a pan-cancer clinical study of personalized neoantigen ......precision therapy. so far, a few...
TRANSCRIPT
CLINICAL CANCER RESEARCH | CLINICAL TRIALS: IMMUNOTHERAPY
A Pan-cancer Clinical Study of Personalized NeoantigenVaccine Monotherapy in Treating Patients with VariousTypes of Advanced Solid Tumors A C
Yong Fang1, Fan Mo2,3,4,5, Jiawei Shou1, Huimin Wang2, Kai Luo2, Shanshan Zhang2,6, Ning Han2,Hongsen Li1, Shengli Ye7, Zhan Zhou3, Rongchang Chen2, Lin Chen2, Liang Liu2, Huina Wang8,Hongming Pan1, and Shuqing Chen2,3,6
ABSTRACT◥
Purpose: Because of their high tumor specificity and immuno-genicity, neoantigens have been considered as ultimate targets forcancer immunotherapy. Neoantigen-based vaccines have demon-strated promising efficacy for several cancer types. To furtherinvestigate the antitumor potentials for other types of solid tumors,we designed a peptide-based neoantigen vaccine, iNeo-Vac-P01,and conducted a single-arm, open-labeled, investigator-initiatedclinical trial (NCT03662815).
Patients and Methods: Personalized neoantigen vaccines weredesigned and manufactured according to our bioinformatics anal-ysis results from the whole-exome sequencing of tumor and periph-eral blood cell DNAs. Patients were scheduled to be vaccinatedsubcutaneously with adjuvant on days 1, 4, 8, 15, and 22 (primephase), and days 78 and 162 (boost phase). Additional immuniza-tionswere administrated every 2–3months as per patient's potentialbenefit. The safety and efficacywere assessed through adverse events
(AE), progression-free survival (PFS), overall survival (OS), andother parameters.
Results: Of the 22 patients enrolled with advanced malignan-cies, 20 had no or mild AEs, while 2 had grade 3 or 4 acuteallergic reactions only after their sixth boost vaccination. Thedisease control rate was 71.4%. The median PFS was 4.6 months,whereas the median OS was not reached (12-month OS ¼55.1%). Around 80% of individual peptides or peptide poolselicited measurable specific immune response. In addition, ourfindings revealed several potential biomarkers for the predictionof better response.
Conclusions: iNeo-Vac-P01 as monotherapy is feasible andsafe for patients with advanced solid tumors. It could elicit T-cell–mediated immune response targeting tumor neoantigens, andmight have promising antitumor efficacy.
See related commentary by Filderman and Storkus, p. 4429
IntroductionIn the past decade, immunotherapy has attracted intensive attention
as an effective alternative cancer therapy. In particular, immune check-point blockade (ICB) has shown remarkable clinical responses with lowtoxicity and few side-effects in several cancer types, including advancednon–small cell lung cancer, melanoma, bladder cancer, gastric cancer,hepatocellular carcinoma, and colorectal cancer with DNA mismatchrepair deficiency (1–5). Further intensive analysis of those patients with
tumor regression after ICB treatment showed that high tumormutationburden (TMB) could be related to better prognosis. Therefore, neoanti-gens, derived from tumor somatic mutations, are considered as criticaland optimal targets for immunologic recognition of cancer cells.
It has been widely accepted that ICBs show better clinical response intumors with high levels of infiltrating T cells and more antigens (“hot”tumor) rather than those lacking tumor-reactive infiltratingTcells (“cold”tumor). Neoantigen vaccines are designed to present tumor-specificneoantigens and activate cytotoxic T cells to recognize and infiltrate intotumor cells, turning “cold” tumor into “hot” tumor. Also, the processinvolves the training of immune system to target and kill tumor cells. Asgenerated mostly by nonsynonymous mutations in cancer cells (6–8),neoantigens are exempted from central tolerance. Therefore, neoantigenvaccines are more likely to generate robust immune responses (9, 10),functioning as bona fide antigens to facilitate tumor regression (11).
Therapeutic neoantigen cancer vaccines are safe, tolerable, andcapable of eliciting robust T-cell responses to kill tumor cells (12, 13).Plenty of related clinical trials have been launched in the past 5 years.Two studies on melanoma demonstrated that neoantigen peptide orRNA vaccines could not only benefit for the regression of advancedmelanoma, but also provide long-term protection against tumorrelapse and metastasis (12, 13). In subsequent investigation, neoanti-gen vaccine along with adjuvant could induce predominantly T-cellresponses against predicted neoepitopes in patients with newly diag-nosed glioblastoma (14, 15), and increase the number of tumor-infiltrating T cells (15). In addition, adoptive transfer of mutantprotein–specific tumor-infiltrating lymphocytes have been applied tomediate complete durable regression ofmetastatic breast cancer. Thesepersonalized neoantigen vaccines could elicit sustained responses ofT cells and display great potentials for further development (14, 15).
1Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou,Zhejiang, China. 2Hangzhou Neoantigen Therapeutics Co., Ltd., Hangzhou,Zhejiang, China. 3College of Pharmaceutical Sciences, Zhejiang University,Hangzhou, Zhejiang, China. 4Vancouver Prostate Centre, University of BritishColumbia, Vancouver, British Columbia, Canada. 5Hangzhou AI-Force Thera-peutics Co., Ltd., Hangzhou, Zhejiang, China. 6Zhejiang California InternationalNanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, China. 7Shulan(Hangzhou) Hospital, Hangzhou, Zhejiang, China. 8AcornMed BiotechnologyCo., Ltd., Beijing, China.
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Y. Fang, F. Mo, and J. Shou contributed equally to this article.
Corresponding Authors: Shuqing Chen, Zhejiang University, Yuhangtang Road866, Hangzhou, Zhejiang 310058, China. Phone: 8657-1882-08411; Fax: 8657-1882-08410; E-mail: [email protected]; and Hongming Pan, Sir Run RunShaw Hospital, Hangzhou, China. Phone: 8657-1860-06926; E-mail:[email protected]
Clin Cancer Res 2020;26:4511–20
doi: 10.1158/1078-0432.CCR-19-2881
�2020 American Association for Cancer Research.
AACRJournals.org | 4511
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
Asmentioned above, all of these exploratory studies were done witha few patients (no more than 17) of a single cancer type, andcombination therapies were applied in these studies to improvepatients' response. Despite the relatively promising clinical results,the antitumor potential of neoantigen vaccine has not been proven inbroader cancer types. In principle, the antitumor potential of neoanti-gen vaccine was not limited to certain tumor type as long as appro-priate tumor neoantigens were identified. Therefore, a pan-cancerclinical study focused on neoantigen vaccine monotherapy instead ofcombination therapy was conducted to demonstrate the antitumorefficacy of neoantigen vaccine in various types of advanced solidtumors. Preliminary results indicated iNeo-Vac-P01 monotherapycould elicit specific T-cell activation and induce broad spectrum ofantitumor effects, without limitation to tumor type. In addition,several biomarkers potentially predictive for patient's better responsewere revealed.
