1
Molecular Analyses Support the Safety and Activity of Retroviral Replicating
Vector Toca 511 in Patients
Daniel J. Hogan1*, Jay-Jiguang Zhu2, Oscar R. Diago1, Dawn Gammon1, Ali Haghighi1, Guangrong
Lu2, Asha Das1, Harry E. Gruber1, Douglas J. Jolly1and Derek Ostertag1*
1. Tocagen Inc., 4242 Campus Point Court, Suite 500 San Diego, CA 92121
2. The University of Texas Health Science Center at Houston, McGovern Medical School, 6400
Fannin St, Houston, TX 77030
*Corresponding Authors: [email protected], [email protected]
Tocagen Inc., R&D Discovery Medicine, 4242 Campus Point Court, Suite 500 San Diego, CA
92121
Running title: Toca 511 molecular monitoring in patients
Keywords: Toca 511, retroviral replicating vector, gene therapy, immuno-oncology,
glioblastoma, insertional mutagenesis, APOBEC
Authors’ disclosures of potential conflicts of interest:
DJH, ORD, DG, AH, AD, HEG, DJJ and DO are employees of Tocagen Inc. and own stock in the company.
HEG and DJJ are founders of Tocagen Inc.
DJH, HEG, DJJ and DO have filed patents related to Toca 511 that are owned by Tocagen Inc.
Abbreviations:
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5-FU: 5-Fluorouracil, 5-FC: 5-Fluorocytosine, Toca FC: Toca 5-Fluorocytosine, yCD2: yeast
cytosine deaminase version 2, HGG: high grade glioma, gRV: gamma-retrovirus, RRV: retroviral
replicating vector, RNV: retroviral non-replicating vector, MLV: murine leukemia virus, HSC:
hematopoietic stem cell, IV: intravenous, SCID: Severe combined immunodeficiency, LTR: long-
terminal repeat, qPCR: quantitative PCR, TSS: transcription start site, LLOQ: Lower Limit of
Quantification, SNV: single nucleotide variant, indel: insertion and deletion, GBM: glioblastoma
multiforme, CSF: cerebral spinal fluid, MOI: multiplicity of infection
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ABSTRACT
Purpose: Toca 511 is a gammaretroviral replicating vector encoding cytosine deaminase that
selectively infects tumor cells and coverts the antifungal drug 5-fluorocytosine into the
antineoplastic drug 5-fluorouracil, which directly kills tumor cells and stimulates anti-tumor
immune responses. As part of clinical monitoring of Phase 1 clinical trials in recurrent high
grade glioma we have performed extensive molecular analyses of patient specimens in order to
track vector fate.
Experimental Design: Toca 511 and Toca FC (extended-release 5-fluorocytosine) have been
administered to 127 high grade glioma patients across three phase I studies. We measured
Toca 511 RNA and DNA levels in available body fluids and tumor samples from patients to
assess tumor specificity. We mapped Toca 511 integration sites and sequenced integrated Toca
511 genomes from patient samples with detectable virus. We measured Toca 511 levels in a
diverse set of tissue samples from one patient.
Results: Integrated Toca 511 is commonly detected in tumor samples and is only transiently
detected in blood in a small fraction of patients. There was no believable evidence for clonal
expansion of cells with integrated Toca 511 DNA, or preferential retrieval of integration sites
near oncogenes. Toca 511 sequence profiles suggest most mutations are caused by APOBEC
cytidine deaminases acting during reverse transcription. Tissue samples from a single whole-
body autopsy affirm Toca 511 tumor selectivity.
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Conclusions: Toca 511 and Toca FC treatment was not associated with inappropriate integration
sites and clonal expansion. The vector is tumor selective and persistent in patients who
received Toca 511 injections.
Statement of Translational Relevance
Retroviral replicating vectors (RRVs) are potential therapies for a wide range of
oncological malignancies, with ongoing durable responses and multiyear survival outcomes for
some recurrent high grade glioma patients in a phase 1 trial. Detailed characterization including
viral presence in biological fluids and cancer cells, viral insertion site location and clonality, as
well as viral sequence stability are important to support future approval of biologic therapies
built on RRVs. Previous treatment-related adverse events, including lymphomas, in some
clinical trials with distinct non-replicating retroviral vectors were preceded by clonal expansion
of infected cells due to integration near a proto-oncogene. As part of our ongoing development
of RRV Toca 511 in recurrent high grade glioma, we performed extensive monitoring of the
virus in patient tumors and body fluids, including Toca 511 quantitation, integration site
identification and mutation profile characterization. Our results support the favorable safety
profile of Toca 511 and expansion of clinical trials into other indications. Moreover, this work
provides a broad framework for molecular monitoring of RRV therapies during clinical
development.
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INTRODUCTION
Given the limits of conventional cancer treatments, retroviral replicating vectors (RRVs)
have emerged as a potential backbone for useful therapies across a wide range of oncological
malignancies. RRVs selectively infect tumor cells without directly lysing them. This differentiates
them from directly oncolytic and highly inflammatory viruses such as adenovirus and herpes
viruses (1-3). Thus, RRVs provide a platform for therapies based on tumor-specific gene delivery
strategies without the inherent limitation of rapidly killing infected cells. RRV are selective for
tumor cells partially due to virus-selective advantages in the tumor microenvironment from
blunted innate immune responses as well as suppressed adaptive immune responses relative to
normal dividing cells (3-6). Viral dependency on mitosis for integration contributes to cancer
cell selectivity (7), and the non-inflammatory nature of the infection (and replication
competency) allows subsequent spread (8).
Toca 511 (vocimagene amiretrorepvec) is a RRV derived from a murine gammaretrovirus
(gRV), with an amphotropic envelope to allow infection of human cells. Such viruses are known
to be non-pathogenic to humans (9), and all adult humans so far tested appear to be
seropositive (10,11). The modified Toca 511 virus encodes a transgene for an optimized yeast
cytosine deaminase (yCD2) that converts the orally available prodrug 5-fluorocytosine (5-FC)
into cytotoxic 5-fluorouracil (5-FU) (12-14).
Three phase I clinical trials, NCT01156584 (Study 8), NCT01470794 (Study 11) and
NCT01985256 (Study 13), evaluated safety of Toca 511 and Toca FC (an extended-release
formulation of 5-FC) therapy in patients with recurrent high grade glioma (HGG) by different
routes of Toca 511 administration, followed by oral Toca FC dosing. In Study 8, Toca 511 was
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administered by injection into the tumor, without resection. In Study 11, tumor resection was
performed, followed by multiple injections of Toca 511 into the tissue walls of the resection
cavity (9). In Study 13, intravenous (IV) administration of Toca 511 (bolus injections for one,
three or five consecutive days) was followed by surgical resection plus additional injections of
Toca 511 into the tissues of the resection cavity eight to fourteen days later (clinical results
from Study 13 will be presented elsewhere). For all trials, four to six weeks after the final Toca
511 administration, patients were treated with Toca FC for approximately one week, in
repeated cycles, every four to eight weeks, for six months, or until radiological evidence of
tumor progression, clinical progression, or termination by treating physicians (Fig. 1A).
Historical safety concerns are associated with different retroviral non-replicative vectors
(RNVs). For instance, RNV preparations grossly contaminated with replicative murine leukemia
virus (MLV) derived from recombination during serial passage through ecotropic and
amphotropic murine cell derived packaging cell lines were employed for ex vivo transduction of
hematopoietic stem cells (HSCs) followed by autologous transplant into monkeys (15,16). Three
of ten monkeys so treated, that were unable to develop antiviral immune responses, developed
lymphomas containing multiple copies of an array of MLV related sequences in the tumor
genomes. A second source of concern was the observation, following transplant of autologous
cytokine-stimulated HSCs transduced ex vivo with RNV, of delayed occurrence of lymphoma in
some clinical trial patients several years after treatment (17-19). T-cell lymphoma was linked to
treatment of X-linked Severe Combined Immune Deficiency Syndrome (X-SCIDS) and Wisckott-
Aldrich Syndrome (WAS), which in turn was linked to insertions near the promoter for LMO2,
leading to its ectopic expression. Myelodysplastic Syndrome was linked to treatment of Chronic
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Granulomatous Disease and insertion and activation of MDS-EVI-1 oncogene (19,20). However,
a therapy based on RNV transduction and reimplantation of autologous HSCs for ADA deficient
SCIDS has not shown such side effects, and was recently approved for sale in Europe (Strimvelis,
GSK Ltd.) (18). Additionally, long-term outcomes with persistent RNV transduced T-cells have
also failed to show such side effects (21). These observations suggest that such adverse
outcomes are influenced by factors including multiplicity of infection, nature of the transgene,
indication, clinical condition and age of the recipient, expected mechanism of action of the
transgene and target tissue (17,22).
Several methodologies have been developed to track the fate of viral vectors in
patients. Viral RNA and DNA levels can be measured over a broad dynamic range with
quantitative PCR (qPCR). Viral integration sites, originally identified by Sanger sequencing of
cloned PCR products (23), can be systematically mapped via next-generation sequencing (24-
26).
While previous gRV trials used RNVs, often transducing autologous cells ex vivo, Toca
511 is a RRV used to infect cancer cells in vivo. Thus, host immunity to viral proteins and virus-
host restriction factor interactions may influence Toca 511 spread and activity (27). For
instance, APOBEC proteins target a number of retroviruses in humans, causing G to A
hypermutation via processive cytidine deamination during reverse transcription (28,29). The
influence of APOBEC proteins on Toca 511 in patient samples can be measured via next-
generation sequencing. Additionally, RNVs (unlike RRVs) usually lack genes to produce viral
proteins that could eventually stimulate immune responses against infected cells. Thus, Toca
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511 therapy requires an overlapping but distinct set of assays for molecular monitoring than
used in RNV trials.
