ChAd mRNA Solid Tumors

Articles
https://doi.org/10.1038/s41591-022-01937-6
Individualized, heterologous chimpanzee

adenovirus and self-amplifying mRNA neoantigen
vaccine for advanced metastatic solid tumors:
phase 1 trial interim results
Christine D. Palmer 1, Amy R. Rappaport1, Matthew J. Davis1, Meghan G. Hart1, Ciaran D. Scallan1,
Sue-Jean Hong1, Leonid Gitlin1, Lauren D. Kraemer1, Sonia Kounlavouth1, Aaron Yang1, Lindsey Smith1,
Desiree Schenk1, Mojca Skoberne1, Kiara Taquechel 1, Martina Marrali1, Jason R. Jaroslavsky1,
Charmaine N. Nganje1, Elizabeth Maloney 1, Rita Zhou1, Daniel Navarro-Gomez1,
Adrienne C. Greene1, Gijsbert Grotenbreg1, Renee Greer1, Wade Blair 1, Minh Duc Cao1,
Shawn Chan1, Kyounghwa Bae1, Alexander I. Spira2, Sameek Roychowdhury3, David P. Carbone 3,
Brian S. Henick4, Charles G. Drake4, Benjamin J. Solomon5, Daniel H. Ahn6, Amit Mahipal7,
Steve B. Maron 8, Benny Johnson9, Raphael Rousseau1, Roman Yelensky 1, Chih-Yi Liao10,
Daniel V. T. Catenacci10, Andrew Allen1, Andrew R. Ferguson1 and Karin Jooss1 ✉
Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing
vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recom-
mended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA
(samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an
ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regi-
men was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included
pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia,
duodenitis, increased transaminases and hyperthyroidism. The RP2D was 1012 viral particles (VP) ChAd68 and 30 µg sam-
RNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manu-
facturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with
microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circu-
lating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analy-
ses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.
C
ancer immunotherapies have shown promise in harnessing Accordingly, peptide-based neoantigen vaccine platforms have
the immune system to target and destroy cancers, leading to to date failed to consistently induce robust neoantigen-specific
clinical benefit enriched in patients with a high mutational CD8 T cell responses in the majority of patients13–15. Although
burden1–5. Multiple studies indicate that cytotoxic CD8 T cells tar- more immunogenic, a homologous prime boost messenger RNA
geting tumor neoantigens are critical to tumor control and clear- (mRNA)-based vaccination approach elicited predominantly CD4
ance in response to immunotherapies targeting CTLA-4 or PD-16–10. T cell responses16–18. Cumulatively, previous findings suggest that a
Clinical responses to CPI therapy rely mostly on reinvigorating pre- successful cancer vaccine should (1) target tumor-specific neoanti-
existing tumor-specific T cell responses11, and active vaccination gens, (2) use highly immunogenic vaccine platform(s), (3) expand
to expand preexisting and prime de novo tumor-specific T cells is and prime T cells, (4) be combined with CPI therapy19 and (5) gen-
anticipated to overcome this limitation. erate long-term memory responses to ensure continuous tumor
The limited success of cancer vaccines in the past can be attrib- control for durable clinical benefit.
uted to a number of factors, including selection of poorly immuno- Viral vector-based vaccine platforms, such as recombinant ade-
genic self-antigens12, insufficiently immunogenic vaccine platforms novirus, are able to prime robust T cell responses20–24. Although high
and immunosuppressive milieus in patients with advanced cancers4. seroprevalence of anti-adenoviral antibodies in human populations
Gritstone bio, Inc., Emeryville, CA, USA. 2Virginia Cancer Specialists, Virginia Cancer Specialists, VA, Fairfax, USA. 3The Ohio State University Medical Center,
1
Columbus, OH, USA. 4Columbia University Medical Center, New York, NY, USA. 5Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. 6Mayo Clinic,
Phoenix, AZ, USA. 7Mayo Clinic Cancer Center, Rochester, MN, USA. 8Memorial Sloan Kettering Cancer Center, New York, NY, USA. 9The University of Texas
MD Anderson Cancer Center, Houston, TX, USA. 10University of Chicago Comprehensive Cancer Center, Chicago, IL, USA. ✉e-mail: [email protected]
Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1619

Articles Nature Medicine
can lead to diminished immune responses to antigens delivered via memory T cells (Extended Data Fig. 1b). Accordingly, interferon-γ
adenoviral-based vaccines20,25, preexisting anti-vector immunity can (IFN-γ) ELISpot at week 106 revealed positive responses to four or
be circumvented by using non-human adenoviral species, such as more of the SIV antigens in all six animals, and samRNA adminis-
chimpanzee adenovirus (ChAd). RNA virus-based vaccines have tration at week 106 resulted in an ~8-fold and ~17-fold increase in
been tested in humans in a range of cancer and infectious disease antigen-specific T cell response detectable at week 107 ± s.c. ipilim-
indications26,27, and samRNA vectors are of particular interest due to umab, respectively (Fig. 1d and Extended Data Fig. 1c). Finally, the
their ability to express high and durable antigen levels28. ability of the boosted antigen-specific CD8 T cells to kill target cells
In this study, the quality of antigen-specific T cell responses presenting SIV epitopes was determined. CD8 T cells from all six
induced by a heterologous vaccine regimen consisting of a ChAd animals showed cytotoxic activity against their cognate targets, with
vector (ChAd68) and a fully synthetic Venezuelan equine encepha- CD8 T cells from four of six animals achieving killing of >70%, even
litis virus-based samRNA vector was assessed in non-human pri- at the lower effector-to-target ratio of 10:1 (Extended Data Fig. 1d).
mates (NHPs) and patients with advanced cancers in combination
with CPIs. The potency and longevity of the immune response Individualized vaccine regimen is safe and well tolerated. After
against simian immunodeficiency virus (SIV) model antigens29 establishing the potency and longevity of T cell responses induced
and the impact of subcutaneous (s.c.) anti-CTLA-4 administra- by this vaccine in NHPs, the vaccine was assessed as an individual-
tion were assessed in NHPs. Immunogenicity against individualized, heterologous regimen expressing patient-specific neoantigens
ized (patient-specific) predicted cancer neoantigens30 and in-depth in an ongoing phase 1/2 clinical study in patients with advanced
analysis of T cell responses elicited by vaccination are being assessed cancers (Extended Data Fig. 2a; NCT03639714). The primary
in an ongoing first-in-human phase 1/2 clinical trial (GRANITE, objectives for the phase 1 portion of this study were assessment of
NCT03639714) in patients with metastatic MSS-CRC, non-small safety, tolerability and RP2D. A single priming dose of ChAd68 was
cell lung cancer (NSCLC) or gastroesophageal adenocarcinoma administered i.m. followed by multiple i.m. samRNA boosts with
(GEA). Primary endpoints of the phase 1 part of the study were escalating doses of samRNA (Fig. 2a). Eligible patients included
safety and tolerability and RP2D. Secondary and exploratory end- those with metastatic MSS-CRC, NSCLC or GEA who were begin-
points include immunogenicity, OS, feasibility of manufactur- ning treatment with standard-of-care chemotherapy during vaccine
ing patient-specific vaccines and changes in ctDNA. Induction manufacturing and had tissue available for neoantigen prediction,
of neoantigen-specific CD8 T cell responses was observed in all normal organ function and no contraindications for treatment
patients, including patients with MSS-CRC whose tumors lacked with immunotherapy (Methods). As of data cutoff, enrollment in
immune reactivity before vaccination. Decreases in ctDNA and phase 1 had been completed, and the study is ongoing. For each
stabilization of disease and/or reduction in tumor burden in these patient meeting a threshold for cumulative predicted neoantigen
patients suggest early signs of potential therapeutic benefit in this score30 (Extended Data Fig. 2a), the 20 highest ranking mutations
CPI-refractory patient population31–33. were selected for inclusion in a codon-optimized vaccine expres-
sion cassette. The same expression cassette was inserted into both
Results patient-specific vaccine vectors for manufacturing. Patient-specific
Vaccination induces durable CD8+ T cell responses in NHPs. To vaccines were successfully manufactured for 15 patients enrolled
assess the potency and durability of the immune responses induced for vaccine manufacturing, confirming manufacturing feasibility
by this heterologous vaccine regimen preclinically in NHPs, a (a secondary endpoint) of this individualized vaccination approach
model antigen cassette encoding six well-defined NHP-specific (one patient subsequently elected not to receive study treatment).
MAMU-A*01-restricted SIV antigens29 was inserted into the Fourteen patients received vaccination and monthly intravenous
ChAd68 and samRNA vaccine platforms. MAMU-A*01-positive (i.v.) anti-PD-1 antibody nivolumab administration (480 mg every 4
rhesus macaques were immunized intramuscularly (i.m.) with weeks). Patients at dose level 3 (DL3) and DL4 also received s.c. ipi-
ChAd68 prime (n = 11), followed by i.m. samRNA boosts at 4, 12 limumab (30 mg) with each vaccination (Fig. 2a and Table 1). Four
and 20 weeks, with (n = 6) or without (n = 5) concomitant admin- patients (G1, G8, G9 and G10) received continued standard-of-care
istration of s.c. anti-CTLA-4 antibody ipilimumab (Fig. 1a and chemotherapy with study treatment, and eight patients received
Supplementary Table 1). ChAd68 induced immune responses a single ChAd68 boost ≥24 weeks after the prime vaccination
against all six antigens (mean 923 and 313 spot-forming units (Extended Data Table 1). Planned dosing intervals and timepoints
(SFU)/106 cells at week 4 ± s.c. ipilimumab, respectively; Fig. 1b). with blood draws for immunogenicity are shown in Extended Data
Primed T cell responses were boosted following administration of Fig. 2b. The median age of the 14 phase 1 patients was 59 years;
samRNA, with T cell responses still detectable 32 weeks after prime tumor types included metastatic MSS-CRC (n = 7), GEA (n = 6) or
in both groups. Concomitant administration of s.c. ipilimumab NSCLC (n = 1); and patients had received one or two prior lines of
resulted in maintenance of an overall higher T cell response (mean therapy (Table 1 and Extended Data Table 1). The individualized
843 and 277 SFU/106 cells ± s.c. ipilimumab, respectively; Fig. 1b). vaccine regimen was safe and well tolerated, with no dose-limiting
ChAd68 readministration at week 32 boosted T cell responses toxicities. The primary safety endpoint of this vaccine regimen was
~15-fold and ~12-fold ± concomitant ipilimumab, respectively reflective of a viral-based vaccine regimen with TRAEs consistent
(Fig. 1c). Immunization resulted in the generation and maintenance with a vaccine-induced immune response (Fig. 2b). TRAEs >10%
of antigen-specific effector memory T cells (Extended Data Fig. 1a), included pyrexia (86%), fatigue (43%), musculoskeletal and injec-
which have been demonstrated to be predictive of providing thera- tion site pain (both 36%) and diarrhea (29%). Serious TRAEs were
peutic benefit to patients with cancer34. largely consistent with the known safety profile of CPI therapy and
In both therapeutic and prophylactic vaccine settings, the gen- included one count each of pyrexia, duodenitis, increased transami-
eration and maintenance of long-lived memory responses is highly nases and hyperthyroidism (Fig. 2b). Based on the totality of the
desirable to maintain disease control or provide protection from safety and immunogenicity data (secondary endpoint; see next sec-
future infections. To investigate whether antigen-specific memory tion) from the phase 1 part of the study, the primary endpoint of
T cells were detectable in NHPs up to 2 years after the ChAd68 prime RP2D was determined to be 1012 VP ChAd68 and 30 µg samRNA.
vaccination, six animals that had not received additional treatment
and remained available from the initial study were assessed. Tetramer Vaccine induces neoantigen-specific CD8+ T cells. The
analysis of peripheral blood mononuclear cells (PBMCs) 80 weeks key secondary endpoint of immunogenicity was assessed for
after ChAd68 prime identified the presence of antigen-specific central neoantigen-specific T cells. To preferentially stimulate CD8 T cells
1620 Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine

Nature Medicine Articles
a
Shown in panel c
~1 year ChAd68 (1012 VP)
0 4 12 20 32 33 80 106 107 108 samRNA (300 µg)
Shown in panel b Shown in panel d
b c
ChAd68 boost
10,000 15,000
Pol SV9 ChAd68 prime + samRNA boosts + Ipilimumab
Gag LW9 – Ipilimumab
Env CL9
7,500 Env TL9
IFN-γ (SFU per 106 cells)

Gag CM9
10,000
Tat TL8
5,000
5,000
2,500
0 0
32 33
0
2
4
5
6
8
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15
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28
29
30
31
32
7
17
27
ChAd68 prime + samRNA boosts + ipilimumab d samRNA boost
10,000 30,000 + Ipilimumab
– Ipilimumab
7,500

20,000
5,000
10,000
2,500
0 0
106 107
0
2
4
5
6
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10
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28
29
30
31
32
7
17
27
Weeks after ChAd68 prime Weeks after ChAd68 prime
Fig. 1 | Heterologous vaccination with ChAd68 and samRNA induces broad, durable CD8+ T cell responses in NHPs that are detectable long-term
and can be boosted ≥2 years after prime. a, Schematic of study treatment schedule. b, Antigen-specific T cell response (IFN-γ SFU per 106 cells) at
each measured timepoint for duration of study for each group (mean ± standard error of the mean for each antigen stacked), n = 6 per group up to week
20 (n = 5 for group without ipilimumab at weeks 24–107; Supplementary Table 1). c, Antigen-specific T cell response at weeks 32 and 33 (before and
after ChAd68 boost) for each group (n = 5 without ipilimumab, n = 6 with ipilimumab), average response per animal (SFU per 106 cells) for all antigens
stacked. d, Antigen-specific T cell response at weeks 106 and 107 (before and after samRNA boost) for each group (n = 5 without ipilimumab, n = 6 with
ipilimumab), average response per animal (SFU per 106 cells) for all antigens stacked.
via major histocompatibility complex (MHC) class I presenta- ELISpot following heterologous prime boost vaccination in 100% of
tion in immune assays30,35,36, individual patient minimal epitope patients (13/13), with a ChAd68 boost further increasing peripheral
(8–11mer) peptide pools were designed to include the 40 highest T cell levels in 67% of patients (4/6) with evaluable post-boost sam-
ranked predicted neoepitopes (CD8 pool; Supplementary Table 2). ples (Fig. 2c and Extended Data Fig. 3a). Only 15% of patients (2/13)
Because this exploratory minimal epitope pool design does not had responses above LOD to their cognate neoantigen peptide pool
include all potential neoantigens per mutation and can dispro- at baseline, which were increased following vaccine administra-
portionately favor some mutations with high prediction scores, tion (Fig. 2c). Similarly to observations in NHPs, vaccine-induced
additional patient-specific peptide pools consisting of overlapping T cell responses were long-lived, and T cell levels of 700–5,000
15mers spanning each vaccine cassette were also used to interrogate SFU/106 cells were maintained for >52 weeks in three patients
T cell responses more broadly (15mer pool; Supplementary Table 2). with available longitudinal samples (Extended Data Fig. 3a,b).
Posttreatment PBMC samples were available for 13 of 14 patients Notably, readministration of ChAd68 resulted in elevated T cell lev-
(all except patient G5). Stimulation of PBMCs with patient-specific els detectable 12–24 weeks after ChAd68 boost (patients G1, G3,
peptide pools resulted in IFN-γ responses detectable by ex vivo G8 and G11; Fig. 2b and Extended Data Fig. 3a). Dose escalation

a b
Treatment-related AEs >10%
Phase 1: dose selection based on safety data Hypotension Grade 1/2
Rash Grade 3/4
Adaptive design (n = 14); all patients received nivolumab
Injection site reaction
12
Arthralgia
Dose level 4: GRT-C901 (IM: 10 VP) + 30 mg ipilimumab Decreased appetite
GRT-R901 (IM: 300 µg) + 30 mg ipilimumab Diarrhea
12
Injection site pain
Dose level 3: GRT-C901 (IM: 10 VP) + 30 mg ipilimumab Muskoskeletal pain
GRT-R901 (IM: 100 µg) + 30 mg ipilimumab
Fatigue
12
Pyrexia
Dose level 2: GRT-C901 (IM: 10 VP) Treatment-related serious AE by patient
GRT-R901 (IM: 100 µg)
Hyperthyroidism
12 Transaminases increased
Dose level 2: GRT-C901 (IM: 10 VP) Duodenitis
GRT-R901 (IM: 30 µg)
Pyrexia
0 5 10 15
Number of events
c
G1
2,000 2,000 6,000
ULOQ
1,500 1,500 4,000
1,000 1,000
2,000
500 500
500 500
1,000
400 400 800
300 300 600
200 200 400
100 100 200

