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e16264 Publication Only A pilot trial of personalized neoantigen pulsed autologous dendritic cell vaccine as adjuvant treatment in hepatocellular carcinoma. Qiuji Wu, Qiu Li; Cancer Center, West China Hospital, Sichuan University, Chengdu, China Background: The primary treatment for hepatocellular carcinoma (HCC) is radical surgery. However, HCC has a high propensity to recur postoperatively and no standard adjuvant ther- apies have been approved. This Phase I trial aims to evaluate the safety and efficacy of an adjuvant personalized neoantigen pulsed autologous dendritic cell vaccine (Neo-DCVac-02) inpatients with HCC. Methods: Eligible patients had histologically confirmed high-risk HCC after radical surgery. After obtaining the necessary baseline biopsy specimens and PBMCs of ap-propriate quality for DNA and RNA sequencing, neoantigen prediction, screening and synthesiswere performed. Subsequently, leukapheresis was performed and DCs were isolated and cul-tured. DCs were then pulsed with neoantigen peptides to produce Neo-DCVac-02. On day 0,patients were treated with cyclophosphamide at a dose of 250 mg/m2. The prepared Neo- DCVac-02 vaccine was administered subcutaneously to the axillary and inguinal regions bi- laterally on day 1, followed by daily administration of GM-CSF at a dose of 0.075 mg for 5 days(days 2-6). The primary objectives of this study were to assess feasibility and safety/tolerability.Secondary objectives included evaluation of immune responses (via vaccine response: IFN g ELISpot), relapse-free survival (RFS) and overall survival (OS). Results: A total of 32 patients provided informed consent to initiate the process of personalized neoantigen discovery, ofwhich 26 (81%) met the requirements for successful product selection for clinical manufactur-ing. Feasibility was demonstrated with 13 patients receiving Neo-DCVac-02. 13 patients did not receive the neoantigen vaccine (7 due to progressive disease, 6 due to withdrawal of informed consent because of COVID-19). The treatment was well tolerated with no grade 2+ treatment-related adverse events (TRAEs). The most common Grade 1 TRAEs were injection site reactions(75%), rash (16.7%), fatigue (8.3%), neutropenia (8.3%), and headache (8.3%). Five of 13evaluable patients showed increased antigen-specific T-cell activity and more than 50% of theneoantigen peptides in Neo-DCVac-02, and sustained cellular responses were observed up toone year. Increases in T-cell activation/co-stimulation seen after treatment with Neo-DCVac-02, as demonstrated by flow cytometric analysis, suggest immune priming. At a median follow- up of 29.7 months, median RFS was not reached, with 1- and 2-year RFS of 84.6% and 60%, respectively, and all patients are alive. Patients with a strong immune response had a longermedian relapse-free survival (not reached) compared to patients with a weak immune response(17.6 months, P = 0.003). Conclusions: These data demonstrate that Neo-DCVac-02 is safe, well tolerated and capable of inducing durable antigen-specific lymphocyte immune responses thatmay correlate with delayed HCC recurrence. Clinical trial information: NCT04147078.

ResearchSponsor: The China Postdoctoral Science Foundation.GASTROINTESTINAL CANCER —GASTROESOPHAGEAL, PANCREATIC, AND HEPATOBILIARY Downloaded from ascopubs.org by 190.236.76.167 on December 10, 2024 from 190.236.076.167 Copyright © 2024 American Society of Clinical Oncology. All rights reserved.

Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 1 of 24 04MAY2018 ABBREVIATED FINAL ANALYSIS STATISTICAL ANALYSIS PLAN Aduro Biotech, Inc. Protocol Number: ADU- CL-02 A Phase 1B Study to Evaluate the Safety and Induction of Immune Response of CRS- 207 in Combination with Pemetrexed and Cisplatin as Front -line Therapy in Adults with Malignant Pleural Mesothelioma Protocol Number: ADU- CL-02 Protocol Version: Version 7, 15 November 2016 Name of Test Drug(s): CRS -207 Phase: 1B Sponsor: Aduro Biotech, Inc. 740 Heinz Avenue Berkeley, CA 94710 SAP Author: SAP D ate: 04MAY2018 SAP Version: Abbreviated Version Notice of Confidential and Proprietary Information: The information contained in this document is confidential belonging to Aduro Biotech, Inc.

Acceptance of this document constitutes agreement by the recipient that no information contained herein will be published or disclosed without prior written authorization from an official of Aduro Biotech, Inc. However, this document m ay be disclosed to appropriate Institutional Review Board and Ethics Committees or duly authorized representatives of a national regulatory authority under the condition that they are requested to keep it confidential. In the event of an actual or suspected breach of this obligation, Aduro Biotech, Inc. should be notified promptly. NCT Number: NCT01675765 Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 3 of 24 04MAY2018 TABLE OF CONTENTS Section Page SIGNATURE PAGE ................................ ................................

............................... 2 TABLE OF CONTENTS ........................................................................................ 3 ABBREVIATIONS .................................................................................................. 5 1. INTRODUCTION AND OBJECTIVES OF ANALYSIS ........................ 7 1.1. Introduction ...................................................................................