Patients and MethodsPatients
Eligible patients were at least 18 years old with advanced malignanttumors confirmed histologically or cytologically. Patients had diseaseprogression after two or more lines of standard treatment; at least onemeasurable lesion as per investigator-assessed RECIST version 1.1; anEastern Cooperative Oncology Group (ECOG) performance status of0 or 1; good functioning main organs, such as heart, liver, and kidney;and could provide sufficient tumor tissue and blood samples for DNA-sequencing assay or qualified genome/exome-sequencing data oftumor tissues and normal tissues.
Key exclusion criteria included: having other malignant tumor,except for cured basal cell carcinoma, thyroid carcinoma, or cervicaldysplasia; lack of identified neoantigen in the sequencing data; receivedbone marrow or stem cell transplants; and allergic to any drug,polypeptide, or other potential immunotherapies.
Trial design and treatmentThis was a single-arm, open-label, investigator-initiated clinical
study at Sir Run Run Shaw Hospital, School of Medicine, Zhejiang
University (Hangzhou, Zhejiang, China). The final study protocol wasapproved by institutional review board and independent ethics com-mittee, and conducted in accordance with Declaration of Helsinki andthe International Conference on Harmonisation Guidelines for GoodClinical Practice. All patients had signed informed consent formsbefore treatment. The primary endpoints of the study were safety andfeasibility, and the secondary endpoints were efficacy based on pro-gression-free survival (PFS), overall survival (OS), and neoantigen-specific immune responses. The safety of the study was assessed on thebasis of occurrence of adverse events (AE). The feasibility of this trialwas assessed by whether neoantigen could be identified and thevaccines could be synthesized for clinical use.
iNeo-Vac-P01 comprises 5–20 peptides at lengths of 15–35amino acids. The peptides were grouped into 2–4 pools (based onHLA typing, affinity, and allele frequency) and then injectedsubcutaneously at a quantity of 0.1 or 0.3 mg per peptide in upperarms and para-umbilical area, respectively. Patients were scheduledto receive iNeo-Vac-P01 with GM-CSF as adjuvant on days 1, 4, 8,15, and 22 (i.e., prime phase), as well as on days 78 and 162 (i.e.,boost phase; refs. 12, 15–18). The adjuvant GM-CSF was injectedsubcutaneously 30 minutes before the administration of iNeo-Vac-P01 at a quantity of 40 mg per injection nearby the injection site ofiNeo-Vac-P01. Additional boost vaccines might be administereddepending on ethics and patients' potential benefit according to theclinical research protocol.
Clinical assessment, monitoring, and follow-up in this researchwere conducted, including physical examination, ECOG performance,vital sign, blood test, and urinalysis to assure the safety of eachimmunization; imaging examination at baseline and approximatelyevery 8 weeks postvaccination to assess clinical efficacy; and enzyme-linked immunospot (ELISpot), T-cell receptor (TCR) sequence, andflow cytometry (T-cell subsets and cytokines) conducted pretreatmentand every 8–12 weeks after treatment for the detection of specificimmune response.
Tumors were assessed by investigators according to RECIST v1.1criterion at baseline and approximately every 8 weeks thereafter.Patients' conditions were monitored while receiving neoantigenvaccine treatment and every 3 months after treatment discontin-uation. The related AEs were recorded and graded for safetyevaluation according to the NCI Common Terminology Criteriafor Adverse Events (CTCAE version 4.0) throughout whole treat-ment period.
Generation of personalized neoantigen vaccinesTo identify mutation-derived neoantigens, some tumor tissues
and blood samples were obtained directly by surgery, while otherswere obtained by biopsy or intravenous blood sampling. Whole-exome sequencing (WES) with coverage depths of 500� for tumorand 100� for blood cells was conducted on these samples usingHiseq 4000 NGS platforms (Illumina; AcornMed BiotechnologyCo., Ltd.; the raw sequence data has been deposited in GenomeSequence Archive under accession number HRA000171 at http://bigd.big.ac.cn/gsa-human; refs. 19–23). In case of unavailability offresh tumor samples, formalin-fixed, paraffin-embedded (FFPE)samples were used instead.
The bioinformatics analysis, which consists modules of sequencingread filtering, genome alignment, mutation calling, HLA typing, MHCaffinity prediction, gene expression profiling, vaccine peptide sequencedesign, and mutation-centered prioritization based on therapeuticpotency, was performed by our in-house pipeline iNeo-Suite (Sup-plementary Materials and Methods; Supplementary Fig. S1).
Translational Relevance
Neoantigens, a class of antigens that derive from tumor-specificmutations, have been long envisioned as optimal targets to distin-guish tumor cells from normal cells while bypass central immunetolerance. The aim of neoantigen immunotherapy is to trainpatient's own immune system to recognize and eliminate cancercells through the use of neoantigens. Thus, the precise identifica-tion of neoantigens becomes the foundation for the success of thisprecision therapy. So far, a few clinical studies have demonstratedthe antitumor potentials of neoantigen-based personalized vac-cines in cancers such as melanoma and glioblastoma. To furtherevaluate the anticancer efficacy of neoantigen vaccine for patientswith advanced solid tumors, we conducted a pan-cancer clinicalstudy of neoantigen vaccine as monotherapy. Our preliminaryresults have proved the feasibility, safety, and efficacy of neoantigenpeptide-based vaccine. In general, our data indicated the greatpotential of iNeo-Vac-P01 as a competitive candidate for furtherdevelopment, alone or in combination with other therapies such ascheckpoint blockade or radiation frequency ablation.
Fang et al.
Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH4512
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
To generate personalized neoantigen vaccines, the customizedclinical grade long peptides were manufactured through chemicalsynthesis at GMP-like standard (bacteria-free, >95.0% purity, andquantities of bacterial endotoxin less than 10 EU/mg). The watersolubility of peptides was tested after synthesis, followed by theremoval of insoluble peptides from iNeo-Vac-P01.
IFNg ELISpot assayTo confirm the immunogenicity of iNeo-Vac-P01, ELISpot assays
were performed for each patient at a series of time points pre- andpostvaccination. Peripheral blood mononuclear cells (PBMC) wereisolated from the peripheral blood (10–30 mL) collected from eachpatient and coincubated (2 � 105 cells per well) with peptides for 16–24 hours using Human IFNg precoated ELISpot kit according to thestandard protocol. An automatic plate reader with appropriate para-meters was used to count the spots in ELISpot plates (SupplementaryMaterials and Methods).
TCR sequenceTo observe the change of T-cell population after vaccination, TCR b
chain was sequenced for each patient before and after vaccination.Peripheral blood (10 mL) was collected from each patient for theextraction of RNA from PBMCs. Samples were analyzed by high-throughput sequencing of TCRusing ImmuHubTCRProfiling Systemat a deep level (ImmuQuad Biotech; Supplementary Materials andMethods).
