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Supplementary Materials for
A single dose of peripherally infused EGFRvIII-directed CAR T cells
mediates antigen loss and induces adaptive resistance in patients with
recurrent glioblastoma
Donald M. O’Rourke, MacLean P. Nasrallah, Arati Desai, Jan J. Melenhorst,
Keith Mansfield, Jennifer J. D. Morrissette, Maria Martinez-Lage, Steven Brem,
Eileen Maloney, Angela Shen, Randi Isaacs, Suyash Mohan, Gabriela Plesa,
Simon F. Lacey, Jean-Marc Navenot, Zhaohui Zheng, Bruce L. Levine, Hideho Okada,
Carl H. June, Jennifer L. Brogdon, Marcela V. Maus*
*Corresponding author. Email: [email protected]
Published 19 July 2017, Sci. Transl. Med. 9, eaaa0984 (2017)
DOI: 10.1126/scitranslmed.aaa0984
The PDF file includes:
Materials and Methods
Fig. S1. Sample stain of peripheral blood T cells for CART-EGFRvIII.
Fig. S2. Lack of correlation between engraftment and absolute lymphocyte count.
Fig. S3. Histology and CD3 immunohistochemistry stain of pre- and post-CART
infusion tumor specimens.
Fig. S4. Validation of RNAscope ISH.
Fig. S5. Validation of PD-L1 staining.
Table S1. Individual patient characteristics.
Table S2. Individual product characteristics.
Table S3. Individual post-CART infusion events.
Table S4. Adverse events at least possibly related to CART-EGFRvIII.
Table S5. Immunohistochemical antibodies and ISH probes used.
Legend for table S6
Other Supplementary Material for this manuscript includes the following:
(available at
www.sciencetranslationalmedicine.org/cgi/content/full/9/399/eaaa0984/DC1)
Table S6. Primary cytokine data (separate Excel file).
www.sciencetranslationalmedicine.org/cgi/content/full/9/399/eaaa0984/DC1
Materials and Methods
Reagents and protocol for flow cytometry of CART-EGFRvIII cells
Antibodies for T cell detection panels were anti-CD45 V450 (clone HI30), anti-CD14
V500 (clone M5E2), anti-CD56 Ax488 (clone B159), anti-CD4 PerCP-Cy5.5 (clone
RPA-T4), anti-CD8 APC-H7 (clone SK1) (all from BD Bioscience). Also, anti-CD3
BV605 (clone OKT3), anti-HLA-DR BV711 (clone L243), anti-CD19 PE-Cy7 (clone
H1B19) were used from Biolegend. CAR-EGFRvIII expression was assessed by using a
bis-biotinylated EGFRvIII peptide (916-biotin, Novartis) and the secondary staining
reagent Streptavidin-PE from BD Bioscience (cat#554061). Cells were resuspended in
100 µL PBS containing 1% fetal bovine serum, 0.02% sodium azide and bis-biotinylated
EGFRvIII peptide and incubated for 30 min on ice, washed, resuspended in 100 µL PBS
containing 1% fetal bovine serum, 0.02% sodium azide, surface antibodies and SA-PE,
and incubated for 30 minutes on ice, washed, resuspended in 250ul PBS containing 1%
fetal bovine serum and 0.02% sodium azide and acquired using a Fortessa flow cytometer
equipped with a violet (405 nm), blue (488 nm), a green (532 nm), and a red (628 nm)
laser. Data were analyzed using FlowJo software (Version 10, Treestar). Compensation
values were established using eBiosciene UltraComp eBeads (eBioscience cat#01-222-
42) and DIVA software. The gating strategy for T cells was as follows: Intact cells (FSC-
A/SSC-A) / single cells (FSC-A/FSC-H) / CD14- CD16- CD19- CD3+/ CD3+.
Tumor specimen processing and staining
Hematoxylin and eosin staining and immunohistochemistry were conducted on 5-µm-
thick FFPE tissue sections mounted on Leica Surgipath slides following drying at 70 °C
for 60 min. Immunohistochemistry for the anti-CD3 mouse monoclonal antibody (clone
LN10, Leica #PA0553) was performed on a Leica Bond III instrument using the Bond
Polymer Refine Detection System (Leica Microsystems AR9800) following heat-induced
epitope retrieval with epitope 2/EDTA for 20 minutes.
Reagents and protocols for immunohistochemistry and RNAscope ISH
The reagents utilized were: 1) RNAscopeVS probe sets including positive control probe
PPIB (Cat#313906-C2) and DapB negative control probe (Cat#310048), 2) RNAscopeVS
FFPE reagent kit (Cat#320600) including Pretreat A, pretreat B, and Amp1 through 7, 3)
RNAscopeVS FFPE accessory kit (Cat#320630) and 4) RNAscopeVS FFPE offline CC
Kit (Cat#320043) including 10x pretreat 2 solution. For the Ventana automated system
the reagents used were: 1) mRNA DAB detection kit (Cat#760-224), 2) mRNA probe
amplification kit (Cat#760-222), 3) mRNA pretreatment kit (Cat# 760-223) and 4) Probe
dispensers for ACD probes (Cat# 960-76X). The ISH method followed protocols
established by ACD Bio and Ventana systems. Briefly, the sections were baked at 60
degrees for 30 minutes. The rehydration protocol was 3 steps xylene for 3 minutes; 2
times 100% alcohol for 3 minutes; once 95% alcohol for 3 minutes; followed by one step
80% alcohol for 3 minutes; distilled water rinse for one minute; and tap water for 2
minutes. Rehydration was follow by offline tissue conditioning at 99 degree for 15
minutes. Finally, the slides were transferred to Ventana Ultra for finishing the ISH
procedure including protease pretreatment; hybridization and amplification for three
hours; and detection with HRP and hematoxylin counter stain.
