Supplementary Materials and Methods
Plasmid vectors
DNA plasmids SB11-pIRES2-EGFP codes for SB11 and EGFP under control of CMV
promoter in a modified bicistronic reporter construct (pIRES2-EGFP, Clontech, Palo Alto,
CA). To generate CD19+ T cells as target cells, we generated the SB plasmid
ΔCD19(CoOp)-F2A-HyTK/pSBSO coding for the extracellular domain of truncated
human CD19 (amino acid 1 to 313)(23) by ligating F2A self-cleavage element and coexpressed with CoOp hygromycin phosphotransferase (Hygro) fused to CoOp thymidine
kinase (TK, Supplementary Figure 1d). To generate CD19+ NSO target cells, we
generated the SB plasmid ΔCD19(CoOp)-F2A-Neo-TK/pSBSO by replacing Hygro in
ΔCD19(CoOp)-F2A-HyTK/pSBSO with a neomycin resistance gene cassette encoding
aminoglycoside phosphotransferase (Supplementary Figure 1e).
Isolation of CD3+ T cells
To isolate CD3+ T cells, 107 PBMC were mixed with 20 uL of human NK-cell isolation
MACS microbeads (Miltenyi Biotec, Auburn, CA, catalog #130-092-657) in a total
volume of 100 uL and incubated on ice for 30 minutes. During incubation a LS column
(Miltenyi Biotec, catalog #130-042-401) was primed by washing twice with MACS buffer
(Miltenyi Biotec, catalog #130-091-221) and the MACS microbeads labeled cell
suspension was loaded onto the LS column followed by washing twice with MACS
buffer. The flow through (unlabeled cells) were collected, pelleted at 200g for 10
minutes, mixed with 20 uL of CD3+ T-cell isolation MACS microbeads (Miltenyi Biotec,
catalog #130-050-101) in a total volume of 100 uL, and incubated on ice for 30 minutes.
Following incubation labeled cell suspension was loaded onto a fresh LS column
followed by washing twice with MACS buffer. CD3+ T cells retained in the column were
eluted as the positively selected fraction.
Electroporation and propagation to generate CAR+ T cells
The electroporated cells were transferred to 12-well plates containing 3 to 4 mL of
phenol-free RPMI culture media supplemented with 20% FBS; rested for 2 to 3 hours at
37°C; and cultured overnight in 6 to 7 mL of 10% phenol-free RPMI supplemented with 2
mM L-glutamine and 10% FBS. After overnight culture, the T cells were co-cultured with
thawed irradiated clone 4 aAPC (Figure 1a and 1b) (Clone 4 aAPC γ-irradiated with 100
Gy) at a 1:2 ratio (CAR+ T cell: aAPC) based on CAR+ (Fc+) expression. Artificial APC
(clone #4) were re-added every 7 days at the same ratio. Soluble recombinant human IL21 (eBiosciences, San Diego, CA, cat # 34-8219-85) was added at a concentration 30
ng/mL beginning on Day 1 of culture and soluble recombinant IL-2 (Chiron, Emeryville
CA) was added at a concentration of 50 units/mL beginning on Day 7 of culture. IL-21
and IL-2 were re-added on a Monday, Wednesday, and Friday schedule after addition of
aAPC. T cells were enumerated every 7 days and viable cells were counted by Trypan
blue dye exclusion using a Cellometer automated cell counter (Nexcelom Bioscience,
Lawrence, MA) with 0.4% Trypan blue at a 1:1 ratio. If the percentage of CD3negCD56+
(NK cells) exceeded 5%, the cell culture was depleted of CD56+ cells using a CD56specific antibody and paramagnetic beads (Miltenyi Biotec, cat # 130-050-401) over a
LS column.
Generating CD19+ NS0 cells
To generate target CD19+ mouse cells, 106 NSO cells were re-suspended in 100 µL of
mouse B cell Nucleofector solution (Lonza, catalogue # VCA-1003), mixed with 2 µg of
supercoiled ΔCD19(CoOp)-F2A-Neo-TK/pSBSO and 0.5 µg of supercoiled pKan-CMVSB11, and electroporated using Nucleofector Program Z-001 (Lonza) in a Nucleofector II
device (Lonza). Beginning 2 days after electroporation neomycin sulfate (InvivoGen,
Cat# ant-gn) was added to the culture at 800 g/mL and this drug was re-added every
other day for a month. Genetically modified NS0 cells underwent fluorescence activated
cell sorting (FACS), (BD Biosciences, FACSAria) for homogeneous expression of CD19
using mouse antibody specific for anti-human CD19 (BD Pharmingen; cat#555413).
