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SLC46A3 As a Potential Predictive Biomarker for Antibody–Drug

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SLC46A3 As a Potential Predictive Biomarker for Antibody–Drug Published OnlineFirst August 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1300 Cancer Therapy: Preclinical Clinical Cancer Research SLC46A3 as a Potential Predictive Biomarker for Antibody–Drug Conjugates Bearing Noncleavable Linked Maytansinoid and Pyrrolobenzodiazepine Warheads Krista Kinneer1, John Meekin1, Arnaud C. Tiberghien2, Yu-Tzu Tai3, Sandrina Phipps4, Christine Mione Kiefer4, Marlon C. Rebelatto5, Nazzareno Dimasi4, Alyssa Moriarty6, Kyriakos P. Papadopoulos6, Sriram Sridhar5, Stephen J. Gregson2, Michael J. Wick6, Luke Masterson2, Kenneth C. Anderson3, Ronald Herbst1, Philip W. Howard2, and David A. Tice1 Abstract Purpose: Antibody–drug conjugates (ADC) utilizing non- also examined in patient-derived xenograft and in vitro cleavable linker drugs have been approved for clinical use, and models of acquired T-DM1 resistance and multiple myeloma several are in development targeting solid and hematologic bone marrow samples by RT-PCR. malignancies including multiple myeloma. Currently, there Results: Loss of SLC46A3 expressionwasfoundtobea are no reliable biomarkers of activity for these ADCs other than mechanismofinnateandacquiredresistancetoADCs presence of the targeted antigen. We observed that certain cell bearing DM1 and SG3376. Sensitivity was restored in refrac- lines are innately resistant to such ADCs, and sought to tory lines upon introduction of SLC46A3, suggesting that uncover the underlying mechanism of resistance. expression of SLC46A3 maybemorepredictiveofactivity Experimental Design: The expression of 43 lysosomal than target antigen levels alone. Interrogation of primary membrane target genes was evaluated in cell lines resistant multiple myeloma samples indicated a range of SLC46A3 to ADCs bearing the noncleavable linker, pyrrolobenzodiaze- expression, including samples with undetectable levels like pine payload SG3376, in vitro. The functional relevance of multiple myeloma cell lines resistant to BCMA-targeting SLC46A3, a lysosomal transporter of noncleavable ADC DM1andSG3376ADCs. catabolites whose expression uniquely correlated with Conclusions: Our findings support SLC46A3 as a potential SG3376 resistance, was assessed using EPHA2-, HER2-, and patient selection biomarker with immediate relevance to BCMA-targeted ADCs and isogenic cells overexpressing or clinical trials involving these ADCs. Clin Cancer Res; 1–13. genetically inactivated for SLC46A3. SLC46A3 expression was Ó2018 AACR. Introduction treatment of erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2)- positive metastatic breast cancer (3), inotuzumab ozogamicin for Antibody–drug conjugates (ADC) combine a mAb with a the treatment of acute lymphoblastic leukemia (4), and gemtu- cytotoxic drug (warhead) to preferentially eliminate antigen-pos- zumab ozogamicin for the treatment of CD33-positive acute itive cells for the treatment of cancer (1). Four ADCs are approved myeloid leukemia (5). Upon binding to the targeted antigen on for clinical use: brentuximab vedotin for the treatment of Hodgkin the cell surface, ADCs prepared with enzymatically cleavable lymphoma (2), ado-trastuzumab emtansine (T-DM1) for the linkers (e.g., brentuximab vedotin) or acid-labile hydrazone lin- kers (e.g., inotuzumab ozogamicin and gemtuzumab ozogami- cin) are internalized and processed within the cell, releasing the 1Oncology Research, MedImmune, Gaithersburg, Maryland. 2Spirogen, QMB Innovation Centre, London, United Kingdom. 3The Jerome Lipper Multiple cytotoxic warhead after linker cleavage. Warheads released in this Myeloma Center and LeBow Institute for Myeloma Therapeutics, Dana-Farber manner are typically membrane permeable and capable of Cancer Institute, Harvard Medical School, Boston, Massachusetts. 4Antibody bystander killing (6–8). In contrast, ADCs with noncleavable Discovery and Protein Engineering, MedImmune, Gaithersburg, Maryland. linkers, such as T-DM1, rely upon proteolytic degradation of the 5 6 Translational Medicine, MedImmune, Gaithersburg, Maryland. South Texas antibody in the lysosome to release an amino acid linker warhead Accelerated Research Therapeutics, San Antonio, Texas. (9). These catabolites are generally not membrane permeable (10, Note: Supplementary data for this article are available at Clinical Cancer 11) and therefore require transport from the lysosome to reach Research Online (http://clincancerres.aacrjournals.org/). their intracellular target (12). Several ADCs utilizing noncleavable Corresponding Author: Krista Kinneer, MedImmune, 1 MedImmune Way, linkers are currently in clinical development and target both solid Gaithersburg, MD 20878. Phone: 