LOD LOD
LOD
0 0 0
1
2
3
4
6
8
9
10
11
12
13
14
1
2
3
4
6
8
9
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11
7
7
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Patient ID
d 1,500
CD8enr (CD4 depl) CD4enr (CD8 depl) Total PBMC
10 6 6 CD8enr (CD4 depl)
70.1% 1.27% 10 5.71% 0.30% CD4enr (CD8 depl)
IFNγ (SFU per 106 cells)
105 105
1,000
104 104
G2
103 103 500

0 0
–103 –103
23.9% 4.69% 14.5% 79.5% LOD
0
0 103 105 0 103
10 5 DMSO CD8 pool 15mer pool
1,000 Total PBMC

106 106
90.1% 0.11% 1.18% 0.02% CD8enr (CD4 depl)
5
IFNγ (SFU per 106 cells)
10 105 800 CD4enr (CD8 depl)
104 104 600

CD8-PerCP-Cy5.5
G8
103 103 400

0 0
–103 –103 200
7.00% 2.77% 3.60% 95.2%
LOD
0 104 105 106 0 104 105 106 0
CD4-APCeF780 DMSO CD8 pool 15mer pool
Fig. 2 | First-in-human data show the vaccine regimen is safe and induces CD8+ T cell responses against predicted patient-specific cancer neoantigens.
a, Phase 1 study schematic outlining dose escalation. b, Safety data: TRAEs >10% and all treatment-related serious adverse events (AEs) are shown (n = 14
patients). Graph shows counts of grade 1/2 (blue bars) and 3/4 (orange bars) events. c, Patient PBMC ex vivo IFN-γ ELISpot responses to patient-specific
peptide pools (Supplementary Table 2) are shown for baseline (left panel), maximum response following ChAd prime and samRNA boosts (middle panel)
and maximum response post ChAd boost (right panel). Graphs show mean SFU per 106 PBMCs ± standard deviation (s.d.) for triplicate ELISpot wells.
Assay limit of detection (LOD) and upper limit of quantitation (ULOQ) are indicated by dotted lines. d, Flow plot showing relative CD8 and CD4 T cell
frequencies in CD4- or CD8-depleted (depl) PBMC populations. Corresponding ELISpot data (mean SFU of technical replicates ± s.d.) are shown for whole
PBMCs (black bars), CD8enr PBMCs (navy blue bars) and CD4enr PBMCs (orange bars) stimulated with dimethylsulfoxide (DMSO), patient-specific CD8 or
15mer pools for patients G2 and G8.

responses to at least four different mutations. Antigen-specific
Table 1 | Baseline and disease characteristics by DL expansion of T cells in short in vitro stimulation (IVS) cultures30,37
DL1 DL2 DL3 DL4 Total confirmed expansion of preexisting and priming of de novo T cell
(n = 3) (n = 2) (n = 3) (n = 6) (n = 14) responses after treatment (Extended Data Fig. 4b). All 12 patients
tested had detectable T cell responses to multiple mutations at post-
Age (yr), 72 70 51 55 59 treatment timepoints (Fig. 3a). Voluntary leukapheresis and pooled
median (59–74) (63–76) (50–53) (38–61) (38–76)
posttreatment timepoints enabled full deconvolution of T cell
(range)
responses against all 40 minimal epitopes included in the CD8 pool
Female 1 (33) 1 (50) 1 (33) 1 (17) 4 (29) for patients G1, G2 and G8, revealing positive responses to ≥50%
gender, n (%) of vaccine mutations in multiple HLA class I alleles for all three
ECOG, n (%) patients (Fig. 3b,c and Extended Data Fig. 5). These data indicate
0 2 (67) 1 (50) 1 (33) 3 (50) 7 (50) that partial deconvolution via minipools (often necessitated due
to limitations in sample availability) likely underestimates the true
1 1 (33) 1 (50) 2 (67) 3 (50) 7 (50)
breadth of neoantigen-specific responses induced by this heter-
Number of prior therapies, n (%) ologous vaccine. Peptide-HLA (pHLA) tetramers were generated
1 2 (67) 1 (50) 1 (33) 2 (33) 6 (43) to assess the breadth and memory phenotype of antigen-specific
2 1 (33) 1 (50) 2 (67) 4 (67) 8 (57) T cells. pHLA binding was detected in the context of multiple HLA
class I alleles for patients G1, G2 and G8. Tetramer-positive CD8
Prior anti-PD(L)1 therapy, n (%)
T cells were characterized by 1.3- to 1.5-fold higher frequencies of
Yes 1 (33) 0 (0) 0 (0) 0 (0) 1 (7) effector memory and up to 20-fold lower frequencies of naive T cells
No 2 (67) 2 (100) 3 (100) 6 (100) 13 (93) when compared to total CD8 T cells (Fig. 3d and Extended Data
Tumor types, n (%) Fig. 6a). ChAd68 boost in patient G1 resulted in a 10-fold increase
in tetramer positive cells, with post-boost tetramer-positive CD8
MSS-CRC 0 1 (50) 2 (67) 4 (67) 7 (50) T cells consisting of ≥93% effector memory cells in the context of
GEA 2 (67) 1 (50) 1 (33) 2 (33) 6 (43) both class I alleles tested (Fig. 3e and Extended Data Fig. 6b).
NSCLC 1 (33) 0 0 0 1 (7)
CD8+ T cells are polyfunctional and kill target cells. In addition
DL1, GRT-C901/GRT-R902 30 μg + i.v. nivolumab; DL2, GRT-C901/GRT-R902 100 μg + i.v.
nivolumab; DL3, GRT-C901/GRT-R902 100 μg + i.v. nivolumab + s.c. ipilimumab; DL4, GRT-C901/ to driving broad T cell responses, a potent vaccine regimen should
GRT-R902 300 μg + i.v. nivolumab + s.c. ipilimumab. ECOG, Eastern Cooperative Oncology Group. induce cytotoxic T cells that are able to kill target cells presenting
cognate epitopes in the context of MHC class I. Secretion of cyto-
toxic markers assessed by Meso Scale Discovery (MSD) and tri-
of samRNA suggested that more consistent boosts in T cell responses plex FluoroSpot was confirmed in eight patients (Extended Data
were observed in patients receiving 30-µg and 100-µg doses of sam- Fig. 6c,d). Notably, readministration of ChAd68 resulted in
RNA (Extended Data Fig. 3c). Prolonged samRNA dosing intervals increased granzyme B secretion following CD8 pool stimulation in
of 8–16 weeks occurring due to logistical constraints during coro- four of six patients with evaluable post-ChAd68 boost timepoints
navirus disease 2019 (COVID-19) restrictions revealed interest- (Extended Data Fig. 6e). To assess whether this cytotoxic signature
ing insights into the kinetics of samRNA-boosted T cell responses was driven by CD8 T cells, cytokine secretion was analyzed via intra-
(Extended Data Fig. 3a,b). In patient G1 (GEA, concomitant che- cellular cytokine staining (ICS). Stimulation of ex vivo PBMCs with
motherapy), T cells expanded 1.5-fold during an 8-week interval CD8 pool resulted in detectable frequencies of IFN-γ+, CD107α+,
between samRNA boosts and remained consistently >700 SFU/106 tumor necrosis factor α (TNF-α)+ and interleukin-2 (IL-2)+ CD8
cells during a 16-week period following samRNA administration T cells in patients G2 and G8, which were increased ~2-fold to
at week 52. A sevenfold increase in CD8 T cell responses between ~700-fold following IVS expansion (Fig. 4a and Extended Data
samRNA doses at weeks 24 and 36 was observed in patient G3 Fig. 7a). Boolean gating confirmed the presence of quadruple-,
(NSCLC). Patient G2 (GEA) T cell responses showed ~1.5-fold triple-, and double-positive polyfunctional CD8 T cells for both
contractions and expansions of the T cell response during a 9-week patients (Fig. 4b, Extended Data Fig. 7b). Polyfunctional CD4 T cell
samRNA dosing interval between weeks 4 and 13. These data sug- responses following cognate 15mer pool stimulation were also
gested that lower doses and longer boost intervals may be beneficial detectable in patients G2 and G8 following IVS culture (Extended
in driving T cell responses with this new samRNA platform. This Data Fig. 7c). The ability of antigen-specific CD8 T cells to kill
led to the selection of 30 µg samRNA as the RP2D, a primary end- target cells presenting neoantigen was determined. Co-culture
point (see previous section) and longer boost intervals (8 weeks) of expanded neoantigen-specific patient T cells with single HLA
being assessed in the ongoing phase 2 part of this study. allele-expressing target cells pulsed with cognate neoantigen pep-
tides resulted in a 2.5-fold to 6.4-fold decrease in live target cells over
CD8+ T cells recognize neoantigens in multiple HLA alleles. time compared to controls, indicating antigen-specific T cell-driven
Responses to cognate CD8 pools and 15mer pools were analyzed killing of target cells presenting cognate pHLA for both patients G2
for CD8 versus CD4 T cell bias in five patients with available and G8 (Fig. 4c and Extended Data Fig. 7d).
samples (G1, G2, G8, G11 and G14). As expected based on pep-
tide length, IFN-γ responses following CD8 pool stimulation were T cells are increased in tumors following vaccination. To exert
driven by CD8 T cells, whereas responses to 15mer pool stimula- anti-tumor effects and clinical benefit in patients, vaccine-induced
tion were either CD8-driven or mixed CD4/CD8 T cell responses T cells need to infiltrate tumors and lyse cells presenting their
(Fig. 2d, Extended Data Fig. 4a). Active vaccination against tumor cognate pHLA antigen through recognition via the T cell recep-
neoantigens should lead to expansion of preexisting and priming tor (TCR). Paired TCR sequencing and gene expression profiling
of de novo tumor-specific T cell responses. To assess the breadth of via single-cell RNA sequencing was performed on baseline and
responses with limited available PBMC samples, 4 minipools cov- on-treatment PBMC timepoints from patients with available cor-
ering the 20 patient-specific vaccine neoantigens (Supplementary responding tumor biopsies (G1 and G3; Extended Data Fig. 8a).
Table 2) were used to partially deconvolute T cell responses in 12 of Neoantigen-reactive CD8 T cells were increased 1.6-fold to 6.4-fold in
13 patients. Positive responses to four different minipools indicate on-treatment samples compared to baseline (Extended Data Fig. 8b).

a b Individual peptide deconvolution

Mini pool responses
ZNF233 ZBTB46 TTBK1
Ex vivo Post-IVS TEP1 YKT6 TGM6
100 100 TAX1BP1 SUN2 SURF6
Mini_4 SPG11 SLC27A3 PLEKHG1
Mini_3
Percentage of total response
SOSTDC1 SAP130 PIGO

80 80 Mini_2 RAB11B RPL3L ONECUT2
Mini_1 PRUNE2 QTRT1 NRP1
Mutated gene
OGT POLR1E KRAS
NKAIN1 PHF8 GLDN
60 60 MYH14 MYO5B GGA2
MUC2 MAP2K2 EXPH5
LMNB2 IGSF23 ERF
40 40 DYNC1H1 GIN1 EHBP1L1
DNAJC10 FKBP15 DNAJC11
CHP2 FILIP1 DNAH2
20 20 CCNH DHX37 CUL4A
C1orf50 CTR9 CSMD3
BNC2 CHD6 CHD9
BICD1 CENPM AP1B1
0 0 AHNAK ADAR ABI1
1
2
3
4
7
8
9
10
11
12
13
14
0 1 2 3 0 1 2 3 4 0 1 2 3
G
G
G
G
G
G
G
G
G
G
G
Patient ID G Number of positive epitopes per mutation
c d Patient G2 leukopak
Predicted HLA coverage
of positive epitopes Total CD8+ A*30:04 Tet+ A*02:01 Tet+ B*08:01 Tet+
105 21.5% 105 0.23% 105 105 0.01%
CD8-PerCP-Cy5.5
0.33%
104 104 104 104

3 HLA-A*02:01
G1 3 HLA-B*18:01 103 103 103 103
6 HLA-B*44:05
1 HLA-C*02:02 0 0 0 0
–103 –103 –103 –103
3
3
10 3
4
10 4
5
10 5
0
0
0
10 K
15 K
20 K
25 K
0K
0
10
10
10
10
10
10
5
0
0
0
–1
–1
Total = 13 FSC-A Tet-APC Tet-PE Tet-PE
105 1.85% 14.5% 105 0.34% 0.68% 105 1.92% 6.00% 105 2.63% 1.32%
104 104 104

CCR7-PE-Cy7
4
4 HLA-A*02:01 10
4 HLA-A*30:04
G2 1 HLA-B*08:01 103 103 103 103
8 HLA-B*57:01 0 0 0
2 HLA-C*06:02 0
40.1% 43.5% 41.8% 57.2% 30.7% 60.5%
–103 –103 –103 61.4%
–103 35.5%
3
3
4
4
5
5
0
0
0
10
10
10
10
10
10
10
10
10
10
10
10
–1
–1
–1
–1
Total = 19
CD45RA-SB702
0.68% 6.0% 1.32%

