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
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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
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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.
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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
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1 These results support further investigation of lenalidomide and anti-BCMA CAR T cells
2 in the clinic.
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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
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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
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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 Methods). Human materials used in this
4 research were received by the researchers in a fully de-identified manner from
5 commercial repositories or under unrelated IRB-approved clinical studies from adults
6 who consented to testing of their donated samples for future research purposes.
7 Cultures were established with an effector-to-target ratio of 0.3:1 or 1:1 with OPM-2 or
8 RPMI-8226 multiple myeloma target cells. Co-cultures with increased effector cell
9 counts relative to target cell counts results in rapid clearance of BCMA+ target cells.
10 Therefore, E:T ratios of 1.0 and 0.3 were selected to ensure that more CAR T cells
11 would receive an activating stimulus for evaluation with lenalidomide. Lenalidomide
12 (Sigma) was titrated across and above the clinical maximum concentration (1.9 µmol/L
13 for a 25-mg oral dose in patients with multiple myeloma) to assess the functional range
14 of the drug (13). Cell-free supernatants were collected after 24 hours. Experiments were
15 performed 2 to 3 times in 4 donors.
16 Unless noted, anti-BCMA CAR T cells were stimulated with 50 µg/mL BCMA beads for
17 the time indicated at a bead:CAR+ T cell ratio of 1:1. For PD-L1 experiments, 50 µg/mL
18 PD-L1 or control immunoglobulin G was coupled along with 50 µg/mL BCMA. For
19 prestimulation experiments, after 7 days of incubation, cells were debeaded, washed,
20 and cocultured with RPMI-8226-NucLight Red target cell lines in the presence of 1
21 µmol/L lenalidomide or vehicle control. Experiments were performed twice in 3 donors.
22 For surface phenotype analysis, anti-BCMA CAR T cells were cultured with BCMA
23 beads for 7 days, stained with a live/dead dye (Invitrogen; Thermo Fisher Scientific,
8
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1 Carlsbad, CA), BCMA–fractured crystallizable (Fc), antibodies for surrogate CAR
2 marker, CD3, CD4, CD8, CD25, PD-1, TIM3, and LAG3 (BD Biosciences, Franklin
3 Lakes, NJ) and then analyzed on a Fortessa flow cytometer (BD Biosciences). For
4 intracellular cytokine staining, cells were stimulated on BCMA beads for 24 hours, with
5 protein transport cocktail (BD Biosciences) added in the final 4 hours of incubation.
6 Cells were then stained with live/dead dye and surface markers (surrogate CAR marker,
7 CD3, CD4, CD8, CD25, PD-1), fixed/permeabilized (BioLegend, San Diego, CA), and
8 stained for intracellular interleukin (IL) 2, interferon-γ (IFN-γ), and tumor necrosis factor
9 α (TNF-α) (BioLegend). Experiments were performed twice in 3 donors.
10 Serial stimulations
11 Anti-BCMA CAR T cells were plated with irradiated MM1.S target cells at an effector-to-
12 target ratio of 1:2 in the presence of lenalidomide (0.1 µmol/L). Every 3 to 4 days, CAR+
13 cells were enumerated, phenotyped by flow cytometry, and replated with freshly thawed
14 and irradiated MM1.S target cells and lenalidomide. Twenty-four hours following
15 replating on days 5, 8, and 15, cell-free supernatant was assessed for cytokine levels by
16 Meso Scale Discovery (Rockville, MD). Following incubation on day 4, a sample was
17 stained with a live/dead dye and panel of surface markers (epidermal growth factor
18 receptor [EGFR], CD3, CD4, CD8). Fluorescently labeled count-bright beads were
19 added to each sample to get the absolute counts of the total CD3+, Erb+, BCMA+, and
20 CAR+ cells. Replating was maintained for 28 days or until the cell count was < 50,000
21 cells. Experiments were performed 3 times in 3 donors.
22 RNA-seq and ATAC-seq assays
9
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1 Anti-BCMA CAR T cells were cultured in the presence or absence of BCMA beads for
2 either 24 hours or 7 days with or without 1 µmol/L lenalidomide. Strand-specific, poly-A-
3 selected RNA-seq reads were trimmed, mapped to hg38, and quantified using
4 ArrayStudio (OmicSoft, Cary, NC). ATAC-seq was performed according to published
5 methodology (14) (See Supplementary Methods). Experiments were performed twice in
6 3 and 4 donors.
7 In vivo OPM-2 tumor model
8 All animal studies were conducted in accordance with protocols approved by the
9 Institutional Animal Care and Use Committee. NOD.Cg-PrkdcscidIL-2rgtm1Wjl/SzJ mice
10 (NSG; Jackson Laboratory, Bar Harbor, ME) were injected intravenously with 2 × 106
11 OPM-2/luciferase cells and allowed to engraft for 14 days prior to intravenous CAR T
12 infusion. One day prior to or 14 days following injection with 1 × 106 CAR T or mock
13 control T cells, animals were dosed (intraperitoneally) daily for 50 days with 10 mg/kg
14 lenalidomide in phosphate-buffered saline. Blood was collected for quantitation of
15 circulating CAR T cells, and cells were stained with antibodies to exclude mouse-
16 specific cells (H-2kd, TER-119, and muCD45) and analyzed by flow cytometry (See
17 Supplementary Methods).
18 Statistics
19 Linear fixed-effect or mixed-effect models (15), a more flexible technique related to
20 more traditional nested or repeated measures ANOVA methods, were used to assess
21 the significance of lenalidomide treatments on cytolytic activity and cytokine production,
22 with treatment, and time treated as fixed effects and animal and donor treated as
10
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1 random effects, nested with time when repeated measurements were derived from the
2 same animal. P values were obtained by likelihood ratio tests comparing the full model
3 with the effect of interest against the model without the effect of interest. Flow-cytometry
4 median fluorescence intensity values were log2 transformed and bioluminescence
5 values were log10 transformed to better approximate normality. Survival analyses were
6 performed (16) with log-rank testing to determine the significance of the effect of
7 lenalidomide treatments on survival curves.