301-398-4219. Fax: 301-398-9219. E-mail: tumors and hematologic malignancies, including diffuse large B- [email protected] cell lymphoma and multiple myeloma (refs. 13–16). doi: 10.1158/1078-0432.CCR-18-1300 Although T-DM1 therapy has shown significant clinical benefit, Ó2018 American Association for Cancer Research. most patients eventually relapse despite continued treatment www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1300 Kinneer et al. TNFRSF17 (BCMA) antibody was prepared using VH and VL Translational Relevance sequences retrieved from patent application US 2014/0105915 Although antibody–drug conjugates (ADC) utilizing the A1. Trastuzumab drug conjugate, T-DM1 (ado-trastuzumab payload DM1 have been approved for clinical use, and several emtansine), was purchased from Blue Door Pharma. All antibo- are in clinical development that target both solid tumors and dies used in this study, except for those conjugated to DM1 and hematologic malignancies, predictive biomarkers beyond monomethyl auristatin F (MMAF), were engineered for the site- antigen expression have yet to be identified. Herein, we show specific conjugation of two drugs per antibody as described by that loss of lysosomal transporter SLC46A3 expression is a Dimasi and colleagues (32). mechanism of innate and acquired resistance to ADCs bearing The PBD dimer SG3249 was synthesized at Novasep. mcMMAF the payloads DM1 or SG3376 (a PBD dimer) with nonclea- and DM1–succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- vable linkers, making SLC46A3 a potential biomarker for carboxylate (SMCC) were purchased from ALB Technology. patient selection in ongoing trials with ADCs bearing these SG3541 was prepared at Spirogen as described in the Supple- payloads. mentary Materials. SG3376 was prepared at Spirogen according to published methods (31). Conjugation of SG3249 (27), SG3376 (31), and SG3541 was performed as described previously (32, 34). (17–19). Currently, the only predictive biomarker for response to To generate the mcMMAF ADCs with a drug to antibody ratio T-DM1 is overexpression of HER2 (20), which is detected by (DAR) of 4 at the antibodies' native cystines, the antibodies were measurement of HER2 protein levels by IHC or of HER2 gene reduced using 2.5-molar equivalent (MEq) tris(2-carboxyethyl) – amplification by fluorescent or chromogenic in situ hybridization phosphine in PBS pH 7.2 1 mol/L EDTA for 1 hour at 37 C. The (21). Acquired resistance to T-DM1 has been evaluated in several reaction mixture was then incubated with 4 equivalents of – – preclinical studies, and due to the complexity of this molecule, mcMMAF for 1 hour at 25 C in PBS pH 7.2 1 mmol/L EDTA several potential mechanisms have emerged: (i) antigen loss and/ 10% v/v dimethylsulfoxide. The reaction was quenched by the or downregulation, (ii) increased expression of drug transporters addition of 4 equivalents (over the mcMMAF) of N-acetyl MDR1 (ABCB1) and MRP1 (ABCC1), (iii) defects in ADC traf- cysteine. ficking, and/or (iv) changes in receptor and signaling pathways To generate the DM1 ADCs with DAR 4 at the antibody lysines, (22). In addition, aberrations in lysosomal pH and proteolytic the antibodies at 2 mg/mL in 1 mmol/L borate buffer, pH 8.5, activity (23) and loss of the lysosomal transporter solute carrier were incubated at room temperature with 8 MEq DM1. The family 46 member 3 (SLC46A3; refs. 12, 24) have been observed conjugation reaction was monitored with reduced reverse-phase in T-DM1–resistant cell lines, highlighting a potential role for the LC/MS as described previously (32, 34), and the reaction was lysosome in T-DM1 resistance. stopped when a DAR of 4 was reached (total reaction time, fi Until recently, the cytotoxic warheads used for most ADCs in approximately 1 hour). The ADCs were puri ed and characterized clinical development were based on antimitotic agents, namely, analytically as described previously (32, 34). the auristatins and maytansines (1). ADCs prepared with the cleavable linker dimeric pyrrolobenzodiazepine (PBD) payloads Human cells talirine (SGD-1910; refs. 25, 26) or tesirine (SG3249; ref. 27) are Primary multiple myeloma samples were obtained from now entering the clinic. PBD dimers are a class of compounds that patients after informed consent was obtained, in accordance with form sequence-selective DNA crosslinks in the minor groove of the Declaration of Helsinki and under the auspices of a protocol DNA, leading to cell death (28, 29) and thereby offering an approved by the Dana-Farber Cancer Institute Review Board. fi alternative mechanism of action for tumor targeting. New classes Myeloma cells were puri ed before RNA isolation as described of PBD payloads with noncleavable linkers (30), including the previously (16). RT112, HT29, and KYSE-410 cells
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