Laura M. Bartle

A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation

We have identified a human cytomegalovirus cell-death suppressor, denoted vICA, encoded by the viral UL36 gene. vICA inhibits Fas-mediated apoptosis by binding to the pro-domain of caspase-8 and preventing its activation. vICA does not share significant sequence homology with FLIPs or other known suppressors of apoptosis, suggesting that this protein represents a new class of cell-death suppressors. Notably, resistance to Fas-mediated apoptosis is delayed in fibroblasts infected with viruses that encode mutant vICA, suggesting that vICA suppresses death-receptor-induced cell death in the context of viral infection. Although vICA is dispensable for viral replication in vitro, the common targeting of caspase-8 activation by diverse herpesviruses argues for an important role for this antiapoptotic mechanism in the pathogenesis of viral infection in the host, most likely in avoiding immune clearance by cytotoxic lymphocytes and natural killer cells.

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

SAR650984, A Novel Humanized CD38-Targeting Antibody, Demonstrates Potent Antitumor Activity in Models of Multiple Myeloma and Other CD38+ Hematologic Malignancies

Purpose: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984. Experimental Design: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice. Results: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells. Conclusion: These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies. Clin Cancer Res; 20(17); 4574–83. ©2014 AACR.

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.

Mirvetuximab Soravtansine (IMGN853), a Folate Receptor Alpha–Targeting Antibody-Drug Conjugate, Potentiates the Activity of Standard of Care Therapeutics in Ovarian Cancer Models

Elevated folate receptor alpha (FRα) expression is characteristic of epithelial ovarian cancer (EOC), thus establishing this receptor as a candidate target for the development of novel therapeutics to treat this disease. Mirvetuximab soravtansine (IMGN853) is an antibody-drug conjugate (ADC) that targets FRα for tumor-directed delivery of the maytansinoid DM4, a potent agent that induces mitotic arrest by suppressing microtubule dynamics. Here, combinations of IMGN853 with approved therapeutics were evaluated in preclinical models of EOC. Combinations of IMGN853 with carboplatin or doxorubicin resulted in synergistic antiproliferative effects in the IGROV-1 ovarian cancer cell line in vitro. IMGN853 potentiated the cytotoxic activity of carboplatin via growth arrest and augmented DNA damage; cell cycle perturbations were also observed in cells treated with the IMGN853/doxorubicin combination. These benefits translated into improved antitumor activity in patient-derived xenograft models in vivo in both the platinum-sensitive (IMGN853/carboplatin) and platinum-resistant (IMGN853/pegylated liposomal doxorubicin) settings. IMGN853 co-treatment also improved the in vivo efficacy of bevacizumab in platinum-resistant EOC models, with combination regimens causing significant regressions and complete responses in the majority of tumor-bearing mice. Histological analysis of OV-90 ovarian xenograft tumors revealed that concurrent administration of IMGN853 and bevacizumab caused rapid disruption of tumor microvasculature and extensive necrosis, underscoring the superior bioactivity profile of the combination regimen. Overall, these demonstrations of combinatorial benefit conferred by the addition of the first FRα-targeting ADC to established therapies provide a compelling framework for the potential application of IMGN853 in the treatment of patients with advanced ovarian cancer.

Metabolites of Antibody–Maytansinoid Conjugates: Characteristics and in Vitro Potencies

Several antibody-maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical studies in mice, the cytotoxic metabolites initially formed in vivo are further processed in the mouse liver to give several oxidized metabolic species. In this work, the primary AMC metabolites were synthesized and incubated with human liver microsomes (HLMs) to determine if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how these impurities would be metabolized by HLM.