8 Results
9 Anti-BCMA CAR T function is enhanced by lenalidomide in the presence of BCMA-
10 expressing myeloma cell lines
11 Cytolytic activity and cytokine production of lenalidomide-treated CAR T cells
12 transduced with the anti-BCMA CAR were evaluated in vitro in the presence of BCMA-
13 expressing multiple myeloma cell lines with varying sensitivity to lenalidomide
14 (Supplementary Methods). Multiple donors were assessed to evaluate donor-dependent
15 effects of lenalidomide, including CAR T product manufactured from an
16 immunomodulatory drug-refractory patient. An additional scfv was tested in the
17 presence of lenalidomide to confirm the effects were not binder-specific (Supplementary
18 Figure 1B). Increased anti-BCMA CAR T cytolytic activity against OPM-2 target cells
19 titrated with increased concentrations of lenalidomide across all donors at the 0.3:1 E:T
20 ratio (P = 6.2 × 10-5; Figure 1A). Donor 1 and 2 demonstrated the least efficient target
21 cell killing for OPM-2 cells (Supplementary Figure 1C) and had increased activity with
22 lenalidomide at a 1:1 ratio (Supplementary Figure 1D). In addition, all CAR T donors
23 (baseline shown in Supplementary Figure 1E) had significantly increased IFN-γ, IL-2,
11
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1 and TNF-α production in a lenalidomide concentration–dependent manner upon
2 coculture with OPM-2 cells (P < .002; Figure 1B). The treatment effect of lenalidomide
3 on CAR T cytolytic activity appeared to be donor dependent in coculture with RPMI-
4 8226 (Figure 1C), with the patient donor showing a significant increase in cytolytic
5 activity (P = 1.9 × 10-8). Notably, cytokine production by CAR T cells in RPMI-8226
6 coculture (baseline shown Supplementary Figure 1F) was significantly increased across
7 all donors and cytokines upon treatment with lenalidomide (P < .003; Figure 1D).
8 Importantly, addition of lenalidomide did not alter BCMA target expression
9 (Supplementary Figure 2). These results demonstrated that when anti-BCMA CAR T
10 cells were stimulated with 0.3:1 and 1:1 effector-to-target ratios, addition of lenalidomide
11 increased effector functionality of CAR T cells across several metrics of CAR T function,
12 including cytolytic activity and cytokine production.
13 Lenalidomide potentiation of anti-BCMA CAR T cytokine expression is dependent on
14 stimulation strength
15 The CAR T–intrinsic treatment effects of lenalidomide on cytokine production for the
16 CD4+ and CD8+ CAR T populations were next evaluated in the absence of target cells.
17 As lenalidomide can directly limit multiple myeloma cell viability, a CAR-specific
18 stimulation reagent of recombinant human BCMA was used to assess the direct effect
19 of lenalidomide on activated CAR T cells in the absence of BCMA-expressing target
20 cells. To this end, recombinant human BCMA–labeled beads were developed to
21 stimulate CAR T cells and provide the means to titrate both the magnitude of stimulation
22 (low [5 µg/mL], medium [50 µg/mL], and high [200 µg/mL]) and the concentration of
23 lenalidomide (0.1 and 1.0 µmol/L). At a medium stimulation condition, we measured
12
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1 secreted cytokine production and observed a mean 200% increase in IL-2 and TNF-α
2 concentrations compared with vehicle control, with donor-dependent increases in IFN-γ
3 (Supplementary Figure 3). Anti-BCMA CAR T cells activated with BCMA beads showed
4 stimulation level–dependent effects, with the low, 5- µg BCMA beads causing lower
5 CAR T CD25 expression and intracellular IFN-γ cytokine staining compared with
6 medium- (50 µg) and high- (200 µg) BCMA beads (Figure 2A-B, left). Lenalidomide
7 significantly (P < .05) increased the percentage of IFN-γ+ and TNF-α+ intracellular
8 staining at multiple stimulation levels for both CD4+ (Figure 2A) and CD8+ (Figure 2B)
9 CAR T cells. In the absence of stimulation, lenalidomide had no effect on CAR T
10 cytokine staining, indicating that cytokine enhancement provided by lenalidomide
11 requires stimulation.
12 Inhibitory receptors can alter T cell receptor–mediated activation and limit the effector
13 functionality of T cells (17). We explored whether the lenalidomide-induced potentiation
14 of CAR T activation and cytokine production could override PD-L1–mediated inhibition.
15 Evaluation of both healthy and patient donor CAR T cells demonstrated that addition of
16 recombinant PD-L1 to recombinant BCMA beads significantly reduced IFN-γ, IL-2, and
17 TNF-α levels (P< .006; Figure 2C-D). Importantly, lenalidomide treatment potentiated
18 secreted cytokine levels beyond those from CAR T cells treated with vehicle in the
19 presence of PD-L1 (P < .007).
20 Anti-BCMA CAR T function during serial and chronic stimulation was prolonged by
21 lenalidomide
22 Previous studies indicate that performance in a serial stimulation assay may represent
23 CAR T fitness and in vivo efficacy (18). Notably, after repeated stimulation with MM1.s
13
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1 target cells, lenalidomide increased CAR T expansion on average by 0.82 population
2 doublings over 28 days across 5 donors relative to controls (P = 2.8 x 10-8; Figure 3A).
