and schedule resulted in good efficacy with both conjugates in a BJAB sc xenograft model. Thus, the K7153A-SMCC-DM1 conjugate was highly active against lymphoma xenograft tumors and, based on preclinical experience, is expected to have comparable, if not better, therapeutic index to that of the SPP-linked conjugate. Taken together, these data support the selection of the K7153A antibody and the SMCC-DM1 design as the optimal anti-CD37 antibody-maytansinoid conjugate for clinical development (designated IMGN529). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2830. doi:10.1158/1538-7445.AM2011-2830

Abstract 4565: IMGN529: A therapeutic maytansinoid conjugate of an anti-CD37 antibody with multiple mechanisms of action for B-cell lymphoma and leukemia

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD37 is a B-cell surface antigen that is an attractive target for antibody and antibody-drug conjugate mediated therapies due to its restricted expression profile. It is expressed on malignant B-cells in NHL and CLL, but on normal tissue its expression is highly restricted to B-cells present in blood and lymphoid tissues. A large panel of anti-CD37 murine monoclonal antibodies were generated and screened for their specific CD37 binding affinity, direct anti-proliferative activity and pro-apoptotic activity against lymphoma cell lines. Selected antibodies were humanized by variable domain resurfacing and one antibody, designated K7153A, demonstrated the best overall activity in terms of direct antibody activity as well as effector function. K7153A demonstrated much stronger pro-apoptotic activity against Ramos and Raji cells than either of two reference compounds, the anti-CD37 SMIP TRU-016 or the anti-CD20 antibody rituximab, and did not require cross-linking to achieve this effect. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. IMGN529 retains the high specific binding affinity of the K7153A antibody, with an EC50 of 0.5 nM. IMGN529 also demonstrated the same strong pro-apoptotic activity as the K7153A antibody against Ramos cells, with an EC50 of 0.1 nM. Antibody-dependent cell-mediated cytotoxicity (ADCC) assays, using purified human NK cells as effector cells, showed that K7153A and IMGN529 have similar potent ADCC activity against Ramos and Daudi cells with an EC50 of less than 10 pM. In addition, both K7153A and IMGN529 demonstrated comparable complement-dependent cytotoxicity (CDC) in the presence of human complement against Ramos cells. These results indicate that IMGN529 retains the intrinsic functions of the K7153A antibody. IMGN529 was highly cytotoxic in vitro against NHL cell lines such as Daudi, BJAB, Namalwa and SU-DHL-4 with a greater degree of cell killing and lower EC50 value (19 – 36 pM) than the K7153 antibody alone. In contrast, TRU-016 showed no effect on any of these cell lines and rituximab was only active against SU-DHL-4 cells. In vivo, IMGN529 showed markedly higher efficacy against established SU-DHL-4 and BJAB xenograft tumors than the K7153A antibody alone, with significant anti-tumor activity at single doses of 5 mg/kg or lower. Together, these results demonstrate that IMGN529 combines the strong pro-apoptotic activity, CDC and ADCC activity of its anti-CD37 antibody component with the potent cytotoxic activity provided by the targeted delivery of its maytansinoid payload. IMGN529 is a highly active antibody-drug conjugate with a unique combination of anti-tumor activities and is therefore a promising therapeutic candidate for the treatment of CD37-positive lymphomas and leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2011-4565

Abstract 5467: IMGN289, an EGFR-targeting antibody-maytansinoid conjugate with potent activity against non-small cell lung cancer (NSCLC) regardless of dependency on EGFR pathway.

NSCLC accounts for approximately 85% of all lung cancers. Based on tumor histology, NSCLC can be subdivided into adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC), which account for 40%, 25-30% and 10-15% of NSCLC cases, respectively. In a fraction of AC cases, driver oncogenes have been identified that enable effective treatment with targeted therapies. For most NSCLC cases, however, etiology is still unknown and effective targeted therapies have yet to be achieved. Additionally, even cancers that initially respond to targeted therapies eventually develop resistance, creating the need for other therapeutic approaches. EGFR is one of the best characterized driver oncogenes in NSCLC and also is frequently expressed at high levels in this indication. In house immunohistochemical analysis found EGFR to be highly expressed in 23.8% of AC (n = 21), 60% of SCC (n = 10) and 50% of LCC (n = 8), confirming published data that EGFR represents an attractive therapeutic target for NSCLC. As a novel approach to targeting EGFR-expressing tumors, we have developed IMGN289, an antibody-“drug” conjugate (ADC) consisting of the J2898A antibody (Ab), which selectively binds to EGFR and inhibits EGFR-driven tumor cell growth, conjugated to the maytansinoid DM1, a potent anti-microtubule agent, via the SMCC thioether linker. IMGN289 showed significantly enhanced cytotoxic activity, relative to cetuximab or unconjugated J2898A, against a panel of NSCLC cell lines dependent on EGFR signaling. In vitro, anti-EGFR Abs typically inhibit less than 70% of tumor cell growth at 30 nM concentration, whereas IMGN289 exposure approached 100% inhibition at a significantly lower concentration. Enhanced potency of IMGN289 against EGFR-dependent tumors was demonstrated in vivo in the H292 xenograft tumor model, where the minimally effective doses of IMGN289 and its J2898A Ab were 1 and 3 mg/kg, respectively. In addition, IMGN289 was effective against EGFR-expressing NSCLC cells that are not dependent on EGFR signaling and therefore resistant to anti-EGFR Abs. For example, IMGN289 was active against H226 and H1703 mesenchymal cell lines in vitro and H1703 xenograft tumor in vivo, while neither cetuximab nor J2898A alone was active. IMGN289 was also active against EGFR mutant HCC827 cell lines that have acquired resistance to EGFR tyrosine kinase inhibitors through T790M EGFR mutation or MET gene amplification. Thus, EGFR-targeting IMGN289 achieves a distinct anti-tumor mechanism that is independent of EGFR pathway inhibition. In summary, IMGN289 is highly active against EGFR-positive NSCLC cells regardless of dependency on the EGFR pathway and represents a promising therapeutic candidate for NSCLC. Citation Format: Thomas D. Chittenden, Yulius Y. Setiady, Peter U. Park, Jose F. Ponte, Ling Dong, Anna Skaletskaya, Christina N. Carrigan, Alfred A. Villaluz, Jan Pinkas, Robert J. Lutz, John M. Lambert. IMGN289, an EGFR-targeting antibody-maytansinoid conjugate with potent activity against non-small cell lung cancer (NSCLC) regardless of dependency on EGFR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2013-5467