Fig. S1. Sample stain of peripheral blood T cells for CART-EGFRvIII. Staining is
shown for subject 201, 7 days following CART-EGFRvIII infusion. Cells were gated on
forward and side scatter, and CD3 positivity; doublets and CD14/CD16/CD19 staining
macrophages, NK cells, and B cells were excluded. Negative controls included FMO and
addition of secondary antibody without biotinylated EGFRvIII-peptide (left panel).
CART-EGFRvIII engraftment by flow cytometry as graphed in Figure 3a was calculated
by subtracting CART-EGFRvIII positively gated cells in the no-peptide control from
CART-EGFRvIII positively staining cells with biotinylated peptide (i.e., in this example,
9.4 – 0.3 = 9.1).
FMO CART-EGFRvIII
Fig. S2. Lack of correlation between engraftment and absolute lymphocyte count.
Spearman correlation of absolute lymphocyte count with peak expansion of CART-
EGFRvIII cells in peripheral blood as measured by flow cytometry (left) or quantitative
PCR (right). No significant correlation is observed.
400 600 800 1200LLN (1000)0
2
4
6
8
10
ALC (x103 cells/µL)
Pea
k E
xpan
sion
(% C
D3+
)
Rho = -0.024P = 0.94(n = 10)
400 600 800 1200LLN (1000)0
1000
2000
3000
4000
5000
ALC (x103 cells/µL)
Pea
k co
pies
/µg
geno
mic
DN
A
Rho = -0.26P = 0.46(n = 10)
Fig. S3. Histology and CD3 immunohistochemistry stain of pre- and post-CART
infusion tumor specimens. Data are shown for the first five subjects who underwent
post-CART tumor resection. Subject ID numbers are indicated at left (35313 was the
institutional protocol number, followed by the subject number). Hematoxylin and eosin
(H&E) staining are shown at left, CD3 immunohistochemistry staining is shown at right
for each subject. Day numbers are relative to CART-EGFRvIII infusion, which was
designated as Day 0.
Fig. S4. Validation of RNAscope ISH. Representative fields demonstrating infiltration
of CAR positive cells in human xenograft (top row) and absence of signal in xenograft
from mice treated with un-transduced (UTD) cells (bottom row). No signal is apparent
with the negative control probe directed at a bacterial dihydrodipicolinate reductase
(DAPB) gene. Diffuse signal is apparent with the positive control probe directed against
the peptidylprolyl isomerase B (PPIB) housekeeping gene.
Fig. S5. Validation of PD-L1 staining. PD-L1 expression was measured by IHC with
the Ventana immunohistochemistry assay, an approved companion diagnostic assay for
nivolumab. (a) shows representative staining of glioblastoma tumor sample at high and
low power (inset); staining is observed in a subset of tumor, glial, and infiltrating
inflammatory cells. (b) concurrent irrelevant IgG negative control, (c) thymus positive
control tissue, both of which were included in each run of the assay.
PD-L1 IHC PD-L1 IHC IgG A B C
Table S1. Individual patient characteristics.
Table S2. Individual product characteristics.
CART-EGFRvIII
CART-EGFRvIII
ID# %Transd. Dose
201 12.3 4.99 x 108
202 4.8 1.75 x 108
204 21.1 5 x 108
205 14 5 x 108
207 21.6 5 x 108
209 18.4 5 x 108
211 23 5 x 108
213 25.6 5 x 108
216 21.4 5 x 108
217 12.8 5 x 108
Table S3. Individual post-CART infusion events.
ID number Grade 3 or higher Adverse Events
Treatment Following Study
Withdrawal/Disease Progression
No
surgery
201 None Bevacizumab
202 Day 9: Gr.3 seizure; Dex 4q6;
Day 15: Siltuximab Bevacizumab
204 None Bevacizumab
Late
surgery
205 None Bevacizumab and MLN0128 (clinical trial)
207 None Carboplatin and bevacizumab
209 None Not applicable
Early
surgery
211
Day 27: Gr.3 headache, weakness;
Day 29: Siltuximab; dex10q6; Gr.4
cerebral edema
Bevacizumab with lomustine followed by
bevacizumab with irinotecan
213 Day 14: hemorrhage in operative bed Bevacizumab alone followed by
bevacizumab with irinotecan
216 None
Bevacizumab alone followed by
bevacizumab with lomustine and
rindopepimut vaccine (expanded access
vaccine trial)
217 None Bevacizumab
Table S4. Adverse events at least possibly related to CART-EGFRvIII. Data cutoff 8/31/2016.
Table S5. Immunohistochemical antibodies and ISH probes used.
Table S6. Primary cytokine data (separate Excel file).
Target Antibody/probe type source Final concentration
CD3 790-4341 Monoclonal Ventana predilute
CD134 TA307365 Monoclonal Origene 19ug/ml
CD25 LS-C152559 Monoclonal LifeSpan BioSciences
16ug/ml
PDL1 SP263 Monoclonal Ventana predilute
EGFRvIII hEGFRVIII B_D rabbit ab
(IPROT:102817 pPL3052)
N/A Internal 1.25ug/ml
CD8 790-4460 Monoclonal Ventana predilute
3’UTR CAR Cat#438086 N/A ACD Bio N/A
PPIB Cat#313906-C2 ISH Probe ACD Bio N/A
DAPB Cat#310048 ISH Probe ACD Bio N/A
IFN-γ Cat#310501 N/A ACD Bio N/A
GRZMB ab134933 Monoclonal Abcam 6.8ug/ml