multiple myeloma is enhanced in the presence of lenalidomide · 1 1 Title: Anti–B-cell maturation...
Transcript of multiple myeloma is enhanced in the presence of lenalidomide · 1 1 Title: Anti–B-cell maturation...
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Title: Anti–B-cell maturation antigen chimeric antigen receptor T cell function against 1
multiple myeloma is enhanced in the presence of lenalidomide 2
Authors: 3
Melissa Works, Neha Soni, Collin Hauskins, Catherine Sierra, Alex Baturevych, Jon C. 4 Jones, Wendy Curtis, Patrick Carlson, Timothy G. Johnstone, David Kugler, Ronald J. 5 Hause, Yue Jiang, Lindsey Wimberly, Christopher R. Clouser, Heidi K. Jessup, Blythe 6 Sather, Ruth A. Salmon, and Michael O. Ports 7
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Affiliations: 9
Juno Therapeutics, A Celgene Company, Seattle, Washington. 10
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Running title: 12
Lenalidomide enhances anti-BCMA CAR T function 13
Keywords: multiple myeloma; lenalidomide; CAR T; BCMA; Immunology 14
Financial support: This study was funded by Juno Therapeutics, A Celgene Company. 15
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Corresponding author: Melissa Works 17
400 Dexter Ave N Suite 1200 18
Seattle, WA 98109 19
Phone: 206-566-5731 21
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Disclosure of conflicts of interest: All authors are employed by and have equity interest 23
in Juno Therapeutics, Inc, A Celgene Company. 24
Abstract: 246/250 words 25
Manuscript: 4540/5000 26
Figures: 6/7 27
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References: 32/50 1
Supplementary Document: Yes 2
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Portions of this work were presented in poster form at the 59th annual meeting of the 4
American Society of Hematology; Atlanta, GA; December 9, 2017. 5
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Key points 1
Anti-BCMA CAR T cell function is enhanced by lenalidomide during acute or chronic 2
stimulation. 3
RNA- and ATAC-seq data support elements of T-effector and memory-cell molecular 4
signatures. 5
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Abstract 1
Anti–B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells have 2
shown promising clinical responses in patients with relapsed/refractory multiple 3
myeloma. Lenalidomide, an immunomodulatory drug, potentiates T cell functionality, 4
drives antimyeloma activity, and alters the suppressive microenvironment; these 5
properties may effectively combine with anti-BCMA CAR T cells to enhance function. 6
Using an anti-BCMA CAR T, we demonstrated that lenalidomide enhances CAR T cell 7
function in a concentration-dependent manner. Lenalidomide increased CAR T effector 8
cytokine production, particularly under low CAR stimulation or in the presence of 9
inhibitory ligand programmed cell death 1 ligand 1. Notably, lenalidomide also enhanced 10
CAR T cytokine production, cytolytic activity, and activation profile relative to untreated 11
CAR T cells in chronic stimulation assays. This unique potentiation of both short-term 12
CAR T activity and long-term functionality during chronic stimulation prompted 13
investigation of the molecular profile of lenalidomide-treated CAR T cells. Signatures 14
from RNA sequencing and assay for transposase-accessible chromatin using 15
sequencing indicated that pathways associated with T-helper 1 response, cytokine 16
production, T cell activation, cell-cycle control, and cytoskeletal remodeling were altered 17
with lenalidomide. Finally, study of lenalidomide and anti-BCMA CAR T cells in a 18
murine, disseminated, multiple myeloma model indicated that lenalidomide increased 19
CAR T cell counts in blood and significantly prolonged animal survival. In summary, 20
preclinical studies demonstrated that lenalidomide potentiated CAR T activity in vivo in 21
low-antigen or suppressive environments and delayed onset of functional exhaustion. 22
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These results support further investigation of lenalidomide and anti-BCMA CAR T cells 1
in the clinic. 2
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Introduction 1
Despite improvements in the treatment of newly diagnosed multiple myeloma, it remains 2
uncured, and nearly all patients relapse and become resistant to available treatments 3
(1). Based on the encouraging activity of chimeric antigen receptor (CAR) T cells 4
targeting CD19 in non-Hodgkin lymphoma, CAR T cells targeting plasma cells 5
expressing B-cell maturation antigen (BCMA) have been developed for multiple 6
myeloma (2). Although BCMA CAR T cells have shown promise in the clinic, an 7
immunosuppressive myeloma tumor microenvironment, including programmed cell 8
death 1 ligand 1 (PD-L1) expression (3), and the potential for activation-induced 9
exhaustion may limit the durable responses of CAR T cells in some patients (4). 10
Lenalidomide is an immunomodulatory drug indicated for the treatment of multiple 11
myeloma (5); it has pleiotropic effects that directly impair primary tumor growth and 12
modulate the immunosuppressive tumor microenvironment to help facilitate a more 13
robust antitumor inflammatory response (6,7). Studies have shown that lenalidomide 14
can directly increase in vitro T cell function, even in heavily treated patients with 15
progressive disease, irrespective of immunomodulatory-drug refractory status (8). 16
Lenalidomide has also been shown to increase acute in vitro and in vivo CAR T cell 17
functionality in several model systems with varying CAR constructs and indications 18
(9,10). For example, lenalidomide in combination with CS1-directed CAR T cells with a 19
CD28z endodomain was associated with increased secretion of T-helper (Th) 1–20
associated cytokines, decreased secretion of Th2–associated cytokines, and increased 21
survival in tumor-bearing mice (10). Additional studies are required to refine 22
lenalidomide’s mechanism of action and investigate its application in a chronic CAR T–23
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stimulation setting in conditions of varying antigen density or in the presence of 1
inhibitory ligands such as PD-L1. 2