3 Multiple samples at intermediate time points were collected to assess cytokine
4 production during serial stimulation until a donor had insufficient cells to continue the
5 assay in the vehicle-treated group. Increased cell counts were associated with a
6 significant increase in IL-2, IFN-γ, and TNF-α production per cell in the media (P < 1.2 x
7 10-9; Figure 3B-D).
8 We also developed a novel long-term chronic stimulation assay designed to diminish
9 anti-BCMA CAR T effector function. We observed a limited increase in BCMA CAR T
10 cytolytic activity against RPMI-8226 cells in the presence of lenalidomide in an acute
11 assay (Figure 1); however, CAR T prestimulation appears to exhaust the cells and
12 results in decreased functionality. Prestimulated CAR T cells showed decreased
13 cytolytic activity (P = 2.1 × 10-4) and IFN-γ cytokine production (P = .03) compared with
14 freshly thawed anti-BCMA CAR T cells (Figure 4A), indicating that chronic
15 prestimulation leads to functional impairment. CAR T cells were also prestimulated with
16 BCMA beads with 1 µmol/L lenalidomide prior to analysis of cytolytic activity and
17 cytokine production. Notably, the presence of lenalidomide during the prestimulation
18 period preserved cytolytic function (P = .04), and a trend was observed toward
19 increased cytokine production compared with cells exposed to vehicle during the
20 prestimulation period (Figure 4B-D).
21 The phenotype of anti-BCMA CAR T cells stimulated for 7 days with BCMA beads was
22 assessed, and the addition of lenalidomide significantly increased CAR+ viability of anti-
23 BCMA CAR T material across 3 healthy donors (P = .04; Figure 4E). The addition of
14
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1 lenalidomide did not alter the total cell count across all donors (Figure 4E) in this 7-day
2 period, and no significant differences were observed in percentage CAR+ between
3 vehicle- and lenalidomide-treated CAR T cells or among CAR+ cells in memory
4 subtypes by classification with CD45RA or CD27 by CCR7 (Supplementary Figure 4).
5 Flow cytometric analysis across CAR T donors indicated that the addition of
6 lenalidomide functionally altered the balance between activation and immunoregulatory
7 markers by increasing the surface expression of TIM3 in the CD8+ population (P = 4.0 ×
8 10-4), with mixed effects on the CD4+ CAR+ population (Figure 4F). Across all donors
9 and in both the CD4+ and CD8+ CAR+ populations, lenalidomide increased CD25 (CD4+
10 and CD8+; P = 2.2 × 10-16) and the percentage positive for LAG3 expression (CD8+ P <
11 .03; CD4+ P = .002). Notably, a decrease in the percentage of PD-1+ cells was also
12 observed in the CD4+ population (P = .04), with 2 of 3 donors showing a decrease in the
13 CD8+ population as well.
14 Anti-BCMA CAR T RNA-seq and ATAC-seq profiles were altered by lenalidomide after
15 short-term and chronic stimulation
16 Because few phenotypic changes were noted by fluorescence-activated cell sorting
17 following the addition of lenalidomide in the context of antigen-specific stimulation, we
18 decided to employ unbiased transcriptomic and epigenomic analyses to further assess
19 features that could underlie the enhanced functionality. Investigation of the molecular
20 signature of lenalidomide-treated anti-BCMA CAR T cells was assessed following short-
21 term (24-hour) or chronic (7-day) stimulation, as described above. Principal component
22 analysis demonstrated clustering based on stimulation (stimulation or no stimulation)
23 and time (24 hours or 7 days) for both the RNA-seq (Figure 5A; GSE113281) and
15
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1 ATAC-seq (Figure 5C; GSE113853) data sets. Next, we examined the role of
2 lenalidomide after 24 hours or 7 days of stimulation after accounting for donor-to-donor
3 variability. RNA-seq analysis showed alteration of a small set of genes (214) at 24
4 hours, and a larger number of genes (583) changed after 7 days of stimulation in the
5 presence of lenalidomide (Figure 5B). Notably, ATAC-seq analysis revealed a limited
6 set of chromatin accessibility changes associated with lenalidomide treatment after 24
7 hours of stimulation, with a dramatic change in profile and an increase in the number of
8 sites with changes in chromatin accessibility after 7 days of stimulation in the presence
9 of lenalidomide (Figure 5D). To further identify specific transcriptional changes
10 associated with lenalidomide treatment, gene ontology analysis was applied to the
11 RNA-seq data set. Pathways associated with T cell chemotaxis (leukocyte
12 extravasation, integrin, integrin-linked kinase, and C-X-C motif chemokine receptor 4–
13 associated gene sets), intracellular signaling, and cytoskeleton (Rac/Rho/Cdc42) were
14 upregulated in the presence of lenalidomide within 24 hours of stimulation compared
15 with vehicle controls (Figure 5E). These data support an increase in inducible
16 costimulator (ICOS)–related signaling pathways—a finding that is in line with previous
17 publications demonstrating an increase in ICOS and ICOS ligand in the CD3+
18 population of peripheral blood mononuclear cells treated with lenalidomide ex vivo (19).
19 After 7 days of stimulation, lenalidomide upregulated pathways associated with Th1 T
20 cell response and costimulation while decreasing Th2-associated gene signatures
21 (Figure 5F).