Abstract 4576: IMGN853, an anti-Folate Receptor I antibody-maytansinoid conjugate for targeted cancer therapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Previously we reported (O. Ab; EORTC, 2010) that an antibody-maytansinoid conjugate (AMC) composed of an anti-FOLR1 antibody conjugated to the cytotoxic maytansinoid, DM4, via the disulfide-containing linker, SPDB, was potent in killing FOLR1-expressing cancer cells in vitro and in vivo. In light of the favorable results noted, we assessed the optimal antibody, linker, and maytansinoid agent for an AMC targeting FOLR1, as reported here. Antibody selection. Anti-FOLR1 antibodies were generated by immunizing mice with human FOLR1-expressing cells, and a panel of FOLR1-specific antibodies was identified by flow cytometry binding assay. Several FOLR1-antibodies with high binding affinity to both human and monkey FOLR1 were chosen for further evaluation and were humanized using ImmunoGens resurfacing technology. Antibodies were conjugated to DM1 via the non-cleavable SMCC linker and the conjugates tested for activity against FOLR1-positive KB cells in vitro and in vivo. All conjugates had comparable cytotoxic potencies in vitro. However, the in vivo anti-tumor activity of one conjugate, M9346A-SMCC-DM1, was significantly better than that of SMCC-DM1 conjugates of other FOLR1 antibodies. Based on this finding, the M9346A antibody was chosen for further development. Linker/maytansinoid selection. The M9346A antibody was linked to DM1 or DM4 via the disulfide-containing cleavable linkers SPP, SPDB or sulfo-SPDB, or via the non-cleavable SMCC linker. We compared the in vitro cytotoxic activities of these conjugates on KB, Igrov-1 and Jeg-3 cell lines. The conjugates with cleavable linkers displayed markedly greater in vitro activities than the SMCC conjugate. We then examined the in vivo activities of the conjugates in FOLR1-positive KB- and Ovcar 3-tumor models. Again, we found that the conjugates with cleavable linkers were more active in vivo than the noncleavable conjugate. Among the conjugates with cleavable linkers, the sulfo-SPDB-DM4 conjugate was the most active conjugate against the Ovcar-3 model, it had activity comparable to that of the SPDB-DM4 conjugate against KB tumors, and both were more active than the SPP-DM1 conjugate in the two xenograft models. Taking into consideration that sulfo-SPDB-DM4 was the most efficacious design in vivo and the potential of the hydrophilic sulfo-SPDB-linker to enable better activity against PgP-expressing cells (previously reported data), M9346A-sulfo-SPDB-DM4 was selected to be the candidate for development and designated IMGN853. IMGN853 is a promising candidate for the treatment of FOLR1-expressing tumors. 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 4576. doi:10.1158/1538-7445.AM2011-4576

Abstract 2967: In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs)