CAR T cells have unique functional properties that can be altered by the characteristics 3
of the single-chain variable fragment, choice of transmembrane and endodomain, and 4
manufacturing process—all of which determine CAR T fitness during long-term 5
stimulation (11). In this study, we sought to characterize a novel anti-BCMA CAR 6
construct and determine short- and long-term effects of lenalidomide on CAR T function. 7
In addition, immunomodulatory drugs have been shown to directly induce CD28 tyrosine 8
phosphorylation (12), suggesting that these drugs impinge on costimulatory signaling 9
pathways; however, the role of a 41BBz endodomain during immunomodulatory drug 10
application is unclear. To understand the complex nature of lenalidomide’s mechanism 11
of action, we applied RNA sequencing (RNA-seq) and assay for transposase-accessible 12
chromatin using sequencing (ATAC-seq) technology to determine whether these 13
functional differences were associated with changes in the regulatory networks involved 14
in T cell function and activation. Finally, we examined concurrent and delayed 15
administration of lenalidomide with a subcurative dose of anti-BCMA CAR T injection to 16
further identify potential clinical applications and dosing strategies. 17
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Materials and methods 19
In vitro cytolytic, cytokine, and flow cytometry CAR T assessment 20
T cells obtained from peripheral blood samples from consenting healthy adult donors 21
and a patient with multiple myeloma refractory to pomalidomide were transduced to 22
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express a construct containing an extracellular BCMA-binding single-chain variable 1
fragment and intracellular 41BBz endodomain (65%-76% CAR+, Supplementary Figure 2
1A, Supplementary Table 1, Supplementary Methods). Human materials used in this 3
research were received by the researchers in a fully de-identified manner from 4
commercial repositories or under unrelated IRB-approved clinical studies from adults 5
who consented to testing of their donated samples for future research purposes. 6
Cultures were established with an effector-to-target ratio of 0.3:1 or 1:1 with OPM-2 or 7
RPMI-8226 multiple myeloma target cells. Co-cultures with increased effector cell 8
counts relative to target cell counts results in rapid clearance of BCMA+ target cells. 9
Therefore, E:T ratios of 1.0 and 0.3 were selected to ensure that more CAR T cells 10
would receive an activating stimulus for evaluation with lenalidomide. Lenalidomide 11
(Sigma) was titrated across and above the clinical maximum concentration (1.9 µmol/L 12
for a 25-mg oral dose in patients with multiple myeloma) to assess the functional range 13
of the drug (13). Cell-free supernatants were collected after 24 hours. Experiments were 14
performed 2 to 3 times in 4 donors. 15
Unless noted, anti-BCMA CAR T cells were stimulated with 50 µg/mL BCMA beads for 16
the time indicated at a bead:CAR+ T cell ratio of 1:1. For PD-L1 experiments, 50 µg/mL 17
PD-L1 or control immunoglobulin G was coupled along with 50 µg/mL BCMA. For 18
prestimulation experiments, after 7 days of incubation, cells were debeaded, washed, 19
and cocultured with RPMI-8226-NucLight Red target cell lines in the presence of 1 20
µmol/L lenalidomide or vehicle control. Experiments were performed twice in 3 donors. 21
For surface phenotype analysis, anti-BCMA CAR T cells were cultured with BCMA 22
beads for 7 days, stained with a live/dead dye (Invitrogen; Thermo Fisher Scientific, 23
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Carlsbad, CA), BCMA–fractured crystallizable (Fc), antibodies for surrogate CAR 1
marker, CD3, CD4, CD8, CD25, PD-1, TIM3, and LAG3 (BD Biosciences, Franklin 2
Lakes, NJ) and then analyzed on a Fortessa flow cytometer (BD Biosciences). For 3
intracellular cytokine staining, cells were stimulated on BCMA beads for 24 hours, with 4
protein transport cocktail (BD Biosciences) added in the final 4 hours of incubation. 5
Cells were then stained with live/dead dye and surface markers (surrogate CAR marker, 6
CD3, CD4, CD8, CD25, PD-1), fixed/permeabilized (BioLegend, San Diego, CA), and 7
stained for intracellular interleukin (IL) 2, interferon-γ (IFN-γ), and tumor necrosis factor 8
α (TNF-α) (BioLegend). Experiments were performed twice in 3 donors. 9
Serial stimulations 10
Anti-BCMA CAR T cells were plated with irradiated MM1.S target cells at an effector-to-11
target ratio of 1:2 in the presence of lenalidomide (0.1 µmol/L). Every 3 to 4 days, CAR+ 12
cells were enumerated, phenotyped by flow cytometry, and replated with freshly thawed 13
and irradiated MM1.S target cells and lenalidomide. Twenty-four hours following 14
replating on days 5, 8, and 15, cell-free supernatant was assessed for cytokine levels by 15
Meso Scale Discovery (Rockville, MD). Following incubation on day 4, a sample was 16
stained with a live/dead dye and panel of surface markers (epidermal growth factor 17
receptor [EGFR], CD3, CD4, CD8). Fluorescently labeled count-bright beads were 18
added to each sample to get the absolute counts of the total CD3+, Erb+, BCMA+, and 19
CAR+ cells. Replating was maintained for 28 days or until the cell count was < 50,000 20
cells. Experiments were performed 3 times in 3 donors. 21
RNA-seq and ATAC-seq assays 22
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Anti-BCMA CAR T cells were cultured in the presence or absence of BCMA beads for 1
either 24 hours or 7 days with or without 1 µmol/L lenalidomide. Strand-specific, poly-A-2
selected RNA-seq reads were trimmed, mapped to hg38, and quantified using 3
ArrayStudio (OmicSoft, Cary, NC). ATAC-seq was performed according to published 4
methodology (14) (See Supplementary Methods). Experiments were performed twice in 5
3 and 4 donors. 6
In vivo OPM-2 tumor model 7
All animal studies were conducted in accordance with protocols approved by the 8
Institutional Animal Care and Use Committee. NOD.Cg-PrkdcscidIL-2rgtm1Wjl/SzJ mice 9
(NSG; Jackson Laboratory, Bar Harbor, ME) were injected intravenously with 2 × 106 10
OPM-2/luciferase cells and allowed to engraft for 14 days prior to intravenous CAR T 11
infusion. One day prior to or 14 days following injection with 1 × 106 CAR T or mock 12