22 To determine whether chromatin accessibility correlated with the transcriptional
23 changes observed during lenalidomide treatment, we integrated the ATAC-seq and
16
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1 RNA-seq data from our chronic 7-day analysis (Figure 5G). Across donors, we
2 observed a significant increase in chromatin accessibility across multiple loci, including
3 those associated with IFN-γ and IL-2RA (CD25), and these changes were correlated
4 with a significant increase in transcription. Importantly, the upregulation of IFN-γ and
5 CD25 were concordant with previous findings from chronic stimulation experiments
6 where lenalidomide treatment resulted in significantly higher proportions of cells
7 expressing these markers. We also observed a decrease in CD69 and CCR7 chromatin
8 accessibility and gene transcription on lenalidomide treatment. Last, we analyzed the
9 ATAC-seq data set for motif enrichment and observed enrichment for a number of
10 motifs bound by multiple factors associated with T cell activation, including AP-1/Jun
11 and nuclear factor κB (Figure 5H) (20).
12 Subcurative dose of anti-BCMA CAR T demonstrated improved tumor clearance and
13 survival in vivo when concurrently dosed with lenalidomide
14 Finally, in order to assess in vivo CAR T function by lenalidomide, mice implanted with
15 OPM-2 tumors were dosed with a subcurative dose of anti-BCMA CAR T cells. Mice
16 with established tumors were dosed daily with lenalidomide 1 day prior or 14 days
17 following injection with a subcurative dose of 1 × 106 anti-BCMA CAR T cells (Figure
18 6A). The addition of concurrent lenalidomide led to a significant decrease in tumor
19 burden for donor 1 (P = .02) and increased survival for donor 1 (P = .057) and donor 2
20 (P = .04) compared with vehicle-treated animals injected with anti-BCMA CAR T alone
21 (Figure 6B-E). Animals on the concurrent lenalidomide dosing regimen also showed
22 increased CAR T counts in the peripheral blood after 7 days (P = 7.3 × 10-6) but not at
23 later time points (Figure 6F-G). Lenalidomide had a small but significant mock CAR T
17
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1 effect on tumor burden for donor 1 alone (P = .003). The addition of delayed dosing of
2 lenalidomide did not improve tumor clearance and survival for either CAR T donor,
3 suggesting that the benefit of this combinatorial approach relied on concurrent
4 administration.
5 Discussion
6 Our studies further explore the mechanism of action and applications of lenalidomide in
7 combination with CAR T cells. In anti-BCMA CAR T cells with a 41BB/CD3z-containing
8 endodomain, we observed a rapid increase in cytokine production following stimulation
9 with either myeloma target cells or direct CAR stimulation via antigen-coated beads.
10 Notably, lenalidomide increased cytokine production and activation across multiple
11 assays at a clinically relevant concentration, indicating an increase in effector function.
12 In addition, an immunomodulatory drug–refractory sample derived from a patient with
13 multiple myeloma also demonstrated increased in vitro functionality in the presence of
14 lenalidomide, indicating that refractory tumor status may be independent of the effects
15 of lenalidomide on CAR T cells. Previous studies demonstrated that increased T cell
16 cytokine production associated with lenalidomide was partly due to the degradation of
17 the transcription factors Ikaros and Aiolos by Cereblon (21). In addition, Ikaros has been
18 shown to alter the threshold for activation of T cells downstream of T cell receptor and
19 IL-2 receptor signaling as well as protein kinase C, phosphatidylinositol 3-kinase, and
20 calcineurin signaling (22). Furthermore, our studies support the hypothesis of
21 immunomodulatory drug–lowered activation threshold in functional assays controlling
22 CAR stimulation. We also demonstrated that, in the presence of PD-L1 engagement,
23 the addition of lenalidomide potentiated cytokine production beyond control levels,
18
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1 suggesting that this combinatorial approach may override suppressive inputs from the
2 microenvironment to sustain antitumor functionality.
3 Chronic and serial stimulation assays may recapitulate the repeated stimulation to
4 which a CAR T cell is exposed in patients’ tumors and allow for examination of an
5 exhaustion-like state of the CAR T cell. Our functional data support a prolonged period
6 for CAR T cell cytolytic activity and cytokine production in the presence of lenalidomide.
7 Prolonged lenalidomide treatment period increased IL-2 across serial stimulation and
8 chronic stimulation assays. The increased IL-2 production over time may provide a
9 mechanism to sustain T cell effector function over chronic stimulation; this mechanism
10 agrees with previous studies demonstrating that low IL-2 production by CAR T cells was
11 associated with exhaustion (23). Exposure to lenalidomide in chronic or serial
12 stimulation assays also resulted in increased TNF-α and IFN-γ, increased viability, and
13 decreased PD-1 expression on the CAR T cell surface. Notably, characterization of
14 CD19-directed CAR T cells determined that the PD-1–negative CAR T population was
15 associated with therapeutic response (24). These results were observed in the
16 presence of increased TIM3 and LAG3—markers previously shown to be associated
17 with T cell exhaustion (25). The duality of these exhaustion-associated makers may
18 indicate that the addition of lenalidomide to CAR T cells leads to an alternative
19 differentiation or activation state outside the canonical states of T cell differentiation. In
20 other words, lenalidomide may affect these “exhaustion” signaling pathways
21 independently of PD-1, or, alternatively, these markers are not indicative of functional
22 exhaustion in these anti-BCMA CAR T cells. In support of these data, previous studies
19
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1 have shown that a dissociation between surface markers and functional assessment of
2 exhaustion and molecular dissection of cell state may be more informative (26).
3 In addition to functional assays, the RNA- and ATAC-seq studies resulted in a number
4 of insights into possible mechanisms for lenalidomide-induced increases in CAR T
5 function. First, the number of transcriptional and chromatin accessibility changes
6 associated with stimulation and time were predominant compared with the effects of
7 lenalidomide, indicating a relatively subtle effect of lenalidomide on transcriptional
8 networks. Second, the changes associated with lenalidomide were broad, including
9 early changes in transcripts associated with cytoskeletal remodeling and chemotaxis.