Site-specific attachment of cell-killing agents to antibodies directed against tumor-associated antigens has continued to be an active area of innovation in the field of ADCs. Most reports focus on homogeneous ADCs that have a DAR (cytotoxic molecules per antibody ratio) of 2. Here we describe the preparation, biochemical characterization, and biological evaluation of ADCs made through conjugation of maytansinoids (DM1, DM4) to aldehydes derived from chemically oxidized N-terminal serines (SeriMab) engineered onto the antibody heavy chain (2 DAR) or both light and heavy chain simultaneously (4 DAR). ADCs prepared with a non-cleavable linker or a cleavable disulfide linker were homogeneous 2 or 4 DAR by MS analysis, and were produced in high yield with a monomer content of >98%. Despite conjugation at the N-termini of both the light and heavy chain variable regions, FACS analysis showed the 4 DAR SeriMab conjugates maintained binding to the target antigen. The ADCs showed antigen-specific potency in vitro on a panel of target-expressing cancer cell lines. In the disulfide cleavable linker series, the 2 DAR SeriMab conjugate was 2-5 fold less active than lysine-conjugated Ab-SPDB-DM4 (3.4 DAR), while the 4 DAR SeriMab conjugate was comparably active on an antibody basis. The SeriMab conjugates also displayed strong bystander killing. Surprisingly, in the non-cleavable linker series, the 2 DAR SeriMab conjugate was up to 17-fold more active (depending on cell line) than lysine-conjugated Ab-SMCC-DM1 (3.5 DAR), and the 4 DAR SeriMab conjugate was up to 100-fold more potent than the SMCC-DM1 conjugate on an antibody concentration basis. In a P-gp-positive multi-drug resistant cell line, the non-cleavable 4 DAR SeriMab-maytansinoid conjugate was highly active while the 2 DAR SeriMab ADCs and lysine-conjugated maytansinoid ADCs were >100-fold less potent. The unique oxime bond formed with the non-cleavable SeriMab-maytansinoid conjugate was found to be stable in circulation in mice for >3 days as assayed by affinity capture LC-MS. Polar carboxylic acid containing metabolites were identified which may lead to high cellular retention of maytansinoid species in cancer cells, yielding higher in vitro potency than lysine-linked ADCs in some cell lines. The in vivo anti-tumor activity of disulfide cleavable 2 and 4 DAR SeriMab-DM4 ADCs was evaluated in a clinically relevant cancer xenograft model. The 4 DAR conjugate was active at 60 μg/kg (maytansinoid payload dose) and was more active than the 2 DAR conjugate at this same payload dose. Using the SeriMab conjugation platform we show that in vitro and in vivo activity of site-specific ADCs can be dependent on amount of cytotoxic agent attached per antibody. Citation Format: Luke Harris, Leanne Lanieri, Jose Ponte, Erin Maloney, Laura Bartle, Olga Ab, Juliet Costoplus, Lingyun Rui, Jan Pinkas, Ravi Chari, Thomas Keating, Daniel Tavares, Nathan Fishkin. In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs). [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 2967.

Abstract 667: IMGN853, a folate receptor (FR) α-targeting antibody-drug conjugate (ADC), is highly effective against xenograft models with clinically relevant levels of receptor expression

IMGN853 comprises the anti-FR-α antibody M9346A conjugated to the cytotoxic maytansinoid molecule DM4 via a stable disulfide-containing linker, sulfo-SPDB. IMGN853 is currently in phase I clinical testing; promising clinical results were reported at ASCO 2013. To analyze determinants of the sensitivity of FR-α positive cells to IMGN853, we examined antigen density on the cell surface, internalization and processing of IMGN853, and sensitivity of the cells to IMGN853 and to non-conjugated maytansinoid, S-methyl-DM4, in vitro. Antigen density, internalization and processing were analyzed with 3[H] M9346A. Preliminary experiments demonstrated that 3[H] M9346A is an appropriate surrogate reagent to analyze binding and processing of IMGN853. Sensitivity of the cells to IMGN853 and to the unconjugated maytansinoid was analyzed by a cytotoxicity assay. A panel of seven FR-α positive cell lines was used for the study. Three of the cell lines were sensitive to IMGN853 in an antigen-dependent manner (the cytotoxic effect was blocked in the presence of a saturating excess of the non-conjugated antibody), while four lines were insensitive (the same low cytotoxic activity of IMGN853 was observed with and without the non-conjugated antibody). The proportion of cell surface-bound IMGN853 that was internalized and processed by the sensitive and insensitive cell lines was comparable. The cell lines differed in their FR-α expression, and there was a strong positive correlation between the sensitivity of cells towards IMGN853 and antigen expression on their surface. The cell lines were similar in their sensitivity to S-methyl-DM4. Taken together, the data indicate that the FR-α density on the surface of a cell is a key determinant of its sensitivity to IMGN853. Having observed a correlation between antigen density and sensitivity of the cells to IMGN853, we set out to test IMGN853 activity in xenograft models expressing FR-α at levels comparable to those on patient tumor cells. First, we developed an IHC assay with the use of anti-FR-α antibody BN3.2 (Novocastra Laboratories, UK) that detected various levels of receptor expression in tumor samples. Next, we compared FR-α expression in ovarian carcinoma (OvCa), non-small cell lung carcinoma (NSCLC) samples and in human xenograft models, and examined five models that represented antigen densities of the majority of FR-α positive OvCa and NSCLC patients. We evaluated the anti-tumor activity of IMGN853 in SCID mice bearing these xenograft models after single intravenous injections of 5, 2.5 or 1.2 mg/kg. IMGN853 was highly active with a minimally effective dose from 1.2 to 2.5 mg/kg depending on the model. There was no activity of the conjugate against a FR-α negative xenograft model. Thus, IMGN853 is highly effective against xenograft models with clinically relevant levels of FR-α expression. Citation Format: Olga Ab, Laura M. Bartle, Xiuxia Sun, Rui Wu, Holly A. Johnson, Kathleen R. Whiteman, Alyssa LaBelle, Victor S. Goldmacher. IMGN853, a folate receptor (FR) α-targeting antibody-drug conjugate (ADC), is highly effective against xenograft models with clinically relevant levels of receptor expression. [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 667. doi:10.1158/1538-7445.AM2014-667