In this study we report results of Toca 511 monitoring in blood and tumor samples from
patients who received Toca 511 through direct injection into the resection cavity (Study 11) and
IV delivery (Study 13) 8 to 14 days prior to tumor removal (additional Toca 511 is delivered via
direct injection into the resection cavity), as well as analyses of tissue from a full autopsy of a
patient that participated in a phase 2 clinical study (NCT02414165) and received Toca 511
injection into the HGG resection cavity. Our monitoring methods include Toca 511
quantification, identification of genome integration sites and mutation profiles of Toca 511
genomes from available Toca 511-positive tumor tissue and blood cells. This work provides
molecular and genomic corollaries to traditional safety assessments and presents a framework
for clinical monitoring of RRV therapies during initial development, allowing for continued
confidence in using such vectors in patients.
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MATERIALS AND METHODS
Toca 511 quantification
DNA and RNA Purification
DNA and RNA were extracted from the same source (tumor or whole blood) using a
modified version of Promega’s Maxwell 16 Purification System as communicated by the
manufacturer. Tumor sections were minced in petri plates on dry ice and transferred into 1.5
mL microfuge tubes. 500 L of homogenization/1-thioglycerol solution were added into each
tube and the test articles were agitated with pestles (VWR: Cat #47747-366) followed by
vortexing at full speed for 30 sec. For whole blood DNA and RNA purification, up to 200 L of
whole blood (brought to 200 L with PBS if necessary) was transferred into 1.5 mL microfuge
tubes. 300 L of homogenization/1-thioglycerol solution was added to the tube and vortexed at
full speed for 30 sec. In each case 300 L of the homogenate was transferred into Maxwell 16
DNA Purification cartridges and subjected to automated DNA purification. Purified DNA was
eluted in 300 L of nuclease-free water. For RNA purification, 200 l of lysis buffer was added
into the 1.5 mL microfuge tubes containing the remaining 200 L of the original 500 L
homogenate. Test articles were vortexed for 30 sec and the full volume was transferred into
Promega’s Maxwell 16 simplyRNA Purification cartridges. Automated RNA purification was
carried out and purified RNA was eluted in 50 L of nuclease-free water. RNA purification from
plasma, urine, and saliva and IHC of CD gene were described previously (4).
qPCR
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TaqMan probe-based qPCR was performed as single-targeted 20 μL reactions for Study
11 test articles and as multiplex reactions for Study 13 test articles. The reactions were
prepared in triplicate, on a CFX96 or CFX384 Real Time System (Bio-Rad) using primers and
probes annealing to sequences in the long terminal repeat region of Toca 511 or in the yCD2
transgene (monoplex) or in sequences in the amphotropic MLV4070A ENV, POL and yCD2
transgene (multiplex). Primers and probes were designed with PrimerQuest software and
synthesized by Integrated DNA Technologies. For qPCR detection of gRV-specific sequences,
primers were used at final concentration of 300 nM each of MLV-F (5’- AGC CCA CAA CCC CTC
ACT C-3’) and MLV-R (5’- TCT CCC GAT CCC GGA CGA-3’), and 100 nM of MLV hydrolysis probe
(5’- FAM-CCC CAA ATG AAA GAC CCC CGC TGA CG-3’BHQ_1) with iQ PCR Supermix (Bio-Rad).
For qPCR detection of yCD2, primers were used at 600 nM each of yCD2-F (5’-ATC ATC ATG TAC
GGC ATC CCT AG-3’) and yCD2-R (5’-TGA ACT GCT TCA TCA GCT TCT TAC-3’), and 100 nM of
yCD2 hydrolysis probe (5’-FAM/TCA TCG TCA ACA ACC ACC ACC TCG T/3’BHQ_1). For
simultaneous qPCR detection of POL, ENV and yCD2 the following concentrations were used:
300 nM each of Pol2-F (5’-CAA GGG GCT ACT GGA GGA AAG-3’) and Pol2-R (5’-CAG TCT GGT
ACA TGG AGG AAA G-3’); 100 nM of Pol2 hydrolysis probe (5’-HEX/TAT CGC TGG ACC ACG GAT
CGC AA/ 3’BHQ_1); 300 nM each of Pol3-F (5’-CGA CAC CAG ACT AAG AAC CTA G-3’) and Pol3-R
(5’-CGA TGC CGT CTA CTT TGA GG-3’); 100 nM of Pol3 hydrolysis probe (5’-HEX/CCT CGC TGG
AAA GGA CCT TAC ACA/ 3’IABkFQ); 300 nM each of Env2-F (5’-ACC CTC AAC CTC CCC TAC AAG
T-3’) and Env2-R (5’-GTT AAG CGC CTG ATA GGC TC-3’); 100 nM of Env2 hydrolysis probe (5’-
TEX615/AGC CAC CCC CAG GAA CTG GAG ATA GA/3’BHQ_2); 300 nM each of yCD2-F (5’-ATC
ATC ATG TAC GGC ATC CCT AG-3’) and yCD2-R (5’-TGA ACT GCT TCA TCA GCT TCT TAC-3’); and
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100 nM of yCD2 hydrolysis probe (5’-FAM/TCA TCG TCA ACA ACC ACC ACC TCG T/3’BHQ_1).
Either the Pol2 or Pol3 primer/probe set was used as a component in the triplex reaction.
Thermal cycling conditions consisted of 95°C for 5 min, followed by three cycles of 95°C
for 15 sec and 65°C for 10 sec, followed by 38 cycles of 95°C for 15 sec and 65°C for 30 sec. CFX
Manager 3.0 software (Bio-Rad) was used to calculate threshold cycle (Ct) values. Technical
replicates were averaged and absolute quantification was determined from linear regression
using a six-log serial dilution standard curve (25 to 2.5e6 copies/reaction) from a Toca 511-
containing plasmid, pAZ3-yCD2.
RT-qPCR
TaqMan probe-based RT-qPCR was performed as single-targeted 20 l reactions for
Study 11 test articles and as multiplex reactions for Study 13 test articles. The reactions were
prepared in triplicate using primers and probes annealing to sequences in POL (monoplex) or to
sequences in the amphotropic MLV4070A ENV, POL and yCD2 (multiplex).
For simultaneous RT-qPCR detection of POL, ENV and yCD2 in a single reaction, primers
were used at 300 nM each of Pol2-F (5’-CAA GGG GCT ACT GGA GGA AAG-3’) and Pol2-R (5’-
CAG TCT GGT ACA TGG AGG AAA G-3’); 100 nM of Pol2 hydrolysis probe (5’-HEX/TAT CGC TGG
ACC ACG GAT CGC AA/ 3’BHQ_1); 300 nM each of Pol3-F (5’-CGA CAC CAG ACT AAG AAC CTA G-
3’) and Pol3-R (5’-CGA TGC CGT CTA CTT TGA GG-3’); 100 nM of Pol3 hydrolysis probe (5’-
HEX/CCT CGC TGG AAA GGA CCT TAC ACA/ 3’IABkFQ); 300 nM each of Env2-F (5’-ACC CTC AAC
CTC CCC TAC AAG T-3’) and Env2-R (5’-GTT AAG CGC CTG ATA GGC TC-3’); 100 nM of Env2
hydrolysis probe (5’-TEX615/AGC CAC CCC CAG GAA CTG GAG ATA GA/3’BHQ_2); 300 nM each
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of yCD2-F (5’-ATC ATC ATG TAC GGC ATC CCT AG-3’) and yCD2-R (5’-TGA ACT GCT TCA TCA GCT
TCT TAC-3’); and 100 nM of yCD2 hydrolysis probe (5’-FAM/TCA TCG TCA ACA ACC ACC ACC TCG
T/3’BHQ_1) were used with AgPath-ID One-Step RT-PCR Reagent (Life Technologies) (either the
Pol2 or Pol3 primer/probe set was used as a component in the triplex reaction).
Thermal cycling conditions consisted of 46°C for 20 min, followed by 95°C for 10 min,
followed by 40 cycles of 95°C for 15 sec and 55°C for 45 sec. Absolute quantification was
determined from linear regression on a seven-log serial dilution RNA standard curve (2.55e2 to
2.55e8 copies/mL) purified from Toca 511 viral vector containing the corresponding targets that
underwent RT-qPCR in parallel with the test articles. The Toca 511 standard was originally
qualified by comparison with an in vitro synthesized RNA standard quantified by absorbance at
260 nm.
Identification of Toca 511 Integration Sites
Preparation of Sequencing Libraries
100-1000 ng of genomic DNA (isolated as described above) was sheared using Covaris
E220 to 300 bp peak size. Following cleanup with Ampure XP beads (Beckman Coulter), DNA
was end repaired (End-It™ DNA End-Repair Kit, Epicentre), a 3’ overhang A was added
(NEBNext® dA-Tailing Module, New England Biolabs), and partially double stranded adaptors
(Adaptor1 + Adaptor 2) were ligated with T4 ligase (Quick Ligation™ Kit, NEB). Two rounds of
PCR were performed using NEBNext® High-Fidelity 2X PCR Master Mix. The second round of
PCR included primers with barcoded Illumina adaptors. Following clean-up with Ampure XP
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beads, libraries were quantified by qPCR and Qubit (Thermo Fisher) and pooled for paired-end
sequencing on Miseq or Hiseq 2000.
Oligonucleotides (ordered from IDT):
Adaptor1: 5’-
GTAATACGACTCACTATAGGCTTTCAGACGTGTGCTCTTCCGATCT(NNNNNNN)GCTCCGCTTAAGGGA
CT-3’
Adaptor2: 5’-/5Phos/GTC CCT TAA GCG GAG /3AmMO/-3’
Adaptors 1 and 2 were mixed at equimolar concentrations, annealed and used in ligation
reactions at ~10X molar excess to sheared DNA.