Multiplexed immunofluorescenceTo examine tumor infiltrated T cells, multiplexed immuno-
fluorescence (IF) was performed by staining 4-mm thick FFPEwhole-tissue sections with standard primary antibodies sequen-tially, and pairing with a unique fluorochrome before DAPIstaining. Slides were air dried, mounted with Prolong DiamondAnti-fade Mounting Medium (#P36965, Thermo Fisher Scientif-ic), and observed with Aperio Versa 8 Tissue Imaging System(Leica). Sample images were analyzed using Indica Halo Software(Version 2.3.2089.52; refs. 24, 25; Supplementary Materials andMethods).
Cytometric analysis of T lymphocyte and cytometric bead arrayanalysis of cytokines
To quantify the activation of T cells after vaccinations, patients'peripheral T cells were extracted and labeled with several anti-bodies for cytometry analysis of the proportions of various types ofT cells. To examine the cytokines secreted from the T cellsactivated by iNeo-Vac-P01, the concentrations of cytokines inpatients' peripheral blood were measured by cytometric beadarray according to the manufacturer's protocol (SupplementaryMaterials and Methods).
Statistical analysisData from the patients who received at least one dose of iNeo-Vac-
P01 were included in the safety and clinical effects analysis. Descriptivestatistics were used to determine the characteristics of baseline andassess vaccine's safety. Disease control rate (DCR) was defined as theproportion of complete response (CR), partial response (PR), and stabledisease (SD) for best clinical response. Standard RECISTv1.1 guidelinewas applied for the analysis of all clinical responses, except for onedata showing apparent pseudoprogression, for which iRECIST wasapplied instead. The survival curves were plotted with GraphPadPrism 5 (v5.01).
ResultsPatients and demographics
A total of 22 patients with cancer with advanced stage of varioustumor types, including non–small cell lung cancer (NSCLC), colorectalcancer, melanoma, pancreatic cancer, biliary tract cancer, ovariancancer, small-cell lung cancer, adrenal sebaceous adenocarcinoma,breast cancer, parotid carcinoma, and gastric carcinoma, were enrolledin the trial fromFebruary 7, 2018. All these patients failed to respond orwere unable to tolerate the standard treatment. The patient demo-graphics, baseline disease characteristics, and previous treatments arepresented in Table 1, indicating that 5 (22.72%) patients had livermetastases, 4 (18.18%) patients had lung metastases, 6 (27.27%)patients had both, and 4 (18.18%) patients had bone metastases(Supplementary Table S1). All the data in this article were collectedand summarized before May 31, 2019.
Feasibility of iNeo-Vac-P01 manufacturing for pan-cancerpatients
WES was conducted on patients' tumor fresh tissue and peripheralblood cells as normal control (Supplementary Table S2). Neoantigenswere predicted and prioritized using our in-house pipeline iNeo-Suite(Supplementary Materials and Methods), which leveraged informa-tion including the allelic frequency of mutation, gene expression, theaffinity between the mutated peptide and HLA complex class I and II,
Table 1. Demographic and disease characteristics at baseline.
Characteristics Patients (N ¼ 22)
Age, yearMean 59 � 10Range 28–79
Age category, no. (%)<65 years 16 (72.73)≥65 years 6 (27.27)
Sex, no. (%)Male 12 (54.55)Female 10 (45.45)
Metastatic sites, no. (%)Liver 5 (22.72%)Lung 4 (18.18%)Both liver and lung 6 (27.27%)Bone 4 (18.18%)
ECOG performance status score, no. (%)a
0 7 (31.82)1 15 (68.18)
Radiotherapy, no. (%)Yes 11 (50.00)No 11 (50.00)
Lines of prior systematic therapy, no. (%)2 7 (31.82)≥3 15 (68.18)
Tumor type, no. (%)Melanoma 4 (18.18)Colon cancer 4 (18.18)NSCLC 3 (13.64)Pancreatic cancer 2 (9.09)Biliary tract cancer 2 (9.09)Ovarian cancer 2 (9.09)Others 5 (22.73)
aECOG performance status scores range from 0 to 5, with 0 indicating nosymptoms and higher scores indicating increasing disability.
Neoantigen Vaccination Trial for Advanced Tumor Patients
AACRJournals.org Clin Cancer Res; 26(17) September 1, 2020 4513
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
as well as the feasibility of peptide synthesis. Clinical grade longpeptides were synthesized at lengths of 15–35 amino acids incorpo-ratingmultiple neoepitopes of bothHLA class I and II (SupplementaryTables S3–S6). The turn-around time of the whole process was mostlybetween 1.5 and 3 months, and no preference in tumor type waswitnessed. In total, 91.7% (22/24) patients received iNeo-Vac-01immunization, while 2 other patients dropped out because of rapiddisease progression.
Although patients enrolled were bearing various types of tumorswith the purity estimated by WES data ranging from 12.4% to 83.7%,sufficient neoantigens were predicted for the successful manufacturingof long peptide vaccines for each patient. To be specific, there was amedian of 14 long peptides immunized, which comprised a median of10 class I neoepitopes and 26 class II neoepitopes per peptide. Mostpatients (17/22) received vaccines containing more than 10 peptides(Supplementary Tables S5 and S7).
Study treatmentEach patient's peptides were pooled into 2–4 groups withmaximum
five peptides per pool. The vaccine was administrated subcutaneouslyin two flanking sites of navel and tail-end of arms followingthe administration of GM-CSF as adjuvant. Peripheral cytokine andT-cell subtypes monitoring, TCR sequencing, and IFNg ELISpot assayin vitro were detected at a series of time points before and aftervaccination according to the study protocol and recorded in a casereport form.
Patients were scheduled to receive iNeo-Vac-P01 with GM-CSF asadjuvant on days 1, 4, 8, 15, and 22 (i.e., prime phase), as well as on days78 and 162 (i.e., boost phase). Additional boost vaccines might beadministered depending on ethics and patients' potential benefit(Fig. 1A). The median duration of follow-up was 9.8 months, rangingfrom 0.9 to 14.5 months before the deadline, May 31, 2019. Twenty-one patients completed prime phase vaccinations except for P014because of the withdrawal of informed consent form. Seventeen of22 patients received additional boost vaccines (Fig. 1A). As shown, themedian duration of treatment was 4.15 months (range, 16 days to12.1 months). Patients P005 and P015 had SD ever since enrolled,while P005 dropped out because of grade 3 acute allergic reactions. Noprogress for 2 patients (P001 and P011) was recorded until death. TillMay 31, 2019, 20 patients had ceased iNeo-Vac-P01, while 2 patients(P015 and P019) were still receiving the assigned treatment.
Safety and tolerabilityDefined by NCI CTCAE 4.03, treatment-related AEs occurred in
54.55% of the patients (Table 2). Most witnessed AEs were in grade 1–2, mainly including fatigue (36.36%), chill (18.18%), and fever(13.64%). However, grade 3–4 treatment-related acute allergic reac-tions were only observed in 2 patients (9.09%) after their sixth round ofboost vaccinations, leading to their drop out from the study. The rest ofthe cases of AE observed throughout the treatment were reversiblewithout particular nursing or treatment. No apparent association wasfound between the presence or the type of AEs and the tumor type. Notreatment-related serious AE (SAE) and death was witnessed.