10 After chronic stimulation, a distinct transcriptional signature emerged that included a
11 decrease in transcripts associated with the Th2 response, G2/M checkpoint, and ATM
12 along with an increase in Th1, peroxisome proliferator–activated receptor γ, and actin
13 cytoskeleton–associated genes. These effects may support a role for lenalidomide
14 treatment and cell-cycle control and T cell activation (27). Previous studies have also
15 demonstrated the effects of immunomodulatory drugs on Th1- and Th2-associated
16 signatures as well as changes in elements associated with cytoskeletal remodeling and
17 T cell migration (10,28). The demonstrated early alterations in cytokine production by
18 lenalidomide may contribute to an altered T cell state that is able to simultaneously
19 enhance aspects of both memory and effector function (29). Overall, these results
20 suggest that additional factors beyond those previously reported are involved in the
21 lenalidomide-induced prolongation of CAR T function, including possible changes in
22 cell-cycle control.
20
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1 The application of ATAC-seq provided further insights into potential mechanisms of
2 action of lenalidomide. Although both stimulation and time were the predominant drivers
3 of chromatin accessibility changes, lenalidomide treatment was associated with
4 increases in chromatin accessibility in loci enriched in motifs associated with T cell
5 activation and function after chronic stimulation. These epigenetic changes were
6 coincident with the marked functional changes in CAR T cells incubated with
7 lenalidomide. Alterations in chromatin accessibility signatures have been associated
8 with T cell exhaustion and may be a more robust indicator of exhaustion compared with
9 T cell surface ligand expression (30). These data demonstrated that chronic stimulation
10 with lenalidomide resulted in increased chromatin accessibility and gene expression of
11 IL-2 and CD25 and decreased gene expression and chromatin accessibility of CCR7
12 and CD69. Previous studies suggested that CCR7-expressing cells produced higher
13 levels of IL-2 (31); however, the current study indicates that the IL-2 pathway could be
14 altered independently by lenalidomide, resulting in an alternative T cell state. CD69, a
15 marker of T cell activation, has a nuclear factor κB–responsive element that is required
16 for the CD69 response to TNF-α (32). The closing of CD69-associated chromatin and
17 decrease in transcripts may be a reaction to sustained increases in TNF-α production by
18 CAR T cells cultured with lenalidomide, or it may be a T cell response to increased
19 activation in the presence of lenalidomide. Lenalidomide-treated cells demonstrated
20 increased transcription factor motif enrichment of T cell activation–associated factors,
21 supporting the idea that these cells are exposed to sustained activation signaling.
22 Overall, the lenalidomide-induced CAR T cell state has elements of both effector T cell
23 function, including increased IFN-ɣ and TNF-α production, and memory T cell function,
21
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1 including increased IL-2 and long-term proliferation. Additional studies are underway to
2 determine more about the functional consequences of this alternative CAR T cell state
3 and the associated gene expression and epigenetic changes.
4 Finally, we observed an increase in function by a subcurative dose of anti-BCMA CAR T
5 cells in the OPM-2 orthotopic animal model with the addition of lenalidomide. Early
6 pharmacokinetic (PK) measurements indicated an increase in CAR T counts in the
7 blood, which were associated with improved tumor control and survival. The
8 combination of increased CAR T PK and increased functionality of CAR T cells, as
9 observed in in vitro studies, may have led to improved control over tumor growth
10 following a subcurative dose of CAR T cells. Because lenalidomide may also be applied
11 in a delayed administration setting, possibly weeks after CAR T administration due to
12 toxicity challenges with lymphodepletion and immunomodulatory drugs, we investigated
13 the feasibility of delayed lenalidomide administration. Interestingly, the addition of
14 lenalidomide following peak CAR T expansion at 14 days did not result in improved
15 tumor clearance or survival. These results suggest several possibilities. First, the CAR T
16 cells may have been functionally exhausted at 14 days following injection and were
17 unable to be enhanced by delayed lenalidomide administration. Second, the improved
18 tumor clearance was observed because of early CAR T function and circulating
19 numbers, and tumor clearance cannot be improved or rescued at such a delayed
20 progression. Additional studies should be undertaken to determine whether a window
21 for delayed administration of lenalidomide with CAR T cells exists that is more proximal
22 to CAR T administration.
22
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1 In sum, these studies provide novel insights into the mechanism of functional changes
2 in CAR T cells following lenalidomide addition in vitro and in vivo. The changes
3 associated with lenalidomide were intrinsic to anti-BCMA T cells because precise
4 control of CAR stimulation alone in the presence of lenalidomide led to increased
5 functionality, particularly at lower levels of stimulation. In addition, the transcriptional
6 and epigenetic changes associated with lenalidomide treatment suggest that an
7 alternative CAR T cell state of both enhanced memory and effector T cell functions is
8 induced with long-term lenalidomide treatment. Overall, the administration of
9 lenalidomide, a standard of care for patients with multiple myeloma, in combination with
10 anti-BCMA CAR T in the clinic may be warranted based on the potential for a
11 combination of effects, including tumoricidal effects, a more permissive tumor
12 microenvironment for CAR T function, and the observed intrinsic effects on CAR T
13 function.