control T cells, animals were dosed (intraperitoneally) daily for 50 days with 10 mg/kg 13
lenalidomide in phosphate-buffered saline. Blood was collected for quantitation of 14
circulating CAR T cells, and cells were stained with antibodies to exclude mouse-15
specific cells (H-2kd, TER-119, and muCD45) and analyzed by flow cytometry (See 16
Supplementary Methods). 17
Statistics 18
Linear fixed-effect or mixed-effect models (15), a more flexible technique related to 19
more traditional nested or repeated measures ANOVA methods, were used to assess 20
the significance of lenalidomide treatments on cytolytic activity and cytokine production, 21
with treatment, and time treated as fixed effects and animal and donor treated as 22
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random effects, nested with time when repeated measurements were derived from the 1
same animal. P values were obtained by likelihood ratio tests comparing the full model 2
with the effect of interest against the model without the effect of interest. Flow-cytometry 3
median fluorescence intensity values were log2 transformed and bioluminescence 4
values were log10 transformed to better approximate normality. Survival analyses were 5
performed (16) with log-rank testing to determine the significance of the effect of 6
lenalidomide treatments on survival curves. 7
Results 8
Anti-BCMA CAR T function is enhanced by lenalidomide in the presence of BCMA-9
expressing myeloma cell lines 10
Cytolytic activity and cytokine production of lenalidomide-treated CAR T cells 11
transduced with the anti-BCMA CAR were evaluated in vitro in the presence of BCMA-12
expressing multiple myeloma cell lines with varying sensitivity to lenalidomide 13
(Supplementary Methods). Multiple donors were assessed to evaluate donor-dependent 14
effects of lenalidomide, including CAR T product manufactured from an 15
immunomodulatory drug-refractory patient. An additional scfv was tested in the 16
presence of lenalidomide to confirm the effects were not binder-specific (Supplementary 17
Figure 1B). Increased anti-BCMA CAR T cytolytic activity against OPM-2 target cells 18
titrated with increased concentrations of lenalidomide across all donors at the 0.3:1 E:T 19
ratio (P = 6.2 × 10-5; Figure 1A). Donor 1 and 2 demonstrated the least efficient target 20
cell killing for OPM-2 cells (Supplementary Figure 1C) and had increased activity with 21
lenalidomide at a 1:1 ratio (Supplementary Figure 1D). In addition, all CAR T donors 22
(baseline shown in Supplementary Figure 1E) had significantly increased IFN-γ, IL-2, 23
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and TNF-α production in a lenalidomide concentration–dependent manner upon 1
coculture with OPM-2 cells (P < .002; Figure 1B). The treatment effect of lenalidomide 2
on CAR T cytolytic activity appeared to be donor dependent in coculture with RPMI-3
8226 (Figure 1C), with the patient donor showing a significant increase in cytolytic 4
activity (P = 1.9 × 10-8). Notably, cytokine production by CAR T cells in RPMI-8226 5
coculture (baseline shown Supplementary Figure 1F) was significantly increased across 6
all donors and cytokines upon treatment with lenalidomide (P < .003; Figure 1D). 7
Importantly, addition of lenalidomide did not alter BCMA target expression 8
(Supplementary Figure 2). These results demonstrated that when anti-BCMA CAR T 9
cells were stimulated with 0.3:1 and 1:1 effector-to-target ratios, addition of lenalidomide 10
increased effector functionality of CAR T cells across several metrics of CAR T function, 11
including cytolytic activity and cytokine production. 12
Lenalidomide potentiation of anti-BCMA CAR T cytokine expression is dependent on 13
stimulation strength 14
The CAR T–intrinsic treatment effects of lenalidomide on cytokine production for the 15
CD4+ and CD8+ CAR T populations were next evaluated in the absence of target cells. 16
As lenalidomide can directly limit multiple myeloma cell viability, a CAR-specific 17
stimulation reagent of recombinant human BCMA was used to assess the direct effect 18
of lenalidomide on activated CAR T cells in the absence of BCMA-expressing target 19
cells. To this end, recombinant human BCMA–labeled beads were developed to 20
stimulate CAR T cells and provide the means to titrate both the magnitude of stimulation 21
(low [5 µg/mL], medium [50 µg/mL], and high [200 µg/mL]) and the concentration of 22
lenalidomide (0.1 and 1.0 µmol/L). At a medium stimulation condition, we measured 23
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secreted cytokine production and observed a mean 200% increase in IL-2 and TNF-α 1
concentrations compared with vehicle control, with donor-dependent increases in IFN-γ 2
(Supplementary Figure 3). Anti-BCMA CAR T cells activated with BCMA beads showed 3
stimulation level–dependent effects, with the low, 5- µg BCMA beads causing lower 4
CAR T CD25 expression and intracellular IFN-γ cytokine staining compared with 5
medium- (50 µg) and high- (200 µg) BCMA beads (Figure 2A-B, left). Lenalidomide 6
significantly (P < .05) increased the percentage of IFN-γ+ and TNF-α+ intracellular 7
staining at multiple stimulation levels for both CD4+ (Figure 2A) and CD8+ (Figure 2B) 8
CAR T cells. In the absence of stimulation, lenalidomide had no effect on CAR T 9
cytokine staining, indicating that cytokine enhancement provided by lenalidomide 10
requires stimulation. 11
Inhibitory receptors can alter T cell receptor–mediated activation and limit the effector 12
functionality of T cells (17). We explored whether the lenalidomide-induced potentiation 13
of CAR T activation and cytokine production could override PD-L1–mediated inhibition. 14
Evaluation of both healthy and patient donor CAR T cells demonstrated that addition of 15
recombinant PD-L1 to recombinant BCMA beads significantly reduced IFN-γ, IL-2, and 16
TNF-α levels (P< .006; Figure 2C-D). Importantly, lenalidomide treatment potentiated 17
secreted cytokine levels beyond those from CAR T cells treated with vehicle in the 18
presence of PD-L1 (P < .007). 19
Anti-BCMA CAR T function during serial and chronic stimulation was prolonged by 20
lenalidomide 21
Previous studies indicate that performance in a serial stimulation assay may represent 22
