Gillian Payne

Growth Factors Inactivate the Cell Death Promoter BAD by Phosphorylation of Its BH3 Domain on Ser155

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-xL and related cell death suppressors. When BAD is phosphorylated on either Ser112 or Ser136, it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-xLat the mitochondria. Here we show that phosphorylation of a distinct site Ser155, which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-xL. Protein kinase A functioned as a BAD Ser155 kinase both in vitro and in cells. BAD Ser155 was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser112/Ser136- and a Ser155-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.

Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

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.

Development and Characterization of a Neutralizing Anti-idiotype Antibody Against Mirvetuximab for Analysis of Clinical Samples

Antibody-drug-conjugates (ADCs) are an emerging class of biological therapeutics. Mirvetuximab soravtansine is a novel folate receptor alpha (FRα)-targeting ADC which represents a potential new treatment for patients with ovarian and other FRα-positive cancers. Since patient immune responses to biological therapeutics may negatively affect drug efficacy and patient safety, regulatory authorities require rigorous monitoring of patient samples. Taking advantage of the immune system’s ability to generate highly specific antibodies, the field has turned to anti-idiotype antibodies as powerful tools for the development of sensitive and specific bioassays. Here, we report the generation and characterization of a highly specific neutralizing anti-idiotype antibody directed against M9346A, the antibody moiety of mirvetuximab soravtansine. The anti-idiotype antibody recognizes M9346A with double-digit picomolar affinity, competes with folate receptor antigen for binding to M9346A, and can be used to develop both anti-drug-antibody and neutralizing antibody assays.

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

Abstract 2513: Evaluation of CD37 expression by calibrated IHC identifies non-Hodgkin's lymphoma as a therapeutic target for IMGN529, an anti-CD37-maytansinoid conjugate

Background: CD37 is a B-cell surface antigen reported to be widely expressed on malignant B cells in patients with non-Hodgkin9s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Expression of CD37 in normal tissues is mainly restricted to B cells in blood and lymphoid tissues. This suggests that CD37 may be an attractive target for development of a therapeutic antibody drug conjugate (ADC). The antibody maytansinoid conjugate, IMGN529 consists of a CD37-binding monoclonal antibody, K7153A, with the DM1 maytansinoid cytotoxic agent attached via an SMCC linker. IMGN529 has demonstrated potent in vitro and in vivo anti-tumor activities supporting its development as a treatment for CD37-expressing lymphomas and CLL. Objective: The objectives of this study were to assess the potential utility of IMGN529 for treatment of lymphoma patients by: (1) determining the extent of CD37 expression in NHL tissue samples relative to CD20, a well-validated target for antibody-directed therapies against B-cell malignancies; (2) identifying xenograft models with similar CD37 expression as seen in NHL patient samples; and (3) evaluating IMGN529 efficacy in these xenograft models. Methods: CD37 expression in human lymphoma cell lines was determined by quantitative flow cytometry. Cell pellet controls were prepared from human lymphoma cell lines that represented a range of CD37 expression to establish a calibrated IHC method. Using this IHC method, CD37 and CD20 expression levels were evaluated on a panel of FFPE biopsy samples from patients with NHL and from NHL-derived tumor xenograft models. For all stained biopsy samples, the extent and intensity of immunostaining was evaluated and antigen expression levels were estimated by comparing to the quantified cell pellet controls. In vivo efficacy was evaluated in subcutaneous xenograft models in SCID mice. Results: Biopsy samples from patients with B-cell NHL including subtypes such as follicular lymphoma, diffuse large B-cell lymphoma, Burkitt9s lymphoma, and mantle cell lymphoma showed similar prevalence for CD37 and CD20. Most biopsy samples from patients with NHL showed strong and uniform expression levels of the CD37 antigen. As expected, no positive CD37 staining was seen in multiple myeloma and T-cell lymphoma biopsies. IMGN529 was highly active at a single dose of 10 mg/kg in tumor xenografts with CD37 expression levels similar to NHL patient samples. Conclusion: CD37 is expressed widely in B-cell lymphomas, including all major subtypes, which confirms CD37 as target for development of a therapeutic antibody drug conjugate. The CD37-targeting ADC, IMGN529, shows strong in vivo efficacy in lymphoma xenograft models with comparable CD37 expression levels as seen in lymphoma patients. Together these data support the development of IMGN529 for treatment of NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2513. doi:1538-7445.AM2012-2513

Abstract 3617: Evaluation of folate receptor 1 (FOLR1) expression by calibrated immunohistochemistry identifies candidate tumor subtypes for targeting by IMGN853, an anti-FOLR1-maytansinoid conjugate