14
15 Acknowledgments
16 We thank Kimberly Harrington for her scientific contributions to this project. The authors
17 thank Peter Simon, Chris Carter, and Jenna Quigley-Lee, of MediTech Media, Ltd, for
18 medical writing assistance, which was sponsored by Juno Therapeutics, Inc., A Celgene
19 Company.
20
21 Authorship contributions: MW, NS, CH, AB, JJ, WC, PC, DK, and HJ designed and
22 performed the experiments and analyzed the data. TJ, YJ, and RH analyzed the data.
23
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1 LW, CC, and CS designed and performed the experiments. BS and RS provided
2 scientific guidance and participated in the manuscript review. MW and MP drafted the
3 manuscript. All authors contributed to the writing and revision of the manuscript and
4 approved the final version.
5
6
24
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1 Figure Legends
2 Figure 1. Anti-BCMA CAR T cytolytic activity and cytokine production increased
3 with lenalidomide in a concentration-dependent manner. Anti-BCMA CAR T
4 materials from 3 healthy donors and 1 patient donor were assessed for cytokine
5 production using 2 multiple myeloma cell lines. Cytolytic activity (A, C) and cytokine
6 production were measured after 24 hours (B, D) against OPM-2 (A, B) and RPMI-8226
7 (C, D). Cultures were incubated at a ratio of 0.3:1 (effector to target) for cytolytic activity
8 and 1:1 for cytokine production. Data were normalized to dimethyl sulfoxide vehicle
9 control; error bars represent standard error of the mean. For all functional assessments,
10 dose-response modeling indicated a significant effect for lenalidomide for each donor
11 and across all donors (P < .001), except healthy donors for RPMI-8226 cytolytic assay. *
12 indicates P < 0.05 for lenalidomide across each donor.
13 Figure 2. Anti-BCMA CAR T cytokine production was increased by lenalidomide
14 (Len) within 24 hours and across a range of stimulation intensities. Analysis of
15 CD25 and intracellular cytokine levels (left, white bars indicate baseline effects of bead
16 stimulation) for healthy CAR T donors after 24 hours of BCMA bead stimulation (gated
17 on transduced live CD3+ CAR+) for CD4+ (A) and CD8+ (B) subsets. Gray bars
18 demonstrate relative change for (Len) compared to vehicle alone. (C-D) Analysis of
19 effector cytokine production following CAR-specific stimulation on 50 µg BCMA and 50
20 µg PD-L1 beads for 24 hours in the presence of 1 µmol/L Len. * indicates P < 0.05
21 effect of Len for each stimulation condition.
30
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1 Figure 3. Anti-BCMA CAR T cell count, cytokine production, and activation were
2 increased by lenalidomide after repeated stimulation in vitro. (A) Analysis of cell
3 counts following serial stimulation in an MM1.S cell line in the presence or absence of
4 0.1 µmol/L Len. Data represent population doublings across 5 donors; error bars
5 represent standard deviations across 3 technical replicates. Linear mixed-effects
6 modeling indicated a significant effect for Len for each donor across time (P = 2.8 x 10-
7 8). (B-D) Analysis of bulk cytokine production 24 hours following serial stimulation
8 replating at the indicated time points for 5 separate donors. Cytokine production was
9 normalized to cell number at each reset to account for differences in cell replating
10 density; error bars represent standard deviations. Linear-mixed models indicated a
11 significant effect for Len across donors and time points for IFN-γ (P < 2.9 x 10-10), IL-2
12 (P = 1.3 × 10-13), and TNF-α (P = 1.2 × 10-9). * indicates P < 0.05 compared to vehicle
13 for each donor.
14 Figure 4. Lenalidomide reduced functional exhaustion and altered surface
15 phenotype of anti-BCMA CAR T cells. Cells were treated for 7 days on 50 µg BCMA-
16 coated beads in the presence or absence of 1 µmol/L Len. (A-D) Representative
17 healthy donor–derived, freshly thawed anti-BCMA CAR T cells (vehicle [Veh], Len) or
18 CAR T cells prestimulated with 7 days of BCMA bead stimulation and then cultured with
19 RPMI-8226 cells to measure cytolytic activity (over 7 days; A) and cytokine production
20 (24 hours; B-D). Percentage killing was normalized to anti-BCMA CAR T cells
21 prestimulated on beads in the presence of vehicle. Prestimulated CAR T cells showed
22 decreased cytolytic activity (P = 2.1 × 10-4) and cytokine production (P = .03 for IFN-γ)
23 compared with freshly thawed anti-BCMA CAR T cells. Len during the prestimulation
31
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1 period increased cytolytic function (P = .04). Significance was determined using t tests
2 from linear regression coefficients. Three anti-BCMA CAR T donors (each column) were
3 assessed for (E) overall viability and cell count and by (F) flow cytometry for median
4 fluorescence intensity (MFI; CD25 and TIM3) or percentage positive PD-1 and LAG3 on
5 the surface of T cell markers in CD4+ CAR+ and CD8+ CAR+ subsets (gated on live
6 CD3+ cells). Values shown are percentage baseline (Veh) MFI, viability, or count. *
7 indicates P < 0.05.
8 Figure 5. Anti-BCMA CAR T RNA-seq and ATAC-seq profiles were altered by
9 lenalidomide after short- and long-term stimulation. (A) Principal component
10 analysis of expression (RNA-seq) and (C) chromatin accessibility peaks (ATAC-seq).
11 (B) Volcano plots of differentially expressed genes or (D) peaks ± Len at 24 hours and 7
12 days. Directionality and significance of expression changes in selected, enriched
13 biological pathways at (E) 24 hours and (F) 7 days in CAR T cells ± 1 µmol/L Len. (G)
14 RNA expression compared with chromatin accessibility changes for selected T cell loci.
15 (H) Top enriched motif predictions in ATAC-seq loci with increased accessibility along
16 with their enrichment significance and prevalence at 7 days ± 1 µmol/L Len. FC, fold
17 change; FGF, fibroblast growth factor; NF-κB, nuclear factor κB; Ox, oxidative.