7 1.2. Objectives of the Abbreviated Statistical Analysis Plan (ASAP) ............................................................................................. 7 2. STUDY OBJECTIVES ............................................................................... 8 2.1. Primary Objectives ......................................................................... 8 2.2. Secondary Objectives ...................................................................... 8 2.3. Exploratory Objectives ................................................................... 8 3. STUDY DESIGN ......................................................................................... 9 3.1. Synopsis of Study Design ......................................................... 9 3.2. Randomization and Blinding ................................................... 10 3.3. Study Procedures ...................................................................... 10 3.4. Study Endpoints ......................................................................... 10 3.4.1. Primary Endpoints ........................................................... 10 3.4.2. Secondary Endpoints ....................................................... 10 3.4.3. Exploratory Endpoints ..................................................... 11 4. SUBJECT POPULATIONS .................................................................... 12 4.1. Population Definitions .............................................................. 12 4.2. Protocol Deviations / Violations ............................................. 12 5. STATISTICAL METHODS ...................................................................... 13 5.1. Sample Size Justification ......................................................... 13 5.2. Statistical Methods .................................................................... 13 5.2.1. General Methods .............................................................. 13 5.2.2. Definitions ........................................................................ 14 5.2.3. Adjustments for Covariates .............................................. 15 5.2.4. Multiplicity ...................................................................... 15 5.2.5. Subgroup Analyses .......................................................... 15 Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 4 of 24 04MAY2018 Section Page 5.2.6. Interim Analyses ................................

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15 5.2.7. Missing, Unused and Incomplete Data ............................ 15 5.3. Timing of Analyses .................................................................... 16 5.4. Subject Disposition ................................................................... 16 5.5. Demographic and Baseline Characteristics ....................... 16 5.6. Prior Anti -Cancer Therapies .................................................... 17 5.7. Medical History ........................................................................... 17 5.8. Concomitant Medications ........................................................ 17 5.9. Other Assessments ................................................................... 17 5.10. Treatment Exposure .................................................................. 17 5.11. Efficacy Evaluation .................................................................... 18 5.11.1. Tumor Evaluations and Overall Tumor Response ........... 18 5.11.2. Exploratory Endpoints ..................................................... 18 5.12. Safety Analyses .......................................................................... 18 5.12.1. Adverse Events ................................................................ 18 5.12.2. Deaths .............................................................................. 19 5.12.3. CRS -207 Infusion- related Reactions ............................... 19 5.12.4. Dose Limiting Toxicities ................................................. 20 5.12.5. Laboratory Data ............................................................... 20 5.12.6. Vital Signs and Physical Examinations ........................... 20 5.12.7. Electrocardiogram ............................................................ 21 5.12.8. ECOG Performance Status .............................................. 21 6. CHANGES TO PLANNED ANALYSES ............................................... 22 7. REFERENCES ......................................................................................... 23 8. LIST OF TABLES, FIGURES AND LISTINGS FOR FINAL ANALYSIS ................................................................................................ 24 Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 5 of 24 04MAY2018 ABBREVIATIONS Abbreviation Definition AE Adverse Events ALT Alanine aminotransferase ANC Absolute neutrophil count AST Aspartate aminotransferase ATC Anatomic Therapeutic Chemical BMI Body Mass Index BSA Body Surface Area BV Boost Vaccination CFU Colony -forming units CI Confidence Interval CSR Clinical Study Report CR Complete Response CRF Case Report Form Cy Cyclophosphamide DCR Disease control rate DLT Dose L imiting Toxicity DMC Data Monitoring Committee DPFU Diseas e Progression Follow -Up Period ECG Electrocardiogram ECOG Eastern Cooperative Oncology Group ELISPOT Enzyme -Linked Immunosorbent Spot EOC End of Course EOS End of Study FAS Full Analysis Set FDA Food and Drug Administration FEV1 Forced Expiratory Volume in 1 Second FVC Forced Vital Capacity HLA Human leukocyte antigen ICH International Council on Harmonisation IFN-γ Interferon gamma irRC Immune -Related Response Criteria iDAP Immune Data Analysis Plan MedDRA Medical Dictionary for Regulatory Activities MPM Malignant Pleural Mesothelioma mRECIST Modified Response Evaluation Criteria in Solid Tumors MV Maintenance Vaccinations NCI CTCAE National Cancer Institute Common Toxicity Criteria for Adverse Ev ents OS Overall Survival PBMC Peripheral blood mononuclear cell (s) PD Progressive Disease PFS Progression -free Survival PFTs Pulmonary Function Tests PR Partial Response PT Preferred Term Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 6 of 24 04MAY2018 Abbreviation Definition PV Prime Vaccination RBC Red blood cell RECIST Response Evaluation Criteria in Solid Tumors SAE Serious Adverse Event SAP Statistical Analysis Plan SD Stable Disease , Standard Deviation SI International System of Units SOC System Organ Class TEAE Treatment -emergent Adverse Event TESAE Treatment -emergent Serious Adverse Events TTP Time to Progression ULN Upper limit of normal VC Vital Capacity WBC White blood cell WHO World Health Organization Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 7 of 24 04MAY2018 1. INTRODUCTION AND OBJ ECTIVES OF ANALYSIS 1.1. Introduction ADU- CL-02 is a phase 1b, multicenter, open- label study in adults with malignant pleural mesothelioma (MPM) who are not eligible for curative surgery. The study consists of two cohorts which received CRS- 207 in combination with pemetrexed and cisplatin chemotherapy (Cohort 1) or low -dose cyclo phosphamide (Cy) prior to each CRS -207 infusion (Cohort 2). The study is intended to determine the safety of CRS -207 (with or without Cy) when administered in combination with pemetrexed and cisplatin and to evaluate the induction of immune response to mesothelin. 1.2. Objectives of the Abbreviated Statistical Analysis Plan (ASAP) This is an abbreviated statistical analysis plan ( ASAP) designed to outline the methods to be used in the analysis of study data for the abbreviated final analysis. The derivation and analysis of selected immunological / tumor marker endpoints will be discussed in another standalone document. The statistical analyses and summary tabulations described in this A SAP will provide the basis for the abbreviated reporting of the final anal ysis results from this trial . Populations for analysis, data handling rules, statistical methods, changes from the study protocol , and formats for data presentation are provided. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 8 of 24 04MAY2018 2. STUDY OBJECTIVES 2.1. Primary Objectives To determine the safety of CRS -207 (with or without low -dose cyclophosphamide [Cy] given one day prior to CRS- 207) when administered in combination with pemetrexed and cisplatin and to evaluate the induction of immune response to mesothelin as measured by IFN -γ ELISPOT assay prior to treatment and at time points during and after treatment . 2.2. Secondary Objectives The secondary objectives are to evaluate: • Tumor response • Progression free survival • Time to progression • Overall survival • Predictive value of serum mesothelin for therapeutic response for each treatment regimen 2.3. Exploratory Objectives The exploratory objectives are to: • conduct immune subset analysis (e.g., CD4, CD8, T reg) and gene expression profiling of tumor tissue pre- and post -vaccination; • to assess induction of anti -mesothelin humoral immune response; • to measure tumor marker kinetics as biomarkers of tumor response; • evaluate the association between pulmonary function improvement and tumor response; • evaluate the relationship of tumor response to overall survival. This ASAP will not include the analysis of : immune response to mesothelin, predictive value of serum mesothelin; or any secondary and exploratory objectives. The analysis of mesothelin related objectives will be discussed in another standalone document. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 9 of 24 04MAY2018 3. STUDY DESIGN 3.1. Synopsis of Study D esign Up to 16 subjects were originally planned to be enrolled in this study. Up to an additional 44 subjects were then subsequently planned to be enrolled in this study (for a total of up to 60 subjects) to obtain additional safety, immune, and efficacy data. Subjects were enrolled into two mutually exclusive cohorts described below. Cohort 1: A total of 38 subjects receive d 2 prime vaccinations (PV) of CRS -207 (1 × 109 colony -forming units ( CFU) given intravenously (IV) over approximately 1 hour) 2 weeks apart , followed 2 weeks later by up to 6 cycles of pemetrexed and cisplatin 3 weeks apart. Three weeks after completion of chemotherapy, subjects received an additional 2 infusions (boost vaccinations; BV) of CRS- 207 3 weeks apart. Subjects enrolled in Cohort 1 included the 16 subjects originally planned plus a minimum of the first 16 subjects additionally enrolled. Cohort 2: The remaining subjects enrolled ( 22 total) received CRS- 207 in combinati on with chemotherapy at the dose and schedule described above; however, these subjects also received low-dose Cy (200 mg/m2) over 30 minutes 1 day prior to each CRS -207 infusion (i.e., prior to each PV and BV infusion). All subjects were to return to the clinic 4 weeks after their 2nd boost vaccination for an End of Course (EOC) visit. Subjects then had follow- up visits 4 weeks after the EOC visit and every 8 weeks thereafter until treatment discontinuation. Subjects continued to receive maintenance vaccinations (MV) with (Cohort 1) or without Cy (Cohort 2) at each follow- up visit ( following their original schedule) if they were clinically stable and continued to meet dosing eligibility. Subjects could continue on treatment with radiographic disease progression if clinically stable and the investigator believed the treatment may be providing benefit. Subjects were to return to the clinic for follow -up approximately 4 weeks after their EOC visit and every 8 weeks thereafter until disease progression or the investigator determined the subject was no longer receiving benefit from treatment (if a subject continues treatment beyond progression). All subjects were to complete an End of Study ( EOS) visit no more than 4 weeks following the final dose of study medication or prior to receipt of other cancer -related treatment. Following participation in this study, subjects could consent to long- term follow -up in the ADU- CL-03 study , which would follow them every 3 months until death. Death was captured from public record for subjects who did not enroll in ADU -CL-03. To monitor initial safety of the sequential vaccine regimen, no more than one subject was enrolled per week for the first six subjects. If at any point during the study more than 33% of subjects , cumulat ive, experienced a dose limiting toxicity (DLT), the dose would have been lowered from 1 x 109 CFU to 3 x 108 CFU for all subsequent dosing with CRS- 207. Subjects already receiving treatment would have continued to receive CRS- 207 dosing at the lower dose and all subsequent subjects would have received CRS- 207 at the lower dose. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 10 of 24 04MAY2018 Subjects who withdr ew consent or were removed from study medication before completing at least two cycles of chemotherapy were considered dropouts and replaced at the discretion of the lead investigator, sponsor, and medical monitor. 3.2.