CAR T fitness and in vivo efficacy (18). Notably, after repeated stimulation with MM1.s 23
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target cells, lenalidomide increased CAR T expansion on average by 0.82 population 1
doublings over 28 days across 5 donors relative to controls (P = 2.8 x 10-8; Figure 3A). 2
Multiple samples at intermediate time points were collected to assess cytokine 3
production during serial stimulation until a donor had insufficient cells to continue the 4
assay in the vehicle-treated group. Increased cell counts were associated with a 5
significant increase in IL-2, IFN-γ, and TNF-α production per cell in the media (P < 1.2 x 6
10-9; Figure 3B-D). 7
We also developed a novel long-term chronic stimulation assay designed to diminish 8
anti-BCMA CAR T effector function. We observed a limited increase in BCMA CAR T 9
cytolytic activity against RPMI-8226 cells in the presence of lenalidomide in an acute 10
assay (Figure 1); however, CAR T prestimulation appears to exhaust the cells and 11
results in decreased functionality. Prestimulated CAR T cells showed decreased 12
cytolytic activity (P = 2.1 × 10-4) and IFN-γ cytokine production (P = .03) compared with 13
freshly thawed anti-BCMA CAR T cells (Figure 4A), indicating that chronic 14
prestimulation leads to functional impairment. CAR T cells were also prestimulated with 15
BCMA beads with 1 µmol/L lenalidomide prior to analysis of cytolytic activity and 16
cytokine production. Notably, the presence of lenalidomide during the prestimulation 17
period preserved cytolytic function (P = .04), and a trend was observed toward 18
increased cytokine production compared with cells exposed to vehicle during the 19
prestimulation period (Figure 4B-D). 20
The phenotype of anti-BCMA CAR T cells stimulated for 7 days with BCMA beads was 21
assessed, and the addition of lenalidomide significantly increased CAR+ viability of anti-22
BCMA CAR T material across 3 healthy donors (P = .04; Figure 4E). The addition of 23
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lenalidomide did not alter the total cell count across all donors (Figure 4E) in this 7-day 1
period, and no significant differences were observed in percentage CAR+ between 2
vehicle- and lenalidomide-treated CAR T cells or among CAR+ cells in memory 3
subtypes by classification with CD45RA or CD27 by CCR7 (Supplementary Figure 4). 4
Flow cytometric analysis across CAR T donors indicated that the addition of 5
lenalidomide functionally altered the balance between activation and immunoregulatory 6
markers by increasing the surface expression of TIM3 in the CD8+ population (P = 4.0 × 7
10-4), with mixed effects on the CD4+ CAR+ population (Figure 4F). Across all donors 8
and in both the CD4+ and CD8+ CAR+ populations, lenalidomide increased CD25 (CD4+ 9
and CD8+; P = 2.2 × 10-16) and the percentage positive for LAG3 expression (CD8+ P < 10
.03; CD4+ P = .002). Notably, a decrease in the percentage of PD-1+ cells was also 11
observed in the CD4+ population (P = .04), with 2 of 3 donors showing a decrease in the 12
CD8+ population as well. 13
Anti-BCMA CAR T RNA-seq and ATAC-seq profiles were altered by lenalidomide after 14
short-term and chronic stimulation 15
Because few phenotypic changes were noted by fluorescence-activated cell sorting 16
following the addition of lenalidomide in the context of antigen-specific stimulation, we 17
decided to employ unbiased transcriptomic and epigenomic analyses to further assess 18
features that could underlie the enhanced functionality. Investigation of the molecular 19
signature of lenalidomide-treated anti-BCMA CAR T cells was assessed following short-20
term (24-hour) or chronic (7-day) stimulation, as described above. Principal component 21
analysis demonstrated clustering based on stimulation (stimulation or no stimulation) 22
and time (24 hours or 7 days) for both the RNA-seq (Figure 5A; GSE113281) and 23
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ATAC-seq (Figure 5C; GSE113853) data sets. Next, we examined the role of 1
lenalidomide after 24 hours or 7 days of stimulation after accounting for donor-to-donor 2
variability. RNA-seq analysis showed alteration of a small set of genes (214) at 24 3
hours, and a larger number of genes (583) changed after 7 days of stimulation in the 4
presence of lenalidomide (Figure 5B). Notably, ATAC-seq analysis revealed a limited 5
set of chromatin accessibility changes associated with lenalidomide treatment after 24 6
hours of stimulation, with a dramatic change in profile and an increase in the number of 7
sites with changes in chromatin accessibility after 7 days of stimulation in the presence 8
of lenalidomide (Figure 5D). To further identify specific transcriptional changes 9
associated with lenalidomide treatment, gene ontology analysis was applied to the 10
RNA-seq data set. Pathways associated with T cell chemotaxis (leukocyte 11
extravasation, integrin, integrin-linked kinase, and C-X-C motif chemokine receptor 4–12
associated gene sets), intracellular signaling, and cytoskeleton (Rac/Rho/Cdc42) were 13
upregulated in the presence of lenalidomide within 24 hours of stimulation compared 14
with vehicle controls (Figure 5E). These data support an increase in inducible 15
costimulator (ICOS)–related signaling pathways—a finding that is in line with previous 16
publications demonstrating an increase in ICOS and ICOS ligand in the CD3+ 17
population of peripheral blood mononuclear cells treated with lenalidomide ex vivo (19). 18
After 7 days of stimulation, lenalidomide upregulated pathways associated with Th1 T 19
cell response and costimulation while decreasing Th2-associated gene signatures 20
(Figure 5F). 21
To determine whether chromatin accessibility correlated with the transcriptional 22
changes observed during lenalidomide treatment, we integrated the ATAC-seq and 23
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RNA-seq data from our chronic 7-day analysis (Figure 5G). Across donors, we 1
observed a significant increase in chromatin accessibility across multiple loci, including 2
those associated with IFN-γ and IL-2RA (CD25), and these changes were correlated 3
with a significant increase in transcription. Importantly, the upregulation of IFN-γ and 4