Abstract 3503: A method for quantifying soluble folate receptor 1 in IMGN853 0401 clinical trial patients.

IMGN853, an antibody-“drug” conjugate (ADC), consists of a human folate receptor 1 (FOLR1)-binding monoclonal antibody, M9346A, conjugated to the maytansinoid, DM4, via the cleavable sulfo-SPDB linker. A phase I clinical study has been initiated to identify the maximum tolerated dose, safety, pharmacokinetics (PK), and preliminary efficacy of IMGN853 in patients with FOLR1-positive tumors, including ovarian cancer, non-small cell lung cancer, and other epithelial malignancies. The levels of soluble FOLR1 antigen (sFOLR1) in plasma may vary depending on factors such as tumor antigen expression level, tumor type, and disease stage, and may affect the PK of IMGN853and response to treatment. Previously reported studies describing the detection of sFOLR1 in patient samples are limited and not quantitative. The aim of this study was to develop a sandwich ELISA to quantify levels of sFOLR1 present in plasma samples from cancer patients for use as a potential biomarker of IMGN853 PK and/or clinical response. A panel of murine anti-FOLR1 antibodies was generated at ImmunoGen by standard hybridoma technology. Two clones were chosen for use as capture (FR1-9) and detector (FR1-13) antibodies in the ELISA due to their high affinity and ability to bind to FOLR1 even in the presence of IMGN853 or the FOLR1 ligand, folate. The extracellular domain of FOLR1 fused to murine IgG2a Fc region (FOLR1-Fc) was used as a reference standard. A sandwich ELISA was developed using FR1-9 antibody to capture sFOLR1 from patient plasma, and bound sFOLR1 was detected using biotinylated FR1-13 antibody and streptavidin-HRP with TMB as the substrate. The ELISA was used to quantify sFOLR1 in ovarian carcinoma patient plasma using a reference standard curve generated with FOLR1-Fc antigen. A robust sandwich ELISA was developed to quantify sFOLR1 in patient plasma. The quantitation range of the assay was determined to be 0.2 to 90 nanomolar. Coated plates stored frozen from 0-14 days and protein reagents freeze-thawed over two cycles gave repeatable results. The ELISA was able to detect sFOLR1 in 5 of 10 ovarian cancer patient plasma samples, where the levels of detectable sFOLR1 ranged from 0.7-31 nM. This ELISA is considered suitable to quantify levels of sFOLR1 in patients enrolled in the phase 1 trial over the course of IMGN853 treatment, and resulting data will be assessed for correlations with IMGN853 PK and with clinical response. Citation Format: Nathan Testa, Laura Bartle, Olga Ab, Judy Cheng, Gillian Payne, Robert J. Lutz, Ben Wolf, Christina Carrigan. A method for quantifying soluble folate receptor 1 in IMGN853 0401 clinical trial patients. [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 3503. doi:10.1158/1538-7445.AM2013-3503