Randomization and Blinding This is a non- randomized, open- label study. Subjects were assigned to each cohort sequentially. 3.3. Study Procedures The schedule of assessments is outlined in Section 5.1 of the study protocol. 3.4.

Study Endpoints 3.4.1. Primary Endpoint s The primary efficacy endpoint is the change in number of mesothelin- specific T cells producing interferon gamma ( IFN-γ) by ELISPOT ( enzyme- linked immunosorbent spot) assay from baseline to: • immediately after CRS- 207 (with or without Cy); • after receiving chemotherapy; • and after receiving two subsequent doses of CRS -207 (with or without Cy) following chemotherapy. Safety will be assessed by evaluation of the fol lowing : • Adverse events (AEs) • Deaths • Vital signs • Physical examination findings • Clinical chemistry and hematology laboratory findings 3.4.2. Secondary Endpoints The following secondary endpoints will be used to assess efficacy: • Objective tumor response • Progression free survival (PFS) • Time to progression (TTP) • Overall survival (OS) • Measure of serum mesothelin to assess predictive value for therapeutic response Tumor response and progression will be assessed using modified Response Evaluation Criteria in Solid Tumors (mRECIST )3 for assessment of response in MPM and irRC4. This ASAP will not include the analysis of primary or secondary efficacy endpoints. Tumor and overall response data, based on mRECIST only, will be listed (irRC based Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 11 of 24 04MAY2018 outcomes will not be provided); the handling of the primary endpoint will be as described in Section 2 and Section 3.4.3. 3.4.3. Exploratory Endpoints The exploratory endpoints described in this analysis plan are: • Pulmonary function tests (PFTs) measured by forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1) and vital capacity (VC) [for Cohort 2 only] Additional endpoints, corresponding to exploratory objectives, will be evaluated based on a separate, standalone document . This ASAP will not include the analysis of the exploratory endpoints. PFTs will be listed as described in Sectio n 5.11.2. The analysis of the other exploratory objectives will be discussed in another standalone document . Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 12 of 24 04MAY2018 4. SUBJECT POPULATIONS 4.1. Population Definitions The following subject populations will be evaluated and used for presentation and analysis of the data: • Full Analysis Set (FAS): A ll enrolled subjects who received at least one dose of study treatment . The FAS will be used for all analys es of efficacy and safety . The final SAP version 1.0 used FAS as the analysis dataset. This definition is the same as the definition of the Safety Analysis Set. The FAS now is replaced by the Safety Analysis Set. 4.2.