0.34% 1.92% 2.63%
9 HLA-A*03:01 1.85%
14.5%
G8 4 HLA-A*33:01
1 HLA-C*04:01 30.7%
43.5% 41.8%
57.2% 35.5%
60.5%
40.1% 61.4%
Total = 14
Naive Central memory Effector memory Effector memory RA+
e G1: pre-ChAd68 boost G1: After-ChAd68 boost

3.94% 0.33%
B*44:05 tet+ Tet+ memory B*44:05 Tet+ Tet+ memory 6.01% 0.39%
3.54% 5
5 105
105 10 0.39% 0.33%
10 3.54% 3.94%
CD8-PerCP-Cy5.5
CD8-PerCP-Cy5.5
104 104 104

104
CCR7-PE-Cy7
CCR7-PE-Cy7
33.9%
103 58.7% 103 103

103 93.3%
0.92% 0 0
9.44% 0
0 6.01%
–103 58.7 33.9% –103 –103 93.3%
–103
3
5
3
0
3
5
3
10
10
10
0
0
3
5
0
–1
0
10
10
10
0
10
10
10
0
10
10
10
–1
–1
–1
Tet+ PE CD45RA-SB702 Tet+ PE CD45RA-SB702
Fig. 3 | Vaccine induces polyclonal effector memory CD8 T cell responses across multiple neoantigens and HLAs. a, Patient PBMCs from available
posttreatment timepoints (G1 W12, G2 W5, G3 W8, G4 W6, G7 W4, G8 W12, G9 W8, G10 W16, G11 W16, G12 W12, G13 W12, G14 W13 and G15 W12)
were stimulated overnight in ex vivo or post-IVS IFN-γ ELISpot with patient-specific minipools or DMSO. Graphs show percentage of total response
of mean IFN-γ responses to minipool 1 (turquoise), minipool 2 (light blue), minipool 3 (medium blue) and minipool 4 (navy blue) for each patient.
b, Number of positive minimal epitopes detected (positive by ex vivo or post-IVS ELISpot) for each mutated gene. Undetectable responses against
epitopes tested are indicated by ‘x’. c, Pie charts showing patient-specific predicted HLA class I presentation of positive neoantigen responses shown
in panel b. d, Density plots showing recognition of pHLA (A*30:04, A*02:01, B*08:01) and corresponding T cell memory phenotypes for patient G2. Pie
charts showing frequencies of naive (CD45RA+CCR7+; purple), central memory (CM; CD45RA−CCR7+; blue), effector memory (EM; CD45RA−CCR7−;
green) and T effector memory RA+ (TEMRA; CD45RA+CCR7−; orange) cells in total CD8+ population (left-most chart) and CD8+tetramer+ populations
from corresponding dot plots. e, Density plots and corresponding pie charts showing recognition of pHLA (B*44:05) and corresponding T cell memory
phenotypes before and after ChAd68 boost for patient G1.

Analysis of transcriptional activation signatures confirmed an (Extended Data Table 1 and Supplementary Table 4). For patients
enrichment for CD8 T cells in on-treatment samples (Extended with MSS-CRC, 3 of 7 (42.9%) remained alive (median OS of 8.7
Data Fig. 8c). TCR analysis revealed a total of 12 and 30 distinct months; Extended Data Table 1), with an OS rate at 12 months of
TCR clonotypes (α-β pairs) detected in PBMCs following vaccina- 42.9% reflecting multiple patients experiencing durable benefit.
tion for patients G1 and G3, respectively (Extended Data Fig. 8b). Emerging evidence suggests changes in ctDNA levels
Analysis of IFNG, GZMB, TNFA and IL2 transcripts in T cell clo- on-treatment relative to baseline are a useful biomarker to stratify
notypes revealed 12- to 98-fold increases in frequencies of T cells patients as molecular responders or nonresponders and differen-
expressing two or more cytokine transcripts in on-treatment sam- tiate patients with a good prognosis, especially those with SD per
ples compared to baseline for both patients (Extended Data Fig. RECIST v1.1 criteria38. Of these patients, 100% (3/3) with treat-
8d,e and Supplementary Table 3). Baseline and on-treatment tumor ment durations <6 months had increases in ctDNA concentration,
biopsy samples from patients G1 and G3 were analyzed for the pres- whereas 75% of patients (3/4) with treatment durations ≥6 months
ence of TCR-β CDR3 sequences via bulk DNA sequencing. Analysis demonstrated a molecular response (<50% from baseline; Fig. 5c
of TCR-β CDR3 sequences obtained independently via single-cell and Supplementary Table 4). Two patients had complete ctDNA
RNA sequencing in PBMCs versus DNA sequencing in tumor clearance while on treatment (G8 and G14; Fig. 5c). Most nota-
samples indicated overlap of putative neoantigen-specific T cells in bly, these decreases in ctDNA were more frequent in patients with
both the blood and tumor. For patient G1, 341 TCR-β CDR3 clo- prolonged OS (median OS not reached versus 7.8 months; Fig. 5d).
notypes were identified across baseline and on-treatment tumor Patient G8, a 51-year-old patient with MSS-CRC who received che-
samples with an enrichment of nine clones in the on-treatment motherapy for 21 months, had progressed on first-line chemother-
biopsy. Two of these nine clonotypes were also found in PBMCs apy before study treatment initiation but continued concomitant
sampled at the time of the on-treatment biopsy (Fig. 4d). In patient standard of care chemotherapy during study treatment (Extended
G3, a total of 294 TCR-β CDR3 clonotypes were identified via DNA Data Table 1) and showed clear evidence of tumor regression fol-
sequencing in both tumor samples, with 40 clonotypes enriched in lowing vaccine administration via both ctDNA and radiologic
the on-treatment sample. Five of the 40 TCR-β CDR3 clonotypes assessments. Computed tomography scans showed size reduction
enriched in the on-treatment biopsy overlapped with the activated in multiple lung lesions within 16 weeks and persistent reduction
TCR clonotypes in PBMCs at the time of the on-treatment biopsies in multiple lung and one liver lesion through 48 weeks (Fig. 5e).
(Fig. 4d). Representation of clonotypes from neoantigen-specific Fluorodeoxyglucose-positron emission tomography imaging of the
CD8 T cells in tumor tissue that were also detected in PBMCs pro- stable liver lesions showed a lack of hypermetabolic cells follow-
vides proof-of-concept that vaccine-induced neoantigen-specific ing study treatment administration, suggesting anti-tumor activity
T cells in the periphery are able to infiltrate tumors. of this vaccine regimen not evidenced by computed tomography
imaging (Fig. 5e). In totality, the safety, immunogenicity, OS and
Prolonged survival and decreases in ctDNA in MSS-CRC. ctDNA signals observed in this trial warrant further investigation
Multiple measures of activity were assessed, including OS and of this vaccine platform in patient populations that are refractory to
changes in tumor size via imaging (objective response rate and CPI monotherapy31–33.
progression-free survival per RECIST v1.1, all secondary endpoints),
as well as transcriptional profiling and changes in ctDNA (both Discussion
exploratory endpoints). Standard genomic correlates of responsive The quality of antigen-specific T cell responses induced by a het-
tumors to CPIs (namely, tumor mutational burden, CD274 (PD-L1) erologous vaccine regimen, consisting of ChAd68 and samRNA
and IFNG RNA sequencing signatures) were analyzed in patient vectors in combination with CPI, was assessed in NHPs and
pretreatment biopsies and compared to relevant cancer types from patients with advanced cancers. The potency and longevity of T cell
The Cancer Genome Atlas (TCGA). Pretreatment patient genomic responses to model antigens following vaccination were studied in
data demonstrate that patients with MSS-CRC and GEA in this NHPs, whereas safety, immunogenicity and feasibility of an indi-
study had low tumor mutational burden and PD-L1 expression vidualized patient-specific vaccine were assessed in patients with
and that these tumors had low immune reactivity (Fig. 5a,b and late-stage cancer. This vaccine regimen was well tolerated and
Supplementary Table 4). Such cold tumors are known to be refrac- induced neoantigen-specific CD8 T cell responses in all patients.
tory to CPI therapy. Objective response rate and progression-free Vaccine-driven T cell responses were (1) polyclonal across multiple
survival per RECIST v1.1 show a patient with GEA with a complete mutations and HLA alleles, (2) polyfunctional and (3) able to kill tar-
response and several patients across tumor types with stable dis- get cells presenting cognate peptide. Of note, more consistent boosts
ease (SD) (Extended Data Table 1). Given the observation of SD in T cell responses were observed in patients receiving lower doses
in several patients with MSS-CRC, we focused on analyzing these of samRNA. This could be due to less innate immune activation
seven patients with MSS-CRC for the secondary endpoint of OS via mRNA-sensing pattern recognition receptors that might inter-
and an exploratory endpoint of changes in ctDNA from baseline fere with samRNA amplification and antigen expression. Increased
Fig. 4 | Neoantigen-reactive CD8 T cells are polyfunctional, kill targets presenting cognate peptides in multiple HLA alleles and are increased in
on-treatment tumor samples. a, Representative density plots for patient G2 ICS for CD8+ cells producing IFN-γ, CD107α, TNF-α and IL-2 in ex vivo or
IVS-expanded PBMCs stimulated overnight with DMSO or patient-specific CD8 pool. Cells were gated on PBMCs (forward scatter (FSC)-A versus side
scatter (SSC)-A), singlets (FSC-A versus FSC-H), live cells (FSC-A versus Live/Dead), CD8 or CD4 T cells (CD8 versus CD4) and cytokine+ cells.
b, Polyfunctionality was assessed via Boolean gating of CD8+cytokine+ populations (background subtracted). Pie charts show polyfunctionality of ex vivo
PBMCs and post-IVS PBMCs. Pie arcs depict individual cytokines in red (CD107α), lime green (IFN-γ), turquoise (TNF-α) and navy blue (IL-2). Pie sections
depict frequencies of cells expressing multiple cytokines as outlined in pie category legend. Main functional pie slices are colored red (quadruple positive
for CD107α, IFN-γ, TNF-α and IL-2), orange (triple positive for CD107α, IFN-γ and TNF-α) and yellow (double positive for CD107α and IFN-γ). c, Target
cell killing by IVS-expanded PBMCs from patients G2 (B*08:01, B*57:01, A*30:04). Graphs show relative viability based on NucRed count and normalized
to effector-to-target ratio of 10:1 DMSO control over time. Data are shown as mean ± s.d. from duplicate wells for targets alone stimulated with DMSO
(gray circles) or cognate peptide (black squares) and for 10:1 effector/target co-cultures stimulated with DMSO (light blue circles) or cognate peptide
(navy blue squares). d, DNA sequencing of TCR-β chains in tumor samples in baseline and on-treatment samples from patients G1 and G3. TCR-β chains
expanded in PBMCs, in both PBMCs and tumors, and contracted are shown.

a G2: ex vivo G2: post-IVS b

G2: ex vivo + + + + G2: post-IVS
CD107α IFN-γ IL-2 TNF-α
DMSO CD8 pool DMSO CD8 pool
5 5
10
5
10
5
10 10
Cy5-5-A :: CD8
Comp-PerCP-
4 4 + + +
10
4
10
4 10 10 CD107α IFN-γ TNF-α
3 3
10
3
10
3 10 10
0 0 0 0
0.0008% –10
3 0.58% –10
3 36.3%
–10
3
–10
3
0.20%
3 3 4 5 3 3 4 5
–10 0 10
3 3
10 10
4 5
–10 0 10
3 3 4
10 10
5 –10 0 10 10 10 –10 0 10 10 10
IFNγ-APC IFNγ-APC + +
CD107α IFNγ
5 5
10
5
10
5
10 10
Cy5-5-A :: CD8
Comp-PerCP-
4 4
10
4
10
4
10 10
3 3
3 3 10 10
10 10 Pie arcs
0 0 0 0
9.0% 47.7%
0.20% 0.32% 3 3
CD107α+
CD8-PerCP-Cy5.5
–10
CD8-PerCP-Cy5.5
–10
3
–10
3 –10
IFN-γ+
3 3 4 5 3 3 4 5
–10 0 10
3 3
10 10
4 5
–10 0 10
3 3 4
10 10
5 –10 0 10 10 10 –10 0 10 10 10
CD107α-BV421 CD107α-BV421
TNF-α+
5 5
5
10
10 10 IL-2+
5
Cy5-5-A :: CD8
10
Comp-PerCP-
4 4
4
10
4
10 10
10 Pie categories
3 3
3
10
3
10 10
10 CD107α IFN-γ TNF-α IL-2 CD107α IFN-γ TNF-α IL-2
0 0 0
0 0.30% 1.66% 32.6%
–10
3 0.04% –10
3
–10
3
–10
3
+ + + + – + + +
3 3 4 5 3 3 4 5
–10 0 10
3 3 4
10 10
5
–10 0 10
3 3 4
10 10
5
–10 0 10 10 10 –10 0 10 10 10 + + + – – + + –
TNFα-BV786 TNFα-BV786
+ + – + – + – +
5 5 5 5
10 10 10 10 + + – – – + – –
Cy5-5-A :: CD8
Comp-PerCP-
4 4 4 4
10 10 10 10 + – + + – + +
–
3
10
3
10
3
10
3
10 + – + – – – + –
0 0 0 0
3 0.016% 3 0.25% 3 0.61% –10
3 11.8% + – – + – – – +
–10 –10 –10
–10 0 10
3 3 4
10 10
5 3 3 4 5 3
–10 0 10
3 4
10 10
5 3
–10 0 10
3 4
10 10
5 + – – –
–10 0 10 10 10
IL-2-PE IL-2-PE
Cytokine
c d Expanded TCR-β in IVS (n = 12)

2 Expanded in tumor and IVS 2
50 G1
G2: HLA-B*08:01
Expanded in tumor 7 Phenotype
1 Not significant 334 40
G1 CD8
On-treatment biopsy TCR-β frequency (%)
Relative viability
0 30 CD4
Undetermined
20
Proportion of filtered T cells (%)

–1 Also expanded
10 in tumor
–2
10–2 0
VAF <1%
–3 Baseline On-
treatment
–4 Expanded TCR-β in tumor (n = 9)
0 8 16 24 32 40 48 56 64 72 8
G1
7
2 6
G2: HLA-B*57:01
5
0 10–3
4
Relative viability
3
–2 10–5 10–4 10–3 10–2 2
Baseline biopsy TCR-β frequency (%) 1
–4 VAF 10% 0
Baseline On-
treatment
–6 Expanded in tumor and IVS 5
Expanded in tumor 35 Expanded TCR-β in IVS (n = 27)
Contracted in tumor 11 30
On-treatment biopsy TCR-β frequency (%)
–8 Not significant 243 G3 G3

