Embed Size (px)
Citation preview
Supplementary Materials for
Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of
CD8+ and CD4+ CD19-specific chimeric antigen receptor–modified T
cells
Cameron J. Turtle,* Laïla-Aïcha Hanafi, Carolina Berger, Michael Hudecek,
Barbara Pender, Emily Robinson, Reed Hawkins, Colette Chaney, Sindhu Cherian,
Xueyan Chen, Lorinda Soma, Brent Wood, Daniel Li, Shelly Heimfeld,
Stanley R. Riddell, David G. Maloney
*Corresponding author. Email: [email protected]
Published 7 September 2016, Sci. Transl. Med. 8, 355ra116 (2016)
DOI: 10.1126/scitranslmed.aaf8621
This PDF file includes:
Fig. S1. Plan of CD19 CAR-T cell manufacturing.
Fig. S2. CAR-T cell product characterization.
Fig. S3. Anti-CAR transgene product immune response.
Fig. S4. Tumor regression after a second CAR-T cell infusion in patients who
received Cy/Flu lymphodepletion.
Fig. S5. Delayed normalization of imaging after CD19 CAR-T cell infusion.
Table S1. Variable CD4+/CD8+ T cell ratio in blood of NHL patients.
Table S2. Summary of anti-CAR transgene product immune responses.
Table S3. CRS and neurotoxicity.
Table S4. Multivariate analyses.
Table S5. Simultaneous and sequential testing of cytokines to predict toxicity.
Table S6. P values for Figs. 4 (B and E) and 5 (A and B).
Reference (39)
www.sciencetranslationalmedicine.org/cgi/content/full/8/355/355ra116/DC1
SUPPLEMENTARY MATERIALS
Figure S1: Plan of CD19 CAR-T cell manufacturing. Leukapheresis PBMCs were divided into
two aliquots for selection of T cell subsets. CD4+ T cells were selected from 1 aliquot by positive
immunomagnetic selection using the CliniMACS CD4 Reagent System (Miltenyi Biotec). CD8+
TCM cells were enriched from the second aliquot using a 2-step CliniMACS selection procedure
involving depletion of CD4+, CD14+, and CD45RA+ cells followed by selection of CD62L+ cells
from the CD4-/CD14-/CD45RA- fraction 23. In patients with an absolute CD8+ TCM cell count of
<20/μL, severe lymphopenia, or circulating tumor, either the CD62L+ selection was omitted or
bulk CD8+ T cells were selected using the CliniMACS CD8 Reagent System. The enriched CD4+
and CD8+ T cell subsets were separately stimulated with anti-CD3/anti-CD28 paramagnetic
beads and transduced with a lentivirus encoding a CD19-specific CAR 24 and EGFRt, which
serves as a marker of transgene expression. Cells were cultured in medium supplemented with
IL-2. After anti-CD3/anti-CD28 paramagnetic bead removal, CAR-T cells were stimulated once
with a clinically qualified irradiated allogeneic LCL line, followed by expansion in culture and
formulation in a 1:1 ratio of CD3+/CD4+/EGFRt+: CD3+/CD8+/EGFRt+ cells for infusion
approximately 15-20 days after initial anti-CD3/anti-CD28 paramagnetic bead stimulation.
Figure S2: CAR-T cell product characterization. A) The percentage of CD4+ T cells in a
viable CD45+/CD3+ gate. B) The percentage of EGFRt+ cells in the CD4+ T cell gate. C) The
percentages of CD45RA+/CD62L+, CD45RA-/CD62L+, CD45RA-/CD62L-, and CD45RA+/CD62L-
cells in the CD4+/EGFRt+ gate. D) The percentage of CD8+ T cells in a viable CD45+/CD3+ gate
is shown for CAR-T cell products manufactured from bulk CD8+ T cells (CD8) and CD8+ central
memory T cells (CD8TCM). E) The percentage of EGFRt+ cells in the CD8+ T cell gate is shown
for CAR-T cell products manufactured from bulk CD8+ T cells (CD8) and CD8+ central memory
T cells (CD8TCM). F) The percentages of CD45RA+/CD62L+, CD45RA-/CD62L+, CD45RA-
/CD62L-, and CD45RA+/CD62L- cells in the CD8+/EGFRt+ gate are shown for CAR-T cell
products manufactured from bulk CD8+ T cells (left) and CD8+ central memory T cells (right).