Protocol Deviations / Violations Protocol deviations will be identified and classified as major (violations) before the database is locked. Major protocol deviations may include but are not limited to: • Received the wrong study medication • Dose not properly administered, including o Administrations in which Cy was not administered within 24 hour s of CRS- 207 o Administrations in which protocol required pre- medications were not given • Did not complete at least 2 chemotherapy cycles • Received prohibited concomitant medications • Violation of inclusion or exc lusion criteria , including o Misdiagnosis Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 13 of 24 04MAY2018 5.

STATISTICAL METHODS 5.1. Sample Size Justification Up to 60 subjects in total could be enrolled, based on the original planned cohort of 16 subjects and the study was then expanded to enroll up to an additional 44 subj ects. A total of 32 subjects were planned for Cohort 1 (CRS- 207 in combination with chemotherapy ) (Cohort 1), and up to 28 subjects were planned for Cohort 2 (Cy 1 day prior to each CRS -207 administration combined with chemotherapy ). Ultimately, a total of 38 subjects were in Cohort 1, and 22 subjects were in Cohort 2 (for a total of 60 subjects). The originally planned sample size calculation was based on the primary endpoint of mesothelin -specific T cell responses as measured by ELISPOT (enzyme- linked immunosorbent spot) assay . With one primary parameter (ELISPOT) to be measured with respect to a change from baseline at three post -baseline time points, the sample size was selected to allow each test to be performed using a 0.017 two- tailed significance level, in order to allow the overall set of three tests to be very conservatively performed as if at an overall 0.05 level using a Bonferroni adjustment. Assuming 14 subjects with complete measurements of the main parameter at baseline and the three subsequent time points, there would be 80% power to detect a change from baseline to each time point equal to 1.0 standard deviations of the change (1.0 effect size) using a two -tailed 0.017 level paired t -test. This stringent multiplicity adjustment is for power computation only. In practice, for analysis purposes, instead of requiring that each test achieve a 0.017 level in order to be declared significant, a less overly stringent Hochberg adjustment will be used. In order to allow for a small number of non- evaluable subjects, up to 16 subjects could be e nrolled. The planned expanded cohort of up to 44 additional subjects (Cohort 2) was intended to obtain additional safety, immune, and efficacy data for future study planning. The sample size was based on practical rather than statistical considerations.

5.2.

Statistical Methods 5.2.1. General Methods • All statistical analysis and data summarization will be generated using SAS version 9.3 or later . • Unless otherwise stated, summary tables will be presented by treatment regimen (cohort) and overall . • No formal comparisons across treatment regimen (Cohort 1 vs. Cohort 2) will be performed. • Confidence intervals (CIs) will be presented at the 95% significance level • Continuous variables will be summarized using the number of observations (n), mean, standard deviation (SD), median, minimum, first quartile, third quartile, and maximum . Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 14 of 24 04MAY2018 • Categorical variables will be summarized using number of observations (n), frequency and percentages of subjects (percentages will not be shown when cell count is zero). • AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA), version 15.0. • Concomitant medications will be coded using World Health Organization (WHO) Drug V ersion 1Q 2012. 5.2.2. Definitions • Age: (Informed Consent Date – Date of Birth)/365.25, rounded down to the nearest integer • First Dose Date: Date of first exposure to any study medication ( CRS- 207, Cy, or chemotherapy) • Last Dose Date: The last date which the subject was exposed to any study medication as recorded on the case report form ( CRF) • Study Day : Elapsed time from First Dose Date measured in days and defined as: • Study Day = Event Date – First Dose Date + 1, for events occurring on or after the First Dose Date • Study Day = Event Date – First Dose Date, for events occurring before the First Dose Date • Baseline: Last observation prior to f irst dose of study medication • Treatment Course 1: T reatment Course 1 is defined as the sequence of two CRS- 207 prime vaccinations (with or without low -dose Cy); chemotherapy treatments (up to six cycles); two post -chemotherapy boost vaccinations; EOC follow- up • End of Course : End of the treatment course for subjects that do not receive MV; for subjects that receive MV, the visit following the last administration of MV • End of Study: May occur when a subject discontinues the study. The EOS visit is to take place 28 days (4 weeks) after last dose of study medication. • Study Periods: • Treatment Period: The Treatment Period includes the time from first dose through the EOS safety visit occurring 28 days after the final study medication dose. • Duration of Treatment Exposure: Latest of (Date of EOS visit, Last Dose Date + 28) – First Dose Date + 1. It is assumed that EOS visit occurs approximately 28 days after the final dose of study medication; the EOS visit represents the latest date for which a new AE can be observed/recorded for a subject. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 15 of 24 04MAY2018 • Percentage Change from Baseline in Tumor Thickness per mRECIST : (total tumor thickness at post -baseline visit – baseline total tumor thickness) / (baseline total tumor thickness) 5.2.3. Adjustments for Covariates Not applicable.