Toca511_PCR1: 5’-CCTTGGGAGGGTCTCCTCTGAGT-3’
Linker_PCR1: 5’-GTAATACGACTCACTATAGGCT-3’
Toca511_PCR2:5’-
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACCATGACTAC
CCGTCAGCGGGGGTC-3’
Linker_PCR2: 5’-CAAGCAGAAGACGGCATACGAGAT(6mer barcode)
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’
Processing of Sequencing Data
Paired fastq files were processed using Cutadapt 1.1 (48):
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cutadapt -e 0.05 -g TGACCATGACTACCCGTCAGCGGGGGTCTTTCATTAGTCCCTTAAGCGGAGC --
discard-trimmed -O 17 -o tmp1.1.fastq -p tmp1.2.fastq fastq1 fastq2
cutadapt -e 0.05 -g TGACCATGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGG --discard-trimmed
-O 17 -o tmp2.1.fastq -p tmp2.2.fastq tmp1.1.fastq tmp1.2.fastq
cutadapt -e 0.05 -g TGACCATGACTACCCGTCAGCGGGGGTC --discard-untrimmed -O 17 -o
tmp3.1.fastq -p tmp3.2.fastq tmp2.1.fastq tmp2.2.fastq
cutadapt -e 0.00 -g ^TTTCA --no-indels --discard-untrimmed -o tmp4.1.fastq -p tmp4.2.fastq
tmp3.1.fastq tmp3.2.fastq
cutadapt -e 0.05 -a AGTCCCTTAAGCGGAGC -O 10 -m 11 -o tmp5.1.fastq -p tmp5.2.fastq
tmp4.1.fastq tmp4.2.fastq
cutadapt -e 0.05 -g GCTCCGCTTAAGGGACT --discard-untrimmed -O 10 -m 11 -o tmp6.2.fastq -p
tmp6.1.fastq tmp5.2.fastq tmp5.1.fastq
cutadapt -e 0.05 -a TGAAAGACCCCCGCTGACGGGTAGTCATGGTCA -O 10 -m 11 -o tmp7.2.fastq -
p tmp7.1.fastq tmp6.2.fastq tmp6.1.fastq
Trimmed and filtered fastq files were mapped to the human (hg19) and Toca 511
genomes using Bowtie2 (for human genome: -q -X 2000 --no-mixed --no-discordant --no-unal --
score-min L,0,-0.4)(49). Sorted BAM files were filtered to remove read pairs in which at least
one of the reads contained at least two mismatches to the reference genome in the first ten
bases using RSamtools (50). Filtered BAM files were converted to BED files (Bedtools) (51) and
read pairs with the same start and end positions (+/- 2 bp) were collapsed. We removed read
pairs that shared the same integration site but that had a different fragmentation site in cases
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in which the fragmentation read site accounted for less than 1% of total integration events at
this site; we did this because we found that most (or all) cases were likely due to spurious PCR
products created from PCR duplicates of a single integration event. Integration sites that
contained sequence motifs enriched in sequences adjacent to integration sites from negative
controls, identified using HOMER2, were removed (52). These motifs contained matches to the
3’ end of Toca511 PCR_2 primer and/or the Toca 511 integration footprint (TTTCA). Finally, we
filtered out integration events in which another sample had the same integration site and
fragmentation site in at least 100X greater abundance, which was likely due to index primer
misidentification during sequencing. The locations of integration sites can be found in Dataset
S2.
Other Integration Analyses
We used Bedtools to identify EMSEMBL transcripts adjacent to integration sites. Gene
functional enrichment analyses were performed with Metascape (53). Cancer gene lists were
downloaded from supplementary material associated with Vogelstein et al. (54) and from
Bushman lab website: http://www.bushmanlab.org/links/genelists. The hypergeometric density
distribution was used to test for the significance of overlap between gene lists.
Toca 511 Sequencing
Full-Length PCR for Sequencing
PCR for full-length amplification of Toca 511 was performed as primary reactions
followed by nested reactions using LongRange PCR Kit (Qiagen). Primary PCRs (9197 bp) were
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executed using primers targeting the flanking long terminal repeat regions of Toca 511 at 400
nM each: Tri Set 1-Forward (5’- GACTTGTGGTCTCGCTGTTCCTT-3’) and Tri Set 3-Reverse (5’-
GAGTGAGGGGTTGTGGGCTCT-3’). Primers for nested PCRs (8349 bp) were designed to anneal
internally to the primary amplicons and were used at 400 nM each: 5’ Long PCR Sequencing
Primer-Forward (5-TGGTAGGAGACGAGAACCTAAA-3’) and 3’ UCLA 3-37 IRES-Reverse (5’-
CCCCTTTTTCTGGAGACTAAATAA-3’). Genomic DNA (up to 400 ng) for the primary PCR or
primary PCR reaction (1 L) for the nested PCR, Long Mix buffer (containing 25 mM MgCl2),
dNTPs (500 μM each), DMSO (2%), primers (400 nM), and LongRange Enzyme Mix (1 U) were
combined and PCRs initiated. Primary PCR: thermal cycling conditions consisted of one cycle of
93°C for 3 min, 20 cycles of 93°C for 15 sec, 55°C for 30 sec, 68°C for 9 min, followed by one
cycle of 93°C for 15 sec, 55°C for 30 sec, 68°C with 20 sec incremental increases from previous
time, for an additional 15 cycles. Nested PCR: thermal cycling conditions consisted of one cycle
of 93°C for 3 min, 35 cycles of 93°C for 15 sec, 60°C for 30 sec, 68°C for 8 min.
Tripartite PCR for Sequencing
PCR for tripartite amplification of Toca 511 was performed with three sets of
overlapping primers. Primers were utilized at 150 or 400 nM each: upstream sequence (2356
bp), 713_LTR Set 2-forward (5’-CGGGGGTCTTTCATTTGGGG-3’) and Tri Set 1-Reverse
(5’ACAGTCTGGTACATGGAGGAAAG-3’); middle sequence (4842 bp), 5' 2F2569-Forward (5’-
GGACAGAGGATGAGCAGAAAGA-3’) and TriSet2-Reverse (5’- GCGGTGGAATGATTGGTATAAGTG-
3’); and downstream sequence (3797 bp), Set 14-Forward (5’- AGC CTT CCC AAC CAA GAA AGA-
3’) and Set 14-Reverse (5’- AGC TAG CTT GCC AAA CCT ACA-3’). Reactions were prepared as 25
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L aliquots with LongRange PCR Kit as described above. Thermal cycling conditions consisted of
one cycle of 93°C for 3 min, followed by 30 cycles of 93°C for 15 sec, 60°C for 30 sec, and 68°C
for 4 min.
Preparation for Sequencing
Following the nested PCR, products were separated on 1% TAE ethidium bromide
agarose gels and bands corresponding to the expected size were excised and purified according
to the manufacturer’s instructions (Gel PCR Purification Kit: Qiagen). Quantification of purified
PCR products was performed using QuantiFluor dsDNA (Promega) on an Infinite M200 Plate
Reader (Tecan).
Purified PCR products were prepared for sequencing using Nextera XT kit (Illumina)
according to manufacturer’s instructions. Pooled libraries were sequenced on Illumina MiSeq (2
x 75 base reads) at University of California San Diego IGM facility. Fastq files are available from
NCBI SRA database (SRP149280).
Toca 511 Sequence Analyses
PCR duplicates were removed with PRINSEQ-lite 0.20.4 (55). Read pairs were quality
trimmed with Cutadapt (-q 30,30 --minimum-length 30) and mapped to the Toca 511 genome
using bwa mem 0.7.15-r1140 using default parameters (56). SNVs and indels were identified
with Varscan 2.3 (--p-value 0.01 --min-coverage 500 --min-avg-qual 25 --min-var-freq 0.01) (36)
from Samtools (57) generated mpileup files (-d 20000). The resulting VCF files were parsed,
combined and analyzed in R using custom scripts. Plots for figures were made with ggplot2.
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yCD2 Sequencing
Primers used to amplify the yCD2 coding region included Illumina Nextera XT universal
primer sequences at their 5’ ends: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
CACGGGGACGTGGTTTTCCTT-3’ and 5’-
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTACAGGTGGGGTCTTTCATTCC-3’. Primary PCR was
performed with 400 nM of each primer using Qiagen Long Range PCR kit components. PCR products
were gel purified and a secondary PCR was performed with Nextera XT dual-indexed primers to prepare
samples for sequencing. Samples were pooled and sequenced on Illumina MiSeq (2 x 300 base
reads). Adaptors were removed from sequencing reads with Cutadapt. Reads were quality
trimmed with Trimmomatic v0.32 (SLIDINGWINDOW:30:30) (58) and then mapped to Toca 511
genome with bwa mem as described above. SNVs were identified with Varscan2 as described
above. Fastq files are available from NCBI SRA database (SRP149351).
Patient Autopsy Samples
The whole-body autopsy patient was a 65 year old male with a past medical history of
left temporal anaplastic astrocytoma with transformation to GBM status post resection. The
patient completed Toca 511 dosing and a single cycle of Toca FC. Main study informed consent
was reviewed and received approval from Western Institutional Review Board, which allows
Tocagen to request tissues from autopsy case. Full autopsy consent was obtained by the
investigators at UTHealth. Samples were collected approximately 40 days after the last dose of
Toca FC. Tissue and biofluids samples were collected as follows:, brain -adjacent, tumor – non-
injected site A, tumor – non-injected site B, tumor – non-injected site C, tumor injection site A,
tumor injection site B, tumor injection site C, 1 mL blood, 1 mL urine, skin, lower GI, testes,
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liver, spleen, lymph nodes, bone marrow, spinal cord, lung - left lower lobe, lung - Lobe
Consolidation, CSF.
The brain autopsy was from an 80 year old male with a past medical history of GBM and
recurrent GBM post-resection. Samples from the following locations were collected:
contralateral temp lobe (amygdala), ipsilateral frontal cortex (sagittal), anterior half (putamen
and internal capsule), posterior half, posterior tumor, posterior tumor (slightly more anterior),
anterior tumor (presumed injection site), anterior tumor (presumed injection site, slightly more
posterior).