0 8 16 24 32 40 48 56 64 72
20
3 10–2
G2: HLA-A*30:04
Proportion of filtered T cells (%)
2 10
VAF 9%
Relative viability
1
0
0 Baseline On-
10–3 treatment
–1
Expanded TCR-β in tumor (n = 40)
–2 4
G3
–3 3
10–4
0 8 16 24 32 40 48 56 64 72
10–4 10–3 10–2 2
Time (h)
Baseline biopsy TCR-β frequency (%)
Targets only + DMSO Targets only + peptide VAF 17% 1
E:T 10:1 + DMSO E:T 10:1 + peptide
0
Baseline On-
treatment

a b
200 Study patient
Study patient population TCGA 4 population TCGA data
NSCLC MSS-CRC
GEA MSI-CRC
150 MSS-CRC Gastric
2
Melanoma
IFN-γ signature
PD-L1 RNA-seq
Lung Ad.
Lung Sq.
0
100
–2
50
–4
MSS-CRC
GEA
NSCLC
MSS-CRC
MSI-CRC
Gastric
Lung Ad.
Lung Sq.
Melanoma
0
0.01 0.1 1 10 100 1,000 10,000
Tumor mutational burden
c d
1,500 100
PD per RECIST at <24 weeks
SD per RECIST for ≥24 weeks
Probability of survival
1,250
Percent change in ctDNA %
Unconfirmed PD per RECIST 75 ctDNA decrease (n = 3)

for ≥24 weeks
( mutant hGE ml–1)
1,000
100 50
50
25
0
No ctDNA decrease (n = 4)
–50 0
0 20 40 60 80
–100
Weeks after prime
G4 G10 G6 G11 G9 G8 G14
ctDNA decrease (n = 3) No ctDNA decrease (n = 4)
e Baseline 16 weeks 48 weeks
PET scan
Lung
Pretreatment
Liver
On treatment
Liver
Fig. 5 | Early signals of clinical efficacy in patients with MSS-CRC in advanced disease setting. a, Scatter plot showing tumor sample RNA sequencing
(RNA-seq) for patients before study treatment. PD-L1 normalized RNA-sequencing expected counts (y axis) versus tumor mutational burden (x axis) for
patients and relevant TCGA cancer types. b, Patient RNA-sequencing IFN-γ signature average z-scores (x axis) calculated across patients and relevant
TCGA cancer types (y axis). TCGA samples: MSS-CRC (n = 286), MSI-CRC (n = 62), gastric (n = 406), lung adenocarcinoma (Lung Ad.)_ (n = 498),
lung squamous (Lung Sq.) (n = 463) and melanoma (n = 436). GRANITE patients: MSS-CRC (n = 7), GEA (n = 6) and NSCLC (n = 1). c, Percent change
in ctDNA following initiation of study treatment relative to baseline levels shown as best response and colored by RECIST-based response and time on
treatment. Patients in blue were all on study treatment >24 weeks with two patients treated beyond RECIST-based progressive disease (PD) that was
not confirmed by 24 weeks and continued on study treatment. d, Probability of survival in MSS-CRC patients with (n = 4) or without (n = 3) reduction in
ctDNA since ChAd68 prime. Data cutoff 31 January 2022. e, Radiological images showing tumor lesions in the lung and liver of patient G8 before initiating
study treatment (baseline) and following study treatment (16 and 48 weeks). Fluorodeoxyglucose-positron emission tomography imaging of tumor lesions
in the liver when patient was diagnosed in February 2018 and 7 months after starting study treatment. Arrows indicate relevant target lesions.

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38. Zhang, Q. et al. Prognostic and predictive impact of circulating tumor DNA published maps and institutional affiliations.
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Cancer Disco. 10, 1842–1853 (2020). Springer Nature or its licensor holds exclusive rights to this article under a publishing
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Methods AdjustedSpots = RawSpots + 2 ∗ (RawSpots ∗ saturation/ (100 − saturation)

Ethics statement. The research presented in this paper complies with all the
relevant ethical regulations. Animal studies were performed according to
Each sample was background corrected by subtracting the average value of the
institutional animal care and use (IACUC)-approved protocol at the California
negative control peptide wells. Data are presented as SFU per 106 PBMCs. Wells
National Primate Research Center at the University of California, Davis. The with well saturation values greater than 50% were labeled as too numerous to
clinical study and all related analyses were carried out in accordance with the count, and their value was set to the maximum measured SFU for that experiment.
Declaration of Helsinki and Good Clinical Practice guidelines and was approved
by the appropriate institutional review board (IRB) or ethics committees at each ICS assay (NHP study). Freshly isolated PBMCs were distributed at 106 per well
participating site (University of Chicago Comprehensive Cancer Center, Chicago,
into V-bottom 96-well plates. Cells were pelleted and resuspended in 100 μl
IL; Columbia University Medical Center, New York, NY; The Ohio State University
complete RPMI containing 1 μg ml−1 anti-CD28 mAb (CD28.2, BD Biosciences),
Medical Center, Columbus, OH; Virginia Cancer Specialists, Fairfax, VA; Memorial 1 μg ml−1 anti-CD49d mAb (9F10, BD Biosciences) and 10 μg ml−1 pooled
Sloan Kettering Cancer Center, New York, NY; Peter MacCallum Cancer Centre, peptide antigens or human hepatitis B virus S-antigen peptide (WLSLLVPFV)
Melbourne, VIC, Australia; Mayo Clinic, Phoenix, AZ; Mayo Clinic, Rochester, as a negative control stimulant (GenScript). After 1 h of stimulation, Brefeldin
MN; The University of Texas MD Anderson Cancer Center, Houston, TX). All A (Biolegend) was added to a final concentration of 5 μg ml−1 and incubated
patients provided written, informed consent. Further details can be found at for an additional 16 h. Following stimulation, cells were washed with PBS and
https://clinicaltrials.gov/ct2/show/NCT03639714. stained with fixable viability dye (eBioscience). Extracellular staining was
performed in FACS buffer (PBS + 2% FBS + 2 mM EDTA) with anti-CD3,
NHP study. Animal selection and dosing. Study was performed according to anti-CD4, anti-CTLA-4, anti-PD1 and anti-CD8 antibodies. Cells were washed,
IACUC-approved protocol at the California National Primate Research Center at fixed and permeabilized with Transcription Factor fixation/permeabilization kit
the University of California, Davis. Animals did not receive any prior treatment (eBioscience), and then intracellular staining was performed in permeabilization
with immune-modulatory antibodies or vaccination against SIV and had no prior buffer. Samples were stained for IFN-γ, TNF-α, granzyme B and CD69. Samples
exposure to SIV. Animal groups and dosing are outlined in Supplementary Table 1. were collected on the Attune NxT (Thermo Fisher Scientific). Gating strategy was
Briefly, both ChAd68 and samRNA were administered as bilateral i.m. injections as follows (Supplementary Fig. 1): lymphocytes (SSC-A versus FSC-A), single
into the quadriceps muscle. All ChAd68 doses were at 1012 VP with 2 ml total cells (SSC-H versus SSC-A), viable cells (SSC-A versus LD-506), CD4+ or CD8+
injection volume (1 ml per leg). All samRNA doses were at 300 μg with 1 ml total (CD4-phycoerythrin (PE) versus CD8-e450), cytokine+ (TNF-α-BV421 versus
injection volume (500 μl per leg). Anti-CTLA-4 (ipilimumab) was administered IFN-γ-APC-R700).
as either an i.v. injection of 5 mg kg−1 or a flat dose of 50 mg per animal injected
subcutaneously bilaterally near each vaccine site draining lymph node. Initial Tetramer staining assay (NHP study). Following overnight rest at 4°C, isolated
weights, doses of ipilimumab and treatment groups are shown below. Animals PBMCs were distributed into 96-well V-bottom plates at a density of 106 cells per
37576 and 42300 were excluded from the study after week 20 with no further 100 µl and stained with a viability dye (Live/Dead e506, eBioscience) in PBS for
immune monitoring or immunizations due to recurrent issues with anemia and 20 min at 4°C. TCR recycling was inhibited by treatment with 50 nM Dasatinib
low-yield blood draws. for 1 h at 4°C. Cells were stained for 1 h at 4°C in FACS buffer with Mamu-A*01
tetramers for each of the peptide antigens with a unique combination of two
SIV model antigen cassettes. A model antigen cassette containing six previously fluorophores per antigen (produced in-house). Cells were co-stained for CD3
published SIV viral epitopes29 (SIV Gag CM9 (CTPYDINGQM), SIV Env TL9 (A700, SP34.2, BD Biosciences), CD8 (e450, SK1, BD Biosciences), CD45RA
(TVPWPNASL), SIV Env CL9 (CAPPGYALL), SIV Tat TL8 (TTPESANL), SIV (FITC, 5H9, BD Biosciences) and CCR7 (SB780, 3D12, eBioscience). Samples were
Gag LW9 (LSPRTLNAW) and SIV Pol SV9 (SGPKTNIIV)) was designed. Each collected on the Attune NxT (Thermo Fisher Scientific). The gating strategy was
minimal epitope is embedded in a 25 amino acid sequence with 8 amino acids as follows (Supplementary Fig. 1): lymphocytes (SSC-A versus FSC-A), single cells
flanking each minimal epitope sequence. The cassette also contains class II helper (SSC-H versus SSC-A), viable cells (SSC-A versus LD-506), CD3+ cells (SSC-A
epitopes. The cassette was synthesized by GenScript and designed to allow cloning versus CD3-A700), CD8+ (SSC-A versus CD8-e450), tetramer double positive
into plasmids containing ChAd and samRNA vectors. The ChAd vector is an E1, (tetramer-UV405 versus tetramer-UV605; tetramer-UV405 versus tetramer-PE;
E3 deleted virus, and the cassette expression was flanked by a cytomegalovirus tetramer-PE versus tetramer-UV675; tetramer-PE-Cy7 versus tetramer-UV405)
promoter and an SV40 polyadenylation signal. The ChAd plasmid was digested and memory populations (CD45RA-FITC versus CCR7-SB780).
with PacI and then transfected into HEK293 cells to generate virus. The virus
for the NHP studies was made by CsCl gradient centrifugation and for the good Cytotoxicity (T cell killing) assay (NHP study). Freshly isolated PBMCs for each
manufacturing practice studies was purified by anion exchange chromatography. sample were divided into two portions. T-cell enrichment was performed on one
The same cassette was cloned into BstB1–PacI sites of a pBAC-VEE portion using the Miltenyi NHP T-cell isolation kit (130-092-143), following
alphavirus-based plasmid that was used to make the samRNA RNA by in vitro manufacturer’s protocol and using MS columns (Miltenyi) to deplete non-T cells.
transcription (TriLink) and then formulated into lipid nanoparticles (Arbutus). The second portion (referred to as PBMCs) was stained with carboxyfluorescein
succinimidyl ester (CFSE) at 1 mM (eBioscience). The CFSE-labeled PBMCs
Test articles (NHP study). Anti-CTLA-4 monoclonal antibody (mAb) (ipilimumab) were then loaded with 50 mg ml−1 SIV peptides or a negative control peptide
was provided by Bristol Myers Squibb. ChAd68-MAG was generated by Gritstone (WLSLLVPFV) by incubation for 1 h at 37˚C in RPMI + 1% FBS. Isolated T cells
Oncology (non-GLP) and prepared at 5 × 1011 VP ml−1 in Adenovirus Reference and peptide-loaded target PBMCs were then counted and plated in a 20:1 ratio
Material buffer (10 mM Tris, pH 8.0, 25 mM NaCl and 2.5% glycerol buffer). (T cells to target cells) in a V-bottom plate. Then, 400,000 T cells and 20,000 target
samRNA was generated by TriLink Biotechnologies and formulated in lipid cells were plated per well. Target cells only were also plated as a control. Cells
nanoparticles by Arbutus. were incubated for 6 h at 37˚C in RPMI + 10% FBS. Following incubation, cells
were stained with 7-AAD (Thermo Fisher Scientific) and immediately analyzed
Isolation of PBMCs and serum (NHP study). For immune monitoring, 10–20 ml by flow cytometry using a Cytoflex LX (Beckman). CFSE+ target cells were gated
blood was collected into vacutainer tubes containing heparin and maintained at and 7-AAD+ cells measured (as a percentage of CFSE+ cells). Remnant T cells were
room temperature until isolation. PBMCs were isolated by density centrifugation also analyzed by flow cytometry to confirm T cell isolation. Samples were collected
using lymphocyte separation medium and Leucosep separator tubes. PBMCs were on the Attune NxT (Thermo Fisher Scientific). The gating strategy was as follows
counted using the Attune NxT (Thermo Fisher Scientific) and resuspended at a (Supplementary Fig. 1): lymphocytes (SSC-A versus FSC-A), single cells (SSC-H
concentration of 4 × 106 cells ml−1 in complete RPMI. versus SSC-A), 7-AAD+CFSE+ (7-AAD versus CFSE).
IFN-γ ELISpot assay (NHP study). IFN-γ enzyme-linked immunospot (ELISpot) Anti-ChAd68 neutralizing antibody assay (NHP and clinical study). Dilutions of
assays were performed using precoated 96-well plates (MAbtech, Monkey IFNγ heat-inactivated human and NHP sera were evaluated for the ability to neutralize
ELISpot PLUS, ALP) following the manufacturer’s protocol. Then, 105 PBMCs a ChAd virus expressing the reporter protein beta-galactosidase (ChAd-LacZ).
per well were plated in triplicate with 10 µg ml−1 peptide stimuli (GenScript) and The reciprocal of the dilution that gives 50% or greater neutralization is used to
incubated overnight in complete RPMI (RPMI + 10% FBS). A human hepatitis B assign a neutralization titer. In brief, pre- and post-vaccination serum samples
virus S-antigen peptide not contained in the cassette (WLSLLVPFV, GenScript) were heat inactivated at 56˚C for 30 min. Sera samples were then diluted twofold
was used as a negative control for each sample. Plates were washed with PBS and in serum-free media (DMEM) starting at 1:20 dilution down to 1:1,280. If needed,
incubated with anti-monkey IFN-γ mAb biotin (MAbtech) for 2 h, followed by an additional dilutions were made to get a titer within the range of the assay. The
additional wash and incubation with Streptavidin-ALP (MAbtech) for 1 h. After diluted samples were then mixed with an equal volume of media containing diluted
final wash, plates were incubated for ten minutes with BCIP/NBT (MAbtech) to virus sufficient to infect 293 A cells at an approximate multiplicity of infection
develop the immunospots and dried overnight at 37°C. Spots were imaged and (m.o.i.) of 1. The diluted sera and virus were incubated for 1 h at 37˚C before
enumerated using AID reader (Autoimmun Diagnostika). For data processing and adding onto 293 A cells plated at 2.5 ×104 cells/well on a 96-well tissue culture
analysis, samples with replicate well variability (variability = variance/(median + 1)) plate 1 day earlier. The cells and neutralizing mix were incubated for 1 h at 37˚C
greater than 10 and median greater than 10 were excluded44. Spot values were and then an equal volume of media with 20% FBS was added. The cells and virus
adjusted based on the well saturation according to the formula45: were then incubated for an additional 18–20 h before lysis and measurement of
Nature Medicine | www.nature.com/naturemedicine