Generating CAR+ Jurkat cells
Jurkat cells (5x106) were suspended in 100 µL of Nucleofector Kit V (Lonza, Rockland,
MD) and mixed with 5 µg of supercoiled CD19RCD28mz (CoOp)/pSBSO and 5 µg of
supercoiled pKan-CMV-SB11, transferred to one cuvette, and electroporated using
Nucleofector Program T-14 (Lonza). The electroporated cells were incubated for 10
minutes at room temperature, transferred to 6-well plates containing 5 mL of complete
RPMI [HyQ RPMI 1640 (Hyclone, Logan, UT) supplemented with 2 mM L-glutamine
(GlutaMAX-1, Life Technologies–Invitrogen, Carlsbad, CA) and 10% heat-inactivated
defined fetal bovine serum (FBS; Hyclone)], rested for 2 to 3 hours at 37°C, and washed
with phosphate-buffered saline. The cells were then re-suspended in complete RPMI
medium.
Two
weeks
after
electroporation,
using
anti-human
Fc
antibody
allophycocyanin-conjugated F(ab’)2 fragment goat anti-human immunoglobulin G, Fc
fragment–specific (Jackson ImmunoResearch, West Grove, PA, catalog #109-136-170)
FACS generated Jurkat cell clones that uniformly expressed CAR and clone #12 was
numerically expanded for further analysis. To generate Jurkat cells that constitutively
expressed SB11 and EGFP, 5 µg of supercoiled CMV-SB11-pIRES2-EGFP was electrotransferred into 5x106 Jurkat cells using conditions described above. Two weeks after
electroporation, FACS was used to generate Jurkat cell clones that uniformly expressed
EGFP. EGFP+ clones were tested for SB11 expression by RT-PCR and clones that were
positive for expression of both SB11 and EGFP were used for further experiments.
Flow cytometry
Fluorochrome-conjugated antibodies specific for CD3 (catalogue #347347), CD4
(catalogue #555349), CD8 (catalogue #341051), CD56 (catalogue #555518), CD19
(catalogue #555413), CD32 (catalogue #555448), CD62L (catalogue #559772), and
antibodies CD28 (catalogue #337181), CD64 (catalogue #558592), CD86 (catalogue
#555658), CD137L (catalogue #559446), and CD19 (catalogue #555413) were obtained
from BD Biosciences (San Jose, CA). EGFP expression was used to monitor expression
of mIL-15. Cell surface expression of CD19-specific CAR was detected using F(ab')2
fragment of PE-conjugated goat anti-human Fc at a 1/20 dilution. Anti-human Fc
antibody allophycocyanin-conjugated F(ab’)2 fragment goat anti-human immunoglobulin
G, Fc fragment–specific (Jackson ImmunoResearch, West Grove, PA, catalog #109136-170) was used to detect CAR expression on Jurkat cells. Nonspecific binding of
antibody was blocked using wash buffer (2% FBS in phosphate-buffered saline).
Monitoring for autonomous T-cell growth
We determined whether electroporated and propagated primary T cells were able to
survive and proliferate in the absence of aAPC and exogenous IL-2 following their 28day culture on aAPC. A defined number of genetically modified T cells were cultured for
up to 14 days without aAPC or IL-2 and the number of T cells was compared to the
number of T cells seeded. T cells propagated on aAPC, pre-loaded with OKT3, and IL-2
served as the positive control for proliferation.