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RESULTS
Toca 511 is transiently detected in blood in a small subset of patients in Study 11
In Study 11, surgical resection was followed by multiple injections of Toca 511 into the
walls of the resection cavity; four to six weeks later, Toca FC dosing started (Supplementary
Table S1)(9). In order to quantitatively measure Toca 511 DNA and RNA levels over a broad
dynamic range, robust (reverse-transcription [RT])-qPCR assays that utilized a six-log standard
curve for absolute quantification were developed (see Materials and Methods). The estimated
Lower Limit of Quantification (LLOQ) for the qPCR assay is 25 viral genome copies per reaction,
whereas the LLOQ for the RT-PCR assay is ~7300 viral RNA copies per mL of plasma. As part of
Study 11, we performed Toca 511 qPCR on DNA isolated from whole blood as well as RT-qPCR
on total RNA isolated from plasma, urine and saliva, sampled longitudinally starting just prior to
Toca 511 treatment and continuing at intervals over the course of treatment, including > 500
DNA and RNA test samples across 56 patients (Fig. 1A – red lines)(9). Samples that were within
the quantitative range of the assay, as defined above, were considered detected.
Quantitative Toca 511 RNA signal was rarely detected in urine or saliva samples but was
detected in 32 plasma samples collected from 22 of 56 patients tested (Supplementary Fig. S1
& Supplementary Dataset S1) and rapidly cleared. Sixteen of the positive RNA samples from
plasma were detected from samples collected one day after surgery (visit 2). This is probably
too early for infection and viral replication to occur and likely represents leakage of the injected
vector into the blood stream following surgery. Thirteen patients had positive RNA signal at
times after visit 2, occurring between day ~10 (visit 3) and week 6(visit 5).
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We detected Toca 511 in DNA isolated from blood in fifteen samples collected from
eleven patients (Fig. 1B and Supplementary Dataset S1). Detection of Toca 511 DNA in whole
blood did not correlate with patients’ Toca 511 dose level (Supplementary Dataset S1). In all
cases quantitative signal occurred between week 4 (visit 4) and week 10 (visit 7) (Fig. 1B).
Patients begin taking Toca FC at week 6 (visit 5) and it is likely this helped clear virus from the
blood. Even in cases in which integrated Toca 511 was detected, overall levels were low; the
maximum DNA signal was 3,400 copies per g of genomic DNA (~2 copies per 100 diploid
genome equivalents) (Fig. 1B & 1D). In summary, virus was well controlled as it was
infrequently and only transiently detected in the patients’ blood with quantitative Toca 511
signal in 6% of plasma samples and 3% of whole blood samples, respectively.
Toca 511 infects patients’ HGG tumors
While it was not feasible to systematically measure Toca 511 levels in residual tumors
from patients in Study 11, we obtained tumor samples from eight patients in Study 11 for
whom their tumor recurred following Toca 511 and Toca FC treatment and the tumor was
subsequently resected (re-tumor samples in the figures). We detected Toca 511 in DNA isolated
from three of seven tumors tested. Two of these patients’ tumors had both quantifiable Toca
511 DNA and RNA signal (Fig. 1C & Supplementary Dataset S1), It is likely viral levels in these
tumors are not representative of the initial uptake in the patient population as Toca FC
treatment is expected to deplete Toca 511 expressing cells; moreover, one reason for tumor
recurrence would be from a paucity of Toca 511-infected cancer cells.
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In Study 13, recurrent HGG tumor was resected eight to fourteen days after the initial
day of IV administration of Toca 511, additional Toca 511 was injected into the resected cavity
followed by Toca FC treatment four to six weeks later (Fig. 1A & Supplementary Table S2).
Appropriate qPCR was performed on DNA and RNA isolated from multiple tumor pieces
(referred to, herein, as IV tumor samples) (Fig. 1A). For eight of the seventeen patients given IV
Toca 511, integrated Toca 511 was detected in at least one tumor piece (Fig. 1C), at levels up to
47,000 copies per g of DNA (~31 copies per 100 diploid genome equivalents) (Fig. 1D &
Supplementary Dataset S1). We detected Toca 511 RNA isolated from tumor samples in nine
patients, including six patients whose tumors also had quantifiable levels of integrated Toca 511
(Supplementary Dataset S1). The first two low dose patients showed no quantifiable signal for
DNA or RNA in their tumors. At the third, fourth and fifth dose levels with one, three or five day
administration respectively, Toca 511 was detected in 11 of 15 tumors. These results suggest
that Toca 511 gains access to the brain tumor in a dose dependent manner after virus infusion
into peripheral blood and can successfully deliver therapeutic transgenes.
Toca 511 integration patterns are tissue specific and consistent with previous analyses
of gammaretroviral integration
Identification of integration sites in samples with few integration events per hundreds of
genomes is challenging and we optimized an integration site enrichment procedure and
sequencing analysis workflow to reliably do so (Supplementary Text, Supplementary Fig. S2, S3
& S4). We distinguished clonal events from PCR duplicates by virtue of having the same
integration site, but different fragmentation site, as is standard (e.g. (30-32)). Nine blood
samples from Study 11, and ten tumor samples from Study 13 with quantifiable Toca 511 signal
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were analyzed along with three negative controls (DNA isolated from human cells not exposed
to Toca 511) (Fig. 2A). The three negative controls yielded zero, three and eight “integration
events“ respectively (from millions of sequencing reads); these events were likely created from
spurious priming by the Toca 511 specific nested primer in locations of the human genome that
happened to be immediately followed by the four bp Toca 511 integration sequence. Patient
samples generally had orders of magnitude more read pairs that mapped to the human
genome than the negative control samples, yielding on average 34 integration events per blood
sample and 165 integration events per tumor sample for a total of 1984 measured integration
events across all samples tested (Fig. 2A and Supplementary Fig. S5).
Given gRVs’ preference to integrate near the 5’ends of transcriptionally active units, as
also observed for Toca 511 in cell culture (Supplementary Fig. S3D), we predicted there would
be enrichment of Toca 511 integration sites near annotated mRNA transcription start sites
(TSSs) from patient samples. For these analyses we combined integration sites from blood and
integration sites from tumor into two pools of data. We plotted the distribution of integration
sites adjacent to annotated TSSs and observed a characteristic enrichment centered around
TSSs in both blood and tumor samples, albeit with a shallower peak than observed in cell
culture (Fig. 2B). For both blood and tumor, 14% of integrations occurred within five kb of the
nearest annotated TSS (vs 5% expected based on random chance; p = 0.03 and p < 1e-5 for
blood and tumor, respectively based on proportions test). We asked whether genes proximal to
integration sites (immediately upstream (within 10 kb) or within the gene) encode proteins that
are functionally linked to the site from which samples were taken; blood or brain tumor.
Utilizing the hundreds of GO and REACTOME gene sets (33,34), we found that brain tumor-
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derived integration sites preferentially occurred near genes involved in neuronal functions and
growth/differentiation, while blood-derived integration sites preferentially occurred near genes
linked to blood functions (Fig. 2C). Thus, Toca 511 integration site preferences from patient
tumor and blood transduced in vivo are congruent with previous work on gRVs showing
preference for sites of active transcription ex vivo and in mice (25,26).
Absence of compelling evidence for clonal expansion of Toca 511-integrated cells
The degree to which integration sites are unique versus subclonal expansion is key in
order to assess the putative risk of insertion mutagenesis leading to hyperproliferation. An
abundance of unique integration events would also indicate viral spread within the tumor
versus expansion of one to a few tumor cells with integrated genomes. For each sample, we
determined the number of sites with two or more integration events and plotted the results as
a fraction of all integration events (Fig. 3A). For samples with at least twenty identified
integration events multiple events from the same site comprised fewer than five percent of the
total, with one exception, from patient 11_33 visit 4a blood (not shown in Figure 3A). Follow-up
analyses suggest this potential clonal expansion, which occurred in an Alu element, was a
technical artifact caused by recombination with other Alu elements during PCR (Supplementary
Text and Supplementary Fig. S6).
Given the precedent for clonal expansion due to integration of gRVs adjacent to
oncogenes, particularly LMO2, we determined if there was a preponderance of sites adjacent to
or within various classes of cancer-related genes, in both blood and tumor. We found some
integration events within ten kb of oncogenic drivers or tumor suppressor genes in blood or
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tumor (Fig. 3B). There was one integration site in blood (11_05 visit 7) near a lymphoma-linked
gene, NKAIN2/TCBA1, which encodes a transmembrane protein that interacts with the beta
subunit of a sodium/potassium-transporting ATPase (35). There was no observed clonal
expansion from this integration event and like all other patients detectable Toca 511 signal was
undetectable after Toca FC treatment. These results are consistent with the lack of
development of lymphoma-like symptoms in surviving patients after Toca 511 delivery to date
and the paucity of Toca 511 infected cells in all patients’ blood samples (9).
Toca 511 genomes are mutated by restriction factors in patients
We previously reported Toca 511 gross genome stability in vitro across multiple
passages of the virus in cell culture (13). In the clinical setting, Toca 511 was PCR-amplified from
patient DNA samples using a single nested PCR that spanned nine kb of the genome including
all coding regions, or three overlapping PCR products that spanned the same region
(Supplementary Fig. S7). PCR products were gel purified and those with sufficient material were
prepared for Illumina sequencing. We obtained quality sequencing results (> 1,000X coverage
across at least 6,000 bp) from three blood samples, six samples from re-resected tumors, six
tumor samples following IV treatment and four nonclinical samples, including two cell lines (HT-
1080 and U-87MG) infected with Toca 511 and two plasmids containing the parental Toca 511
genome.
Following quality filtering and read mapping to the Toca 511 reference genome, we
characterized the mutation profiles of the Toca 511 genomes from each sample using Varscan2
(36), which identifies both single nucleotide variants (SNVs) and short insertions and deletions
(indels) (Fig. 4 and Supplementary Fig. S8). At a mutation frequency threshold of 3%, there
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were four SNVs and zero indels in the two plasmid controls and U-87MG infected cells. These
mutations were previously identified shared silent point mutations from the initial cloned MLV
genome from which Toca 511 was derived (37)and were removed for subsequent analyses.