Immune responseBy performing IFNg ELISpot assay in vitro using autologous PBMCs
after vaccination, T-cell activation induced by iNeo-Vac-P01 wasconfirmed in 19 of 21 patients. Overall, 99 of 125 (79.2%) individuallong peptides and 43 of 51 (84.3%) long peptide pools elicited mea-surable peptide-specific immune response (positive results in ELISpotassay after vaccination) in 11 patients and 10 patients, respectively
(Supplementary Table S8). The IFNg spots per 105 PBMCs of thepeptide or peptide poolwith best response are also been shown (Fig. 1B;SupplementaryTable S8).All thepatients showed statistically significantincrease of ELISpot spots after vaccination (P < 0.05), except patientsP006 andP019 showedno increase after treatmentmainly owning to thenoisy background at baseline (Supplementary Fig. S2). Furthermore,TCR sequencing of peripheral T cells before and after vaccinationrevealed that there were new high abundant clones or clones withconsiderably increased abundance detected posttreatment in 17 (77.3%)or 12 (54.5%) patients (Fig. 1B; Supplementary Table S9).
To detect the activation of both CD8þ and CD4þ T cells in tumormicroenvironment during treatment, multiplexed IF on FFPE sampleswith antibodies of CD4, CD8, and Granzyme B (SupplementaryFig. S3) were applied to analyze the tumor regions of pre- andpostvaccination FFPE samples for 3 patients (P008, P013, andP015). To locate the tumor regions for the analysis, hematoxylin andeosin (H&E) stainingwas performed to analyze the adjacent slices of allmultiplexed IF samples, except the postvaccination sample of P008(H&E staining images obtained from Department of Pathology at SirRunRun ShawHospital, Hangzhou, Zhejiang, China)was used instead(Supplementary Fig. S4). As shown in Fig. 1C, the proportion of bothactivated CD8þ T cells (Granzyme B positive) and activated CD4þ
T cells (Granzyme B positive) were found to be increased aftervaccination in 3 patients (5.5 months postvaccination for P008,7 months postvaccination for P013, and 8 months postvaccinationsfor P015), as well as the densities of these two types of T cells. Thesefindings suggested that iNeo-Vac-P01 had the potential to activatetumor-specific CD8þ T cells and CD4þ T cells, which could subse-quently infiltrate into tumor tissue and generate antitumor effects.
Clinical responseExcept for patient P005, standardRECIST 1.1 criteria were applied to
all other patients during assessment. For P005, iRECIST guideline wasapplied as a result of his pseudoprogression. For 21 of 22 patients, whocompleted five prime immunizations, at least one posttreatment targetlesion was evaluated. Tumor reduction was observed in the assessmentof 8 of 21 patients (38.1%), with a 16.7% maximum reduction of targetlesions compared with baseline (Fig. 2A and B). The calculated DCRwas 71.4% (15/21). In Fig. 2B, 2 patients were excluded because of newlesion appearance (P002) and the progression of nontargeted lesionoccurrence (P006) at first posttreatment assessment, respectively. Fif-teen of 21 patients (71.4%) displayed SD (8 with tumor size reductionranging from 1.9% to 18.2%, 7 with tumor size growth ranging from1.1% to9.0%)with amediandurationof 4.4months (ranging from1.7 to14.5þ months, “þ” indicates some patients maintained SD at cut-offdate). The median PFS of 21 treated patients was 4.6 months [95%confidence interval (CI), 2.5–5.2], and the estimated percentage ofpatients without disease progression at 6th month was 27.3% (95% CI,10.3%–46.8%; Fig. 2C). Seven of all treated patients (N¼ 22) had died.As shown in Fig. 2D, the estimated OS at 12th month was 55.1% (95%CI, 25.9%–76.9%), while the median OS had not been reached yet (95%CI, 9.4–not reached). In particular, no evident differenceswere observedbetween various types of cancer, which indicated that iNeo-Vac-P01 hasbroad spectrum antitumor effects.
Potential biomarkers for clinical responsePatients with SD lasting more than 3 months were considered
to have better response than the others. Several mutant genesand copy-number variants detected by WES were found to bepredictive for better response (Supplementary Fig. S5). The muta-tions of genes MUC (mucins), ZFHX3 (Zinc Finger Homeobox 3),
Fang et al.
Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH4514
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
and ABL1 (ABL Proto-Oncogene 1) seemed to be associated withfaster disease progression and poor response, while copy-numbervariations of genes TNFRSF (tumor necrosis factor receptor super-family), SOX3 (Sex determining region Y-box transcription factor3), and MAGE family members (Melanoma-associated antigens)were relatively associated with better response (SupplementaryFig. S5; Supplementary Results).
The changes of peripheral T cells during treatment might providepotential predictive biomarkers for clinical response because T cellsplay a major role in antitumor response. After treatment, the propor-tions of effector CD8þ T cells to total T cells in patients' peripheral
blood samples all increased to certain extend. Although insignificantlyat most time points after vaccination, the effector CD8þ T cells ofpatients with good response had a relatively higher boost comparedwith those of patients with poor response (Supplementary Fig. S6A).Similarly, the level of IFNg in the patients' peripheral blood samples allincreased at prime phase. Notably, different from patients with poorresponse, patients with better response maintained a relatively highlevel of IFNg at boost phase (Supplementary Fig. S6B). On thecontrary, a continuous increase of IL6 during treatment was observedin the peripheral blood samples of patients with poor response(Supplementary Fig. S6C). These findings implied that the clinical
142 4 6 8 10 12Time since treatment initiation (months)
0
Stable diseaseProgressive disease
Study drop-outDeath
Boost vaccinePrime vaccine
16
Unknown disease
A
B
Res
pons
e ra
te (%
)
C
P002
P003
P004
P005
P006
P007
P008
P009
P 010
P011
P012
P013
P 015
P016
P017
P 018
P019
P 020
P 021
P 02 2
0
20
40
60
80
100
Prevax Postvax
P001
P005P004P008P009P010P012P002P006P013P015P016P017P003P007P018P019P011P020P021P001P022P014
0
50
100
150
200
IFN
γ sp
ots
per 1
05P
BM
Cs
CD8+ GZB+ CD4+ GZB+ Merge
Prevax
Postvax
P015
P008
P013
Prevax
Postvax
Prevax
Postvax
IFNγ spot counts
30
20
10
0
Pro
porti
on (%
)P
ropo
rtion
(%)
Pro
porti
on (%
)
Den
sity
(cou
nts/
mm
2 )D
ensi
ty (c
ount
s/m
m2 )
Den
sity
(cou
nts/
mm
2 )
1,000
800
600
400
200
0
5
4
3
2
1
0
5
4
3
2
1
0
150
100
50
0
150
100
50
0Prevax Postvax
CD4+ GZB+ T cellsCD8+ GZB+ T cells
Figure 1.