5.2.4. Multiplicity Not applicable. 5.2.5. Subgroup Analyses Subgroup analyses are not planned to be performed for the final analysis. 5.2.6. Interim Analyses No formal interim analyses are planned. Safety data are reviewed on an ongoing basis.

5.2.7. Missing, Unused and Incomplete Data All AEs with partial/missing onset dates will be considered treatment -emergent adverse events ( TEAEs ) unless a partial date clearly indicates that it occurred prior to first dose of study treatment or more than 28 days after the last dose of study treatment . Start dates with a missing day but which have month and year populated will be imputed such that: • If the provided month and year match the month and year for that subject’s first dose date, then the first dose date will be used. • In all other cases, the 1st of the month will be used with the provided month and year. Start dates with a missing day and month but which have year populated will be imputed such that: • If the provided year matches the year for that subject’s first dose date, then the first dose date will be used. • In all other cases the 1st of January will be used with the provided year. If the start date is completely missing then the date will be assumed as the subject’s first dose date and the event will be considered as treatment emergent, unless the stop date indicates that the event ended prior to first dose date. No imputation will be performed for subjects who did not receive study treatment. In those cases, the start date will be counted as missing. Stop dates will be imputed as follows: • Missing day with a provided year and month will use the last day of the month. • Missing day and month with provided year will use December 31. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 16 of 24 04MAY2018 • Completely missing stop dates will be treated as missing and no imputation will be done. If the imputed stop date is greater than the last known date for the subject ( e.g., date of last contact, date of death) then the imputed date will be replaced with the last known date. Similar rules will be used for prior/ concomitant medications with incomplete or missing start or stop dates. In order to calculate time from diagnosis , partial dates will be imputed. Missing months will be assumed as July. Missing days will be assumed as the 1 st of the month. If the entire diagnosis date is missing, then no imputation will be performed. No additional imputation for other missing data is planned. Missing values will be listed as represented in the clinical database (e.g. ‘NR’ (not reported), blanks).

5.3. Timing of Analyses On 12 December 2017, Aduro decided to cease development activities of CRS -207 and close out ongoing studies. As of 19 January 2018 every subject had completed their end of treatment visit except for 3 subjects who will have the option to continue study - drug treatment based on agreement between the Investigator and patient. All data through 19 January 2018 will be cleaned and soft -locked for CSR synopsis reporting. Additional data collection will be ongoing for these 3 subjects through to the date of database soft -lock. Additional data following database lock will be captured in an amendment to the final CSR synopsis once the 3 subjects have completed their end of study follow -up. This ASAP details the analysis plans for the CSR synopsis . 5.4. Subject Disposition Subject disposition will be presented by overall and cohort (Cohort 1 and Cohort 2). The number of subjects in Safety A nalysis set; number who completed Treatment Course 1; number who received MV ; number who discontinued Course 1 and the Treatment Period; and reasons for Course 1 and Treatment Period discontinuation will be summarized using frequency count s and percent ages . Time on study (first dose to EOS visit) and duration on treatment will be summarized. 5.5. Demographic and Baseline Characteristics Subject demographic and baseline characteristics will be summarized by cohort and overall based on the Safety Analysis set . Age, sex, ethnicity, race, and baseline weight, height , body mass index (BMI), and body surface area (BSA) will be summarized using descriptive statistics . Summaries will be produced for the FAS. The following formulas will be used for derivation and/or data conversion: Height (in cm) = height (in inches) * 2.54 Weight (in kg) = weight (in lbs) * 0.4536 BMI (kg/m2) = weight(kg)/[height(m)2] Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 17 of 24 04MAY2018 BSA(m2) = √[(height(cm)*weight(kg))/3600] Disease characteristics at baseline will be summarized. Time from diagnosis of primary tumor will be summarized using descriptive statistics methods. Time from the date of diagnosis of primary tumor to consent date (inclusive) will be displayed in months Primary site of cancer, histology, TNM classification at diagnosis and study entry, stage at diagnosis and study entry, extent of disease, baseline tumor measurements (sum of longest diameter in target lesions per RECIST 1.1, total tumor thickness measurements, and total sum of assessments per modified RECIST for MPM), number and percentage of subjects with prior radiation and prior surgery, screening PFTs, and baseline ECOG will be summarized. Additional diagnosis data will be listed.

5.6. Prior Anti -Cancer Therapies Prior cancer -related treatment (surgery and radiotherapy ) will be listed. 5.7. Medical History Medical history data will not be summarized but will be included in the data listings . 5.8. Concomitant Medications Medications are collected throughout the study. Medications will be categorized as a prior or concomitant. If a medication was taken and stopped prior to the first dose date, it will be categorized as a prior medication. Otherwise, a medication identified to have been taken post -dose will be considered concomitant medication. M edications with missing or partial dates will be considered concomitant unless the partial date indicates that the medication ended prior to the first dose date or started after the last dose date. All medications and coded terms will be provided in a by -subject data listing.