There were two additional SNVs in the HT-1080 control sample, both occurring at less than 5%
frequency. In contrast to the plasmid and cell culture samples, there was a wide spectrum of
mutations among patient samples. Total SNVs per sample ranged from 31 (11_33 blood visit 7)
to 742 (11_02 re-tumor section 8) (mean = 204) (Fig. 4A & B). Indels were much less frequent
than SNVs (Supplementary Fig. S8). There were generally more SNVs and indels in Toca 511
genomes from re-resected tumors versus newly resected tumors and blood, which could reflect
more rounds of infection and replication and/or enrichment of non-functional Toca 511
following Toca FC treatment (patient 11_02 took one cycle of Toca FC while patient 11_31 took
three cycles of Toca FC).
Next we asked whether there was a bias in the mutation patterns among SNVs, which
may suggest underlying mechanisms of mutation. For each sample we calculated the fraction of
all SNVs corresponding to each of the twelve possible pairwise combinations. As shown in Fig.
4C, an overwhelming majority of SNVs corresponded to three of four possible transitions: G to
A, A to G and T to C. For eleven of thirteen samples the majority of SNVs were G to A
transitions, which is commonly seen in retroviral mutation profiles due to APOBEC cytidine
deaminase-mediated C to U transitions during reverse transcription (Fig. 4D) (28,29). G to A
transitions were more likely to occur at higher frequency than other transitions in nine samples
(Dunn test, p < 0.001). These results suggest that APOBEC-mediated cytidine deamination
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during reverse transcription contributes to Toca 511 mutation spectrum in both patient blood
and tumors, but that its influence varies among samples.
G to A mutations can create premature stop codons in Toca 511
The abundance of SNVs in integrated Toca 511 genomes from patient samples raises the
question as to what degree these mutations inactivate the vector. We focused our attention on
nonsynonymous mutations caused by G to A transitions due their abundance. What stood out
was the potential the codon encoding tryptophan (TGG) to be converted to stop codons (STOP -
TGA, TAG and TAA) by any combination of G to A at the second or third position. Toca 511
contains 52 tryptophan codons: 12 in gag, 24 in pol, 14 in env and two in yCD2. We calculated
the frequency of G to A mutations in tryptophan codons and plotted the results for each sample
as a heatmap (Fig. 5A). There were a number of sites in the genome with high frequency
tryptophan to STOP that occurred in multiple samples. There were ten samples with at least
one tryptophan to STOP at 30% frequency or greater (Fig. 5B). Coinciding with increased
mutation accumulation and/or selection for nonfunctional Toca 511 genomes, tryptophan to
STOP was more abundant in samples from tumors re-resected following repeated cycles of
Toca FC treatment. The most common tryptophan to STOP mutation was in amino acid 10
(W10) in yCD2, which was mutated in eight samples at > 30% frequency. This mutation,
occurring near the 5’end of yCD2 coding sequence presumably eliminates functional yCD2
protein unless an alternative start codon rescues function; for instance, the closest downstream
methionine is at amino acid 15. W10 is immediately adjacent to the first alpha helix and is
conserved in orthologs from closely related fungal species (Supplementary Fig. S9) (38). The
next two most frequent mutations affected W804 and W1016 in pol. W804 to STOP occurred in
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three re-resected tumors and five tumors resected prior to Toca 511 and Toca FC treatment,
while W1016 to STOP occurred in one blood, three re-resected tumors and two tumors
resected prior to Toca FC treatment.
In order to corroborate these results we performed targeted sequencing on the yCD2
coding region (Supplementary Text & Fig. S10). According to the targeted yCD2 sequencing
results at least 30% of yCD2 coding sequences were functional in ten of sixteen samples
isolated from tumors following IV treatment, one of five samples isolated from blood (three of
five were from one patient) and one of three samples from re-resected tumors (Fig. 5C). While
technical replicates for targeted sequencing of yCD2 were highly concordant (Supplementary
Fig. S11), the concordance between Toca 511 sequencing results and targeted yCD2 sequencing
results varied (Supplementary Text & Fig. S10). Thus, we must be cautious about quantitative
interpretation of specific mutation frequencies, which could be skewed by a small number of
viral copies going into PCR reactions prior to sequencing preparation (39). For instance, even in
tumors resected after Toca 511 delivery and multiple rounds of Toca FC, where deleterious
mutations are observed at high frequency, we still detect yCD2 expression, suggesting a
reservoir of functional virus persists (Supplementary Fig. S12) (9).
Toca 511 preferentially targets tumors in a recurrent GBM patient
While we have characterized the tissue and biofluid distribution of Toca 511 in mice
(12), analogous analyses have not been reported in humans. Twenty-one tissue samples
throughout the body were obtained after the death of a male patient with recurrent
glioblastoma multiforme (GBM) who was treated with Toca 511 via injection into the newly
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resected tumor cavity, as in Study 11. The patient died approximately three months after Toca
511 injection and approximately six weeks after a single cycle of Toca FC as the patient declined
further treatment due to poor quality of life with global aphasia. We obtained three pieces of
recurrent tumor at the Toca 511-injection site, three from a separate non-injected GBM tumor,
two from non-neoplastic brain, one from spinal cord, one from cerebral spinal fluid (CSF) as
well as from sites that are considered potential repositories for gRVs or important organs to
test, including spleen, lymph node, bone marrow, lung, liver, lower GI, testes and skin as well as
whole blood and urine.
We measured Toca 511 levels in DNA and total RNA isolated from each autopsy tissue
sample. Toca 511 was detected in RNA isolated from two of three samples from the injected
tumor site and from one of three samples from neighboring non-injected tumor, but not
elsewhere (Fig. 6A, right panel). Toca 511 was detected in DNA isolated from all three injected-
site tumor pieces, two of three non-injected tumor pieces, immediately adjacent non-
neoplastic brain region, spinal cord and CSF (Fig. 6A, left panel). Outside of the central nervous
system we found observable but not quantifiable (< 100 copies/g, < 1 copy/15,000 diploid cell
equivalents) Toca 511 DNA in blood, liver, spleen, lymph node, lung and bone marrow. No Toca
511 signal was observed in DNA isolated from testes, urine, skin, or lower GI. No Toca 511 RNA
was detected outside of the tumor samples, reinforcing the low probability of live virus
shedding by patients. Toca 511 tumor specificity is corroborated by whole brain autopsy
samples isolated from a patient in Study 8 who underwent one cycle of Toca FC (Supplementary
Table 3).
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We amplified and sequenced integrated Toca 511 from DNA isolated from the injection
site tumor, the non-injected tumor, CSF and blood. For all four samples we obtained quality
results from the 3’ PCR product covering 3,750 bp including yCD2 (Supplementary Fig. S7).
There were more than twice as many SNVs from the two tumor samples and CSF relative to
blood (Fig. 6B). Most mutations in tumor and CSF were G to A (Fig. 6C). Nonetheless the specific
mutation pattern in each sample was unique (Fig. 6D). Thus while the general mutation biases
were similar among the tumor and CSF samples, the locations and frequencies of specific
mutations varied considerably.
DISCUSSION
The logic behind gammaretroviral replicating vector gene therapy for oncology
Gammaretroviral replicating vector gene therapy holds promise for a range of
oncological therapeutic indications due its ability to selectively infect cancer cells without direct
cell lysis and deliver a therapeutic transgene. In animal models, Toca 511-infected tumor cells
are killed by 5-FU converted from 5- FC. The diffusible 5-FU also kills susceptible neighboring
cells, including immune suppressor myeloid cells that contribute to the immune-suppressed
tumor microenvironment (40,41). After several cycles of Toca FC, treated immune competent
animals that clear tumor are resistant to tumor re-challenge (12) and this resistance is T-cell
mediated (40,41).
The potential advantages of a RRV, Toca 511, over RNV center around the premise that
there will be multiple rounds of infection and spread in the tumor over time, leading to a
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greater proportion of tumor cells and geographical regions infected, including non-injected
tumors. The replication of retroviruses, including Toca 511, includes stable integration of viral
genomes into infected cancer cell genomes creating a reservoir of Toca 511. This reservoir
persists during Toca FC treatment in part because non-cycling cancer cells are more resistant to
5-FU killing than replicating cells (42). The reservoir provides a source of Toca 511 production
and spread to newly formed tumor cells between Toca FC doses. This allows for continued
killing of tumor cells over multiple cycles of Toca FC and subsequent breaking of immune
tolerance and re-activation of the immune system against the tumor. This is seen in preclinical
models where several cycles of Toca FC are required to produce durable response in immune-
competent animals (12).
Toca 511 selectively replicates and spreads in patient tumors
Several lines of circumstantial evidence suggest Toca 511 does indeed undergo multiple
rounds of infection in patient samples. The strongest evidence comes from mutational profiles
in which the frequencies of specific mutations vary over 30-fold (Fig. 4). Samples from tumors
isolated after Toca 511 and Toca FC treatment categorically displayed more mutations than
samples from tumors taken before Toca FC treatment, likely due to depletion of functional Toca
511 genomes from yCD2-dependent 5-FU-mediated cell death. Detection of integrated Toca
511 in blood samples emerges weeks after dosing suggesting ongoing viral replication and
spread of productive virus over time (Fig. 1). Toca 511 was quantitatively detected in both the
tumor from the injection site and in a separate non-injected tumor in the brain (Figure 6).