CD25 were concordant with previous findings from chronic stimulation experiments 5
where lenalidomide treatment resulted in significantly higher proportions of cells 6
expressing these markers. We also observed a decrease in CD69 and CCR7 chromatin 7
accessibility and gene transcription on lenalidomide treatment. Last, we analyzed the 8
ATAC-seq data set for motif enrichment and observed enrichment for a number of 9
motifs bound by multiple factors associated with T cell activation, including AP-1/Jun 10
and nuclear factor κB (Figure 5H) (20). 11
Subcurative dose of anti-BCMA CAR T demonstrated improved tumor clearance and 12
survival in vivo when concurrently dosed with lenalidomide 13
Finally, in order to assess in vivo CAR T function by lenalidomide, mice implanted with 14
OPM-2 tumors were dosed with a subcurative dose of anti-BCMA CAR T cells. Mice 15
with established tumors were dosed daily with lenalidomide 1 day prior or 14 days 16
following injection with a subcurative dose of 1 × 106 anti-BCMA CAR T cells (Figure 17
6A). The addition of concurrent lenalidomide led to a significant decrease in tumor 18
burden for donor 1 (P = .02) and increased survival for donor 1 (P = .057) and donor 2 19
(P = .04) compared with vehicle-treated animals injected with anti-BCMA CAR T alone 20
(Figure 6B-E). Animals on the concurrent lenalidomide dosing regimen also showed 21
increased CAR T counts in the peripheral blood after 7 days (P = 7.3 × 10-6) but not at 22
later time points (Figure 6F-G). Lenalidomide had a small but significant mock CAR T 23
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effect on tumor burden for donor 1 alone (P = .003). The addition of delayed dosing of 1
lenalidomide did not improve tumor clearance and survival for either CAR T donor, 2
suggesting that the benefit of this combinatorial approach relied on concurrent 3
administration. 4
Discussion 5
Our studies further explore the mechanism of action and applications of lenalidomide in 6
combination with CAR T cells. In anti-BCMA CAR T cells with a 41BB/CD3z-containing 7
endodomain, we observed a rapid increase in cytokine production following stimulation 8
with either myeloma target cells or direct CAR stimulation via antigen-coated beads. 9
Notably, lenalidomide increased cytokine production and activation across multiple 10
assays at a clinically relevant concentration, indicating an increase in effector function. 11
In addition, an immunomodulatory drug–refractory sample derived from a patient with 12
multiple myeloma also demonstrated increased in vitro functionality in the presence of 13
lenalidomide, indicating that refractory tumor status may be independent of the effects 14
of lenalidomide on CAR T cells. Previous studies demonstrated that increased T cell 15
cytokine production associated with lenalidomide was partly due to the degradation of 16
the transcription factors Ikaros and Aiolos by Cereblon (21). In addition, Ikaros has been 17
shown to alter the threshold for activation of T cells downstream of T cell receptor and 18
IL-2 receptor signaling as well as protein kinase C, phosphatidylinositol 3-kinase, and 19
calcineurin signaling (22). Furthermore, our studies support the hypothesis of 20
immunomodulatory drug–lowered activation threshold in functional assays controlling 21
CAR stimulation. We also demonstrated that, in the presence of PD-L1 engagement, 22
the addition of lenalidomide potentiated cytokine production beyond control levels, 23
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suggesting that this combinatorial approach may override suppressive inputs from the 1
microenvironment to sustain antitumor functionality. 2
Chronic and serial stimulation assays may recapitulate the repeated stimulation to 3
which a CAR T cell is exposed in patients’ tumors and allow for examination of an 4
exhaustion-like state of the CAR T cell. Our functional data support a prolonged period 5
for CAR T cell cytolytic activity and cytokine production in the presence of lenalidomide. 6
Prolonged lenalidomide treatment period increased IL-2 across serial stimulation and 7
chronic stimulation assays. The increased IL-2 production over time may provide a 8
mechanism to sustain T cell effector function over chronic stimulation; this mechanism 9
agrees with previous studies demonstrating that low IL-2 production by CAR T cells was 10
associated with exhaustion (23). Exposure to lenalidomide in chronic or serial 11
stimulation assays also resulted in increased TNF-α and IFN-γ, increased viability, and 12
decreased PD-1 expression on the CAR T cell surface. Notably, characterization of 13
CD19-directed CAR T cells determined that the PD-1–negative CAR T population was 14
associated with therapeutic response (24). These results were observed in the 15
presence of increased TIM3 and LAG3—markers previously shown to be associated 16
with T cell exhaustion (25). The duality of these exhaustion-associated makers may 17
indicate that the addition of lenalidomide to CAR T cells leads to an alternative 18
differentiation or activation state outside the canonical states of T cell differentiation. In 19
other words, lenalidomide may affect these “exhaustion” signaling pathways 20
independently of PD-1, or, alternatively, these markers are not indicative of functional 21
exhaustion in these anti-BCMA CAR T cells. In support of these data, previous studies 22
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have shown that a dissociation between surface markers and functional assessment of 1
exhaustion and molecular dissection of cell state may be more informative (26). 2
In addition to functional assays, the RNA- and ATAC-seq studies resulted in a number 3
of insights into possible mechanisms for lenalidomide-induced increases in CAR T 4
function. First, the number of transcriptional and chromatin accessibility changes 5
associated with stimulation and time were predominant compared with the effects of 6
lenalidomide, indicating a relatively subtle effect of lenalidomide on transcriptional 7
networks. Second, the changes associated with lenalidomide were broad, including 8
early changes in transcripts associated with cytoskeletal remodeling and chemotaxis. 9
After chronic stimulation, a distinct transcriptional signature emerged that included a 10