5.9. Other Assessments Coagulation data, urinalysis data, creatinine clearance, virology data, and human leukocyte antigen ( HLA) typing will be provided in by -subject data listings only. 5.10. Treatment Exposure Exposure to study treatment will be summarized overall and by cohort . Treatment duration summaries will be provided separately for CRS -207, Cy, and chemotherapy. The following study medication administrations will be summarized using descriptive statistics as well as categorical based on the number of administrations received (categories will be 1- 2 infusions, 3 -4 infusions, 5- 6 infusions, and > 6 infusions in the applicable summaries) : • Number of total CRS -207 doses received • Number of Cy administrations ( for Cohort 2 only ) • Number of pemetrexed doses administered (complete or partial) • Number of cisplatin , carboplatin, and total cisplatin/carboplatin doses administered (complete or partial) o The number of subjects who switched between cisplatin/carboplatin Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 18 of 24 04MAY2018 Chemotherapy will also be presented categorically by 1- 2 cycles, 3 - 4 cycles, 5 - 6 cycles. The average volume of CRS -207 administered per subject will be summarized using descriptive statistics. The average will be calculated for each subject across all infusions then summarized overall and by cohort. Dose interruptions, incomplete administrations, and missed administrations will be included in the by -subject data listings. 5.11.

Efficacy Evaluation 5.11.1. Tumor Evaluations and Overall Tumor Response Tumor evaluations and overall tumor response data, based on mRECIST, will be listed by date of assessment . 5.11.2. Exploratory Endpoints Pulmonary Function Tests FVC, FEV1, and VC will be listed by cohort and subject. All parameters collected will be based on the percent predicted values.

5.12. Safety Analyses Safety analyses will be conducted using the Safety Analysis set and will be reported by cohort and overall. The safety parameters collected and monitored during this study include AEs and deaths, hematology, serum chemistry, concomitant medications, physical examinations, vital signs and weight, ECOG performance, and 12- lead electrocardiograms (ECGs) . 5.12.1. Adverse Events Adverse events are assessed according to the National Cancer Institute (NCI) Common Terminology Criteria for AEs (CTCAE) 4.03. An AE is any reaction, side effect, or other untoward event, regardless of relationship to study drug, that occurs any time during the study. Adverse events occurring at any time from the beginning of any study dosing until 28 days after the final dose of any study treatment are considered TE AEs. If the start date is the same as the date of the first dose of study drug and there is no indication that the AE began before dosing, the event will be considered treatment emergent. Partial start and stop dates will be imputed as described in the section for missing data. If start and stop dates are missing and/or partial date(s) are insufficient to determine treatment emergence, then the AE will be considered treatment -emergent . Verbatim adverse events will be mapped to preferred term (PT) and system organ class (SOC) using MedDRA version 15.0. In incidence summary displays, AEs will be counted only once per subject within MedDRA category (e.g., overall, SOC, and PT ), by the worst CTCAE grade, and/or the strongest relationship to study drug (not related, unlikely related, p ossibly related, p robably related, d efinitely related). Any relationship summaries will be produced for CRS -207 and c yclophosphamide in combination (for any relationship to either study drug). The frequency and percentage of subjects with TE AEs will be tabulated by MedDRA SOC and PT. Tabulations will be prepared by SOC and PT within SOC and will include: Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 19 of 24 04MAY2018 • All TEAEs • Related TEAEs • Serious TEAEs • Related Serious TEAEs • CTCAE Grade 3 or higher TEAEs • CTCAE Grade 3 or higher related TEAEs • TEAEs with frequency in any cohort >=5% To account for potential differences in the extent of exposure between the treatment categories, a subject -year adjusted rate will also be presented. The rate is calculated as the number of subjects with an event divided by the total subject -years of exposure, where subject -years of exposure for each subject is defined as D uration of Treatment Exposure in days divided by 365.25. Duration of T reatment Exposure for all subject s is defined per total treatment duration in Section 5.2.2. A comprehensive listing of all AEs (including those which are not treatment -emergent) will be provided in a by -subject data listing. In addition, the following listings will be provided: • SAEs • TEAEs leading to death • TEAEs leading to treatment discontinuation Adverse events specifically recorded as CRS -207 infusion- related reactions will be tabulated by frequency (count and percent) using MedDRA SOC and PT. 5.12.2. Deaths The number and percent of subjects who died along with primary cause of death will be summarized overall (including the post -study survival surveillance period) and within 28 days of last dose of study drug. All death data will be listed.