Analysis of Lysis of CD19+ target cells
The acquired images were analyzed for target cell death using AttoVision software (BD
Biosciences) and the results were plotted. Specific lysis was calculated using BD Image
Data Explorer software (BD Biosciences) based upon the formula: percent specific lysis
= (number of dead cells/total number of cells) x 100. To further test the specificity of
CAR+ T cells, the ΔCD19(CoOp)-F2A-HyTK/pSBSO vector was introduced in autologous
primary human T cells and mouse NSO cells. A 4-hour chromium release assay (CRA)
was used to assess the killing of ΔCD19+ NSO cells and autologous ΔCD19+ T-cell
targets by CAR+ T cells.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was purified with an RNeasy mini kit (Qiagen, Valencia, CA) and treated with
DNase (Invitrogen) to remove any contaminated genomic DNA. Total RNA from Jurkat
cells genetically modified to enforce expression of SB11 (and EGFP) was used as the
positive control for SB11 expression (Supplementary methods). Reverse transcription
(RT) reactions were carried out with 2 µg RNA, Superscript III, and random hexamer
primers (Invitrogen) according to the manufacturer’s instructions. A control reaction was
performed without RT to determine presence of contaminating genomic DNA. One
microliter of first-strand cDNA was used as a template for both SB11 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR. The forward primer 5′ATGGGAAAATCAAAAGAAATC-3′
and
reverse
primer
5′-
CTAGTATTTGGTAGCATTGC-3′ were used for SB11 PCR, and the forward primer 5′TCTCCAGAACATCATCCCTGCCAC-3′
and
reverse
primer
5′-
TGGGCCATGAGGTCCACCACCCTG-3′ were used for GAPDH PCR. PCR was
accomplished with Taq PCR Master mix (Qiagen, CA) with an initial denaturation at
94°C for 2 minutes, followed by 30 cycles of 94°C for 15 seconds, 58°C for 40 seconds,
and 72°C for 1 minute followed by an extension at 72°C for 5 minutes. PCR products
were resolved by 0.8% agarose gel electrophoresis.
PCR with genomic DNA
Total genomic DNA was isolated with QiaAmp DNA Mini Kit (Qiagen). Genomic DNA
from SB11+ Jurkat cells was used as the positive control for the presence of SB11. To
detect SB11, 20 ng of genomic DNA was subjected to PCR with an initial denaturation at
94°C for 2 minutes, followed by 25 cycles of 94°C for 15 seconds, 58°C for 40 seconds,
and 72°C for 1 minute followed by an extension at 72°C for 5 minutes. PCR to amplify
GAPDH gene was used as control. Forward and reverse primers for SB11 and GAPDH
were described for RT-PCR. PCR products were resolved by 0.8% agarose gel
electrophoresis.
Quantification of T-cell receptor Vα and Vβ
Vα and Vβ custom probes were designed and screened to eliminate direct and inverted
repeat elements and evaluated for cross-hybridization against the human RefSeq
database (Nanostring Technologies, Seattle, WA). Potential 50-base probes were then
selected for melting temperatures between 78°C and 83°C. The final nCounter code sets
included 45 TCR Vα genes, 46 TCR Vβ genes, and 3 house-keeping genes. Nucleic
acids were directly detected in multiplexed probe hybridization reactions using reagents
and consumables included in the assay kit. Target molecules were detected by
hybridization to capture and reporter probes, each approximately 50 nucleotides in
length, which targeted a contiguous 100-base region in each Vα and Vβ gene. Capture
and reporter probes were ligated to the synthetic DNA backbones containing barcodes
as described previously.(26) For hybridization, 5 µL (100 ng) of total RNA, 10 µL of
hybridization buffer, 10 µL (40 pM) of reporter probes, and 5 µL of capture probes (200
pM) were combined in PCR tubes and incubated at 65°C for 12 to 18 hours in a
thermocycler (Peltier Thermal Cycler, BIO-RAD) with a heated lid. After hybridization,
the samples were processed in the nCounter PrepStation (NCT-PREP-120) and counted
in a nCounter Digital Analyzer (NCT-DIGA-120). The expression levels of each gene
were normalized to those of the 3 house-keeping genes (ACTB, (GenBank Accession
number NM_001101.2), POLR1B (GenBank Accession number NM_019014.3), and
POLR2A (GenBank Accession number NM_000937.2). Gene expression data was
analyzed according to nCounter
TM
data analysis guidelines. To obtain “specific
hybridization counts” for the positive spike controls, housekeeping genes and test genes,
the average of the negative spike control counts in each lane was subtracted from that of
the raw positive control counts, housekeeping genes counts and test genes counts,
respectively. Specific test gene hybridization counts in each lane were then normalized
to both the positive control and housekeeping genes by the following formula: ([test gene
specific hybridization counts] x [positive spike control normalization factor] x
[housekeeping genes normalization factor]). Normalization factors for the positive control
and housekeeping genes in each lane were derived using the following formula: (sum of
average counts / lane-specific sum of counts).