B-galactosidase activity. Media was aspirated and the cells lysed in 100 µl lysis 3. Availability of formalin-fixed, paraffin-embedded (FFPE) tumor specimen for
buffer from Thermo Fisher Scientific (Galacto-Star System). The cells were then neoantigen assessment.
freeze thawed twice (alternating −80˚C and 37˚C), mixed, and then 50 µl was 4. Measurable disease according to RECIST v1.1.
mixed with an equal volume of Galacto-Star substrate (Thermo Fisher Scientific) 5. Lesions amenable to biopsy.
for B-galactosidase measurement. Wells with media alone were used to determine 6. ≥12 years of age (patients 12–17 years of age must weigh >40 kg).
expression in the absence of sera (100% B-galactosidase activity) and wells with no 7. Life expectancy of > 6 months per the investigator.
virus were used to determine the background of the assay. The percent inhibition 8. ECOG performance status of 0 or 1 (or equivalent for patients 12–17 years of
was calculated for each dilution and plotted to generate a curve of percent age based on Karnofsky or Lansky performance scale).
neutralization versus dilution. A titer was assigned either as a limiting dilution 9. Patient has adequate organ function using values obtained within 4 weeks
titer, which is the reciprocal dilution that gives 50% or greater inhibition, or as before starting routine therapy as defined below. The sponsor may consider
interpolated from the dilution curve. patients eligible despite having a laboratory value that does not meet inclu-
sion criteria provided the Investigator reports the patient is otherwise in good
Phase 1 clinical trial. Study design. The study protocol is available in the health and the history and kinetics of the change in the laboratory value do
Supplementary Information. The main objectives of this phase 1/2 study not raise a substantial safety concern over including the patient in the study
(NCT03639714) were to evaluate the safety and tolerability, immunogenicity and or compromise the integrity of the study data. The sponsor and investigator
early clinical activity to determine and evaluate the RP2D of i.m. administration must agree to include any patients with a laboratory value below the defined
of GRT-C901 (ChAd68) and GRT-R902 (self-amplifying RNA formulated in criteria below:
lipid nanoparticles; samRNA), an individualized neoantigen cancer vaccine, in a. Peripheral white blood cell (WBC) ≥3,000/mm3
combination with i.v. nivolumab and s.c. ipilimumab, in patients with metastatic b. Absolute lymphocyte count ≥800/mm3
NSCLC, MSS-CRC, GEA and metastatic urothelial cancer. The phase 1 portion of c. Absolute neutrophils count (ANC) ≥ 1,500/mm3
the study was designed to find an RP2D using an adaptive dose-escalation method d. Platelets ≥100,000/mm3
and modified toxicity probability interval-2 (mTPI-2) among the planned doses; e. Hemoglobin ≥ 9 g dl−1
DL1 through DL4 are outlined in Fig. 2a, with the goal of determining the highest f. Albumin ≥ 3 g dl−1
vaccine dosage with a dose-limiting toxicity (DLT) rate below 30%.46 All patients g. Calculated creatinine clearance >50 ml min−1 using Cockcroft–Gault
received 1012 VP ChAd68 prime and monthly i.v. anti-PD-1 antibody nivolumab equation
administrations (480 mg every 4 weeks). Patients at DL1 and DL2 received 30 µg h. Alanine aminotransferase (ALT) and aspartate aminotransferase
and 100 µg samRNA boosts, respectively. Patients at DL3 and DL4 received 100 µg (AST) ≤3× upper limit of normal (ULN)
and 300 µg samRNA, respectively, in addition to s.c. ipilimumab (30 mg) with i. Total bilirubin ≤1.5× ULN or direct bilirubin ≤1× ULN (patients with
each vaccination. ChAd68 boost was administered to eight patients with varying Gilbert’s disease may be included if their total bilirubin is ≤3.0 mg dl−1)
intervals at a dose of 1012 VP (Extended Data Table 1). The sample size was not j. International normalized ratio or prothrombin time (PT) or partial
driven by a statistical power due to the nature of an adaptive dose-escalation thromboplastin time (PTT) ≤1.5× ULN
method. Therefore, the sample size of the phase 1 portion of the study ranged
from 11 to maximum of 24 patients which is dependent on occurrence of DLTs. 10. For NSCLC only, a history of smoking, defined as more than five packs, or
This study was carried out in accordance with the Declaration of Helsinki and 100 lifetime cigarettes.
Good Clinical Practice guidelines and was approved by the appropriate IRB or 11. For women of childbearing potential, willing to undergo pregnancy testing
ethics committees at each participating site (University of Chicago Comprehensive and agreeing to use at least one highly effective contraceptive method during
Cancer Center, Chicago, IL; Columbia University Medical Center, New York, NY,; the study treatment period and for 5 months after last investigational study
The Ohio State University Medical Center, Columbus, OH; and Virginia Cancer treatment.
Specialists, Fairfax, VA). All patients provided written, informed consent. Further
details can be found at https://clinicaltrials.gov/ct2/show/NCT03639714. Per Inclusion criteria for study treatment stage. Patients must sign a second ICF and
protocol, patients had the potential to receive study treatment concurrent with meet the following inclusion criteria to be eligible for the study treatment stage:
ongoing standard-of-care chemotherapy.
The first patient enrolled on 24 September 2018 and the last patient enrolled 12. If patient did not participate in the vaccine production part of this study,
on 25 November 2019 for the phase 1 portion of the study. As of the data cutoff patient has been enrolled in the screening protocol GO-003.
date of 31 January 2022, a total of 29 patients (i.e., 14 patients in phase 1 and 13 13. Provide a signed and dated ICF document before initiation of any
patients in phase 2) have been treated, and the study is still ongoing to evaluate treatment-related study-specific procedures.
the efficacy of the RP2D using overall response rate, duration of response, clinical 14. Patient has received routine therapy as follows:
benefit rate, deepening of response, progression of survival and OS. Of the 14 i. Patient with NSCLC, metastatic urothelial cancer or GEA who has
phase 1 patients reported on in this study, patients G8, G9 and G10 received received systemic treatment with cytotoxic, platinum-based chemo-
continued standard-of-care chemotherapy with study treatment. G8 received therapy, has measurable disease according to RECIST v1.1 and:
fluorouracil (5-FU) and bevacizumab (initiated 21 months before study treatment), a. has chemotherapy and has an assessment of SD, partial response or
and G9 and G10 received fluoropyrimidine/irinotecan (FOLFIRI) (initiated 12 complete response: will either receive study treatment in addition to
and 8 months before study treatment). Four clinical sites enrolled and treated continued routine therapy, or will receive study treatment after stop-
patients for the phase 1 part of the study: University of Chicago Comprehensive ping routine therapy, per Investigator discretion; or
Cancer Center, Columbia University Medical Center, The Ohio State University b. has experienced disease progression: will receive study treatment as
Medical Center and Virginia Cancer Specialists. There is no data safety monitoring next line of therapy (i.e., without chemotherapy); or
board for this study. A study committee consisting of the investigators and study c. is intolerant to chemotherapy: will receive study treatment as next
sponsor meets regularly to assess the safety profile of the study treatment and make line of therapy (i.e. without chemotherapy)
recommendations for dose escalation. Patient data were collected via electronic ii. Patient with CRC-MSS who has received second-line systemic therapy
data capture and analyzed as described in the Statistics and reproducibility section. including a fluoropyrimidine and oxaliplatin or irinotecan, has measur-
able disease according to RECIST v1.1 and:
Study population. Inclusion criteria. Inclusion criteria for vaccine production a. has assessment of SD, partial response or complete response: will
stage. Patients must meet the following inclusion criteria to be eligible for vaccine receive study treatment in addition to continued routine therapy per
production stage: investigator discretion; or
b. has experienced disease progression: will receive study treatment as
1. Provide a signed and dated Informed Consent Form (ICF) document before
next line of therapy; or
initiation of study-specific procedures.
c. is intolerant to chemotherapy: will receive study treatment as next
2. Patients with the indicated advanced or metastatic solid tumor as follows:
line of therapy
a. NSCLC who are planned for or have received no more than one cycle
15. Patient agrees to undergo research biopsies before study treatment and ap-
of systemic treatment with cytotoxic, platinum-based chemotherapy
proximately 16 weeks after first vaccine administration (if patient has lesions
(patients receiving anti-PD-(L)1 monotherapy are eligible)
amenable to biopsy).
b. GEA who are planned for or have received no more than one cycle of
16. ECOG performance status of 0 to 2 (or equivalent for patients 12–17 years of
systemic treatment with cytotoxic, platinum-based chemotherapy
age based on Karnofsky or Lansky performance scale).
c. metastatic urothelial cancer who are planned for or have received 17. Patient has adequate organ function as defined below. The sponsor may
no more than one cycle of systemic treatment with cytotoxic, consider patients eligible despite having a laboratory value that does not meet
platinum-based chemotherapy inclusion criteria provided the investigator reports the patient is otherwise
d. CRC-MSS who are receiving first-line systemic therapy or who are in good health and the history and kinetics of the change in the laboratory
planned for or have received no more than one cycle of second-line sys- value do not raise a substantial safety concern over including the patient in
temic therapy including a fluoropyrimidine and oxaliplatin or irinotecan the study or compromise the integrity of the study data. The sponsor and

investigator must agree to include any patients with a laboratory value below 13. Known history of positive test for HIV or known acquired immunodeficiency
the defined criteria below. syndrome.
14. History of pneumonitis requiring systemic steroids for treatment (with the
a. Peripheral WBC ≥2,000/mm3 exception of prior resolved in-field radiation pneumonitis).
b. Absolute lymphocyte count ≥500/ mm3 15. Patient with known central nervous system (CNS) metastases and/or carcino-
c. ANC ≥1,000/mm3 matous meningitis.
d. Platelets ≥75,000/mm3 16. Myocardial infarction within 6 months of study initiation, active cardiac
e. Hemoglobin ≥ 9 g dl−1 ischemia, myocarditis or New York Heart Association grade III or IV
f. Calculated creatinine clearance >40 ml min−1 using Cockcroft–Gault heart failure.
equation 17. Pregnant, planning to become pregnant or nursing.
g. ALT and AST ≤3× ULN unless liver metastases are present in which 18. Medical, psychiatric, cognitive or other conditions that compromise the
case patients are included if ALT and AST ≤5 × ULN patient’s ability to understand the patient information, give informed consent,
h. Total bilirubin ≤1.5× ULN or direct bilirubin ≤1× ULN (patients with comply with the study protocol or complete the study.
Gilbert’s disease may be included if their total bilirubin is ≤ 3.0 mg dl−1).
i. International normalized ratio and PT and PTT ≤1.5× ULN, unless
patient is receiving anti-coagulant therapy, in which case patients are Exclusion criteria for the study treatment stage. Patients meeting any of the of the
eligible if PT and PTT is within therapeutic range of intended use of following exclusion criteria will not be eligible for the study treatment stage:
anti-coagulants.
19. Immunosuppression from:
• concurrent, recent (≤ 4 weeks) or anticipated treatment with systemic
Exclusion criteria. Exclusion criteria for the vaccine production stage. Patients
corticosteroids (>10 mg daily prednisone equivalent) or other immuno-
meeting any of the following criteria are not eligible for enrollment in the vaccine
suppressive medications such as OKT3, ATG/ALG, methotrexate, tacroli-
production stage:
mus, cyclosporine, azathioprine or rapamycin. Inhaled or topical steroids,
1. Tumors with genetic characteristics as follows: physiologic corticosteroid replacement therapy for adrenal or pituitary
a. For NSCLC, patients with a known genetic driver alteration in EGFR, insufficiency, steroid pretreatment for chemotherapy (e.g., pemetrexed),
ALK, ROS1, RET or TRK antihistamines, nonsteroidal anti-inflammatory drugs and aspirin are
b. For CRC and GEA, patients with known microsatellite instability-high permitted in the absence of active autoimmune disease
disease based on institutional standard • conditions such as common variable hypogammaglobulinemia or expo-
c. For CRC, patients with a known BRAF V600E mutation or patients sures such as large field radiotherapy
with peritoneal carcinomatosis, and for GEA, patients with peritoneal 20. Patients who have had history of life-threatening TRAEs with prior immuno-
carcinomatosis as their only evidence of disease therapy or who have not recovered from prior cancer therapy-induced AEs
2. Patient has a known tumor mutation burden more than one nonsynonymous (i.e., any AE that remains ≥ grade 3, or that has not returned to baseline with
mutations/megabase. the exception of alopecia, neuropathy and hypothyroidism if controlled by
3. Known exposure to ChAd or any history of anaphylaxis in reaction to a hormone replacement).
vaccination or allergy or hypersensitivity to study drug components. Only applicable to those AEs not covered by other inclusion and exclusion
4. Bleeding disorder (e.g., factor deficiency or coagulopathy) or history of criteria (e.g., ANC or ALT values).
substantial bruising or bleeding following i.m. injections or blood draws. 21. Active, known or suspected autoimmune disease.
5. Patient has received prior therapy consisting of anti-CTLA-4, anti-PD-1, Patients with type I diabetes mellitus, hypothyroidism if requiring hormone
anti-PD-L1 or any other antibody or drug specifically targeting T cell replacement, skin disorders (such as vitiligo, psoriasis or alopecia) not requir-
co-stimulation or checkpoint pathways. ing systemic treatment, or conditions not expected to recur in the absence of
Patients with NSCLC and GEA who have been, or are currently being, treated an external trigger are permitted. Replacement therapy (e.g., thyroxine, insu-
with anti-PD-(L)1 monotherapy are eligible. lin or physiologic corticosteroid replacement therapy for adrenal or pituitary
6. Immunosuppression that is expected to be present at the time of study treat- insufficiency) is not considered a form of systemic treatment.
ment after vaccine production and coming from: 22. Received radiation therapy within 2 weeks before first dose of study
treatment.
a. Concurrent, recent (≤4 weeks) or anticipated treatment with systemic 23. Any severe concurrent non-cancer disease (including active infection and/or
corticosteroids (>10 mg daily prednisone equivalent) or other immu- uncontrolled hypertension) that, in the judgment of the investigator, would
nosuppressive medications such as OKT3, ATG/ALG, methotrexate, make the patient inappropriate for the current study (study treatment may be
tacrolimus, cyclosporine, azathioprine or rapamycin. Inhaled or topical delayed until patient has recovered from disease (such as an infection) or has
steroids, physiologic corticosteroid replacement therapy for adrenal stabilized).
or pituitary insufficiency, steroid pretreatment for chemotherapy (e.g., 24. Active tuberculosis or recent (<2 week) clinically substantial infection, or
pemetrexed), antihistamines, nonsteroidal anti-inflammatory drugs evidence of active hepatitis B or hepatitis C.
and aspirin are permitted in the absence of active autoimmune disease 25. History of pneumonitis requiring systemic steroids for treatment (with the
b. Conditions such as common variable hypogammaglobulinemia or ra- exception of prior resolved in-field radiation pneumonitis).
diation exposure such as large field radiotherapy 26. Patient with uncontrolled CNS metastases or brain stem metastasis, patient
7. History of allogeneic tissue/solid organ transplant. receiving steroids for CNS disease and/or carcinomatous meningitis; patients
8. Patients who have had a history of life-threatening TRAEs with prior im- with irradiated CNS metastases should be discussed on a case-by-case basis
munotherapy or have not recovered from prior cancer therapy-induced AEs with the sponsor before enrollment.
(i.e., any AE that remains ≥ grade 2 or that has not returned to baseline with 27. Myocardial infarction, active cardiac ischemia, myocarditis or New York
the exception of peripheral neuropathy, alopecia and hypothyroidism that is Heart Association grade III or IV heart failure since inclusion in the study.
controlled with hormone replacement therapy). 28. Pregnant, planning to become pregnant or nursing.
9. Active, known or suspected autoimmune disease. 29. Treatment with botanical preparations (e.g. herbal supplements or traditional
a. Patients with type I diabetes mellitus, hypothyroidism, adrenal or pitui- Chinese medicines) intended for general health support or to treat the disease
tary insufficiency that is controlled with hormone replacement therapy, under study within 2 weeks before treatment that may interfere with study
skin disorders (such as vitiligo, psoriasis or alopecia) not requiring sys- treatment per investigator discretion.
temic treatment, or conditions not expected to recur in the absence of 30. Medical, psychiatric, cognitive or other conditions that compromise the
an external trigger are permitted. Replacement therapy (e.g., thyroxine, patient’s ability to understand the patient information, give informed consent,
insulin or physiologic corticosteroid replacement therapy for adrenal or comply with the study protocol or complete the study.
pituitary insufficiency) is not considered a form of systemic treatment.
10. History of other cancer within 2 years with the exception of basal or squa- Individual patient-specific vaccine cassettes. Patient tissue next-generation
mous cell carcinoma of the skin, superficial bladder cancer or carcinoma sequencing, analysis and neoantigen prediction using the EDGE model were
in situ of the breast, cervix, prostate or other neoplasm that has undergone performed as previously described30. Twenty neoantigens were selected for each
potentially curative therapy with no evidence of disease recurrence following patient, as summarized in Supplementary Table 2. The 20 neoantigen epitopes
agreement with sponsor. were designed with flanking sequences so that each are 25 amino acids in length
11. Any severe concurrent non-cancer disease (including active systemic infec- and arranged as a cassette to minimize generation of new junction epitopes. A
tion and/or uncontrolled hypertension) that, in the judgment of the investiga- class II helper domain consisting of universal class II epitopes PADRE and tetanus
tor, would make the patient inappropriate for the current study. toxoid were included at the C terminus of the cassette. The cassettes were codon
12. Active tuberculosis or recent (<2 week) clinically substantial infection, or optimized for maximum protein expression in humans and for ease of synthesis.
evidence of active hepatitis B or hepatitis C. The cassettes were synthesized from overlapping oligonucleotides (Integrated