5.12.3. CRS- 207 Infusion-r elated Reactions CRS- 207 infusion- related reactions (fever, chills, rigors, nausea, vomiting, hypotension) that occur red within 2 days of the infusion will be summarized based on data recorded on applicable CRF pages . These AEs will be graded and coded in the same manner as general AEs. The tabulation will include a count of the number of subjects with infusions, incidences of maximum grades for infusion- related reactions, as well as numerical summaries of the number of infusions and infusion- related reactions for subjects . An incidence table for infusion- related reactions similar to those specified for general AEs, by PT in decreasing order of overall frequency , will be present ed. Denominators for percentages include subjects with 1 or more CRS- 207 infusions. A comprehensive by -subject data listing of all CRS- 207 infusion- related reactions will be provided. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 20 of 24 04MAY2018 5.12.4. Dose Limiting Toxicities If data allows, CRS- 207 DLTs will be presented in a data listing. As defined in Section 3.6 of the study protocol, DLTs are events determined by the investigator as related to CRS -207 that meet one of the following criteria: • A fever of >40°C that lasts for greater than 24 hours and does not respond t o antipyretics • Clinically significant hypotension unresponsive to intravenous fluids (e.g., systolic blood pressure <90 mmHg or mean arterial pressure <55 mmHg as measured on two separate occasions at least 10 minutes apart) • Grade 3 or greater decreases in leukocytes, absolute neutrophil count ( ANC) , or platelets that persist for more than 4 days • Hemoglobin ≤7.0 g/dL • Alanine aminotransferase ( ALT), aspartate aminotransferase ( AST), or alkaline phosphatase elevations >5 times the upper limit of normal ( ULN) (Grade 3) that persist for more than 7 days • Initiation of antibiotic therapy, coincident with simultaneous isolation of CRS -207 from a normally sterile body site, other than blood (e.g., cerebrospinal fluid, joint fluid) • Unexpected Grade 3 laboratory abnormalities lasting >48 hours • Grade 3 or greater hypophosphatemia or lymphopenia that persist for more than 7 days • Any other Grade 3 or greater event according to National Cancer Institute’s CTCAE Version 4.03 5.12.5. Laboratory Data Hematology and serum chemistry parameters will be converted according to the International System of Units (SI) and will be listed by cohort and subject . Shift tables displaying the shift from baseline to the worst value by NCI CTCAE grade will be presented for the Treatment Period. The worst value is defined as the maximum NCI CTCAE grade based upon the worst observation post -baseline while on study . “Worst” can be defined as “high/hyper ” or “low /hypo” (or bilaterally) and will be specified within the parameter being summarized. Separate shift tables will be prepared for shifts to the worst low toxicity and to the worst high toxicity for lab parameters with bi - directional toxicity grading. 5.12.6. Vital Signs and Physical Examinations Physical examination data will be provided in a by -subject data listing. Vital signs data will be provided in a by -subject data listing. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 21 of 24 04MAY2018 5.12.7. Electrocardiogram Resting 12- lead ECG data will summarized by a shift table of baseline to worst result during the Treatment Period with the categories of ‘normal,’ ‘abnormal, not clinically significant,’ and ‘abnormal, clinically significant .’ 5.12.8. ECOG Performance Status ECOG data will be provided in a by -subject data listing. Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 22 of 24 04MAY2018 6. CHANGES TO PLANNED ANALYSES Changes to the protocol -specified statistical analyses are as follows: • Primary and secondary efficacy endpoints will not be evaluated; tumor and overall response data, based on mRECIST alone, will be listed. Analysis of immune response to mesothelin, the predictive value of serum mesothelin, and all exploratory endpoints corresponding to exploratory objectives identified in Section 2.3, with exception of that corresponding to pulmonary function, will be discussed in a standalone document . • The per protocol population will no longer be evaluated.

• Data are no longer planned to be summarized or evaluated by study phase. • In regard to safety analysis only relevant safety data will be summarized and all will be listed . Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 23 of 24 04MAY2018 7. REFERENCES 1. ICH-E9 Statistical Principals for Clinical Trials, September 1998 2. ICH-E3 Stru cture and Content of Clinical Study Report, July 1996 3. Byrne MJ, Nowak AK. Modified RECIST criteria for assessment of response in malignant pleural mesothelioma. Annals of oncology: official journal of the European Society for Medical Oncology / ESMO 2004;15:257- 60 4. Wolchok JD, Hoos A, O'Day S, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune- related response criteria. Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15:7412- 20 Aduro Biotech, Inc. CONFIDENTIAL Protocol ADU -CL-02 Final Version 1 Page 24 of 24 04MAY2018 8. L IST OF TABLES, FIGURES AND LISTINGS FOR FINAL ANALYSIS All tables, listings and figures will be numbered according to the ICH -E3 Guideline2. The complete table of contents of data tables, figures and listings can be found in the mock tables, figures, and listings document .

Cai et al. Molecular Cancer (2021) 20:164 https://doi.org/10.1186/s12943-021-01467-8 RESEARCH Personalized neoantigen vaccine prevents postoperative recurrence in hepatocellular carcinoma patients with vascular invasion Zhixiong Cai1,2,3†, Xiaoping Su4,5†, Liman Qiu1,2,3†, Zhenli Li1,2,3†, Xiaolou Li1,2,3, Xiuqing Dong1,2,3, Fuqun Wei1,2,3, Yang Zhou1,2,3, Liuping Luo1,2,3, Geng Chen1,2,3, Hengkai Chen1,2,3, Yingchao Wang1,2,3, Yongyi Zeng1,2,3* and Xiaolong Liu1,2,3* Abstract Background: Clinically, prophylactic anti-recurrence treatments for hepatocellular carcinoma (HCC) patients after radical surgery are extremely limited. Neoantigen based vaccine can generate robust anti-tumor immune response in several solid tumors but whether it could induce anti-tumor immune response in HCC and serve as a safe and effec- tive prophylactic strategy for preventing postoperative HCC recurrence still remain largely unclear. Methods: Personalized neoantigen vaccine was designed and immunized for 10 HCC patients with high risk of post - operative recurrence in a prime-boost schedule. The safety and immune response were assessed through adverse events, tissue sequencing, ELISpot, TCR sequencing. The clinical response was evaluated by recurrence-free survival (RFS) and personalized circulating tumor DNA (ctDNA) sequencing. Results: In the 10 enrolled patients, no obvious adverse events were observed during neoantigen vaccinations. Until the deadline of clinical trial, 8 of 10 patients were confirmed with clinical relapse by imaging, the other 2 patients remained relapse-free. From receiving first neoantigen vaccination, the median RFS of 10 patients were 7.4 months. Among 7 patients received all planned neoantigen vaccinations, 5 of them demonstrated neoantigen-induced T cell responses and have significantly longer RFS after radical surgery than other 5 patients without responsive neoanti- gens or only with prime vaccination and propensity scores matching control patients (p = 0.035). Moreover, tracking personalized neoantigen mutations in ctDNA could provide real-time evaluation of clinical response in HCC patients during neoantigen vaccination and follow up. Conclusion: Personalized neoantigen vaccine is proved as a safe, feasible and effective strategy for HCC anti-recur - rence, and its progression could be sensitively monitored by corresponding neoantigen mutations in ctDNA, and thus provided solid information for individualized medicine in HCC. Trial registration: This study was registered at Chinese Clinical Trial Registry; Registration number: ChiCT R1900 020990.