Telomere assay
CD3+ T cells and CD3+CAR+ T cells harvested at time points after propagation were
fixed (3:1 methanol–acetic acid mixture), treated with 0.04 g/mL demecolcine (Gibco
BRL, Carlsbad, CA) for 45 minutes at 37°C, and incubated with a hypotonic solution
(0.075 M KCl) for 20 minutes. The cells were then fixed in a freshly made fixative (3:1
methanol–acetic acid mixture), washed with the fixative, placed on glass slides, and airdried. The slides were pretreated with 2x SSC prepared from 20x SSC (3 M sodium
chloride and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl) stock for 1 hour at
37°C, rinsed with distilled water, serially treated with 70%, 80%, and 100% absolute
ethanol, and then air-dried. The slides were denatured with 70% formamide/2x SSC for
2 minutes in a 74°C water bath and then immediately placed in ice-cold 70% ethanol.
The slides were then washed with 70%, 80%, and 100% ethanol for 2 minutes each, air-
dried, and coded for blinded analysis. Cytologic preparations of experimental CAR+ T
and control unmanipulated CD3+ T cells were hybridized with a telomeric DNA probe
(Dako, Carpinteria, CA) according to the manufacturer's instructions. FISH preparations
were examined under a Nikon 80i photomicroscope and images were captured using a
charge-coupled device camera (Sensys KAF 1400-G2, Photometrics, AZ). The
percentage of telomeric area in the interphase nuclei were quantified using Metaview
Imaging System software version 3.6a (Universal Imaging Co., Westchester, PA). At
least 50 interphase nuclei were quantified from each sample to determine the mean and
median percentages of telomeric nuclear areas.
Southern hybridization analysis
CAR+ Jurkat-cell clone #12 was maintained in continuous tissue culture for up to 6
months without selective pressure and without losing CAR expression (data not shown)
were used for analysis. Genomic DNA was isolated from Jurkat cell clones using the
QIAmp DNA mini kit (Qiagen). Southern hybridization analysis was performed as
previously described(28) on genomic DNA isolated from the Jurkat cell clones using
restriction enzyme combinations that cut both inside and outside the CAR transposon
sequence to generate a fragment of different size for each transposon integrant. Briefly,
10 g of genomic DNA from each Jurkat cell clone was digested overnight with NheI and
NcoI; ClaI and SphI; SacI and NcoI; and NheI and XbaI and resolved using 0.8%
agarose gel. CD19RCD28mz(CoOp)/pSBSO plasmid DNA was digested with the same
enzyme combinations. A 770-bp NheI and NcoI double-digested fragment encoding the
5’
portion
of
the
single-chain
variable
fragment
region
was
isolated
from
CD19RCD28mz(CoOp)/pSBSO DNA plasmid and radiolabeled using a random primer
labeling kit (Boehringer-Manheim, Indianapolis, IN) using 10 uL
P dCTP (250 uCi of α
32
32
P dCTP at 6000Ci/mMol, PerkinElmer,CT; Cat# PE#BLU513Z250UC) for use as a
DNA probe. Removal of unincorporated nucleotides was performed with Quick Spin
Column, Sephadex (G50) (Roche, IN; Cat # 11523023001). During the probe
preparation nylon membrane with DNA was incubated with 10 mL of QuickHyb solution
(Stratagene, CA; Cat# 201220) in a clean hybridization bottle warmed up to 680C for 1
hour. During membrane pre-incubation, the radioactive labeled probe and 100 uL of
salmon sperm DNA (Stratagene, CA; Catalog #201190), denatured at 1000C for 5
minutes, iced 5 minutes, mixed to 1 mL QuickHyb and added to bottle containing prehybing blot. After 4 hours of hybridization at 680C, membrane was washed with 2X SSC
+ 0.1% SDS for 3-4x at room temperature followed by two 30 minutes wash at 620C with
0.1X SSC + 0.1% SDS. Following wash, membrane was wrapped with saran wrap and
exposed to film for 4hrs-overnight.
