Robert J. Lutz

Antibody-Maytansinoid Conjugates Are Activated in Targeted Cancer Cells by Lysosomal Degradation and Linker-Dependent Intracellular Processing

Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen-specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, we used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, we examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibody-maytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma Cells In vitro and In vivo

Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

Antibody-maytansinoid conjugates for the treatment of myeloma.

Despite recent advances in the treatment of multiple myeloma, new agents are still needed to improve the outcome for patients. The established success of monoclonal antibodies in the treatment of some cancers has promoted interest in developing antibody-based therapies for multiple myeloma. Efforts have included the development of antibodies conjugated to potent cytotoxic moieties that combine the specificity of anti-myeloma-targeting antibodies with highly active anti-tumor compounds. Two such immunoconjugates currently in clinical development are composed of antibodies that target cell surface proteins found on multiple myeloma cells, and are coupled to cytotoxic maytansinoids. IMGN901 targets the neural cell adhesion molecule, CD56, which is expressed on the majority of myeloma cells, as well as on other cancers, while BT062 targets CD138, a primary diagnostic marker for multiple myeloma. In this review, we discuss the preclinical and early clinical data for these two promising new antibody-based anti-myeloma agents.

A novel anti-CD37 antibody-drug conjugate with multiple anti-tumor mechanisms for the treatment of B-cell malignancies

CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.

IMGN853, a Folate Receptor-α (FRα)–Targeting Antibody–Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors

A majority of ovarian and non–small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody–drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non–small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochemistry. IMGN853 displayed cytotoxic activity against FRα-negative cells situated near FRα-positive cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clinical development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors. Mol Cancer Ther; 14(7); 1605–13. ©2015 AACR.

Lorvotuzumab mertansine, a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5–10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.

Abstract 5483: Preclinical evaluation of IMGN289, an anti-EGFR antibody-maytansinoid conjugate for the treatment of squamous cell carcinoma of the head and neck.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Squamous cell carcinoma of the head and neck (SCCHN) include cancers that affect the squamous epithelium of the mouth, larynx, nasal passages, sinuses and pharynx. The epidermal growth factor receptor (EGFR) has emerged as an important antigen for novel targeted therapies in SCCHN, where EGFR overexpression correlates with aggressive disease, resistance to standard therapies and a poor prognosis. EGFR expression was evaluated on a panel of SCCHN xenografts and primary SCCHN tumors using a calibrated immunohistochemical (IHC) staining method on formalin-fixed paraffin-embedded sections. The results demonstrated that 83 of 85 (98%) SCCHN tumors stained positive for EGFR and, of the 83 positive tumors, 80 (96%) had high EGFR expression, confirming that EGFR represents an attractive therapeutic target for SCCHN. IMGN289 is an antibody-“drug” conjugate (ADC) consisting of the humanized monoclonal antibody, J2898A, which selectively binds to EGFR, linked to the potent cytotoxic maytansinoid, DM1, via a SMCC thioether linker. The cytotoxic activity of IMGN289 was evaluated against a panel of SCCHN cell lines in vitro. IMGN289 was active in 9 of 13 cell lines assayed, including 5 of 8 that were resistant to cetuximab. Cytotoxic activity was observed against cell lines originating from multiple anatomic sites including the pharynx, oral cavity, larynx and tongue. The anti-tumor activity of IMGN289 was evaluated in EGFR-positive SCCHN xenograft models with expression levels comparable to patient tumors. Immunodeficient mice bearing established subcutaneous xenograft tumors were treated with a single intravenous injection of IMGN289 at 1, 2.5 or 5.0 mg/kg (based on antibody concentration). A group of mice dosed with the J2898A antibody at 5 mg/kg was included in the study. In the FaDu xenograft model, IMGN289 was active with a minimally efficacious dose (MED) of 1 mg/kg, highly active at 2.5, and at 5 mg/kg 5/6 partial regressions (PR) and 2/6 complete regressions (CR) were observed. Conjugation of DM1 to J2898A achieved greater anti-tumor activity than J2898A, which was active in the study but without regressions. In the HSC-2 xenograft model, IMGN289 was also highly active, with a MED of 2.5 mg/kg and tumor regression at 5 mg/kg with 6/6 PR and 4/6 CR. Once again, the activity of IMGN289 was more robust than naked J2898A antibody, which was active with 2/6 PR and 1/6 CR. The strong anti-tumor activity of IMGN289 against SCCHN xenograft tumors with EGFR expression levels comparable to patient tumors suggests that IMGN289 may be a promising compound for the treatment of SCCHN. Citation Format: Jose F. Ponte, Yulius Y. Setiady, Ling Dong, Anna Skaletskaya, Christina N. Carrigan, Alfred Anderson-Villaluz, Robert J. Lutz, Jan Pinkas. Preclinical evaluation of IMGN289, an anti-EGFR antibody-maytansinoid conjugate for the treatment of squamous cell carcinoma of the head and neck. [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 5483. doi:10.1158/1538-7445.AM2013-5483