Multiple lines of evidence presented herein support Toca 511’s selectivity for tumor
cells over normal cells and tissues in humans. Integrated Toca 511 was detected transiently in
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blood but only in 11 of 56 patients treated in Study 11 (Fig. 1). Toca 511 RNA was only detected
in two of the fifteen blood samples with detectable levels of integrated Toca 511 DNA
(Supplementary Dataset S1). While it was not feasible to measure Toca 511 levels in tumor cells
in the resection study prior to Toca FC treatment, we did measure Toca 511 levels in the
context of IV delivery as well as in tumors that recurred post Toca FC treatment. In these
contexts, Toca 511 DNA and Toca 511 RNA were detected in > 40% of tumors, at levels
generally higher than seen in blood (Fig. 1), arguing for active virus production in the tumor
(Supplementary Dataset S1). These results are extended in a more expansive analysis of non-
tumor samples from a patient’s whole body autopsy, where we detected integrated virus and
viral RNA in the injected tumor site as well as in a non-injected brain tumor, but we only
detected non-quantifiable levels of virus DNA in a subset of bodily sites outside of the central
nervous system (Fig. 6). All patients cleared detectable virus signal in blood within the first
couple cycles of Toca FC suggesting that human patients are generally able to control the virus
systemically, even if a sizeable fraction of integrated Toca 511 genomes appear to be
inactivated (Fig. 4 and Fig. 5). We hypothesize blood clearance is due to a combination of yCD2-
dependent apoptosis following 5-FC administration, Toca 511 independent cell turnover and
multiple innate and adaptive defense mechanisms that act naturally to clear MLV, which is not
zoonotic.
APOBEC-mediated cytidine deamination is likely the dominant source of Toca 511
mutations in patients
Most mutations in Toca 511 genomes isolated from patient samples were G to A
transitions (Fig. 4). The human genome encodes a repertoire of APOBEC cytidine deaminases,
some of which are incorporated into retroviral particles via interactions with the core viral
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protein GAG and viral RNA and catalyze C to U transitions on single-stranded DNA during
reverse transcription in the cytoplasm (43), leading to G to A mutations in the viral coding
strand. The most parsimonious explanation for the mutation profiles is that Toca 511 particles
encapsulate one or more APOBEC molecules, which then processively mutate C’s to U’s during
reverse transcription in the subsequent rounds of infection, leading to mutated and often
inactivated integrated virus (Fig. 4). Among G to A mutations, those resulting in conversion of
the tryptophan codon (TGG) to premature stop codons (TGA, TAG and TAA) are expected to be
particularly deleterious to viral functions. The complement of the tryptophan codon is the
preferred sequence context for APOBEC3G, suggesting this paralog as a likely culprit. Generally
though, the sources and identities of APOBECs incorporated into Toca 511 particles in patients
are not known. While in some samples we estimate > 90% frequency of specific G to A stop
mutations, such as mutations leading to conversion of tryptophan at position 10 in yCD2 (Fig. 4,
Fig. 5C & Supplementary Fig. S10), we are cautious about quantitative interpretation of the
mutation frequencies given the observed intra-sample discordance (Supplementary Text & Fig.
S10). Indeed, we detected yCD2 via IHC in some tumor samples with high frequency of
conversion of tryptophan at position 10 to stop codon (Supplementary Fig. S12), suggesting
APOBECs inactivate some, but not all, Toca 511 genomes. However, we are unable to make
quantitative connections between observed yCD2 expression by IHC from one piece of
tumor to Toca 511 DNA and RNA levels in another piece of tumor due to intra-tumor
heterogeneity.
As Toca 511 is derived from a mouse gammaretrovirus, it did not specifically evolve
mechanisms to minimize inactivation by human APOBECs (or other human restriction factors).
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34
In principle, future generations of gRVs could be engineered to minimize tryptophan residues
within the transgene, which could lead to enhanced efficacy in tumors while maintaining the
safety profile.
No evidence for pathology or molecular abnormalities from Toca 511 insertions
A primary safety concern using RRVs has been that infection of blood progenitor cells
leading to oncogenic transformation could occur in some clinical situations as has been
observed with RNVs. Toca 511 integration profiles from blood and tumor samples described
herein, after administration both into the tumor resection cavity and IV, show no compelling
evidence for clonal expansion of infected cells within the time frame of these trials (Study 11
started in 2011)(Fig. 3). These results are consistent with the absence of direct clinical evidence
for this kind of adverse event so far in recurrent HGG patients treated with Toca 511 and Toca
FC in a resection setting (9). However, given the precedence for subsequent malignancies in
patients with HGG and temozolomide-related haematological adverse events (44,45), it is likely
that we will eventually encounter patients treated with Toca 511 and Toca FC that develop
haematological adverse events, including lymphoma. The assays presented herein would
enable us to gauge the contribution, if any, of Toca 511 insertional mutagenesis.
Gammaretrovirus integration is stimulated by physical interactions between integrase
with BET proteins which orchestrate assembly of transcription initiation complexes, leading to
so-called pseudo-random insertion preferentially near transcription start sites, including active
enhancers and promoters (46). Therefore, many gRV integration sites are within gene
regulatory regions, which in turn could influence the transcriptional regulation of linked genes,
as seen for LMO2 in HSCs in some treated X-SCID patients, resulting in the delayed onset of
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35
leukemia in some of these severely immune compromised patients (47). Our analyses of Toca
511 integration sites in blood and brain tumor suggest that while Toca 511 follows pseudo-
random preferences of other gRVs, there was no compelling evidence for clonal expansion as
judged by overrepresentation of specific integration sites, nor was there preferential insertion
near oncogenes (Fig. 3). We did find that insertion sites in blood and brain tumor occurred
preferentially near genes that function in their respective tissue type (Fig. 2).
Conclusions
Toca 511 and Toca FC treatment represents a general novel anti-cancer modality (9).
This study fills in crucial gaps in our understanding of the therapeutic use of Toca 511 and
replicating retroviral vectors in general. The data provided herein provide molecular rationales
for the previously reported lack of Toca 511-related excess tumorigenicity observed in patients
treated with Toca 511 and Toca FC (9) as well as the continued investigation of replicating
retrovirus-based immunotherapies. A randomized phase 3 trial (NCT02414165) in patients with
recurrent GBM and anaplastic astrocytoma and a phase Ib trial investigating treatment in solid
tumors (NCT02576665) are ongoing.
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36
FIGURE LEGENDS
Fig. 1. Toca 511 detection frequency and timing in DNA isolated from patient blood
and tumor samples.
(A) Timeline of resection Study 11 and IV Study 13 treatment regimes. The timelines start at
day 1 and are not to scale. The grey boxes above the timelines show when Toca FC was
taken. The vertical red lines below the timelines indicate regularly scheduled blood draws
for Toca 511 monitoring.
(B) Heatmap representation of Toca 511 DNA signal in blood as a function of time for the
eleven patients of 56 in Study 11 with detectable signal. Visit number is shown above the
heatmap. Visit 3a is 1 week after visit 3. Visit 4a is 1 week after visit 4.
(C) Barplot showing the percentage of patients with detectable Toca 511 signal in DNA: blood =
red, re-resected tumor after Toca 511/FC treatment = magenta, resected tumor after IV
treatment = blue. The numbers on top of the bars show the fraction of patients with
detectable signal.
(D) Boxplot showing the estimated number of copies of Toca 511 per g of genomic DNA for
samples with detectable signal in (C). The horizontal bar shows the median, the lower and
upper hinges correspond to the first and third quartiles respectively, and the whiskers
extend from the hinge to the largest value no further than 1.5 x the interquartile range from
the hinges. There are 15 samples from 11 patients with quantifiable signal in blood (B).
Fig. 2. Toca 511 integration patterns are tissue specific and consistent with previous
analyses of gammaretroviral integration.
(A) Boxplot showing the number of identified integration events obtained from negative
controls (grey), blood samples (red) and IV tumor samples (blue).
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37
(B) Distribution of Toca 511 integration sites in blood samples (red) and tumor samples (blue)
around the nearest annotated transcription start site (TSS). Preferential Toca 511
integration near TSSs is consistent with established integration preferences of gRVs.
(C) Genes that harbor Toca 511 integration sites immediately upstream (within 10 kb) or within
the gene encode proteins whose expression is linked to the site from which samples were
taken. (top-red) Integration sites in blood tend to occur within or near genes encoding
proteins linked to blood-cell functions. (bottom-blue) In contrast, brain tumor integration
sites are enriched for genes linked to neuronal stem cell proliferation and differentiation
and neuronal functions. The significance of enrichment of integration sites near genes
annotated in each category is represented as a bar plot of the p-value, calculated using the
hypergeometric density distribution function. GO = Gene Ontology term, PID = Pathway
Interaction Database.
Fig. 3. Absence of compelling evidence for clonal expansion of Toca 511-integrated
cells.
(A) The number of total integration events for each sample are color-coded by the proportion
of sites represented once (grey) versus the number of sites represented multiple times
(black). Samples in red are from blood: e.g., 11_05_v7 indicates Study 11 patient 05 visit 7
blood draw. Samples in blue are from brain tumors: e.g., 13_03_p5 indicates Study 13
patient 03 tumor piece 5.
(B) Neither blood (red) nor tumor (blue) integration sites preferentially occur near genes linked
to tumorigenesis. The significance of enrichment of integration sites near genes annotated
in each category is represented as a bar plot of the p-value, calculated using the
hypergeometric density distribution function.
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38
Fig. 4. Toca 511 genomes are mutated by restriction factors in patients.
(A) Boxplot showing the total number of single nucleotide variants (SNVs) from different classes
of samples at a mutation frequency threshold of 3%: Toca 511 containing plasmid and
transduced cell lines (black, n=4), blood (red, n=3), brain tumors re-resected after further
progression of disease after Toca 511 delivery (magenta, n=4), brain tumors following IV
dosing prior to Toca FC treatment (blue, n=6).
(B) Mutations occur at a wide-range of frequencies in patient samples. Barplots show the
fraction of mutations across all patients from whom data could be obtained, with frequency
3-10 % (light green), 10-30% (green) and greater than 30% (dark green).
(C) Boxplot showing the percent totals of all possible point mutations from different classes of
samples. For instance, A>C indicates A to C transitions. Nearly all mutations are transitions.
(D) Barplot showing the percentage frequency of the four possible transitions in each patient
sample. G to A mutations predominate for all samples.