Each point represents data from a single patient. The red lines indicate the median of each
group.
Figure S3: Anti-CAR transgene product immune response. A) Cy/Flu reduces CD8+ T cell-
mediated cytotoxicity directed against the CAR transgene. Cryopreserved PBMCs collected
from patients before lymphodepletion chemotherapy and approximately 4 weeks after the first
and/or second CAR-T cell infusion were stimulated twice at 7-day intervals with autologous
irradiated CAR-T cells and IL-2. Pre-infusion and post-infusion cultures from Patient 13 were
evaluated by 51chromium release assay against CD19 CAR-transduced autologous T cells
(black) or non-transduced autologous T cells (white). B-C) Patient 13’s T cell lines that exhibited
specific lysis of autologous CAR-T cells were stimulated with pools of overlapping peptides from
the CAR construct, and peptide pools that induced IFN-γ secretion higher than that induced by T
cells alone in an ELISpot assay were identified. Peptides 50 and 51 are located within the
murine scFv.
Figure S4: Tumor regression after a second CAR-T cell infusion in patients who received
Cy/Flu lymphodepletion. Imaging studies from Patients 14 and 28 (Table S3), who received
Cy/Fly lymphodepletion before their first cycle of CAR-T cells, demonstrate tumor regression
after a second cycle of Cy/Flu lymphodepletion chemotherapy and CAR-T cell infusion at an
equivalent or higher dose of CAR-T cells.
Figure S5: Delayed normalization of imaging after CD19 CAR-T cell infusion. Continued
tumor regression was noted after Cy/Flu lymphodepletion and 2x106 EGFRt+ cells/kg without
additional anti-tumor therapy in a patient with refractory primary mediastinal B cell lymphoma. A)
Integrated CAR transgene in PBMC demonstrates robust CAR-T cell expansion and persistence
after CAR-T cell infusion. B) PET-CT scans show a large FDG-avid renal mass before CAR-T
cell therapy (left) that increased in size on day 28 after CAR-T cell infusion (middle). Without any
additional therapy, there was resolution of the renal mass by day 62 after CAR-T cell therapy
(right). The patient remains in CR 5 months after CAR-T cell infusion. C) A biopsy of the renal
mass on day 34 after CAR-T cell infusion showed infiltration of EGFRt+ CAR-T cells.
Table S1. Variable CD4+/CD8+ T cell ratio in blood of NHL patients.
CD8+ T
cells/L
CD4+ T
cells/L
CD4+:CD8+ ratio
1147 928 0.8
225 409 1.8
313 690 2.2
306 114 0.4
122 198 1.6
810 388 0.5
816 295 0.4
9 21 2.3
624 86 0.1
190 295 1.6
467 361 0.8
310 465 1.5
210 374 1.8
270 289 1.1
331 1088 3.3
3475 997 0.3
120 329 2.7
97 213 2.2
211 150 0.7
545 627 1.2
82 196 2.4
55 287 5.2
264 379 1.4
108 142 1.3
35 59 1.7
66 184 2.8
196 93 0.5
1971 329 0.2
1548 339 0.2
7 58 8.3
49 49 1.0
143 1956 13.7
Table S2. Summary of anti-CAR transgene product immune responses.