Detection of transposon integration by FISH
CD19RCD28mz(CoOp)/pSBSO DNA plasmid was labeled by nick translation with
Spectrum Green (Abbott Molecular, Abbott Park, IL) and purified. Prior to use, the slides
with samples was incubated at 72°C in an incubator for 2 minutes to denature the
chromosome and the labeled DNA probe was denatured in a water bath for 6 minutes at
74°C. Hybridization of the DNA probe to fixed chromosomes was performed at 370C
overnight. Following the hybridization slides were washed in 2XSSC at 500C for 5
minutes, counterstained with DAPI/antifade (vector Laboratories, Inc. Burlingame, CA)
and images of 40 to 50 metaphase spreads were captured and analyzed using the
QUIPS PathVysion System (Santa Clara, CA).
Quantitative real-time genomic PCR and analysis
Genomic DNA from PBMC and CAR+ T cells (harvested at 28 days of propagation on
aAPC) was isolated using a QIAamp DNA mini kit (Qiagen). For the transgenedependent assay, the PCR was established in triplicate with 100 ng of genomic DNA, 10
µL of TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA); 1 µL
(1x primer at 900 nM and 1x probe at 250 nM) of 20 x FAM labeled CAR-specific
TaqMan probe primer set [forward (5’- GAGGGCAACGTCTTTAGCTG-3’) and reverse
(5’-GATGATGAAGGCCACTGTCA-3’) primers and carboxyfluorescein (FAM)-labeled
probe (5’-AGATGTTCTGGGTGCTGGTC-3’)] and 1 µL (1x primer at 900 nM and 1x
probe at 250 nM) of 20 x VIC labeled TaqMan RNase P Probe Primer set (Cat
#4316844, Applied Biosystems) in a total reaction volume of 20 µL. These primers
hybridize to the CAR in IgG4Fc and CD28 trans-membrane domains. Amplification and
detection were performed with a StepOnePlus Real-Time PCR System (Applied
Biosystems): 2 minutes at 50C, 10 minutes at 95C, forty 15-second cycles at 95C,
and 1 minute at 60C. Autosomal RNase P gene present at 2 copies per diploid cell, was
included in each reaction, as an endogenous reference for normalization.(21) A standard
curve of RNase P CT vs. cell number was achieved by a serial dilution of (100 ng, 10 ng,
1 ng, 0.1 ng) of genomic DNA from genetically modified Jurkat clone #12 bearing one
copy of integrated CD19-specific CAR transgene. In parallel, a curve was generated to
describe CAR transgene CT vs. transgene number to determine the number of integrated
transgenes. A PBMC sample (containing no genetically modified T cells) was included in
parallel as a negative control. The cycle numbers at which positive threshold signals
were measured were assigned a cycle threshold (CT) value. Transgene copy number in
the CAR+ T cells was defined as the amount of target normalized to the endogenous
reference RNaseP and relative to the calibrator Jurkat clone #12 as determined by ΔΔCT
method (Applied Biosystems, CA). The transgene-independent method to determine
copy number was performed as described(29) using a 1 µL (1x primer at 900 nM and 1x
probe at 250 nM) of 20 x FAM-labeled SB transposon left IR/DR-specific probe primer
TaqMan
assay
mix
that
included
and
CTCGTTTTTCAACTACTCCACAAATTTCT-3’)
forward
reverse
(5’(5’-
GTGTCATGCACAAAGTAGATGTCCTA-3’) primers (that bind to one unique site in the
left
(5’)
IR/DR
of
pT
and
pT2)
as
well
as
FAM-labeled
probe
(5’-
CTGACTTGCCAAAACT-3’), 1 µL (1x primer at 900 nM and 1x probe at 250 nM) of 20 x
VIC labeled TaqMan RNase P Probe Primer set (Cat #4316844, Applied Biosystems)
and 10 µL of TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City,
CA) in a total reaction volume of 20 µL. Amplification and detection of integrated
transgene with transgene-independent TaqMan probe and primers sets were performed
with StepOnePlus Real-Time PCR System with the same PCR conditions as performed
for CAR-specific TaqMan probe and primers sets. Copy number determination was
performed by ΔΔCT method as described above.