Background: The folate receptor α (FOLR1) is a GPI-linked glycoprotein reported to be highly expressed in cancers including ovarian, renal, brain, lung, and breast carcinomas, while having restricted distribution in normal tissues, including kidney and lung. This expression profile suggests that FOLR1 may be an attractive target for development of a therapeutic antibody-drug conjugate. The antibody maytansinoid conjugate (AMC), IMGN853, consists of a FOLR1-binding monoclonal antibody, M9346A, conjugated to the maytansinoid, DM4, attached via the cleavable sulfo-SPDB linker. IMGN853 exhibited potent anti-tumor activity in preclinical studies, supporting its development as a treatment for FOLR1-expressing cancers. Objective: The aim of the study was to identify potential patient populations for treatment with IMGN853 by determining the level, cellular distribution, and incidence of FOLR1 expression in carcinomas. Methods: A calibrated immunohistochemical (IHC) staining method was developed for assessing FOLR1 expression in formalin-fixed paraffin-embedded (FFPE) tissues. Staining conditions were established wherein the intensity of FOLR1 membrane staining observed in tumor tissues could be related to FFPE cell pellet controls, prepared from human tumor cell lines expressing known levels of FOLR1 (quantified by flow cytometry). This calibrated IHC staining method was applied to FOLR1-positive controls and a panel of tumor tissues including ovarian, renal, brain, lung, and breast carcinomas. The extent, pattern, and intensity of immunostaining were evaluated for each tissue, and the level of FOLR1 expression was estimated using comparison to the cell pellet controls. Results: Biopsy samples from patients with ovarian cancer and non-small cell lung adenocarcinoma or bronchiolalveolar carcinomas of the lung express significant levels of membrane-associated FOLR1. Expression in these indications was frequently observed exhibiting high, homogeneous, and cell membrane-localized patterns – properties considered favorable for AMC-targeting. In particular, a high percentage of the most common subtypes of ovarian cancer, serous (79%) and endometrioid (70%), expressed high (> 260,000 antigen sites per cell) and homogeneous levels of FOLR1. Likewise, a high percentage of the non-small cell lung adenocarcinoma (54%) and bronchioloalveolar carcinoma (100%) subtypes also expressed high (> 260,000 antigens per cell) levels. In contrast, FOLR1 expression in most non-small cell lung squamous carcinomas was undetectable. These findings support the development of IMGN853 for treatment of ovarian carcinomas and NSCL adenocarcinoma and bronchiolalveolar carcinomas, and provide a potential approach for assessing FOLR1 expression in patient tumors during clinical development. 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 3617. doi:10.1158/1538-7445.AM2011-3617

Abstract 5335: The antigen target of IMGN901, CD56, is expressed at significant levels in merkel cell carcinoma (MCC)

Background: IMGN901 consists of a CD56-binding monoclonal antibody, huN901, with a maytansinoid agent, DM1, attached via a stable disulfide linker, SPP. The CD56 antigen, also known as neural cell adhesion molecule (NCAM), is present in a number of malignancies including Merkel cell carcinoma (MCC), small cell lung carcinoma and other neuroendocrine tumors, ovarian cancers and multiple myeloma. Once bound to CD56, IMGN901 is internalized into a cancer cell and its DM1 is released to cause cell death via inhibition of tubulin polymerization. IMGN901 is currently in Phase I testing for the treatment of CD56-positive multiple myeloma and CD56-positive solid tumors. Six patients with MCC have received IMGN901, used as a single agent, in one of the Phase I trials underway. Clinical benefit was observed in 3 of these 6 patients: one complete response (CR), one partial response (PR) and one stable disease (SD). Objective: The goal of this study was to assess the potential utility of IMGN901 as a treatment for MCC by identifying the range of CD56 antigen expression levels in MCC. Methods: A calibrated immunohistochemical (IHC) staining method for CD56 was developed. CD56 staining conditions were established where 4 formalin fixed paraffin embedded (FFPE) MCC samples showed differential staining. FFPE cell pellet controls prepared from human tumor cell lines were next identified which showed CD56 staining in the same range as the MCC samples. The CD56 antigen level for each tumor cell line was subsequently quantified by flow cytometry. The calibrated IHC staining method was then applied to positive cell pellet controls and an extended panel of MCC tissues. For each resulting tissue section, the extent and intensity of immunostaining was evaluated and the CD56 expression level was estimated by comparing to the cell pellet controls. Results: The majority of samples from the MCC panel exhibited heterogeneous or homogeneous staining with significant levels of CD56 expression. Tissues from primary biopsy sites and from metastases showed comparable levels of CD56 expression. To date, biopsy samples have been acquired from 3 of the 6 MCC patients who have received IMGN901 (two IMGN901 non-responders and one responder) and these all showed high and homogeneous levels of CD56 expression. Conclusion: This IHC study indicates that the majority of MCC patient tissues express significant levels of CD56, supporting the development of the CD56-targeting compound, IMGN901, for this indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5335.