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: mvmaus@mgh.harvard.edu

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