Fig. 5. G to A mutations can create premature stop codons in Toca 511.
(A) The tryptophan (W) codon is converted to stop codons (STOP) by G to A transitions. Each
row corresponds to the Toca 511 mutation spectrum from a single DNA sample. The color
bar on the left side indicates each sample’s tissue of origin (colors as in Figure 4A). The
heatmap shows the percentage frequency of conversion of W to STOP along the Toca 511
genome. High frequency inactivating mutations were largely restricted to tumor re-resected
(magenta) after multiple Toca FC treatment cycles.
(B) The number of W to STOP changes (on an amino acid basis) across Toca 511 as a function of
their frequency in the sample.
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39
(C) Boxplot representation of the estimated percentage of “functional” yCD2 based on targeted
yCD2 sequencing. “Functional” is defined as 100 minus the maximum mutation frequency of
the three observed G to A mutations that are expected to inactivate yCD2 (Supplementary
Fig. S10).
Fig. 6. Toca 511 preferentially targets tumors in a GBM patient.
(A) (left) Average Toca 511 signal (copies/g) from qPCR replicates with quantifiable signal. *
indicates samples for which signal was below the LLOQ – the bars are arbitrarily set at 50%
of the LLOQ; (right) Average Toca 511 RNA signal (copies/g) from RT-qPCR replicates with
quantifiable signal. Note the break in the x-axis.
(B) Barplot showing the fraction of SNVs with percentage frequency 3-10 % (light green), 10-
30% (green) and greater than 30% (dark green). The source of DNA is indicated below the
plots.
(C) Barplot showing mutation frequency for the four possible transitions.
(D) Heatmap representation of percentage frequency of SNVs across the 3’ end of Toca 511
genome from the four patient samples. Only positions in Toca 511 in which at least one
sample had a SNV were included. Samples were clustered by Euclidean distance.
ACKNOWLEDGEMENTS
We would like to thank John Wood MBA RAC (Tocagen Inc.) and John M. Coffin Ph.D. (Tufts
University) for critically reading the manuscript. We wish to thank the patients and their
families for participating in Tocagen’s Clinical Trials. We are also deeply grateful to those
patients and families who consented to the autopsy procedures.
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40
Funding
The authors thank the ABC2 Foundation (Washington, DC), the National Brain Tumor Society
(Watertown, MA), the American Brain Tumor Association (Chicago, IL), the Musella Foundation
(Hewlett, NY), and Voices Against Brain Cancer (New York, NY) for their support and
collaborations.
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41
REFERENCES 1. Kaufman HL, Kohlhapp FJ, Zloza A. Oncolytic viruses: a new class of immunotherapy drugs.
Nature reviews Drug discovery 2015;14(9):642-62 doi 10.1038/nrd4663. 2. Kohlhapp FJ, Kaufman HL. Molecular Pathways: Mechanism of Action for Talimogene
Laherparepvec, a New Oncolytic Virus Immunotherapy. Clinical cancer research : an official journal of the American Association for Cancer Research 2016;22(5):1048-54 doi 10.1158/1078-0432.CCR-15-2667.
3. Logg CR, Robbins JM, Jolly DJ, Gruber HE, Kasahara N. Retroviral replicating vectors in cancer. Methods in enzymology 2012;507:199-228 doi 10.1016/B978-0-12-386509-0.00011-9.
4. Critchley-Thorne RJ, Simons DL, Yan N, Miyahira AK, Dirbas FM, Johnson DL, et al. Impaired interferon signaling is a common immune defect in human cancer. Proceedings of the National Academy of Sciences of the United States of America 2009;106(22):9010-5 doi 10.1073/pnas.0901329106.
5. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annual review of immunology 2007;25:267-96 doi 10.1146/annurev.immunol.25.022106.141609.
6. Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nature reviews Immunology 2006;6(10):715-27 doi 10.1038/nri1936.
7. Roe T, Reynolds TC, Yu G, Brown PO. Integration of murine leukemia virus DNA depends on mitosis. The EMBO journal 1993;12(5):2099-108.
8. Lin AH, Burrascano C, Pettersson PL, Ibanez CE, Gruber HE, Jolly DJ. Blockade of type I interferon (IFN) production by retroviral replicating vectors and reduced tumor cell responses to IFN likely contribute to tumor selectivity. Journal of virology 2014;88(17):10066-77 doi 10.1128/JVI.02300-13.
9. Cloughesy TF, Landolfi J, Hogan DJ, Bloomfield S, Carter B, Chen CC, et al. Phase 1 trial of vocimagene amiretrorepvec and 5-fluorocytosine for recurrent high-grade glioma. Science translational medicine 2016;8(341):341ra75 doi 10.1126/scitranslmed.aad9784.
10. Kim S, Park EJ, Yu SS, Kim S. Development of enzyme-linked immunosorbent assay for detecting antibodies to replication-competent murine leukemia virus. Journal of virological methods 2004;118(1):1-7 doi 10.1016/j.jviromet.2004.01.010.
11. Martineau D, Klump WM, McCormack JE, DePolo NJ, Kamantigue E, Petrowski M, et al. Evaluation of PCR and ELISA assays for screening clinical trial subjects for replication-competent retrovirus. Human gene therapy 1997;8(10):1231-41 doi 10.1089/hum.1997.8.10-1231.
12. Ostertag D, Amundson KK, Lopez Espinoza F, Martin B, Buckley T, Galvao da Silva AP, et al. Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector. Neuro Oncol 2012;14(2):145-59 doi nor199 [pii]
10.1093/neuonc/nor199. 13. Perez OD, Logg CR, Hiraoka K, Diago O, Burnett R, Inagaki A, et al. Design and selection of Toca
511 for clinical use: modified retroviral. Molecular therapy : the journal of the American Society of Gene Therapy 2012;20:1689-98 doi 10.1038/mt.2012.83
10.1038/mt.2012.83. Epub 2012 May 1. 14. Twitty CG, Diago OR, Hogan DJ, Burrascano C, Ibanez CE, Jolly DJ, et al. Retroviral Replicating
Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types. Human gene therapy methods 2016;27(1):17-31 doi 10.1089/hgtb.2015.106.
Research. on May 27, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 26, 2018; DOI: 10.1158/1078-0432.CCR-18-0619
42
15. Vanin EF, Kaloss M, Broscius C, Nienhuis AW. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. Journal of virology 1994;68(7):4241-50.
16. Purcell DF, Broscius CM, Vanin EF, Buckler CE, Nienhuis AW, Martin MA. An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer. Journal of virology 1996;70(2):887-97.
17. Yi Y, Hahm SH, Lee KH. Retroviral gene therapy: safety issues and possible solutions. Current gene therapy 2005;5(1):25-35.
18. Cicalese MP, Ferrua F, Castagnaro L, Pajno R, Barzaghi F, Giannelli S, et al. Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency. Blood 2016;128(1):45-54 doi 10.1182/blood-2016-01-688226.
19. Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, et al. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003;101(6):2099-114 doi 10.1182/blood-2002-07-2314.
20. Yi Y, Noh MJ, Lee KH. Current advances in retroviral gene therapy. Current gene therapy 2011;11(3):218-28.
21. Muul LM, Tuschong LM, Soenen SL, Jagadeesh GJ, Ramsey WJ, Long Z, et al. Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial. Blood 2003;101(7):2563-9 doi 10.1182/blood-2002-09-2800.
22. Haviernik P, Bunting KD. Safety concerns related to hematopoietic stem cell gene transfer using retroviral vectors. Current gene therapy 2004;4(3):263-76.
23. Deichmann A, Hacein-Bey-Abina S, Schmidt M, Garrigue A, Brugman MH, Hu J, et al. Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. The Journal of clinical investigation 2007;117(8):2225-32 doi 10.1172/JCI31659.
24. Scholler J, Brady TL, Binder-Scholl G, Hwang WT, Plesa G, Hege KM, et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Science translational medicine 2012;4(132):132ra53 doi 10.1126/scitranslmed.3003761.
25. LaFave MC, Varshney GK, Gildea DE, Wolfsberg TG, Baxevanis AD, Burgess SM. MLV integration site selection is driven by strong enhancers and active promoters. Nucleic acids research 2014;42(7):4257-69 doi 10.1093/nar/gkt1399.
26. De Ravin SS, Su L, Theobald N, Choi U, Macpherson JL, Poidinger M, et al. Enhancers are major targets for murine leukemia virus vector integration. Journal of virology 2014;88(8):4504-13 doi 10.1128/JVI.00011-14.
27. Wolf D, Goff SP. Host restriction factors blocking retroviral replication. Annual review of genetics 2008;42:143-63 doi 10.1146/annurev.genet.42.110807.091704.
28. Yu Q, Konig R, Pillai S, Chiles K, Kearney M, Palmer S, et al. Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome. Nature structural & molecular biology 2004;11(5):435-42 doi 10.1038/nsmb758.
29. Armitage AE, Katzourakis A, de Oliveira T, Welch JJ, Belshaw R, Bishop KN, et al. Conserved footprints of APOBEC3G on Hypermutated human immunodeficiency virus type 1 and human endogenous retrovirus HERV-K(HML2) sequences. Journal of virology 2008;82(17):8743-61 doi 10.1128/JVI.00584-08.
30. Hacein-Bey-Abina S, Pai SY, Gaspar HB, Armant M, Berry CC, Blanche S, et al. A modified gamma-retrovirus vector for X-linked severe combined immunodeficiency. The New England journal of medicine 2014;371(15):1407-17 doi 10.1056/NEJMoa1404588.
Research. on May 27, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 26, 2018; DOI: 10.1158/1078-0432.CCR-18-0619
43
31. Sherman E, Nobles C, Berry CC, Six E, Wu Y, Dryga A, et al. INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes. Molecular therapy Methods & clinical development 2017;4:39-49 doi 10.1016/j.omtm.2016.11.002.