18 Figure 6. In vivo efficacy of subcurative dose of anti-BCMA CAR T and blood anti-
19 BCMA CAR T count was altered by lenalidomide. (A) Two Len dosing regimens,
20 concurrent (C) or delayed (D) daily dosing, were tested in a disseminated NSG mouse
21 OPM-2 tumor model with a single, subcurative dose of anti-BCMA CAR T cells from 2
22 separate donors (8 mice per group). (B-C) Tumor bioluminescent measurement and (D-
23 E) animal survival. Error bars represent standard error of the mean. Concurrent Len led
32
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1 to a significant decrease in tumor burden for donor 1 (P = .02) and increased survival
2 (log-rank test) for donor 1 (P = .057) and donor 2 (P = .04) compared with vehicle
3 (Veh)–treated animals injected with anti-BCMA CAR T alone. Linear mixed-effects
4 models (accounting for repeated mouse measurements over time) were used to
5 estimate treatment effects for the tumor burden analysis, and log-rank testing was used
6 for significance testing for the survival analyses. (F-G) Flow cytometric analysis of blood
7 CAR T cells gated on CD45+ CD3+ CAR+. Error bars represent standard error of the
8 mean. Concurrent Len showed significantly increased CAR T expansion after 7 days in
9 vivo (P = 7.3 × 10-6, t test). * indicates P < 0.05 for concurrent lenalidomide compared to
10 vehicle control.
11
12
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Figure 1
A B OPM-2 IFN-γ IL-2 TNF 250 500 250 Patient 400 * Healthy 1 * 400 200 200 Healthy 2 300 * * * Healthy 3 * 300 * *
150 150 * Killing, % * Baseline, % 200 * Baseline, % * Baseline, % * * * 200 100 100 100 100
1 1 1 1 0 10 0.1 10 1 1 10 .01 0.1 1 0.1 0. 0 0.01 0.01 0.0 vehicle vehicle vehicle vehicle Lenalidomide (µmol/L) Lenalidomide (µmol/L) Lenalidomide (µmol/L) Lenalidomide (µmol/L) C D RPMI-8226 IFN-γ IL-2 TNF 250 140 600 250
500 200 120 * * 200 * * 400 * * 150 300 150 Killing, % * * 100 Baseline, % Baseline, % Baseline, % 200 * * 100 * * 100 80 100 1 0 1 1 1 .01 0.1 1 0.1 10 0.1 10 0.1 10 0 0.01 0.01 0.01 vehicle vehicle vehicle vehicle Lenalidomide (µmol/L) Lenalidomide (µmol/L) Lenalidomide (µmol/L) Lenalidomide (µmol/L)
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. Figure 2
A CD4+ 40000
30000 IFN- γ IL-2 TNF-α 20000 20 5 5 0.1 µmol/L Len CD25, MFI 10000 1 µmol/L Len 15 4 4 0 30 3 3
10 2 2 20 * 5 * 1 * * * 1 10
IFN- γ +, % 0 0 0 A A A 0 M M M A BCMA BCMA BCMA M o stim BC BCMA o stim BC BCMA o stim BC BCMA n g g n g g n g g BC BCMA µg µ µ µg µ µ µ µ µ o stim g BCMA g n µ g 5 0 0 5 0 0 0 µg µ 5 5 5 0 5 0 0 0 0 5 0 5 0 2 2 2 2 B CD8+
40000
30000 IFN- γ IL-2 TNF-α 20 20000 6 5 * CD25, MFI 10000 15 * 4 * 4 * 0 3 * * 40 10 * 2 30 2 5 * * 1 20 * * 0 0 0 10 IFN- γ +, % A A A M M M 0 o stim BC BCMA BCMA o stim BC BCMA BCMA o stim BC BCMA A n g g n g g BCMA M µ µ µ µ µ µg n µ g g g µg µ BC BCMA 5 0 0 0 0 o stim g BCMA 5 5 0 5 0 n µ g 0 5 0 5 0 µg µ 2 2 2 5 0 0 5 0 2 C Healthy Donor #1 γ 60,000 IFN- IL-2 TNF 1000 200 * * * No PD-L1 800 150 PD-L1 40,000 600
, pg/mL 100 400 IL-2, pg/mL 20,000 TNF, pg/mL IFN- γ 50 200
0 0 0
Vehicle Vehicle Vehicle µmol/L Len µmol/L Len µmol/L Len Healthy Donor #2 1 1 1 IFN-γ IL-2 TNF 60,000 * 5000 * 400 * 4000 300 40,000 3000 , pg/mL 200 2000 IL-2, pg/mL IFN- γ 20,000
TNF, pg/mL 100 1000
0 0 0
hicle ehicle e ehicle V V V µmol/L Len µmol/L Len µmol/L Len 1 1 1 D Patient Donor IFN-γ IL-2 TNF 80,000 600 600 * * 60,000 400 400
, pg/mL 40,000
TNF, pg/mL 200 IL-2, pg/mL IFN- γ 200 20,000
0 0 0
Vehicle Vehicle Vehicle Downloaded from mct.aacrjournals.orgµmol/L Len on September 23, 2021.µmol/L © Len 2019 American Association forµmol/L Cancer Len Research. 1 1 1 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.