iNeo-Vac-P01 induced specific T-cell response andantitumor activation. A, Swimmer plot showed thefollow-up information of enrolled patients (N¼ 22) ina time range of 16 months. Light gray lines and yellowdots indicated the period of prime and the time pointsof boost vaccinations, respectively. The depiction ofdisease condition and patient status were indicatedby lines and dots with various colors. Patient numbersare shown as the values of y-axis. B, For each patientmarked in x-axis, green triangle and red diamondrepresented the relative response rates, which areequal to the ratios of peptides (or peptide pools) withpositive ELISpot results to all immunized peptides (orpeptide pools) before and after vaccination, respec-tively. The bar chart with secondary y-axis representsthe IFNg spots per 105 PBMCs of the peptide orpeptide pool with best response for each patient. C,The multiplexed IF images of FFPE samples obtainedfrom patients P008, P013, and P015 pre- (prevax) andpostvaccination (postvax). CD8 (red in P008 andP013, and pink in P015) andGranzymeB (GZB, yellow)double positive T cells, CD4 (green) and Granzyme B(yellow) double positive T cells, and the mergedsignals are shown on the left. The proportion (%) anddensity (counts per mm2) of the CD8 and Granzyme Bdouble positive T cells and CD4 and Granzyme Bdouble positive T cells are shown on the right.
Neoantigen Vaccination Trial for Advanced Tumor Patients
AACRJournals.org Clin Cancer Res; 26(17) September 1, 2020 4515
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
efficacy might be predicted by the peripheral CTLs' proliferation andactivation (Supplementary Results).
Case report of a patient with advanced hepatic biliary tractcancer
In the case of patient P005, a 63-year-old male, initially diagnosedwith intrahepatic biliary tract cancer in 2013, was treated by surgicalexcision in June 2013 followed by postoperative chemotherapy tillApril 2014. Tumor recurrence and metastases were confirmed by CTscan and pathologic tissue biopsy in April 2017. Then he was treatedwith apatinib for 6 months. He enrolled in a clinical trial of a PD-1antibody (IBI308) for six cycles after failing to respond to chemo-therapy again, and dropped out because of progressive disease (inNovember 2017, on a clinical trial in The First Affiliated Hospital,Zhejiang University in China, Hangzhou, China).
On March 22, 2018, he started to receive iNeo-Vac-P01. Thetreatment scheme is displayed in Fig. 3A, including five primevaccinations and six subsequent boost vaccinations. Compared withstandard treatment scheme with only two boost vaccinations, fourmore boost vaccinations were added under patient's consent, based onthe facts that his symptoms were greatly relieved and the level of atumormarker (CarbohydrateAntigen 72–4)was significantly reduced.After the last boost vaccination, a grade 3–4 acute allergic reactionoccurred along with clinical manifestations: nausea, vomiting, andrash. Radiographic imaging was performed at 2nd, 5th, 8th, 10th, and12thmonths after the first vaccination. The CT scans demonstrated an
Table 2. Treatment-related AEs in all treated patientsa.
Patients (N ¼ 22)Any grade Grades 3–4No. % No. %
Any AE 12 54.55 2 9.09Fatigue 8 36.36 0 0Chill 4 18.18 0 0Fever 3 13.64 0 0Emesis 2 9.09 0 0Muscle soreness 2 9.09 0 0Injection site reaction 1 4.55 0 0Dizzy 1 4.55 0 0Nausea 1 4.55 0 0Upper gastrointestinal hemorrhage 1 4.55 0 0Lose weight 1 4.55 0 0Acute allergic reaction 0 0 2 9.09
aIncluding all patientswho received at least one dose of a trial treatment. Eventswere attributed to treatment by the investigator and are listed as indicated bythe investigator on the case report form.
+
+
+ ++
+
+
++ +
A B
Colon cancer
Melanoma
Pancreatic carcinomaBiliary tract cancer
Others
Lung cancer
Nontarget lesion progression% change truncated to 100%
First occurrence of new lesion
Cha
nge
ofta
rget
lesi
ons
from
base
line
(%)
+
806040
0−20−40
100
0 5 10 15 20 25 30 35 40 45 50 55Time since treatment initiation (weeks)
20
No. at risk
100908070605040302010P
rogr
essi
on-fr
ee s
urvi
val (
%)
0 2 4 6 8 10 12 14 16Time (months)
21 18 13 6 4 4 2 2 1
0 2 4 6 8 10 12 14 16Time (months)
100908070605040302010
Ove
rall
surv
ival
(%)
No. at risk22 22 20 16 15 11 7 3 2
Bes
t clin
ical
resp
onse
(%)
C D
P00
3
P00
4
P00
5
P00
7
P00
8P
009
P01
0
P01
1
P01
2
P01
3
P01
5
P01
6P
017
P01
8
P01
9P
020
P02
1
P02
2
P00
1
806040
0−20−40
100
20
−60−80
−100
Colon cancer
Melanoma
Pancreatic carcinomaBiliary tract cancer
Others
Lung cancer
−60−80
−100
Figure 2.
Clinical response induced by iNeo-Vac-P01.A, Percentage changes of tumor lesion size from baselinewere recorded over a period of 55weeks.B, Thewaterfall plotsrecorded the best clinical response of patients enrolled. Dashed lines above and below indicate 20% increase or 30% reduction of the sum of the longest diameter ofthe tumor, respectively, which are in accordance with the cut-off value for progressive disease and PR by RECIST 1.1. Patient P005 was diagnosed aspseudoprogression according to the change of target tumor lesion. The Kaplan–Meier survival curves with PFS (C) and OS (D). Tick marks represent data censoredat the last time that patient was known to be SD or unknown (C) and alive (D).
Fang et al.
Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH4516
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
evident increase of tumor size (maximum diameter of target lesionswas 122.9 mm) at 5th month compared with baseline (maximumdiameter of target lesions was 89.1 mm), and a significant shrinkage oftumor size (maximum diameter of target lesions was 75.4 mm) at 8thmonth, indicating a pseudoprogression previously occurred (Fig. 3B).His disease continually maintained stable at cut-off date (over14.5 months). PBMCs were tested for the reactivity against theimmunizing peptides. Robust de novo immune response againstpredicted neoantigen (mutant PIGK) was generated after 8-weekvaccination, analyzed using ex vivo IFNg ELISpot (Fig. 3C and D).TCR sequencing of peripheral T cells revealed that the abundance oftwo TCR clones (CASSQDSTGAVNEQFF and CAWSVGKYGDT-QYF) considerably increased after vaccination.Meanwhile, a newTCRclone (CASSFITEAKNIQYD)was detected after vaccination (Fig. 3E).
These data suggest that a subset of T cells with specificities induced byiNeo-Vac-P01 can be successfully activated and kill tumor cells.