DNA Technologies (IDT)), that cover both DNA strands of the cassette sequence and surface marker antibodies (CD8-PerCP-Cy5.5, CD3-BV605, CCR7-PE-Cy7
plus sequences in the plasmid backbones and assembled into either ChAd or from BioLegend; CD4-APC-eF780, CD45RA-SuperBright702 from eBioscience;
samRNA plasmid backbones by Gibson assembly (Codex DNA). The assembled PD-1-BUV737 from BD Biosciences) before fixation and permeabilization with
plasmids were then transformed into bacteria and screened by Sanger sequencing FIX & PERM Cell Permeabilization Kit (Thermo Fisher Scientific). Following
for correct clones which served as the templates for good manufacturing practice permeabilization, cells were stained for intracellular cytokines using anti-human
ChAd68 production or for samRNA transcription. ChAd68 plasmids were IFN-γ-APC, TNF-α-BV785, and IL-2-PE antibodies (BioLegend) before data
linearized with PacI and then transfected with TransIt Lenti (Mirus Bio) into acquisition on a BD LSRFortessa flow cytometer (BD Biosciences).
293F (Thermo Fisher Scientific) cells to initiate virus production. The virus was
amplified through successive rounds of infection before the final production run HLA class I fluorophore-conjugated tetramer generation and staining (clinical
at scale. The virus was purified at 48–72 h after infection and purified by anion study). HLA class I tetramers were generated from Flex-T biotinylated HLA
exchange chromatography (Satorius) and formulated in 5 mM Tris, pH 7.9, 1 mM monomers (BioLegend) or MBL International with target peptides loaded onto
MgCl2 and 75 mM NaCl. VP titers were determined by absorbance at 260 nm after the monomers by UV exchange according to the manufacturer’s instructions.
SDS disruption and infectious unit titers were determined by immunostaining Successful loading of target peptides by UV exchange was assessed by ELISA
with an anti-Ad antibody (Abcam). samRNA plasmid was used as a template (BioLegend, according to manufacturer’s instructions). Alternatively, biotinylated
for RNA transcription using T7 polymerase, and the RNA was capped using a monomers were generated in-house and prefolded with target peptides. pHLA
vaccine-capping enzyme and mRNA CAP 2′-O-methyl transferase (NEB). The monomers were assembled into tetramers using fluorophore-conjugated
RNA concentration was measured by Ribogreen quantitation and then formulated Streptavidin (PE from BioLegend; APC from eBioscience). Using generated
into lipid nanoparticles (Genevant). fluorophore-labeled peptide-MHC tetramers, PBMCs were tetramer stained
to help identify antigen-specific T cells. Patient PBMC samples were thawed
Patient PBMC isolation and storage. Patient demographics and dosing schedules and resuspended in a 96-well V-bottom plate with FACS buffer (PBS, 10%
are shown in Table 1 and Extended Data Table 1. Due to COVID-19 restrictions, FBS [v/v]). Before staining, samples were incubated with 50 nM Dasatinib
several blood draws were missed. Ad hoc dosing and blood draw timepoints were (Sigma-Aldrich) in a 5% CO2, 37˚C incubator for 30 min. Subsequently, HLA
added with IRB approval for several patients. Whole blood for immunogenicity class I fluorophore-labeled tetramers loaded with target peptides of interest were
testing was collected before administration of the first dose (ChAd68 prime) to added directly to each corresponding PBMC sample. In instances where multiple
assess baseline levels and at 14 and 28 days (2 and 4 weeks) after ChAd68 prime. tetramers needed to be added to the same sample, a cocktail was made with equal
Subsequent planned sample collection post samRNA boosts include days 7, 14 and amounts of each tetramer, then mixed and added at the same time. Samples were
28 after dose 2 (samRNA boost 1) and at days 7 and/or 28 after each additional incubated at room temperature for 1 h in the dark. Following incubation, samples
samRNA boost. Two patients (G2 and G8) consented to an optional leukapheresis were washed in FACS buffer and stained with an antibody master mix containing
procedure (week 19 for G2, week 25 for G8). PBMCs from whole blood or purified anti-human HLA-A,B,C antibody, anti-human CD8-PerCP-Cy5.5,
leukopak were isolated at local PBMC processing sites according to standardized anti-human CCR7-PE-Cy7, anti-human CD3-BV421 (all BioLegend), anti-human
protocols. Briefly, cells were isolated using density gradient centrifugation on CD45RA-SB702, anti-human CD4-APC-eF780 (both eBioscience) and Live/Dead
Ficoll Paque Plus (GE Healthcare), washed with D‐PBS (Corning), counted and Green (Thermo Fisher Scientific). Sample were mixed and incubated at 4˚C in the
cryopreserved in CryoStor CS10 (STEMCELL Technologies) at 5 × 106 cells ml−1 dark protected from light for 20 min. Samples were washed once with FACS buffer
for whole blood and 2 ×107 cells ml−1 for leukopak. Cryopreserved cells were and then resuspended in FACS buffer prior to acquisition on a BD LSRFortessa
stored in liquid N2, shipped in cryoports and transferred to storage in liquid N2 flow cytometer (BD Biosciences).
upon arrival. Cryopreserved cells were thawed and washed twice in OpTmizer
T Cell Expansion Basal Medium (Gibco) with Benzonase (EMD Millipore) and Flow cytometry analyses (clinical study). Samples acquired on flow cytometers
once without Benzonase. Cell counts and viability were assessed using the Guava were analyzed using FlowJo software (FlowJo). Gating strategies for human
ViaCount reagents and module on the Guava EasyCyte HT cytometer (EMD samples are as follows (Supplementary Figs 2 and 3). For CD4 versus CD8 T cell
Millipore). Cells were rested overnight before use in functional assays. assessments: lymphocytes (SSC-A versus FSC-A), single cells (FSC-H versus
FSC-A), viable cells (FSC-A versus LD-ZombieRed), CD3+ cells (FSC-A versus
Peptides (clinical study). Custom-made, recombinant, lyophilized peptides CD3-BV605), CD4+ and CD8+ (CD4-APCeF780 versus CD8-PerCP-Cy5.5).
specific for each patient were produced by GenScript and reconstituted at 5 mg For tetramer studies: lymphocytes (SSC-A versus FSC-A), single cells (FSC-H
ml−1 per peptide in sterile DMSO (VWR International), aliquoted and stored at versus FSC-A), viable cells (FSC-A versus LD-AF488), CD3+ cells (FSC-A versus
−80˚C (Supplementary Table 2). Control peptides to assess responses to infectious CD3-BV421), CD8+Tet+ (CD8-PerCP-Cy5.5 versus tetramer-PE or tetramer-APC),
disease antigens from cytomegalovirus, Epstein–Barr virus and influenza (CEF memory phenotypes (CD45RA-SB702 versus CCR7-PE-Cy7). For ICS studies:
peptide pool) were purchased from JPT Peptide Technologies. lymphocytes (SSC-A versus FSC-A), single cells (FSC-H versus FSC-A), viable cells
(FSC-A versus LD-ZombieRed), CD3+ cells (FSC-A versus CD3-BV605), CD4+
IVS cultures. Neoantigen-reactive T cells from patient samples were expanded and CD8+ (CD4-APCeF780 versus CD8-PerCP-Cy5.5), CD8+ or CD4+ cytokine+
in the presence of cognate peptides and low-dose IL-2 as described previously30. cells (CD8-PerCP-Cy5.5 versus CD107a-BV421, IFN-γ-APC, TNF-ɑ-BV786,
Briefly, thawed PBMCs were rested overnight and stimulated in the presence of IL-2-PE; CD4-APCeF780 versus CD107a-BV421, IFN-γ-APC, TNF-ɑ-BV786, IL-
minimal epitope peptide pools (10 µg ml−1 per peptide, 40 patient-specific peptides 2-PE). Polyfunctionality analyses were performed using Boolean gating (FlowJo)
per pool) or control peptides (CEF) in ImmunoCult-XF T Cell Expansion Medium and graphed using SPICE software v6 (ref. 48). Results are represented as the
(IC media; STEMCELL Technologies) with 10 IU ml−1 recombinant human IL-2 percentage of positive cell populations (frequency of parent). Data are shown as
(R&D Systems) for 14 days in 48- or 24-well tissue culture plates. Cells were seeded background subtracted where indicated.
at 1-2 ×106 cells/well and fed every 2–3 days by replacing two thirds of the culture
media with recombinant human IL-2. MSD U-plex assay. Detection of secreted IL-2, TNF-α and granzyme B (GRZB)
in ex vivo ELISpot supernatants was performed using a U-plex assay MSD
IFNγ ELISpot assay (clinical study). Detection of IFN-γ-producing T cells was U-PLEX Biomarker assay. Assays were performed according to the manufacturer’s
performed by ex vivo ELISpot assay47. Briefly, cells were harvested, counted and instructions. Data were acquired on the MESO SECTOR S 600 instrument and
resuspended in media at 4 × 106 cells ml−1 (ex vivo PBMCs) or 2 × 106 cells ml−1 DISCOVERY WORKBENCH assay analysis software. Analyte concentrations (pg
(IVS-expanded cells) and cultured in the presence of DMSO (VWR International), ml−1) were calculated using serial dilutions of known standards for each cytokine.
phytohemagglutinin-L (Sigma-Aldrich), CEF peptide pool or cognate peptides For graphical data representation, values below the minimum range of the standard
in ELISpot Multiscreen plates (EMD Millipore) coated with anti-human IFN-γ curve were represented as zero.
capture antibody (Mabtech). Following 18–24 h incubation in a 5% CO2, 37˚C,
humidified incubator, supernatants were collected, cells were removed from the Triplex FluoroSpot assay. A multiplexed FluoroSpot assay was performed
plate and membrane-bound IFNγ was detected using anti-human IFN-γ detection according to manufacturer’s instructions (MABtech) on a subset of patient
antibody (Mabtech), Vectastain Avidin peroxidase complex (Vector Labs) and AEC samples for the detection of IFN-γ, IL-2 and granzyme B-producing T cells.
Substrate (BD Biosciences). ELISpot plates were allowed to dry, stored protected Briefly, patient PBMCs were thawed and rested overnight at 2 × 106 cells ml−1.
from light and sent to Zellnet Consulting for standardized evaluation45. Data are The following day, cells were harvested, counted and resuspended at 4 × 106
presented as SFU per million cells. cells ml−1 before plating. Precoated FluoroSpot plates were washed with PBS and
blocked with culture media before patient-specific peptide pools and cells were
ICS (clinical study). PBMCs or IVS-expanded PBMCs were stimulated with added (2 × 105 PBMCs per well). After incubating in a 5% CO2, 37˚C humidified
either cognate minimal (8–11mer) or long overlapping (15mer) peptides, DMSO incubator for 18–24 h, secreted IFN-γ, IL-2 and granzyme B were detected using
(vehicle control; VWR) or PMA/ionomycin cell stimulation cocktail (positive anti-human IFN-γ-BAM/anti-BAM-490, biotinylated anti-human granzyme B/
control; Affymetrix) in the presence of anti-human CD28/CD49d antibody Streptavidin-550 and anti-human-IL-2-WASP/anti-WASP-650 antibody pairs
(BD Biosciences), anti-human CD107α-BV421 (BioLegend), BD GolgiStop followed by fluorescence enhancer. Plates were imaged and enumerated on an
(BD Biosciences) and Brefeldin A (BioLegend) over a period of 18 h. Following AID iSpot reader (Autoimmun Diagnostika). Data are presented as SFU per
overnight incubation, cells were stained with live/dead ZombieRed (BioLegend) million cells.

