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Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cell Function Against Multiple Myeloma Is Enhanced in the Presence of Lenalidomide

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Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cell Function Against Multiple Myeloma Is Enhanced in the Presence of Lenalidomide Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: Anti–B-cell maturation antigen chimeric antigen receptor T cell function against 2 multiple myeloma is enhanced in the presence of lenalidomide 3 Authors: 4 Melissa Works, Neha Soni, Collin Hauskins, Catherine Sierra, Alex Baturevych, Jon C. 5 Jones, Wendy Curtis, Patrick Carlson, Timothy G. Johnstone, David Kugler, Ronald J. 6 Hause, Yue Jiang, Lindsey Wimberly, Christopher R. Clouser, Heidi K. Jessup, Blythe 7 Sather, Ruth A. Salmon, and Michael O. Ports 8 9 Affiliations: 10 Juno Therapeutics, A Celgene Company, Seattle, Washington. 11 12 Running title: 13 Lenalidomide enhances anti-BCMA CAR T function 14 Keywords: multiple myeloma; lenalidomide; CAR T; BCMA; Immunology 15 Financial support: This study was funded by Juno Therapeutics, A Celgene Company. 16 17 Corresponding author: Melissa Works 18 400 Dexter Ave N Suite 1200 19 Seattle, WA 98109 20 [email protected] 21 Phone: 206-566-5731 22 23 Disclosure of conflicts of interest: All authors are employed by and have equity interest 24 in Juno Therapeutics, Inc, A Celgene Company. 25 Abstract: 246/250 words 26 Manuscript: 4540/5000 27 Figures: 6/7 1 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 References: 32/50 2 Supplementary Document: Yes 3 4 Portions of this work were presented in poster form at the 59th annual meeting of the 5 American Society of Hematology; Atlanta, GA; December 9, 2017. 6 2 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Key points 2 Anti-BCMA CAR T cell function is enhanced by lenalidomide during acute or chronic 3 stimulation. 4 RNA- and ATAC-seq data support elements of T-effector and memory-cell molecular 5 signatures. 3 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Abstract 2 Anti–B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells have 3 shown promising clinical responses in patients with relapsed/refractory multiple 4 myeloma. Lenalidomide, an immunomodulatory drug, potentiates T cell functionality, 5 drives antimyeloma activity, and alters the suppressive microenvironment; these 6 properties may effectively combine with anti-BCMA CAR T cells to enhance function. 7 Using an anti-BCMA CAR T, we demonstrated that lenalidomide enhances CAR T cell 8 function in a concentration-dependent manner. Lenalidomide increased CAR T effector 9 cytokine production, particularly under low CAR stimulation or in the presence of 10 inhibitory ligand programmed cell death 1 ligand 1. Notably, lenalidomide also enhanced 11 CAR T cytokine production, cytolytic activity, and activation profile relative to untreated 12 CAR T cells in chronic stimulation assays. This unique potentiation of both short-term 13 CAR T activity and long-term functionality during chronic stimulation prompted 14 investigation of the molecular profile of lenalidomide-treated CAR T cells. Signatures 15 from RNA sequencing and assay for transposase-accessible chromatin using 16 sequencing indicated that pathways associated with T-helper 1 response, cytokine 17 production, T cell activation, cell-cycle control, and cytoskeletal remodeling were altered 18 with lenalidomide. Finally, study of lenalidomide and anti-BCMA CAR T cells in a 19 murine, disseminated, multiple myeloma model indicated that lenalidomide increased 20 CAR T cell counts in blood and significantly prolonged animal survival. In summary, 21 preclinical studies demonstrated that lenalidomide potentiated CAR T activity in vivo in 22 low-antigen or suppressive environments and delayed onset of functional exhaustion. 4 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 These results support further investigation of lenalidomide and anti-BCMA CAR T cells 2 in the clinic. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 5 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Introduction 2 Despite improvements in the treatment of newly diagnosed multiple myeloma, it remains 3 uncured, and nearly all patients relapse and become resistant to available treatments 4 (1). Based on the encouraging activity of chimeric antigen receptor (CAR) T cells 5 targeting CD19 in non-Hodgkin lymphoma, CAR T cells targeting plasma cells 6 expressing B-cell maturation antigen (BCMA) have been developed for multiple 7 myeloma (2). Although BCMA CAR T cells have shown promise in the clinic, an 8 immunosuppressive myeloma tumor microenvironment, including programmed cell 9 death 1 ligand 1 (PD-L1) expression (3), and the potential for activation-induced 10 exhaustion may limit the durable responses of CAR T cells in some patients (4). 11 Lenalidomide is an immunomodulatory drug indicated for the treatment of multiple 12 myeloma (5); it has pleiotropic effects that directly impair primary tumor growth and 13 modulate the immunosuppressive tumor microenvironment to help facilitate a more 14 robust antitumor inflammatory response (6,7). Studies have shown that lenalidomide 15 can directly increase in vitro T cell function, even in heavily treated patients with 16 progressive disease, irrespective of immunomodulatory-drug refractory status (8). 17 Lenalidomide has also been shown to increase acute in vitro and in vivo CAR T cell 18 functionality in several model systems with varying CAR constructs and indications 19 (9,10). For example, lenalidomide in combination with CS1-directed CAR T cells with a 20 CD28z endodomain was associated with increased secretion of T-helper (Th) 1– 21 associated cytokines, decreased secretion of Th2–associated cytokines, and increased 22 survival in tumor-bearing mice (10). Additional studies are required to refine 23 lenalidomide’s mechanism of action and investigate its application in a chronic CAR T– 6 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 stimulation setting in conditions of varying antigen density or in the presence of 2 inhibitory ligands such as PD-L1. 3 CAR T cells have unique functional properties that can be altered by the characteristics 4 of the single-chain variable fragment, choice of transmembrane and endodomain, and 5 manufacturing process—all of which determine CAR T fitness during long-term 6 stimulation (11). In this study, we sought to characterize a novel anti-BCMA CAR 7 construct and determine short- and long-term effects of lenalidomide on CAR T function. 8 In addition, immunomodulatory drugs have been shown to directly induce CD28 tyrosine 9 phosphorylation (12), suggesting that these drugs impinge on costimulatory signaling 10 pathways; however, the role of a 41BBz endodomain during immunomodulatory drug 11 application is unclear. To understand the complex nature of lenalidomide’s mechanism 12 of action, we applied RNA sequencing (RNA-seq) and assay for transposase-accessible 13 chromatin using sequencing (ATAC-seq) technology to determine whether these 14 functional differences were associated with changes in the regulatory networks involved 15 in T cell function and activation. Finally, we examined concurrent and delayed 16 administration of lenalidomide with a subcurative dose of anti-BCMA CAR T injection to 17 further identify potential clinical applications and dosing strategies. 18 19 Materials and methods 20 In vitro cytolytic, cytokine, and flow cytometry CAR T assessment 21 T cells obtained from peripheral blood samples from consenting healthy adult donors 22 and a patient with multiple myeloma refractory to pomalidomide were transduced to 7 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 express a construct containing an extracellular BCMA-binding single-chain variable 2 fragment and intracellular 41BBz endodomain (65%-76% CAR+, Supplementary Figure 3 1A, Supplementary Table 1, Supplementary
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