decrease in transcripts associated with the Th2 response, G2/M checkpoint, and ATM 11
along with an increase in Th1, peroxisome proliferator–activated receptor γ, and actin 12
cytoskeleton–associated genes. These effects may support a role for lenalidomide 13
treatment and cell-cycle control and T cell activation (27). Previous studies have also 14
demonstrated the effects of immunomodulatory drugs on Th1- and Th2-associated 15
signatures as well as changes in elements associated with cytoskeletal remodeling and 16
T cell migration (10,28). The demonstrated early alterations in cytokine production by 17
lenalidomide may contribute to an altered T cell state that is able to simultaneously 18
enhance aspects of both memory and effector function (29). Overall, these results 19
suggest that additional factors beyond those previously reported are involved in the 20
lenalidomide-induced prolongation of CAR T function, including possible changes in 21
cell-cycle control. 22
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21
The application of ATAC-seq provided further insights into potential mechanisms of 1
action of lenalidomide. Although both stimulation and time were the predominant drivers 2
of chromatin accessibility changes, lenalidomide treatment was associated with 3
increases in chromatin accessibility in loci enriched in motifs associated with T cell 4
activation and function after chronic stimulation. These epigenetic changes were 5
coincident with the marked functional changes in CAR T cells incubated with 6
lenalidomide. Alterations in chromatin accessibility signatures have been associated 7
with T cell exhaustion and may be a more robust indicator of exhaustion compared with 8
T cell surface ligand expression (30). These data demonstrated that chronic stimulation 9
with lenalidomide resulted in increased chromatin accessibility and gene expression of 10
IL-2 and CD25 and decreased gene expression and chromatin accessibility of CCR7 11
and CD69. Previous studies suggested that CCR7-expressing cells produced higher 12
levels of IL-2 (31); however, the current study indicates that the IL-2 pathway could be 13
altered independently by lenalidomide, resulting in an alternative T cell state. CD69, a 14
marker of T cell activation, has a nuclear factor κB–responsive element that is required 15
for the CD69 response to TNF-α (32). The closing of CD69-associated chromatin and 16
decrease in transcripts may be a reaction to sustained increases in TNF-α production by 17
CAR T cells cultured with lenalidomide, or it may be a T cell response to increased 18
activation in the presence of lenalidomide. Lenalidomide-treated cells demonstrated 19
increased transcription factor motif enrichment of T cell activation–associated factors, 20
supporting the idea that these cells are exposed to sustained activation signaling. 21
Overall, the lenalidomide-induced CAR T cell state has elements of both effector T cell 22
function, including increased IFN-ɣ and TNF-α production, and memory T cell function, 23
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22
including increased IL-2 and long-term proliferation. Additional studies are underway to 1
determine more about the functional consequences of this alternative CAR T cell state 2
and the associated gene expression and epigenetic changes. 3
Finally, we observed an increase in function by a subcurative dose of anti-BCMA CAR T 4
cells in the OPM-2 orthotopic animal model with the addition of lenalidomide. Early 5
pharmacokinetic (PK) measurements indicated an increase in CAR T counts in the 6
blood, which were associated with improved tumor control and survival. The 7
combination of increased CAR T PK and increased functionality of CAR T cells, as 8
observed in in vitro studies, may have led to improved control over tumor growth 9
following a subcurative dose of CAR T cells. Because lenalidomide may also be applied 10
in a delayed administration setting, possibly weeks after CAR T administration due to 11
toxicity challenges with lymphodepletion and immunomodulatory drugs, we investigated 12
the feasibility of delayed lenalidomide administration. Interestingly, the addition of 13
lenalidomide following peak CAR T expansion at 14 days did not result in improved 14
tumor clearance or survival. These results suggest several possibilities. First, the CAR T 15
cells may have been functionally exhausted at 14 days following injection and were 16
unable to be enhanced by delayed lenalidomide administration. Second, the improved 17
tumor clearance was observed because of early CAR T function and circulating 18
numbers, and tumor clearance cannot be improved or rescued at such a delayed 19
progression. Additional studies should be undertaken to determine whether a window 20
for delayed administration of lenalidomide with CAR T cells exists that is more proximal 21
to CAR T administration. 22
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23
In sum, these studies provide novel insights into the mechanism of functional changes 1
in CAR T cells following lenalidomide addition in vitro and in vivo. The changes 2
associated with lenalidomide were intrinsic to anti-BCMA T cells because precise 3
control of CAR stimulation alone in the presence of lenalidomide led to increased 4
functionality, particularly at lower levels of stimulation. In addition, the transcriptional 5
and epigenetic changes associated with lenalidomide treatment suggest that an 6
alternative CAR T cell state of both enhanced memory and effector T cell functions is 7
induced with long-term lenalidomide treatment. Overall, the administration of 8
lenalidomide, a standard of care for patients with multiple myeloma, in combination with 9
anti-BCMA CAR T in the clinic may be warranted based on the potential for a 10
combination of effects, including tumoricidal effects, a more permissive tumor 11
microenvironment for CAR T function, and the observed intrinsic effects on CAR T 12
function. 13
14
Acknowledgments 15
We thank Kimberly Harrington for her scientific contributions to this project. The authors 16
thank Peter Simon, Chris Carter, and Jenna Quigley-Lee, of MediTech Media, Ltd, for 17
medical writing assistance, which was sponsored by Juno Therapeutics, Inc., A Celgene 18