Patient
CAR-T
cell infusions
First lymphodepletion
Second lymphodepletion
Anti-CAR CD8+ T cell-mediated lysis
Before
lymphodepletion and first CAR-T cell
infusion
After first CAR-T cell
infusion
After second CAR-T cell
infusion
1 2 Cy/E Cy Negative Equivocal Positive
2 2 Cy/E None Negative Positive Positive
3 2 Cy/E Cy Negative Positive Positive
6 2 Cy/E None Negative Negative Positive
13 2 Cy Cy/Flu Negative Positive Not done
14 2 Cy/Flu Cy/Flu Negative Negative Negative
16 2 Cy/Flu Cy/Flu Negative Negative Negative
18 1 Cy/Flu Not applicable Negative Negative Not applicable
20 2 Cy/Flu Cy/Flu Negative Positive Positive
27 1 Cy/Flu Not applicable Negative Negative Not applicable
28 2 Cy/Flu Cy/Flu Negative Negative Negative
PatientCD8+ T cell isolation
LymphodepletionInfused
CD4+/EGFR+ T cells/kg
Infused CD8+/EGFR+ T cells/kg
ICU care Reason for ICU admissionCRS grade according to Lee et al (39)
Neurotoxicity (CTCAE v4.03)
Tocilizumab (4-‐8 mg/kg IV; days after CAR-‐T cell infusion)
Dexamethasone (10-‐20 mg IV
daily; days after CAR-‐T cell infusion)
Methylprednisolone (1-‐2 g IV daily; days after CAR-‐T cell
infusion)
Duration of hospitalization
(days)*
1 CD8TCM Cy/E 1.00E+05 1.00E+05 No NA 2 0 No 0 0 82 CD8TCM Cy/E 1.00E+05 1.00E+05 No NA 2 0 No 0 0 43 CD8TCM Cy/E 1.00E+06 1.00E+06 No NA 1 0 No 0 0 94 CD8TCM Cy/E 1.00E+06 1.00E+06 No NA 1 0 No 0 0 65 CD8TCM Cy/E 3.50E+04 8.80E+06 No NA 0 0 No 0 0 26 CD8TCM Cy/E 1.00E+06 1.00E+06 No NA 0 0 No 0 0 57 CD8TCM Cy 1.00E+06 1.00E+06 No NA 0 0 No 0 0 08 CD4/14/45RA-‐neg Cy/E 1.60E+06 3.90E+05 No NA 3 3 No 0 0 229 CD8TCM Cy 1.00E+07 1.00E+07 No NA 0 0 No 0 0 010 CD8TCM Cy 1.00E+07 1.00E+07 No NA 0 0 No 0 0 111 CD8TCM Cy/Flu3 1.00E+07 1.00E+07 Yes Cardiac failure 4 3 5 0 0 1812 CD8TCM Cy/Flu3 1.00E+06 1.00E+06 No NA 0 0 No 0 0 013 CD8TCM Cy 1.00E+06 1.00E+06 No NA 1 0 No 0 0 014 CD8TCM Cy/Flu5 1.00E+07 1.00E+07 No NA 2 0 No 0 0 1015 CD8 Cy 1.00E+06 9.10E+05 No NA 3 0 No 0 0 3216 CD8TCM Cy/Flu5 3.34E+06 3.62E+06 No NA 2 0 No 0 0 717 CD8 Cy/Flu3 1.00E+06 1.00E+06 No NA 0 0 No 0 0 018 CD8 Cy/Flu5 1.00E+07 1.00E+07 No NA 3 3 No 0 0 819 CD8 Cy/Flu3 1.00E+07 1.00E+07 Yes Hypotension, cardiac arrhythmia 5 4 4, 18, 21 4-‐6, 13-‐15, 19-‐23 7, 21 3020 CD8 Cy/Flu5 1.00E+06 1.00E+06 No NA 3 3 No 0 0 721 CD8 Cy/Flu5 1.00E+07 1.00E+07 Yes Hypotension, neurotoxicity 5 5 2, 4 4 5-‐12 1322 CD8TCM Cy/Flu3 1.00E+05 1.00E+05 No NA 3 3 No 0 0 723 CD8TCM Cy/Flu3 1.00E+05 1.00E+05 Yes Disease progression 0 0 No 13 14-‐18 2024 CD8TCM Cy/Flu3 1.00E+05 1.00E+05 No NA 0 0 No 0 0 025 CD8 Cy/Flu3 1.00E+06 1.00E+06 No NA 0 0 No 0 0 026 CD8TCM Cy/Flu3 1.00E+06 1.00E+06 No NA 0 0 No 0 0 027 CD8 Cy/Flu3 1.00E+06 1.00E+06 No NA 2 0 No 0 0 528 CD8TCM Cy/Flu3 1.00E+06 1.00E+06 No NA 2 0 No 0 0 329 CD8TCM Cy/Flu3 1.00E+06 1.00E+06 No NA 1 0 No 0 0 530 CD8 Cy/Flu3 1.00E+06 1.00E+06 No NA 1 0 No 0 0 631 CD8 Cy/Flu3 9.99E+05 9.80E+05 Yes Neurotoxicity 3 3 No 9, 10 0 1232 CD8TCM Cy/Flu3 9.95E+05 9.99E+05 No NA 0 0 No 0 0 0