32. Gillet NA, Melamed A, Bangham CR. High-Throughput Mapping and Clonal Quantification of Retroviral Integration Sites. Methods in molecular biology 2017;1582:127-41 doi 10.1007/978-1-4939-6872-5_10.
33. Fabregat A, Sidiropoulos K, Garapati P, Gillespie M, Hausmann K, Haw R, et al. The Reactome pathway Knowledgebase. Nucleic acids research 2016;44(D1):D481-7 doi 10.1093/nar/gkv1351.
34. The Gene Ontology C. Expansion of the Gene Ontology knowledgebase and resources. Nucleic acids research 2017;45(D1):D331-D8 doi 10.1093/nar/gkw1108.
35. Tagawa H, Miura I, Suzuki R, Suzuki H, Hosokawa Y, Seto M. Molecular cytogenetic analysis of the breakpoint region at 6q21-22 in T-cell lymphoma/leukemia cell lines. Genes, chromosomes & cancer 2002;34(2):175-85 doi 10.1002/gcc.10057.
36. Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome research 2012;22(3):568-76 doi 10.1101/gr.129684.111.
37. Shoemaker C, Hoffman J, Goff SP, Baltimore D. Intramolecular integration within Moloney murine leukemia virus DNA. Journal of virology 1981;40(1):164-72.
38. Stolworthy TS, Korkegian AM, Willmon CL, Ardiani A, Cundiff J, Stoddard BL, et al. Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine. Journal of molecular biology 2008;377(3):854-69 doi 10.1016/j.jmb.2008.01.002.
39. Welkers MR, Jonges M, Jeeninga RE, Koopmans MP, de Jong MD. Improved detection of artifactual viral minority variants in high-throughput sequencing data. Frontiers in microbiology 2014;5:804 doi 10.3389/fmicb.2014.00804.
40. Mitchell LA, Lopez Espinoza F, Mendoza D, Kato Y, Inagaki A, Hiraoka K, et al. Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model. Neuro Oncol 2017 doi 10.1093/neuonc/nox037.
41. Hiraoka K, Inagaki A, Kato Y, Huang TT, Mitchell LA, Kamijima S, et al. Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity. Neuro Oncol 2017 doi 10.1093/neuonc/nox038.
42. Richard C, Duivenvoorden W, Bourbeau D, Massie B, Roa W, Yau J, et al. Sensitivity of 5-fluorouracil-resistant cancer cells to adenovirus suicide gene therapy. Cancer gene therapy 2007;14(1):57-65 doi 10.1038/sj.cgt.7700980.
43. Cullen BR. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. Journal of virology 2006;80(3):1067-76 doi 10.1128/JVI.80.3.1067-1076.2006.
44. Villano JL, Letarte N, Yu JM, Abdur S, Bressler LR. Hematologic adverse events associated with temozolomide. Cancer chemotherapy and pharmacology 2012;69(1):107-13 doi 10.1007/s00280-011-1679-8.
45. Li X, Li Y, Cao Y, Li P, Liang B, Sun J, et al. Risk of subsequent cancer among pediatric, adult and elderly patients following a primary diagnosis of glioblastoma multiforme: a population-based study of the SEER database. The International journal of neuroscience 2017:1-7 doi 10.1080/00207454.2017.1288624.
46. Sharma A, Larue RC, Plumb MR, Malani N, Male F, Slaughter A, et al. BET proteins promote efficient murine leukemia virus integration at transcription start sites. Proceedings of the National Academy of Sciences of the United States of America 2013;110(29):12036-41 doi 10.1073/pnas.1307157110.
Research. on May 27, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 26, 2018; DOI: 10.1158/1078-0432.CCR-18-0619
44
47. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302(5644):415-9 doi 10.1126/science.1088547.
48. Martin M. Cutadapt Removes Adapter Sequences From High-Throughput Sequencing Reads. doi Cutadapt Removes Adapter Sequences From High-Throughput Sequencing Reads.
49. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods 2012;9(4):357-9 doi 10.1038/nmeth.1923.
50. Morgan M PH, Obenchain V and Hayden N Rsamtools: Binary alignment (BAM), FASTA, variant call (BCF), and tabix file import R package version 1261. http://bioconductor.org/packages/release/bioc/html/Rsamtools.html.2016.
51. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010;26(6):841-2 doi 10.1093/bioinformatics/btq033.
52. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Molecular cell 2010;38(4):576-89 doi 10.1016/j.molcel.2010.05.004.
53. Tripathi S, Pohl MO, Zhou Y, Rodriguez-Frandsen A, Wang G, Stein DA, et al. Meta- and Orthogonal Integration of Influenza "OMICs" Data Defines a Role for UBR4 in Virus Budding. Cell host & microbe 2015;18(6):723-35 doi 10.1016/j.chom.2015.11.002.
54. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Jr., Kinzler KW. Cancer genome landscapes. Science 2013;339(6127):1546-58 doi 10.1126/science.1235122.
55. Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics 2011;27(6):863-4 doi 10.1093/bioinformatics/btr026.
56. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25(14):1754-60 doi 10.1093/bioinformatics/btp324.
57. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009;25(16):2078-9 doi 10.1093/bioinformatics/btp352.
58. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014;30(15):2114-20 doi 10.1093/bioinformatics/btu170.
Research. on May 27, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 26, 2018; DOI: 10.1158/1078-0432.CCR-18-0619
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(cop
ies/
ug)
D
Toca 511 DNA in blood
patie
nt
visit
Figure 1.
blood IV tumorre-tumor
blood IV tumorre-tumor
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4
32
256
0
10
20
negativecontrol
blood IV tumor −10 −5 0 5 10distance to TSS (kb)
inte
grat
ion
even
ts
inte
grat
ion
even
ts
A B
GO: embryo developmentREACTOME: signalling by NGF
REACTOME: GRB2 events in ERBB2 signalingGO: transmembrane receptor protein tyrosine kinase signaling pathway
GO: regulation of neuron projection developmentGO: protein autophosphorylation
GO: neuron projection morphogenesisPID: NFAT pathway
GO: secretion by cellPID: C−MYB pathway
GO: adenylate cyclase−activating GPCR signaling pathwaycell recognition
PID: IL4 pathwayGO: negative regulation of blood circulation
GO: positive regulation of immune response
1 .01 10-4 10-6
p-value
C
Figure 2.
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1
4
16
64
256
inte
grat
ion
even
ts
multiplesingle
1 0.3 0.1 0.03 0.01p-value
cancer predisposition
driver (amp/del)
driver (subtle)
liquid rearrangement
lymphoma
tumor suppressor
0 20 40 60number of genes
BA11
_05_
v711
_06_
v511
_06_
v711
_09_
v511
_18_
v711
_33_
v411
_33_
v713
_03_
p513
_03_
p25
13_0
7_p3
13_0
8_p5
13_0
8_p1
413
_08_
p23
13_1
0_p1
213
_10_
p14
13_1
1_p1
13_1
1_p3
Figure 3.
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3-10%10-30%30-100%
A B
0
200
400
600
SNVs
control blood re-tumor IV tumor
PAZ3
PAZ3
bH
T-10
80U
-87
11_0
6_v5
11_0
6_v7
11_3
3_v7
11_0
2_p7
11_0
2_p8
11_0
2_p1
211
_31_
01_p
113
_08_
p14
13_1
0_p1
213
_12_
p18
13_1
3_p8
13_1
5_p1
513
_15_
p6
0
200
400
600
SNVs
C
0
25
50
75
100
perc
enta
ge
D
11_0
6_v5
11_0
6_v7
11_3
3_v7
11_0
2_p7
11_0
2_p8
11_0
2_p1
211
_31_
01_p
113
_08_
p14
13_1
0_p1
213
_12_
p18
13_1
3_p8
13_1
5_p1
513
_15_
p6
0
25
50
75
perc
ent o
f tot
al
A>C A>G A>T C>A C>G C>T G>A G>C G>T T>A T>C T>G
100
T > CA > GC > T
frequency
G > A
Figure 4.
Research. on May 27, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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0
10
20
30
W to
STO
P
pol env CDgag
Tryptophan (W) --> STOPTGG TAG
TGATAA
A
B
PAZ3
U-8
7
pAZ3
bH
T-10
80
11_0
6_v5
11_0
6_v7
11_3
3_v7
11_0
2_p7
11_0
2_p8
11_0
2_p1
211
_31_
01_p
113
_08_
p14
13_1
0_p1
213
_12_
p18
13_1
3_p8
13_1
5_p1
513
_15_
p6
40
frequency3-10%10-30%30-100%
< 3 11 45 100
% fr
eque
ncy
0
25
50
75
100
blood re_tumor tumorfu
nctio
nal y
CD
2 (%
)
C
Figure 5.
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skinurine
testeslowerGI
lung LLL consolidationlung LLL nodule
liverbone marrow
lymph nodespleenblood
spinal cordbrain distal
brain adjacentCSF
tumor noninjected 3tumor noninjected 2tumor noninjected 1
tumor 3tumor 2tumor 1
Toca511 injected tumorother sites in CNSsites outside CNS
0 100 200DNA (copies/ug)
10 100 105
RNA (copies/ug)
Blood
Tumor 1
CSF
Tumornoninj 2
B C
D
< 3 11 45 100
% fr
eque
ncy
A
G > AT > CA > GC > T
frequency3-10%10-30%30-100%
0
25
50
75
100Bl
ood
CSF
Tum
or 1
perc
enta
ge
0
20
40
60
SNVs
Bloo
d
Tum
or 1
Tum
or n
onin
ject
ed 2
CSF
Tum
or n
onin
ject
ed 2
pol env CD
*
*
*
*******
* < LLOQ
Figure 6.
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Published OnlineFirst June 26, 2018.Clin Cancer Res Daniel J Hogan, Jay-Jiguang Zhu, Oscar Diago, et al. Replicating Vector Toca 511 in PatientsMolecular Analyses Support the Safety and Activity of Retroviral
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Research. on May 27, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 26, 2018; DOI: 10.1158/1078-0432.CCR-18-0619