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 1781: Lorvotuzumab mertansine (IMGN901) in combination with standard-of-care paclitaxel/carboplatin therapy is highly active in a preclinical xenograft model of ovarian cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The CD56-binding antibody-maytansinoid conjugate, lorvotuzumab mertansine (IMGN901), is currently being evaluated as monotherapy in patients with CD56-positive solid tumors including ovarian cancer, SCLC, and Merkel cell carcinoma. Encouraging results have recently been reported for this clinical trial, including 3 objective responses in second-line or later use (Woll, et al., ESMO 2010). CD56 is expressed on approximately 57% of ovarian cancers, supporting the clinical investigation of lorvotuzumab mertansine for this malignancy. Lorvotuzumab mertansine has been shown to be active as a single-agent against CD56-positive ovarian xenograft tumors, including OVCAR-3 and COLO 720E (Whiteman, et al., AACR 2008). Despite the high response rate of ovarian cancer to the standard-of-care first-line paclitaxel/carboplatin chemotherapy (approximately 60%), more than 80% of patients will relapse within 24 months of starting this treatment. Although advances in diagnosis and treatment have improved outcomes, the 5-year overall survival rate for ovarian cancer is only about 45%. Consequently, there is an unmet clinical need for treatments achieving long-term responses as first-line or second-line therapies for ovarian cancer. The possibility of achieving durable responses with combination of lorvotuzumab mertansine plus paclitaxel/carboplatin therapy was investigated in a preclinical model of ovarian cancer. The activity of lorvotuzumab mertansine in combination with the standard-of-care paclitaxel/carboplatin therapy was evaluated against COLO 720E ovarian xenografts in mice. The two dose levels of paclitaxel (10 or 20 mg/kg weekly × 3) plus carboplatin (100 mg/kg single injection) evaluated without lorvotuzumab mertansine were both active in this model, resulting in complete tumor regressions (CR) in 1 of 6 and 2 of 6 mice, respectively, with all tumors recurring during the study. Lorvotuzumab mertansine administered at a dose of 13 mg/kg weekly × 3 was active as a single agent, resulting in a CR in 1 of 6 mice which recurred during the study. Combination of lorvotuzumab mertansine with either dose level of paclitaxel/carboplatin resulted in CRs in all mice (6 of 6 mice/group), with no tumor recurrence during the study period (123 days). The long-term CRs observed in a preclinical model of ovarian cancer suggest lorvotuzumab mertansine in combination with standard-of-care agents would be a promising combination treatment regimen to evaluate in the clinic due to its potential to achieve an increased response rate and more durable responses compared with standard-of-care agents alone, which could represent a significant therapeutic benefit to ovarian cancer patients. 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 1781. doi:10.1158/1538-7445.AM2011-1781

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