Company. 19
20
Authorship contributions: MW, NS, CH, AB, JJ, WC, PC, DK, and HJ designed and 21
performed the experiments and analyzed the data. TJ, YJ, and RH analyzed the data. 22
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24
LW, CC, and CS designed and performed the experiments. BS and RS provided 1
scientific guidance and participated in the manuscript review. MW and MP drafted the 2
manuscript. All authors contributed to the writing and revision of the manuscript and 3
approved the final version. 4
5
6
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25
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Figure Legends 1
Figure 1. Anti-BCMA CAR T cytolytic activity and cytokine production increased 2
with lenalidomide in a concentration-dependent manner. Anti-BCMA CAR T 3
materials from 3 healthy donors and 1 patient donor were assessed for cytokine 4
production using 2 multiple myeloma cell lines. Cytolytic activity (A, C) and cytokine 5
production were measured after 24 hours (B, D) against OPM-2 (A, B) and RPMI-8226 6
(C, D). Cultures were incubated at a ratio of 0.3:1 (effector to target) for cytolytic activity 7
and 1:1 for cytokine production. Data were normalized to dimethyl sulfoxide vehicle 8
control; error bars represent standard error of the mean. For all functional assessments, 9
dose-response modeling indicated a significant effect for lenalidomide for each donor 10
and across all donors (P < .001), except healthy donors for RPMI-8226 cytolytic assay. * 11
indicates P < 0.05 for lenalidomide across each donor. 12
Figure 2. Anti-BCMA CAR T cytokine production was increased by lenalidomide 13
(Len) within 24 hours and across a range of stimulation intensities. Analysis of 14
CD25 and intracellular cytokine levels (left, white bars indicate baseline effects of bead 15
stimulation) for healthy CAR T donors after 24 hours of BCMA bead stimulation (gated 16
on transduced live CD3+ CAR+) for CD4+ (A) and CD8+ (B) subsets. Gray bars 17
demonstrate relative change for (Len) compared to vehicle alone. (C-D) Analysis of 18
effector cytokine production following CAR-specific stimulation on 50 µg BCMA and 50 19
µg PD-L1 beads for 24 hours in the presence of 1 µmol/L Len. * indicates P < 0.05 20
effect of Len for each stimulation condition. 21
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31
Figure 3. Anti-BCMA CAR T cell count, cytokine production, and activation were 1
increased by lenalidomide after repeated stimulation in vitro. (A) Analysis of cell 2
counts following serial stimulation in an MM1.S cell line in the presence or absence of 3
0.1 µmol/L Len. Data represent population doublings across 5 donors; error bars 4
represent standard deviations across 3 technical replicates. Linear mixed-effects 5
modeling indicated a significant effect for Len for each donor across time (P = 2.8 x 10-6
8). (B-D) Analysis of bulk cytokine production 24 hours following serial stimulation 7
replating at the indicated time points for 5 separate donors. Cytokine production was 8
normalized to cell number at each reset to account for differences in cell replating 9
density; error bars represent standard deviations. Linear-mixed models indicated a 10
significant effect for Len across donors and time points for IFN-γ (P < 2.9 x 10-10), IL-2 11
(P = 1.3 × 10-13), and TNF-α (P = 1.2 × 10-9). * indicates P < 0.05 compared to vehicle 12
for each donor. 13
Figure 4. Lenalidomide reduced functional exhaustion and altered surface 14
phenotype of anti-BCMA CAR T cells. Cells were treated for 7 days on 50 µg BCMA-15
coated beads in the presence or absence of 1 µmol/L Len. (A-D) Representative 16
healthy donor–derived, freshly thawed anti-BCMA CAR T cells (vehicle [Veh], Len) or 17
CAR T cells prestimulated with 7 days of BCMA bead stimulation and then cultured with 18
RPMI-8226 cells to measure cytolytic activity (over 7 days; A) and cytokine production 19
(24 hours; B-D). Percentage killing was normalized to anti-BCMA CAR T cells 20
prestimulated on beads in the presence of vehicle. Prestimulated CAR T cells showed 21
decreased cytolytic activity (P = 2.1 × 10-4) and cytokine production (P = .03 for IFN-γ) 22
compared with freshly thawed anti-BCMA CAR T cells. Len during the prestimulation 23
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32
period increased cytolytic function (P = .04). Significance was determined using t tests 1
from linear regression coefficients. Three anti-BCMA CAR T donors (each column) were 2
assessed for (E) overall viability and cell count and by (F) flow cytometry for median 3
fluorescence intensity (MFI; CD25 and TIM3) or percentage positive PD-1 and LAG3 on 4
the surface of T cell markers in CD4+ CAR+ and CD8+ CAR+ subsets (gated on live 5
CD3+ cells). Values shown are percentage baseline (Veh) MFI, viability, or count. * 6
indicates P < 0.05. 7
Figure 5. Anti-BCMA CAR T RNA-seq and ATAC-seq profiles were altered by 8
lenalidomide after short- and long-term stimulation. (A) Principal component 9
analysis of expression (RNA-seq) and (C) chromatin accessibility peaks (ATAC-seq). 10
(B) Volcano plots of differentially expressed genes or (D) peaks ± Len at 24 hours and 7 11
days. Directionality and significance of expression changes in selected, enriched 12
biological pathways at (E) 24 hours and (F) 7 days in CAR T cells ± 1 µmol/L Len. (G) 13
RNA expression compared with chromatin accessibility changes for selected T cell loci. 14
(H) Top enriched motif predictions in ATAC-seq loci with increased accessibility along 15
with their enrichment significance and prevalence at 7 days ± 1 µmol/L Len. FC, fold 16
change; FGF, fibroblast growth factor; NF-κB, nuclear factor κB; Ox, oxidative. 17
Figure 6. In vivo efficacy of subcurative dose of anti-BCMA CAR T and blood anti-18
BCMA CAR T count was altered by lenalidomide. (A) Two Len dosing regimens, 19
concurrent (C) or delayed (D) daily dosing, were tested in a disseminated NSG mouse 20
OPM-2 tumor model with a single, subcurative dose of anti-BCMA CAR T cells from 2 21
separate donors (8 mice per group). (B-C) Tumor bioluminescent measurement and (D-22
E) animal survival. Error bars represent standard error of the mean. Concurrent Len led 23
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33