Single-cell TCR sequencing and digital gene expression. Single-cell library Transduction, culture and live sorting of single HLA-expressing A375 NucLight
preparation and sequencing was completed according to 10x Genomics v1 5’ Red cell lines. A375 cells were obtained from ATCC (CRL-1619). Endogenous
Chromium Single Cell V(D)J protocol CG000186 Rev A. The only deviation class I HLAs were knocked out using CRISPR-Cas9 ribonucleoprotein complexes.
in library preparation was in V(D)J library step 5.4 with five cycles of PCR. All HiFi S.p. Cas9 Nuclease V3 (1081060), Cas9 Electroporation Enhancer (1075915)
sequencing was completed on the Illumina NovaSeq at a loading concentration of and custom guide RNAs were obtained from IDT. Ribonucleoproteins were
450 pM with 5% PhiX. The 10x Genomics Cell Ranger version 3.1 was used for V(D) electroporated into A375 cells using the Neon transfection system from Thermo
J and Digital Gene Expression library analysis and was current at time of research. Fisher Scientific with a voltage of 1,400 and two pulses for 20 ms. CRISPR-treated
A375 cells were stained using a 1:100 anti-human HLA-A,B,C antibody from
Adaptive TCR sequencing of RNA later-preserved tumor samples. Tumor DNA Biolegend (311405), and HLA-null cells were sorted using the Cell Sorter SH800S
was extracted via Qiagen AllPrep DNA/RNA kit and quantified with both Qubit (SONY Biotechnology) (Supplementary Fig. 4). Single-allele A375 cell lines
high sensitivity dsDNA assay (Thermo Fisher Scientific) and Agilent Genomic were generated with HLA sequences obtained from the coding sequence of the
DNA tape on the 4200 TapeStation system. Between 300 and 1,000 ng extracted human genome (HLA-B*08:01:01:01, HLA-B*57:01:01:01, HLA-A*30:04:01:01,
DNA was sent to Adaptive Biotechnologies for T cell receptor beta sequencing, and HLA-A*03:01:01:01, HLA-C*04:01:01:01). HLAs were cloned into the
results were returned to us via the immunoSEQ Analyzer 3.0. pCDH-EF1α-MCS1 lentiviral expression backbone vector from System Biosciences
(CD502A-1). Lentivirus was generated for each HLA and titer determined using
TCR sequencing analysis. Significant tumor TCR expansion was determined via the Lenti-X qRT-PCR titration kit from Takara Bio (631235). Single HLAs were
a binomial test with Benjamini–Hochberg correction and P value <0.05, analyzing transduced into A375 HLA-null cells at an m.o.i. of 100 with 8 ug ml−1 polybrene
TCRs with five cells or more in either timepoint. IVS TCR expansion was analyzed (EMD Millipore, TR-1003). Cells were spinoculated at 800g for 45 min at room
by filtering 10× V(D)J results for each timepoint for TCRs (1) with a full alpha/ temperature at a density of 8 × 105 cells per ml. Fresh media was added and cells
beta pair, (2) found above an abundance threshold and (3) not found in the CEF were incubated overnight. HLA-positive cells were sorted after at least 48 h using
IVS. The proportion of the resulting TCRs was then tracked from baseline to on the method described above. Fluorescent single-allele A375 cells were generated
treatment, and the polyfunctional activation of these expanded TCR populations with NucLight Red (IncuCyte, 4476) at an m.o.i. of 3 with 8 μg ml−1 polybrene
was also analyzed by searching in the transcriptome for non-zero expression of (EMD Millipore, TR-1003). Cells were spinoculated at 800g for 45 min at room
IFN-γ, granzyme B, TNF and IL-2. The expanded TCRs were cross-referenced with temperature at a density of 8e5 cells ml−1. Fresh media was added and cells were
those expanded in the tumor biopsies, and overlap was statistically significant by incubated overnight. Red fluorescent and HLA-positive cells were sorted after at
Fisher test (P < 0.001). least 48 h using the method described above.
cfDNA collection and isolation. Whole blood was collected in two 10-ml cell-free IncuCyte killing assays. Single HLA-expressing A375 NucLight Red cell lines
DNA (cfDNA) BCT (Streck) starting at the time of the prime, and subsequent were seeded in 96- or 48-well plates at concentrations of 2.5 × 104 or 3.5 ×104 cells
draws were collected at dosing visits. Due to COVID-19 restrictions, several per well in DMEM with 10% heat-inactivated FBS. The plates were placed in the
blood draws were missed. Whole blood underwent a double-spin protocol to first IncuCyte S3 (Essen Biosciences) and 24 h after the seeding, the effector cells were
separate plasma from WBCs and red blood cells before a second spin to remove plated at a concentration of 2.5 × 105 or 3.5 × 105 cells per well in a 96- or 48-well
any remaining cellular debris. The separated plasma was frozen and shipped to plate, respectively, for an effector to target ratio of 10:1. Individual peptides
Gritstone Bio and stored at −80°C until extraction. cfDNA was extracted from the (GenScript; Supplementary Table 2) were added to the treated wells for a final
entire plasma volume of a single draw using the Apostle MiniMax cfDNA Isolation concentration of 10 µg ml−1, and DMSO was used for the control wells. The plates
kit (ApostleBio) and quantified using the Qubit 1x dsDNA High Sensitivity Assay were imaged with the IncuCyte for up to 72 h, after which the data were analyzed
(Thermo Fisher Scientific). using the IncuCyte S3 2018 analysis software. Viability of A375 was assessed by
red cell count, and relative viability was calculated relative to DMSO co-culture
control wells.
ctDNA sequencing and analysis. Custom hybrid capture panels were designed
for each patient enrolled in the study. Each patient panel was designed to capture
Statistics and reproducibility. For the clinical study, the sample size is not
the EDGE-predicted neoantigens and all predicted coding (nonsynonymous)
driven by a statistical power due to the nature of an adaptive dose-escalation
mutations present in the original screening biopsy specimen. Target mutations
method. Therefore, the sample size of the phase 1 portion of the study ranges
for each patient were curated and sent for customized design to IDT as xGen
from 11 to maximum of 24 patients, which is dependent on occurrence of
Lockdown Probes. Each capture pool contained up to nine patients per pool.
DLTs. No data were excluded from the analyses, and experiments were not
Patient-matched genomic DNA was fragmented before library preparation using
randomized. The investigators were not blinded to allocation during experiments
the NEB FS module (NEB). Shotgun libraries for cfDNA (up to 30 ng) and the
and outcome assessment. For the NHP study, the number of animals per group
fragmented, patient-matched genomic DNA (20–30 ng) were prepared using
is n = 3/sex, which is the minimum number of animals needed to do simple
the KAPA HyperPrep (KAPA Biosystems) kit using a customized pool of duplex
statistics. The total number of animals used in this study is considered the
adapters containing unique molecular identifiers for duplex sequencing (IDT).
minimum required to properly characterize the effect of the test articles (i.e.,
Shotgun libraries were captured with patient-specific probes overnight using the
vaccine and anti-CTLA-4) and compare their route of administration (i.e., s.c.
IDT xGen Hybridization and Wash kit. Enriched libraries were sequenced on
versus i.v.) and has been designed such that it does not require an unnecessary
an Illumina NovaSeq to a minimum mean raw depth of 80,000×. Briefly, unique
number of animals to accomplish its objectives. For both the NHP and clinical
molecular identifiers were clipped from the raw sequencing reads before alignment
study, descriptive statistics are described throughout. Where applicable,
to hg38 using BWA-MEM. Using fgbio, aligned reads were grouped by position
statistical analyses applied for specific methods are outlined in the relevant
and duplex identity. Consensus reads were created using a duplex of 3× (three
methods sections.
supporting reads from each strand) and realigned to hg38. Variant calling was
performed using FreeBayes and filtered for the patient-specific variants. Variant
Reporting summary. Further information on research design is available in the
allele frequency was converted to mutated haploid genomic equivalents per
Nature Research Reporting Summary linked to this article.
milliliter plasma (hGE ml−1) using the extracted plasma volume and total cfDNA
yield from the extraction. Percent change in ctDNA was calculated as the change of
the median mutated hGE ml−1 from the baseline sample. Data availability
Deidentified individual participant clinical data that underlie the results reported
Patient tumor biopsy whole-exome and transcriptome analysis and TCGA in this article are available for transfer. Interested investigators can obtain and
data comparison. Pretreatment GRANITE patient FFPE biopsy-derived DNA certify the data transfer agreement and submit requests to the principal investigator
and RNA and matched whole blood-derived DNA were whole-exome sequenced (K.J.). Investigators and institutions who consent to the terms of the data transfer
(IDT xGen V1) and analyzed as described previously38. Relevant tumor types agreement form, including, but not limited to, the use of these data for the
for genomic comparison from TCGA Pan-Cancer Atlas dataset included gastric purpose of a specific project and only for research purposes, and to protect the
(n = 405 stomach adenocarcinoma), colon adenocarcinoma (n = 332, MSS-CRC confidentiality of the data and limit the possibility of identification of participants
(281) and MSI-CRC (51)), lung adenocarcinoma (n = 498), lung squamous cell in any way whatsoever for the duration of the agreement, will be granted access.
carcinoma (n = 463, LUSC) and melanoma (n = 436, skin cutaneous melanoma). Gritstone bio will then facilitate the transfer of the requested deidentified data.
For RNA expression analyses, all GRANITE and TCGA sample RNA-seq by This mechanism is expected to be via a Gritstone Secure File Transfer Service, but
Expectation-Maximization expected counts were normalized utilizing DESeq2 Gritstone bio reserves the right to change the specific transfer method at any time,
(ref. 49). Normalized counts were used to determine patient PD-L1 RNA expression provided appropriate levels of access authorization and control can be maintained.
and the IFN-γ RNA expression signatures, which were calculated as the average of Source data are provided with this paper. Pan-Cancer Atlas data sets were obtained
28 IFN-γ-associated gene specific z-scores across all GRANITE and TCGA samples from the cBioPortal (https://www.cbioportal.org/) and the UCSC Xena TCGA
(n = 2,494)50. Tumor mutational burden was calculated for each GRANITE patient Pan-Cancer Atlas data hub (https://pancanatlas.xenahubs.net), and raw data are
as the total of all somatic variants called >4% variant allele frequency divided by available through the Genomic Data Commons (https://gdc.cancer.gov/). Epitope
the exome bait panel size (39 Mb). For TCGA samples, total mutations called were selection and patient-specific neoantigen selection used a previously published
divided by exome bait panel size used (38 Mb)51 (Supplementary Table 4). algorithm30. Source data are provided with this paper.

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Acknowledgements Bayer (2020) (healthcare companies <10,000 USD). S.R. received honoraria from IDT
We would like to thank the patients and their families; clinical staff and study (2017) and Illumina (2018) (technology companies). S.R. received consulting fees from
coordinators; Bristol Myers Squibb for providing ipilimumab and nivolumab; and QED Therapeutics (2018, 2019) and Merck (2019) (healthcare companies <10,000 USD).
K. Caldwell, A. Dixon, C. Voong, J. Abhyankar, G. Talbot, A. Bezawada, W. Zhai, M. S.R. received travel reimbursement from Incyte Corporation (2019) (<999 USD). C.Y.L.
Zhong, T. Patch and J. Rouhana. We also thank T. Chan for critical review and guidance. received honoraria from Cancer Experts Now and has consulting/advisory board roles
Funding was provided by the study sponsor, Gritstone bio. Employees of Gritstone bio with Transthera, BluePrints Medicine, Genentech, QED Therapeutics, Histosonics and
received salaries for study conceptualization, design, data analyses, decision to publish Ipsen and is part of the Speaker’s Bureau for Eisai and Incyte. S.B.M. received honoraria
and manuscript preparation. No other authors received specific funding for this work from Natera, Bicara, Novartis, Basilea and Daiichi Sankyo and owned stock in Calithera
from the sponsor. outside the submitted work. The remaining authors declare no competing interests.
Author contributions
C.D.P., A.R.R., M.D., M.S., G.G., W.B., R.R., R.Y., A.A., A.F. and K.J. designed the study Additional information
and wrote the manuscript. C.D.P., A.R.R., M.D., M.G.H., C.D.S., L.D.K., S.K., A.Y., L.S., Extended data is available for this paper at https://doi.org/10.1038/s41591-022-01937-6.
D.S., M.S., K.T., M.M., J.R.J., C.N.N., E.M., R.Z., D.N.G., A.C.G., R.G., K.B., M.D.C. Supplementary information The online version contains supplementary material
and R.Y. contributed to experimental design, execution and data analysis. A.I.S., S.R., available at https://doi.org/10.1038/s41591-022-01937-6.
D.C., B.S.H., C.G.D., B.J.S., D.A., A.M., S.B.M., B.J., R.R., C.-Y.L., D.V.T.C. and A.R.F.
contributed to clinical oversight, patient recruitment, enrollment and treatment. C.D.S., Correspondence and requests for materials should be addressed to Karin Jooss.
L.G., S.-J.H., W.B., M.D.C., S.C. and K.J. contributed to vaccine design and production. Peer review information Nature Medicine thanks Robert Seder and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work. Primary
Handling editors: Saheli Sadanand and Joao Monteiro, in collaboration with the Nature
Competing interests
Medicine team.
C.D.P., A.R.R., M.G.H., C.D.S., L.G., S-.J.H., L.D.K., S.K., D.S., M.D., M.M., J.R.J.,
C.N.N., R.Z., D.N.G., A.C.G., M.D.C., S.C., K.B., R.R., A.A., A.R.F., K.J., A.Y., L.S., M.S., Reprints and permissions information is available at www.nature.com/reprints.

Extended Data Fig. 1 | See next page for caption.