Legend:CD8 CliniMACS CD8+ T cell isolationCD8TCM CD4/CD14/CD45RA-‐depletion followed by CD62L positive selection from the CD4/14/45RA-‐negative fractionCD4/14/45RA-‐neg CD4/CD14/CD45RA-‐depleted PBMCCy Cyclophosphamide 2-‐4 g/msq IV on day 1Cy/E Cyclophosphamide 2-‐4 g/msq IV on day 1; Etoposide 100-‐200 mg/msq/day IV on days 1-‐3Cy/Flu3 Cyclophosphamide 30-‐60 mg/kg IV on day 1; Fludarabine 25 mg/msq IV days 2-‐4Cy/Flu5 Cyclophosphamide 60 mg/kg IV on day 1; Fludarabine 25 mg/msq IV days 2-‐6*All cause; from the start of lymphodepletion
Table S4. Multivariate analyses.
Multivariate analysis for the occurrence of severe neurotoxicity was performed using the stepwise
logistic regression method. The model included the following baseline variables: lymphodepletion
regimen, percentage of CD19+ cells in bone marrow before lymphodepletion therapy, disease
subcategory, infused CAR-T cell dose, number of previous therapies, and CD4+, CD8+, and CD8+ central
memory T cell counts before subset isolation. Each post-CAR-T cell infusion variable (peak
CD4+/EGFRt+ cell count in blood, peak CD8+/EGFRt+ cell count in blood, peak serum ferritin, and peak
serum IL-6 concentration) was entered into the model separately to examine its predictability.
Variables p-value
Peak CD4+/EGFRt+ cell count 0.045
Peak CD8+/EGFRt+ cell count 0.042
Peak serum ferritin 0.022
Peak serum IL-6 0.018
Day 1 serum IL-6 0.016
Day 1 serum IL-15 0.031
Table S5. Simultaneous and sequential testing of cytokines to predict toxicity.
Cytokines Simultaneous Sequential
Net Sensitivity Net Specificity Net Sensitivity Net Specificity
IL-15 and TGF-β 0.98 0.61 0.77 0.95
IL-15 and IL-6 0.97 0.63 0.69 0.96
TGF-β and IL-6 0.97 0.63 0.69 0.96
IL-15, TGF-β, and IL-6 1.00 0.49 0.60 0.99
Table S6. P values for Figs. 4 (B and E) and 5 (A and B).
Figure 4B Peak p-value
CRS (severe vs. mild)
IL-6 0.005
IFN-γ 0.046
Ferritin 0.007
CRP 0.002
CRS (mild vs. none)
IL-6 0.002
IFN-γ 0.080
Ferritin 0.005
CRP <0.001
Figure 4E Peak p-value
Neurotoxicity (grade ≥ 3 versus
grade 0-2)
IL-6 0.004
IFN-γ 0.009
IL-15 0.002
IL-2 0.014
IL-18 0.019
TIM3 0.020
Ferritin <0.001
CRP 0.010
TGF-β 0.003
Figures 5A, B Day 1 p-value
CRS (severe vs. not
severe)
IL-8 0.004
IFN-γ 0.006
IL-15 0.005
IL-10 0.011
IL-6 0.031
Neurotoxicity (grade ≥ 3 versus
grade 0-2)
IL-15 0.002
TGF-β 0.003
IL-6 0.006
IL-10 0.016
IL-8 0.039
IFN-γ 0.041