to a significant decrease in tumor burden for donor 1 (P = .02) and increased survival 1
(log-rank test) for donor 1 (P = .057) and donor 2 (P = .04) compared with vehicle 2
(Veh)–treated animals injected with anti-BCMA CAR T alone. Linear mixed-effects 3
models (accounting for repeated mouse measurements over time) were used to 4
estimate treatment effects for the tumor burden analysis, and log-rank testing was used 5
for significance testing for the survival analyses. (F-G) Flow cytometric analysis of blood 6
CAR T cells gated on CD45+ CD3+ CAR+. Error bars represent standard error of the 7
mean. Concurrent Len showed significantly increased CAR T expansion after 7 days in 8
vivo (P = 7.3 × 10-6, t test). * indicates P < 0.05 for concurrent lenalidomide compared to 9
vehicle control. 10
11
12
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Figure 1
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15
20
0
10
20
30
400
10000
20000
30000
40000
0
1
2
3
4
5
0
1
2
3
4
5
0
5
10
15
20
0
10
20
30
Figure 2
IFN- γ IL-2 TNF-α
IFN- γ IL-2
CD4+
CD8+
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
A
B
C
D
IFN
-γ+,
%CD
25, M
FI
IFN
-γ+,
%
0.1 µmol/L Len1 µmol/L Len
no stim
5 µg BCMA
50 µg BCMA
200 µ
g BCMA
TNF-α
No PD-L1
0
50
100
150
200
PD-L1
IFN-γ IL-2 TNFHealthy Donor #1
Patient Donor
Vehicl
e
1 µmol/L
Len
Vehicl
e
1 µmol/L
Len
Vehicl
e
1 µmol/L
Len
Vehicl
e
1 µmol/L
Len
Vehicl
e
1 µmol/L
Len
Vehicl
e
1 µmol/L
Len
0
20,000
40,000
60,000
IFN
-γ, p
g/m
L
IL-2
, pg/
mL
TNF,
pg/
mL
IFN
-γ, p
g/m
L
IL-2
, pg/
mL
TNF,
pg/
mL
0
200
400
600
800
1000
0
20,000
40,000
60,000
80,000
0
200
400
600
0
200
400
600
Healthy Donor #2
Vehicl
e0
20,000
40,000
60,000
1 µmol/L
Len
IFN
-γ, p
g/m
L
Vehicl
e0
1000
2000
3000
4000
5000
1 µmol/L
Len
Vehicl
e0
100
200
300
400
TNF,
pg/
mL
1 µmol/L
Len
IL-2
, pg/
mL
IFN-γ
IFN-γ IL-2
IL-2
TNF
TNF
0
10000
20000
30000
40000CD
25, M
FI
* **
**
**
* * *
* * *
* *
*
* * * *
* * * *
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Time, days (reset time points)
Healthy 1 vehicleHealthy 1 Len
Healthy 2 LenHealthy 3 vehicle
Healthy 2 vehicle
Healthy 3 Len
Figure 3
Healthy 1 vehicleHealthy 1 LenHealthy 2 vehicleHealthy 2 LenHealthy 3 vehicle
Healthy 3 Len
A
B C D24-Hour Secreted Cytokines
Healthy 4 vehicleHealthy 4 Len
Patient vehiclePatient Len
0 4 7 11 14 18 21 25 280
5
10
15
Popu
latio
n do
ublin
gs
xx
x
x
xx
x
Healthy 4 vehicleHealthy 4 Len
Patient vehiclePatient Len
Day 4/
5Day
80.0
0.2
0.4
0.6
0.8
IFN-γ
IFN-γ,
pg/
mL/
cell
Day 4/
5Day
80.000
0.005
0.010
0.015
0.020
IL-2
, pg/
mL/
cell
IL-2
Day 4/
5Day
80.000
0.001
0.002
0.003
0.004
0.005
TNF
TNF,
pg/
mL/
cell
P = 3 x 10 -8
*
*
*
* *
** *
*
*
*
**
*
*
*
* *
*
*
*
*
*
* *
*
*
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Figure 4
10,000
20,000
30,000
40,000
IFN
-γ, p
g/m
L
IL-2
, pg/
mL
20
40
60
80
100
TNF,
pg/
mL
A B
C D
Vehicl
e
Prestim
vehicl
e
Prestim
LenLen
Vehicl
e
Prestim
vehicl
e
Prestim
LenLen
Vehicl
e
Prestim
vehicl
e
Prestim
LenLen
500
0
1000
1500
2000
2500
Vehicl
e
Prestim
vehicl
e
Prestim
LenLen
25
50
75
100
125
K
illin
g, %
Healthy 1 Healthy 2 Healthy 3
CD4
166 120 113
122 103 80
Viability
Count 50
100
150
200 105 135 90
151 227 164
300 191 175
72 78
1 2 3
TIM3
CD25
LAG3
PD-1 56
+
CD8
181 192 132
100 300 300
155 116 110
52 69 105
1 2 3
TIM3
CD25
LAG3
PD-1
+ +
100
200
300
E FVe
hicl
e, %
Vehi
cle,
%
CART
CART
Prestimulation (7 days)
Functional Assessments Len
Len
debead, washCAR
T Phentotypic Assessments
* *
*
*
*
*
*
*
*
*
*
*
BCMA bead
RPMI-8226
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24hr
24hr + stim
d7 + stim
H1 H2 H3 H4
24hr
24hr + stim
d7 + stim
H1 H2 H3 H4
Figure 5
−0.5 0.0 0.5 1.0
−2.0
−1.5
−1.0
−0.5
0.0
0.5
1.0
1.5
RNA Expression vs. ATAC Accessibility for Selected Genes
Chromatin Accessibility Change (Log2FC), Len vs Vehicle
RN
A Ex
pres
sion
Cha
nge
(Log
2FC
),Le
n vs
Veh
icle
CCR7CD69
IFN-g
IL2RA
down up total2 13 15
down up total2510 294 2804
down up total37 177 214
down up total255 328 583
A
B
C
D
ATAC-seq peak associated with locusATAC-seq mean/gene
E F
G H Increased Motif Enrichment
RNA-Seq ATAC-Seq
Len Vehicle Len Vehicle
log2 fold change log2 fold change log2 fold change log2 fold change
-log 10
p-a
dj
-log 10
p-a
dj
-log 10
p-a
dj
-log 10
p-a
dj
Motif Name
% of TargetSequences with MotifMotif Log P Value
Z-score (24h +stim, Len vs Vehicle) Z-score (d7, Len vs Vehicle)-log10(p) -log10(p)
Leukocyte extravasationNFkB
TREM1HGF
Renin-angiotensinActin-based motility by Rho
Acute phase responseHMGB1RhoGDl
cAMP-mediatedGNRH
Dendritic cell maturationG-alpha(s)
ERK/MAPKRac
Pho familyCdc42RhoA
IL-6PPARg/RXRa
Actin cytoskeletonSirtuin
ILKCXCR4Integrin
Wnt/B-cateninEphrin receptor
Thrombin
Signaling Pathway Signaling PathwayTh2
G2/M checkpointiCOS-iCOSL in Th cells
Th1FGFIL-3
NRF2 Ox. stress responseFLT3
GNRHATM
Renin-angiotensinBMP
cAMP-mediatedRhoA
Actin-based motility by RhoG-alpha(s)
PPARg
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1E6 CAR T injec on
D0 D14 D22 D28D8Concurrent lenalidomide (C)
Delayed lenalidomide (D)
OPM-2injec on
CAR T pharmacokinetic assessment
Figure 6
Mock + vehicleMock + LenCAR T + vehicle (C)CAR T + Len (C)CAR T + Len (D)
0 20 40 60 80 100
20
40
60
80
100
Days After CAR T Injection
Surv
ival
, %
Surv
ival
, %
0 20 40 60 80 100
20
40
60
80
100
Days After CAR T Injection
1
2
3
4
CA
R T
/µL
Blo
odC
AR
T/µ
L B
lood
Mock CAR T
1
2 Mock CAR T
0.5
1.0 Mock CAR T
0.5
1.0 Mock CAR T
1
2
3
4
1
2
2
4
6
8
10
1
2
3
Veh Len Veh
Len (C
)
Len (D
)Veh Len Veh
Len (C
)
Len (D
)Veh Len Veh
Len (C
)
Len (D
)Veh Len Veh
Len (C
)
Len (D
)
N/A
0 20 40 60
106
107
108
109
1010Donor 1
Days After CAR T Injection
Bio
lum
ines
cenc
e, p
/s
Bio
lum
ines
cenc
e, p
/s
0 20 40 60
106
107
108
109
1010Donor 2
Days After CAR T Injection
A
B C
D E
F
GN/A
Donor 1
Donor 2
Day 8 Day 14 Day 22 Day 28
*
*
*
*
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Published OnlineFirst August 8, 2019.Mol Cancer Ther Melissa Works, Neha Soni, Collin Hauskins, et al. of lenalidomidefunction against multiple myeloma is enhanced in the presence Anti-B-cell maturation antigen chimeric antigen receptor T cell
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on September 6, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
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