Extended Data Fig. 1 | (A) Representative FACS plots (NHP 4 and NHP 10) and pie charts for each population (% of total CD8+ or % of CD8+IFNγ+, mean
for each group (n = 6 per group), week 13 post prime (one week post samRNA boost). Analysis of antigen-specific (IFNγ+) memory T cell populations,
using CCR7 and CD45RA (Naïve: CCR7+CD45RA+, purple; Central Memory: CCR7+CD45RA-, blue, Effector memory: CCR7-CD45RA-, green; Effector
memory RA+: CCR7+CD45RA+, orange). Pie chart represents mean percent of total tetramer population. (B) Analysis of memory T cell populations at
80 weeks post prime (1 year post last boost). Antigen-specific T cells were identified by combinatorial tetramer staining for six different antigens. Memory
analysis was performed on total tetramer+ population, using CCR7 and CD45RA (Naïve: CCR7+CD45RA+, purple; Central Memory: CCR7+CD45RA-, blue,
Effector memory: CCR7-CD45RA-, green; Effector memory RA+: CCR7+CD45RA+, orange). Pie chart represents mean percent of total tetramer population
(n = 6). (C) Antigen-specific T cell response in each individual animal (n=6 animals) 2 years post prime (week 106) and one week post samRNA boost
(week 107), for six SIV antigens, as measured by IFNγ ELISpot. SFU/106 to each antigen (mean of three technical replicates per antigen with SD; negative
control value subtracted) stacked to provide sum total response. Red asterisk represents values that were too numerous to count and were set to
maximum measured value (11000 for TL8, plated at 2.5x104 cells/well, and 3100 for TL9, plated at 1x105 cells/well). (D) Killing of antigen loaded target
cells by CD8 T cells (collected 2 weeks post-boost, week 108), relative viability of SIV-loaded PBMCs (as determined by 7-AAD staining), normalized to
negative control peptide-loaded PBMCs for each sample, at specified effector to target ratios.

Extended Data Fig. 2 | (A) CONSORT flow diagram of patient enrollment. (B) Schematic outlining personalized vaccine production, planned dosing
schedule, and immune monitoring.


Extended Data Fig. 3 | (A) Data from PBMCs stimulated overnight in ex vivo IFNγ ELISpot with patient-specific peptide pools are shown for all Phase
patients with available samples (all except G5). Individual patient IDs legend is shown below graph. Graphs show mean SFU per 106 PBMCs +/- standard
deviation (SD) for technical triplicates (ELISpot wells) for all patients and timepoints except for G1 (W20 and W72), G2 (W42), G3 (W0, W5 and W8),
G6 (all timepoints), G12 (W12 and W13), G13 (W12), and G14 (all timepoints), where only sufficient cells to test technical duplicates were available. For
patient G3 W1 enough cells were available to test a single well. Assay limit of detection (LOD) and upper limit of quantitation (ULOQ) are indicated by
dotted lines. ChAd68 boost for patients G1, G3, G8, and G11 are indicated by adenovirus symbol. (B) Longitudinal ELISpot data ≥52 weeks from PBMCs
stimulated overnight in ex vivo IFNγ ELISpot with patient-specific CD8 Pools (blue bars) or DMSO (gray bars) are shown for patients G1, G2 and G8.
Graphs show mean SFU per 106 PBMCs +/- standard deviation (SD) for triplicate ELISpot wells. (C) ELISpot data (fold change of mean of technical
triplicates (all except for G6 (pre and post), G12 & G13 (post), where enough cells to test duplicates were available) are shown for all patients relative to
pre-samRNA boost timepoint (week 4, available for all patients except G9), and maximum response post samRNA boost (5-36 week timepoints). Graphs
show paired data from patients receiving 30µg samRNA (light blue), 100µg samRNA (medium blue), and 300µg samRNA (navy blue).

Extended Data Fig. 4 | (A) ELISpot data (mean SFU per 106 PBMCs +/- SD for triplicate wells) are shown for whole PBMCs (black bars), CD4-depleted
(CD8enr) PBMCs (navy blue bars), and CD8-depleted (CD4enr) PBMCs (orange bars) stimulated with DMSO, CD8 Pool, or 15mer pool for patients G1, G11,
and G14 (pooled posttreatment timepoints). (B) Post-IVS ELISpot data for baseline and posttreatment sample PBMCs stimulated with patient-specific
CD8 and minipools are shown for patients G1, G2, G3, G8, G9, G12, G13 and G4. Graphs show mean of technical duplicates (SFU per 106 PBMCs) for all
except G2 CD8 Pool, where enough cells to test triplicates were available, with DMSO background subtracted. ULOQ for post-IVS assay is indicated by
dotted line.


Extended Data Fig. 5 | Data from PBMCs stimulated overnight in ex vivo (left graphs) or post-IVS (right graphs) IFNγ ELISpot with individual patient-
specific peptides are shown for patient G1, G2, and G8. Graphs show individual duplicate values (SFU per 106 PBMCs) for each stimulation condition.
Individual peptides with average responses >LOD (positive response) are shown in color (see mutation-specific coloring in graphs), and responses <LOD
(negative response) are shown in clear circles.


Extended Data Fig. 6 | (A) Density plots showing recognition of pHLA and memory phenotypes in PBMCs from posttreatment timepoints (G1, W48,
HLA-A*02:01-APC and HLA-B*44:05-PE; G8, W25, A*03:01). Pie charts showing frequencies of naïve (CD45RA+CCR7+; purple), central memory (CM;
CD45RA-CCR7+; blue), effector memory (EM; CD45RA-CCR7-; green), and T effector memory RA+ (TEMRA; CD45RA+CCR7-; orange) cells in bulk CD8
and CD8+tetramer+ populations from corresponding dot plots. (B) Density plots showing recognition of peptide-HLA (HLA-A*02:01-APC) pre- and
post- ChAd68 boost for patient G1. Pie charts showing frequencies of naïve (CD45RA+CCR7+; purple), central memory (CM; CD45RA-CCR7+; blue),
effector memory (EM; CD45RA-CCR7-; green), and T effector memory RA+ (TEMRA; CD45RA+CCR7-; orange) cells in CD8+tetramer+ populations from
corresponding dot plots. Cells were gated on lymphocytes (SSC-A vs FSC-A), single cells (FSC-H vs FSC-A), viable cells (FSC-A vs LD-AF488), CD3+ cells
(FSC-A vs CD3-BV421), CD8+Tet+ (CD8-PerCP-Cy5.5 vs tetramer-PE or tetramer-APC), memory phenotypes (CD45RA-SB702 vs CCR7-PE-Cy7). (C)
Supernatants from ex vivo IFNγ ELISpot (G1 W5, G2 W12, G3 W37, G7 W5, G8 W12, G10 W16, G11 W2, G12 W8) were analysed by MSD U-plex assay for
levels of IL-2, TNFα, and Granzyme B (GRZB) following stimulation with patient-specific CD8 Pools or DMSO. Stacked bar graphs show mean cytokine
levels (triplicate wells) in pg/ml (background subtracted) +/- SD for replicate wells for IL-2 (light blue), GRZB (orange), and TNFα (yellow). (D) PBMCs
from optional leukopak donations (G2 W19, G8 W25) were analysed by ex vivo FluoroSpot assay to measure IFNγ, GRZB and IL-2 expression following
overnight stimulation with DMSO or patient-specific CD8 Pools. Graphs show mean SFU per 106 PBMCs +/- SD for triplicate FluoroSpot wells stacked
for IFNγ (navy blue), GRZB (orange), and IL-2 (light blue). E) MSD U-plex analysis of Granzyme B (GRZB) levels in ELISpot supernatants pre- and post-
ChAd68 boost for patients G1, G3, G8, G11, G13 and G14 are shown in pg/ml (background subtracted, mean +/- SD for triplicate wells). No post ChAd68-
boost samples were available for patients G9 and G12.


Extended Data Fig. 7 | (A) Representative density plots for intracellular cytokine staining (ICS) of patient G8 ex vivo and IVS-expanded leukopak PBMCs
stimulated overnight with either DMSO or patient-specific CD8 Pool (Supplementary Table 2) to assess production of IFNγ, CD107α, TNFα, and IL-2 in
CD8+ T cells. Cells were gated on PBMCs (FSC-A vs SSC-A), singlets (FSC-A vs FSC-H), live cells (FSC-A vs Live/Dead), CD8 T cells and cytokine+ cells.
(B) Polyfunctionality was assessed via Boolean gating of CD8+cytokine+ populations (background subtracted). Pie charts show polyfunctionality of ex vivo
PBMCs (left pie chart) and post-IVS PBMCs (right pie chart). Pie arcs depict individual cytokines in red (CD107α), lime green (IFNγ), turquoise (TNFα),
and navy blue (IL-2). Pie sections depict frequencies of cells expressing multiple cytokines as outlined in Pie Category legend. Main functional pie slices
are colored red (quadruple positive for CD107α, IFNγ, TNFα, and IL-2), orange (triple positive for CD107α, IFNγ, and TNFα), and yellow (double positive
for CD107α and IFNγ). (C) Polyfunctionality was assessed via Boolean gating of CD4+cytokine+ populations (background subtracted). Pie charts show
polyfunctionality of post-IVS PBMCs for patient G2 (left pie chart) and G8 (right pie chart). Pie arcs depict individual cytokines in red (CD107α), lime
green (IFNγ), turquoise (TNFα), and navy blue (IL-2). Pie sections depict frequencies of cells expressing multiple cytokines as outlined in Pie Category
legend. Main functional pie slices are colored red (quadruple positive for CD107α, IFNγ, TNFα, and IL-2), orange (triple positive for CD107α, IFNγ, and
TNFα), and yellow (double positive for CD107α and IFNγ). (D) Target cell killing by IVS-expanded PBMCs from patients G8 (A*03:03, C*04:01). Graphs
show relative viability based on NucRed count and normalized to effector:target (E:T) ratio of 10:1 DMSO control over time. Data are shown as mean +/-
SD from duplicate wells for targets alone stimulated with DMSO (gray circles) or cognate peptide (black squares), and for 10:1 E:T co-cultures stimulated
with DMSO (light blue circles) or cognate peptide (navy blue squares).


Extended Data Fig. 8 | (A) Schematic outlining approach for matched PBMC and FFPE tumor tissue TCR analyses. (B) Post-IVS ELISpot (mean SFU per
106 PBMCs +/- SD for duplicate wells) from baseline (W0; orange) and on-treatment (W16; blue) timepoints corresponding to available on-treatment
tumor biopsy samples (W16) for patients G1 and G3. TCR clonotypes (paired α-β chains) present at baseline and expanded on-treatment (expanded;
orange) or absent at baseline and present on-treatment (de novo primed; blue) and the corresponding single-cell mRNA signatures for CD4 or CD8
expression are shown. Patient G1: CD8 expanded (n=1), CD8 de novo (n=10), CD8 preexisting (n=60, CD4 expanded (n=0), CD4 de novo (n=1), CD4
preexisting (n=3). Patient G3: CD8 expanded (n=2, CD8 de novo (n=13), CD8 preexisting (n=5), CD4 expanded (n=2), CD4 de novo (n=9), CD4
preexisting (n=27), undetermined expanded (n=0), undetermined de novo (n=4), undetermined preexisting (n=5). (C) Violin plots showing analyses of
CD3, CD4, and CD8 mRNA expression in cells from baseline (orange) and on-treatment (blue) PBMC samples for patients G1 and G3. (D) UMAP plots
showing individual cells expressing TCR clonotypes present in on-treatment samples (closed circles, n=12 for G1, n=27 for G3) and expressing 0, 1, 2, 3,
or 4 cytokine transcripts (see also Supplementary Table 3). (E) UMAP plots showing individual cells expressing ≤1 (blue) and ≥2 cytokines (red) and TCR
clonotypes of interest (n=12 for G1, n=27 for G3) comparing baseline and on-treatment samples (see also Supplementary Table 3).

Extended Data Table 1 | Listing of patient baseline characteristics, Disposition, and Efficacy by DL (as of 31 January 2022)
GEA: Gastroesophageal adenocarcinoma; NSCLC: Non-Small Cell Lung Cancer; CRC: Colorectal Cancer; 1Dose Level 1=GRT-C901/GRT-R902 30μg+ Nivolumab; Dose Level 2= GRT-C901/GRT-R902
100μg+ Nivolumab; Dose Level 3= GRT-C901/GRT-R902 100μg+ Nivolumab+ SC Ipilimumab; Dose Level 4= GRT-C901/GRT-R902 300μg+ Nivolumab+ SC Ipilimumab 2Weeks from the prime ChAd68
dosing 3Best of response as per RECIST v 1.1; CR=confirmed response; PR=partial response; SD=stable disease (≥ 16 weeks on treatment); PD=progressive disease; NE=no evidence of disease 4Time from
the first dose with GRT-C901 (or GRT-R902 if patient does not receive GRT-C901) to the earliest date of PD or death by any cause 5Time to death (died) or last contact/assessment (alive) from the 1st
study treatment 6This is a confirmed complete response defined as disappearance of target liver lesions and no visible disease on imaging; biopsy at original primary tumor site showed microscopic invasive
adenocarcinoma at 4 months; response duration of 6 months followed by worsening of primary esophageal lesion with dysphagia *Concurrent chemotherapy with study treatment: Patient G1 received 3
doses of 5-FU over the first 8 weeks. Patient G8 received 5-FU/bevacizumab (had been receiving for 21 months prior to study treatment). Patient G9 received FOLFIRI/cetuximab (receiving for 11 months
prior to study treatment). Patient G10 received FOLFIRI (receiving for 11 months prior to study treatment). Patient G12 received Paclitaxel/ramucirumab started after 12 weeks of study treatment.

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Individualized, heterologous chimpanzee

Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1619

1620 Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine

~1 year ChAd68 (1012 VP)

0 4 12 20 32 33 80 106 107 108 samRNA (300 µg)

Shown in panel b Shown in panel d

IFN-γ (SFU per 106 cells)

IFN-γ (SFU per 106 cells)

Weeks after ChAd68 prime Weeks after ChAd68 prime

Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1621

100 100 200

103 103 500

1,000 Total PBMC

10 105 800 CD4enr (CD8 depl)

104 104 600

103 103 400

1622 Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine

Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1623

a b Individual peptide deconvolution

SOSTDC1 SAP130 PIGO

104 104 104 104

104 104 104

0.68% 6.0% 1.32%

Naive Central memory Effector memory Effector memory RA+

e G1: pre-ChAd68 boost G1: After-ChAd68 boost

104 104 104

103 58.7% 103 103

Tet+ PE CD45RA-SB702 Tet+ PE CD45RA-SB702

1624 Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine

Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1625

a G2: ex vivo G2: post-IVS b

c d Expanded TCR-β in IVS (n = 12)

Proportion of filtered T cells (%)

–8 Not significant 243 G3 G3

1626 Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine

Unconfirmed PD per RECIST 75 ctDNA decrease (n = 3)

e Baseline 16 weeks 48 weeks

Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1627

1628 Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine

Nature Medicine | VOL 28 | August 2022 | 1619–1629 | www.nature.com/naturemedicine 1629

Methods AdjustedSpots = RawSpots + 2 ∗ (RawSpots ∗ saturation/ (100 − saturation)

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Extended Data Fig. 1 | See next page for caption.

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Extended Data Fig. 3 | See next page for caption.

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Extended Data Fig. 5 | See next page for caption.

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