Case report of a patient with pancreatic cancerIn the case of patient P008, a 65-year-old female, diagnosed with
pancreatic cancer in July 2016, and later subjected to surgery, pathol-ogy showed low differentiated adenocarcinoma in the tail of herpancreas. A total of six cycles of postsurgery chemotherapy wereconducted based on a gemcitabine regimen. In June 2017, upperabdomenMRI enhancement scan showed relapse with hepatic metas-tases. Soon afterwards, she received chemotherapy and radiofrequencyablation (RFA) for liver metastases.
On April 6, 2018, she started to receive iNeo-Vac-P01, and under-went a total of eight doses of iNeo-Vac-P01, including five prime and
Baseline 5th month 8th month2nd month 10th month
0
2
4
6
8
CASSFITEAKNIQYFCASSQDSTGAVNEQFFCAWSVGKYGDTQYF
0
10
20
30KIAA1191RAB3IPSPEGMAD2L1BPKIF7PIGKTNKS1BP1
IFN
γ sp
ots
per 1
05P
BM
Cs
12th monthB
C D E
A
Pre-treatment Post-treatment
Abu
ndan
ce o
f per
iphe
ral T
cel
ls (%
)
PIGK
KIF7
MAD2L1BP
CEF
DMSO
KIAA1191
RAB3IP
SPEG
TNKS1BP1
PIGK-wild-type
Figure 3.
A case report of patient P005, iNeo-Vac-P01mediated specific T-cell response and tumor regression.A, Figure showed the treatment scheme of patient P005.B, CTscan images showed the series changes of left top lung lesion (top) and left bottom lung lesion (bottom) during the treatment. The red arrows indicate thecorresponding tumor lesions. C andD, Ex vivo IFNg and ELISpot of PBMCswas performedwith peptides at different time points. DMSOwas used as negative controlandmixed peptides from CEF (including peptides of cytomegalovirus, Epstein–Barr virus, and influenza virus) were used as positive control. E, Increased abundanceof peripheral T-cell clones after vaccination was detected by TCR sequencing.
Neoantigen Vaccination Trial for Advanced Tumor Patients
AACRJournals.org Clin Cancer Res; 26(17) September 1, 2020 4517
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
three boost vaccinations. During treatment, the changes of targetlesions were monitored every 2 months. MRI showed liver lesionregression at 2nd and 4th month after vaccination compared withbaseline level (Fig. 4A). Grade 1 and 2 side effects, including chill,fatigue, and muscle soreness were observed and recorded duringregimen. No SAE was noted through the whole treatment period.Ex vivo IFNg ELISpot of PBMCs showed markedly stronger response(except week 18 due to the poor condition of PBMCs) to all peptidepools at week 3 (average 132.8 spots), week 9 (average 213.9 spots), andweek 32 (average 87.1 spots) compared with that of baseline (week 0,average 6.1 spots; Fig. 4B and C). Notably, pretreatment PBMCs also
showed measurable response to all five peptide pools, suggesting thatprevious RFAmight lead to release of tumor neoantigenswhich furtheractivated specific T cells.
DiscussionPatients with various types of advanced solid tumors were enrolled
in this study. Despite the significant differences in their TMBs, forexample, those between P009 (pancreatic cancer) and P011 (ovariancancer) in particular, effective vaccines were designed and preparedfor all patients, following the successful identification of patients'
Figure 4.
A case report of patient P008, iNeo-Vac-P01 mediated specific T-cell response and tumor regression. A, MRI scan was performed at baseline and approximately2months, 4months, after vaccination. The red arrows indicate the target tumor lesions.B andC, Ex vivo IFNg ELISpot of PBMCswas performedwith vaccine peptidesat different time points. DMSO was the negative control and CEF (including peptides of cytomegalovirus, Epstein–Barr virus, and influenza virus) was the positivecontrol.
Fang et al.
Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH4518
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
neoantigens by iNeo-Suite. Here, several different vaccine immuni-zation schemes were applied for the patients' maximum profit andsafety, exploring the impact of peptide dosage (100 mg or 300 mg/peptide) and boost immune cycles (1-month or 2-month boost inter-vals as well as 2–6 boost times) on vaccines' safety and efficacy(Supplementary Table S1). As a result, two cases of acute allergicreaction with similar clinical manifestations occurred in the study,which might result from the increase of boost times, owing to peptide-specific antibody accumulation. This hypothesis needs experimentalvalidation by ELISA. No significant differences in terms of immuno-logic responses, clinical activities, and AEs were observed in this studywhile applying 100 and 300 mg prime doses. However, it was noticedthat patients with more AEs usually had better response, indicating apotential relationship between clinical response and AEs (Supplemen-tary Table S1), which requires further validation.
Although none of the enrolled patients was observed with CR or PR,their DCR was relatively high (71.4%, 15/21), with PFS and OS similarto the reported data of Neon Therapeutics Inc. In addition, the valuesof these indexes could be higher if iRECIST were used instead ofstandard RECIST v1.1 for our data analysis. So far, both advancedpancreatic cancer and biliary tract cancer are known for their poorprognosis and short survival times. For example, the first-line standardtherapy for metastatic pancreatic cancer is nanoliposomal irinotecanplus fluorouracil and folinic acid with median OS of 6.1 months andPFS of 3.1 months (26). However, as of data cutoff, the PFS of ourenrolled 2 patients with pancreatic cancer was 4.2 months and6.3 months, respectively, while their OS was 14.0þ months and13.3þ months, respectively. In addition, the enrolled 2 patients withbiliary tract cancer were under deduced SD for 10.8þ months and14.5þ months, respectively, potentially longer than the 11.7-monthmedian OS of cisplatin/gemcitabine as the first-line standard treat-ment in advanced biliary cancer (27), and these enrolled patients withadvanced pancreatic and biliary tract cancer failed to routine line ofchemotherapy, immunotherapy, or small-molecular target therapy.Although these cases cannot stand for a large cohort, the above datasuggested that the patients with pancreatic cancer and biliary tractcancer in this study might have benefited from our personalizedpeptide vaccine.
In this study, both the median PFS (4.6 months, 95% CI, 2.5–5.2)and OS with a 55.1% estimated percentage at the 12th month (95% CI,25.9%–76.9%). Take some typical clinical trials of cancers with highincidence rates for example. In a phase III clinical trial (CheckMate078) for NSCLC progressed during/after platinum-based doubletchemotherapy, the median OS and PFS of nivolumab (anti-PD-1antibody) treatment were 12.0 months (95% CI, 10.4–14.0) and2.76 months (95% CI, 2.37–3.35), respectively (28). In ATTRAC-TION-2, a phase III trial for patients with advanced gastric or gastro-esophageal junction cancer failed after at least two previous chemo-therapy regimens, the median OS and PFS of nivolumab treatmentwere 5.26months (95%CI, 4�60–6�37) and 1.61months (95%CI, 1.54–2.30), respectively (29). In addition, a phase II study of anti-CTLA-4mAb (tremelimumab) for patients with refractory metastatic colorec-tal cancer demonstrated amedian PFS of 2.3months (95%CI, 2.1–2.6)and a median OS of 4.8 months (95% CI, 4.1–7.7; refs. 30). Althoughthe sample size was small, the personalized neoantigen vaccine iNeo-Vac-P01 had shown promising antitumor efficacy, which needsfurther study with a larger sample size.