Figure 3
A 15 Healthy 1 vehicle P = 3 x 10 -8 Healthy 1 Len Healthy 2 vehicle Healthy 2 Len 10 Healthy 3 vehicle Healthy 3 Len Healthy 4 vehicle Healthy 4 Len Patient vehicle x Patient Len 5 x x Population doublings Population x x x x 0 0 4 7 11 14 18 21 25 28
Time, days (reset time points)
24-Hour Secreted Cytokines B C D Healthy 1 vehicle IFN-γ IL-2 TNF Healthy 1 Len 0.8 0.020 0.005 Healthy 2 vehicle * ** * * * Healthy 2 Len 0.015 0.004 * 0.6 * Healthy 3 vehicle * * * * 0.003 * * Healthy 3 Len 0.4 * 0.010 *
, pg/mL/cell , * 0.002 *
γ Healthy 4 vehicle * * * *
IL-2, pg/mL/cell IL-2, 0.005 * 0.2 pg/mL/cell TNF, * Healthy 4 Len IFN- 0.001 * * * Patient vehicle 0.0 0.000 0.000 Patient Len /5 8 /5 8 8 4 y 4 y 4/5 y y a y a y a a D a D a D D D D
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.
Figure 4
RPMI-8226 Len CAR BCMA bead Len T Functional Assessments CAR T CAR debead, wash T Phentotypic Assessments Prestimulation (7 days)
A 125 * B * 40,000 100 30,000 75 *
20,000 50 , pg/mL Killing, %
25 IFN- γ 10,000
Len Len Vehicle Vehicle
Prestim Len Prestim Len Prestim vehicle Prestim vehicle C 2500 * D 100 2000 * 80 1500 60
1000 IL-2, pg/mL 40 TNF, pg/mL 500 20
0
Len Len Vehicle Vehicle Prestim Len Prestim Len Prestim vehicle Prestim vehicle E F
CD4 1 2 3 CD8 1 2 3 300 Healthy 1 Healthy 2 Healthy 3 200 TIM3 105 135 90 * TIM3 181 192 132
Viability 166 120 113 150 * 200 * CD25 151 227 164 *CD25 100 300+ 300+ 100 Vehicle, % LAG3 300+ 191 175 LAG3 155 116 110 Vehicle, % Count 122 103 80 50 * * 100
PD-1 56 72 78 PD-1 52 69 105 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 reviewedFigure and 5 accepted for publication but have not yet been edited.
RNA-Seq ATAC-Seq A C
24hr d7 + stim
24hr + stim
24hr + stim
24hr d7 + stim
H1 H2 H3 H4 Len Vehicle B H1 H2 H3 H4 Len Vehicle D p-adj p-adj p-adj p-adj 10 10 10 10 -log -log -log -log
log2 fold change log2 fold change log2 fold change log2 fold change down up total down up total down up total down up total 37 177 214 255 328 583 2 13 15 2510 294 2804
E Signaling Pathway F Signaling Pathway Leukocyte extravasation Th2 NFkB TREM1 G2/M checkpoint HGF iCOS-iCOSL in Th cells Renin-angiotensin Actin-based motility by Rho Th1 Acute phase response HMGB1 FGF RhoGDl IL-3 cAMP-mediated GNRH NRF2 Ox. stress response Dendritic cell maturation G-alpha(s) FLT3 ERK/MAPK GNRH Rac Pho family ATM Cdc42 Renin-angiotensin RhoA IL-6 BMP PPARg/RXRa Actin cytoskeleton cAMP-mediated Sirtuin RhoA ILK CXCR4 Actin-based motility by Rho Integrin Wnt/B-catenin G-alpha(s) Ephrin receptor PPARg Thrombin
-log10(p) -log10(p) Z-score (24h +stim, Len vs Vehicle) Z-score (d7, Len vs Vehicle)
G RNA Expression vs. ATAC H Increased Motif Enrichment Accessibility for Selected Genes % of Target ATAC-seq peak associated with locus Sequences ATAC-seq mean/gene IFN-g Motif Name Motif Log P Value with Motif FC), 2 IL2RA
CD69 CCR7 Len vs Vehicle RNA Expression Change (Log −2.0 −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5
−0.5 0.0 0.5 1.0 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Chromatin Accessibility Change (Log2FC), Len vs Vehicle Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1146 Author manuscripts have been peer reviewedFigure and accepted 6 for publication but have not yet been edited.
A 1E6 CAR T OPM-2 injec on CAR T pharmacokinetic assessment injec on
D0 D8 D14 D22 D28 Concurrent lenalidomide (C) Delayed lenalidomide (D)
B C Donor 1 Donor 2 1010 1010 *
109 109
108 108 Mock + vehicle Mock + Len 107 107 CAR T + vehicle (C) CAR T + Len (C)
Bioluminescence, p/s Bioluminescence, p/s CAR T + Len (D)
106 106
0 20 40 60 0 20 40 60 Days After CAR T Injection Days After CAR T Injection D 100 E 100
80 80
60 60 *
40 40 Survival, % Survival, %
20 20
0 20 40 60 80 100 0 20 40 60 80 100 Days After CAR T Injection Days After CAR T Injection Donor 1 F Day 8 Day 14 Day 22 Day 28 4 Mock CAR T 2 Mock CAR T 1.0 Mock CAR T 1.0 Mock CAR T 3 *
2 1 0.5 0.5
1 CAR T/µL Blood N/A G Donor 2 4 2 10 3
8 3 * 2 6 2 1 4 1 1 2 CAR T/µL Blood N/A
Veh Len Veh Veh Len Veh Veh Len Veh Veh Len Veh Len (C)Len (D) Len (C)Len (D) Len (C)Len (D) Len (C)Len (D)
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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
Melissa Works, Neha Soni, Collin Hauskins, et al.
Mol Cancer Ther Published OnlineFirst August 8, 2019.
Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-1146
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