Interestingly, besides patient P008 (Fig. 4B and C), 3 other patients(P004, P014, and P019) of the 9 post-RFA patients (44%) alsodisplayed evident neoantigen-specific T-cell response before vaccina-tion, while there was only 1 of 13 patients (8%) in non-RFA group
found having prevaccination response (Fig. 1B; SupplementaryTable S10). It is probably due to the fact that, different from completesurgical resection, RFA treatment can result in tumor necrosis, whichwill become an immunogenic source, providing proinflammatorysignals (31, 32). After RFA, it may activate and/or generate a largeamount of IFNa and/or neoantigen, while improving the expression ofother costimulating factors and the presenting of tumor antigen to Tcells. In addition, the prevaccination responses seemed unrelated withtumor type, position of RFA, or time interval between last RFA andvaccination (Supplementary Table S10). The iNeo-Vac-P01 vaccina-tion post-RFA could form an effective antitumor T-cell response,which is worth studying further.
Furthermore, compared with non-RFA group, RFA group seemedto have a longer survival (unpublished data). Similarly, in the casestudy of patient P005 who received iNeo-Vac-P01 after PD-1 antibodytreatment, we noticed that the combinatorial therapies of PD-1/L1antibodies and personalized neoantigen vaccinemay be able to providebetter anticancer therapeutic effects. More work needs to be done toevaluate the potential benefits from the combinatorial therapies ofiNeo-Vac-P01 with RFA or PD1/L1 antibodies.
In addition, we also identified several potential biomarkers forclinical response, which required further validation. For example, themutations of genes MUC, ZFHX3, and ABL1 seemed to predict poorresponse, whereas the copy-number variations of genes TNFRSF,SOX3, and MAGE were relatively associated with better response.Meanwhile, the increase of peripheral T cells during treatment impliedpatients' better response. More patients would be enrolled in futurestudy to validate our hypothesis.
In general, the preliminary results demonstrated the feasibility,safety, and efficacy of iNeo-Vac-P01 on patients with various typesof advanced solid tumors. iNeo-Vac-P01 monotherapy could elicitrobust neoantigen-specific T-cell response, significantly increaseT-cell infiltration into tumors, and remodel tumor immune micro-environment alone, suggesting its great potential as cancer immu-notherapy. Until now, 22 patients have participated in this clinicaltrial, more than all of the previous reported patient numbers that wefound in the similar kind of clinical trials. In future, we plan toenroll more patients with early- and intermediate-stage cancers,including those who require recurrence prevention after surgery,making further efforts to explore the antitumor and antirecurrenceefficacy of iNeo-Vac-P01. The combination of iNeo-Vac-P01 withRFA and/or anti-PD-1/L1 antibody is worth further exploring in thefuture for patients with advanced cancer, especially oligometastaticpatients.
Disclosure of Potential Conflicts of InterestF. Mo is an employee for Hangzhou Neoantigen Therapeutics Co., Ltd and
HangzhouAl-Force Therapeutics. H.Wang is an employee forHangzhouNeoantigenTherapeutics Co., Ltd. K. Luo is an employee for Hangzhou Neoantigen TherapeuticsCo., Ltd. N. Han is an employee for Hangzhou Neoantigen Therapeutics Co., Ltd.H. Li is an employee of Sir Run Run Shaw Hospital, Zhejiang University School ofMedicine. R. Chen is an employee for Hangzhou Neoantigen Therapeutics Co., Ltd.L. Chen is an employee for Hangzhou Neoantigen Therapeutics Co., Ltd. L. Liu is anemployee forHangzhouNeoantigenTherapeutics Co., Ltd. S. Chen is an employee forHangzhou Neoantigen Therapeutics Co., Ltd. No potential conflicts of interest weredisclosed by the other authors.
Authors’ ContributionsConception and design: Y. Fang, F. Mo, J. Shou, L. Liu, H. Pan, S. ChenDevelopment of methodology: Y. Fang, F. Mo, H. Wang, K. Luo, S. Zhang, Z. Zhou,L. Liu, H. Wang, H. Pan, S. ChenAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Y. Fang, J. Shou, H. Wang, H. Li, L. Liu, H. Pan
Neoantigen Vaccination Trial for Advanced Tumor Patients
AACRJournals.org Clin Cancer Res; 26(17) September 1, 2020 4519
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Mo, J. Shou, H. Wang, K. Luo, S. Ye, R. Chen, L. LiuWriting, review, and/or revision of the manuscript: Y. Fang, F. Mo, J. Shou,H. Wang, N. Han, L. Chen, L. Liu, H. Pan, S. ChenAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): F. Mo, H. Wang, K. Luo, N. Han, R. Chen, L. Liu, H. PanStudy supervision: Y. Fang, J. Shou, H. Wang, S. Zhang, L. Liu, S. Chen
AcknowledgmentsThe authors thank all patients who participated in the trial and their families, as
well as Sir Run Run ShawHospital as clinical site. The authors also thank Dr. Ye Fengfor suggestions on statistical analysis. This work was supported by National KeyResearch and Development Program of China (2017YFC0908600 to S. Chen),
National Natural Science Foundation of China (81430081 to S. Chen, and81772543 and 81572592 to H. Pan), Zhejiang Medical Innovative Discipline Con-struction Project-2016 (to H. Pan), Project of Health Commission of ZhejiangProvince (2016151029 and 2017197380 to Y. Fang), and Natural Science Foundationof Zhejiang Province (LY17H160029 to J. Shou).
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
Received November 29, 2019; revised March 18, 2020; accepted May 14, 2020;published first May 21, 2020.
References1. Rizvi NA, Mazieres J, Planchard D, Stinchcombe TE, Dy GK, Antonia SJ, et al.
Activity and safety of nivolumab, an anti-PD-1 immune checkpoint inhibitor, forpatientswith advanced, refractory squamousnon-small-cell lung cancer (Check-Mate 063): a phase 2, single-arm trial. Lancet Oncol 2015;16:257–65.
2. Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, et al. MPDL3280A(anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer.Nature 2014;515:558–62.
3. El-KhoueiryAB, Sangro B, YauT, Crocenzi TS, KudoM,HsuC, et al. Nivolumabin patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet2017;389:2492–502.
4. Muro K, Chung HC, Shankaran V, Geva R, Catenacci D, Gupta S, et al.Pembrolizumab for patients with PD-L1-positive advanced gastric cancer(KEYNOTE-012): a multicentre, open-label, phase 1b trial. Lancet Oncol2016;17:717–26.
5. Topalian SL, SznolM,McDermott DF, Kluger HM, Carvajal RD, SharfmanWH,et al. Survival, durable tumor remission, and long-term safety in patients withadvanced melanoma receiving nivolumab. J Clin Oncol 2014;32:1020–30.
6. Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunother-apy for human cancer. Science 2015;348:62–8.
7. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, et al.Cancer immunology. Mutational landscape determines sensitivity to PD-1blockade in non-small cell lung cancer. Science 2015;348:124–8.
8. Anagnostou V, Smith KN, Forde PM, Niknafs N, Bhattacharya R, White J, et al.Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer. Cancer Discov 2017;7:264–76.
9. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science2015;348:69–74.
10. Coulie PG, Van den Eynde BJ, van der Bruggen P, Boon T. Tumour antigensrecognized by T lymphocytes: at the core of cancer immunotherapy. Nat RevCancer 2014;14:135–46.
11. Hu Z, Ott PA, Wu CJ. Towards personalized, tumour-specific, therapeuticvaccines for cancer. Nat Rev Immunol 2018;18:168–82.
12. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, et al. An immunogenicpersonal neoantigen vaccine for patients with melanoma. Nature 2017;547:217–21.
13. Sahin U, Derhovanessian E, Miller M, Kloke BP, Simon P, Lower M, et al.Personalized RNA mutanome vaccines mobilize poly-specific therapeuticimmunity against cancer. Nature 2017;547:222–6.
14. Hilf N, Kuttruff-Coqui S, Frenzel K, Bukur V, Stevanovic S, Gouttefangeas C,et al. Actively personalized vaccination trial for newly diagnosed glioblastoma.Nature 2019;565:240–5.
15. Keskin DB, Anandappa AJ, Sun J, Tirosh I, Mathewson ND, Li S, et al.Neoantigen vaccine generates intratumoral T cell responses in phase Ib glio-blastoma trial. Nature 2019;565:234–9.
16. Weden S, Klemp M, Gladhaug IP, Moller M, Eriksen JA, Gaudernack G, et al.Long-term follow-up of patients with resected pancreatic cancer followingvaccination against mutant K-ras. Int J Cancer 2011;128:1120–8.
17. Gjertsen MK, Buanes T, Rosseland AR, Bakka A, Gladhaug I, Soreide O, et al.Intradermal ras peptide vaccination with granulocyte-macrophage colony-
stimulating factor as adjuvant: clinical and immunological responses in patientswith pancreatic adenocarcinoma. Int J Cancer 2001;92:441–50.
18. Kirner A, Mayer-Mokler A, Reinhardt C. IMA901: a multi-peptide cancervaccine for treatment of renal cell cancer. Hum Vaccin Immunother 2014;10:3179–89.
19. Bais P,Namburi S, Gatti DM, ZhangX, Chuang JH. CloudNeo: a cloud pipeline foridentifying patient-specific tumor neoantigens. Bioinformatics 2017;33:3110–2.
20. Hundal J, Miller CA, Griffith M, Griffith OL, Walker J, Kiwala S, et al. Cancerimmunogenomics: computational neoantigen identification and vaccine design.Cold Spring Harb Symp Quant Biol 2016;81:105–11.
21. Schenck RO, Lakatos E, Gatenbee C, GrahamTA, AndersonARA.NeoPredPipe:high-throughput neoantigen prediction and recognition potential pipeline.BMC Bioinformatics 2019;20:264.
22. Tappeiner E, Finotello F, Charoentong P, Mayer C, Rieder D, Trajanoski Z.TIminer: NGS data mining pipeline for cancer immunology and immunother-apy. Bioinformatics 2017;33:3140–1.
23. Torres-Garcia W, Zheng S, Sivachenko A, Vegesna R, Wang Q, Yao R, et al.PRADA: pipeline for RNA sequencing data analysis. Bioinformatics 2014;30:2224–6.
24. Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Liesko-van S, et al. Mutations associated with acquired resistance to PD-1 blockade inmelanoma. N Engl J Med 2016;375:819–29.
25. Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. VISTA is aninhibitory immune checkpoint that is increased after ipilimumab therapy inpatients with prostate cancer. Nat Med 2017;23:551–5.
26. Wang-Gillam A, Li CP, Bodoky G, Dean A, Shan YS, Jameson G, et al.Nanoliposomal irinotecan with fluorouracil and folinic acid in metastaticpancreatic cancer after previous gemcitabine-based therapy (NAPOLI-1): aglobal, randomised, open-label, phase 3 trial. Lancet 2016;387:545–57.
27. LamarcaA,Hubner RA,DavidRyderW,Valle JW. Second-line chemotherapy inadvanced biliary cancer: a systematic review. Ann Oncol 2014;25:2328–38.
28. Wu YL, Lu S, Cheng Y, Zhou C, Wang J, Mok T, et al. Nivolumab versusdocetaxel in a predominantly chinese patient population with previously treatedadvancedNSCLC: CheckMate 078 Randomized Phase III Clinical Trial. J ThoracOncol 2019;14:867–75.
29. KangYK,BokuN, SatohT, RyuMH,ChaoY,KatoK, et al. Nivolumab in patientswith advanced gastric or gastro-oesophageal junction cancer refractory to, orintolerant of, at least two previous chemotherapy regimens (ONO-4538–12,ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3trial. Lancet 2017;390:2461–71.
30. Chung KY, Gore I, Fong L, Venook A, Beck SB, Dorazio P, et al. Phase II study ofthe anti-cytotoxic T-lymphocyte-associated antigen 4 monoclonal antibody,tremelimumab, in patients with refractory metastatic colorectal cancer. J ClinOncol 2010;28:3485–90.
31. McGahan JP, Brock JM, Tesluk H, GuWZ, Schneider P, Browning PD. Hepaticablation with use of radio-frequency electrocautery in the animal model. J VascInterv Radiol 1992;3:291–7.
32. Widenmeyer M, Shebzukhov Y, Haen SP, Schmidt D, Clasen S, Boss A, et al.Analysis of tumor antigen-specific T cells and antibodies in cancer patientstreated with radiofrequency ablation. Int J Cancer 2011;128:2653–62.
Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH4520
Fang et al.
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881
2020;26:4511-4520. Published OnlineFirst May 21, 2020.Clin Cancer Res Yong Fang, Fan Mo, Jiawei Shou, et al. Solid TumorsMonotherapy in Treating Patients with Various Types of Advanced A Pan-cancer Clinical Study of Personalized Neoantigen Vaccine
Updated version
10.1158/1078-0432.CCR-19-2881doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2020/05/21/1078-0432.CCR-19-2881.DC1
Access the most recent supplemental material at:
Cited articles
http://clincancerres.aacrjournals.org/content/26/17/4511.full#ref-list-1
This article cites 32 articles, 7 of which you can access for free at:
Citing articles
http://clincancerres.aacrjournals.org/content/26/17/4511.full#related-urls
This article has been cited by 1 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/26/17/4511To request permission to re-use all or part of this article, use this link
on April 26, 2021. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 21, 2020; DOI: 10.1158/1078-0432.CCR-19-2881