© The Author(s) 2021.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Open Access *Correspondence: [email protected]; [email protected] †Zhixiong Cai, Xiaoping Su, Liman Qiu and Zhenli Li contributed equally to this work as the joint first authors. 1 The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Xihong Road 312, Fuzhou 350025, Fujian Province, People’s Republic of China Full list of author information is available at the end of the article Page 2 of 13 Cai et al. Molecular Cancer (2021) 20:164 Introduction Hepatocellular carcinoma (HCC) is an increasingly seri - ous global pandemic. Especially in China, where new HCC cases account for 55% of the world’s total cases, approximately 422,100 people die from HCC progression per year [1]. Presently, a large number of HCC patients were diagnosed with different degrees of vascular inva - sion, resulting in low surgical resection rate and poor prognosis. However, previous clinical studies have shown that some HCC patients with vascular invasion in por - tal venous branch can still benefit from surgery treat - ment [2, 3], and such patients could be recommended for surgery treatment following the guidelines for Diag - nosis and Treatment of Primary Liver Cancer in China (2019 Edition) and EASL Clinical Practice Guidelines [4, 5]. Unfortunately, these patients still suffer a high risk of recurrence and metastasis after surgery. At present, pro - phylactic anti-recurrence treatments after surgery are very limited clinically. Transcatheter Arterial Chemoem - bolization (TACE) is the main prophylactic strategy in China, but still lack sufficient supportive evidence while there are no recommended treatments in EASL and NCCN guidelines. Therefore, it is very urgent to develop new and effective strategy for preventing postoperative recurrence. Neoantigens, epitope peptides solely derived from nonsynonymous mutations of malignant tumor cells, could be present to the cell surface with the major his - tocompatibility complex (MHC) on tumor cells and thus could be specific recognized by T cells to elicit strong antitumor immune responses. Comparing with tumor- associated antigen based vaccine, neoantigen based can - cer therapeutic vaccine has been proved as a promising anti-tumor immunotherapy strategy with maximized therapeutic efficacy and minimized risk of autoimmunity, since such neoantigens are only found in tumor tissues [6]. Neoantigens usually originate from somatic DNA point mutations, RNA editing events, insertion-deletion mutations, gene fusions and so on [7]. Recently, numbers of neoantigen vaccine based clinical trials showed that neoantigens derived from somatic point mutations could induce strong antitumor immune responses in patients with melanoma, small cell lung cancer, as well as glio - mas [8–10]. However, whether neoantigen vaccine could induce anti-tumor immune responses in HCC, which tumor mutation burden only ranked in the intermedi - ate position among different types of cancers, with ~ 2.0 mutations/megabase [11, 12], is still in doubt. Previous reports have pointed out that neoantigen burden in HCC patients could reflect tumor evolution during HCC pro - gression and associated with patient’s prognosis, suggest - ing neoantigens might serve as ideal immunotherapeutic targets for HCC immunotherapy [13, 14]. In addition, RNA editing events have been reported with capability to produce neoantigens [15], providing complementary targets for patients with low mutation burden. However, whether it could be used as potential therapeutic targets to induce robust anti-tumor immune responses in HCC patients also needs to be explored. Furthermore, real-time and accurate evaluation of treatment efficacy and prognosis is still a great challenge in HCC surveillance. Due to the limited sensitivity and specificity, imaging (CT/MRI) and serum protein bio - markers fail to acutely provide real-time evaluation of tumor burden, especially in monitoring minimal resid - ual disease (MRD) after radical operation. Our previ - ous study has indicated that circulating tumor DNA (ctDNA), a short DNA fragment derived from tumor cell in plasma, could function as noninvasive and sensi - tive biomarker for monitoring real-time HCC burden by tracking personalized tumor mutations [16]. Jia et al. also indicated that by tracking personalized neoantigen sites in ctDNA could accurately reflect the clinical response during checkpoint blockade immunotherapy in non- small cell lung cancer [17]. Thus, ctDNA might provide a potential strategy to sensitively and accurately assess clinical response during neoantigen based immunother - apy and should be thoroughly investigated. In this prospective clinical study, we enrolled 10 patients with resectable HCC and vascular invasion in portal venous branch, which indicated high risk of recur - rence after radical operation. Following personalized neoantigen identification for each patient, we compre - hensively evaluated whether personalized neoantigen long peptide vaccine could serve as a safe and effective strategy for preventing postoperative recurrence. Fur - thermore, we designed personalized somatic mutation panel, including neoantigen mutation sites, for ctDNA sequencing to monitor the clinical response during neo - antigen based immunotherapy. Methods Clinical trial design and treatment This study was investigator initiated, single-arm, open- label clinical trial at Mengchao Hepatobiliary Hospital of Fujian Medical University in China and was registered at Chinese Clinical Trial Registry (http:// www. chictr.

org.

cn/; ChiCTR1900020990). This trial was aimed to Keywords: Neoantigen vaccine, HCC, Anti-recurrence, Prophylactic treatment, Circulating tumor DNA Page 3 of 13 Cai et al. Molecular Cancer (2021) 20:164 study the safety and feasibility of personalized neoanti - gen vaccine for HCC anti-recurrence after hepatectomy. The corresponding designment, protocol and amend - ment were all approved by the Institution Review Board of Mengchao Hepatobiliary Hospital of Fujian Medical University (Fujian, China). All treatments and sample collections in this trial were in accordance with Decla - ration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice. The informed written consents were signed from all enrolled patients. The safety and feasibility of neoantigen vaccine were the primary endpoints and the correspond - ing immune response and relapse free progression (RFS) were the secondary endpoints. The key inclusion criteria of enrolled patient is seen below: (1) aged 18 to 75 years old male and female, with serum bilirubin not higher than 1.5× upper limit of normal (ULN) and ALT or AST not higher than 2.0× ULN; (2) be diagnosed with resect - able HCC or intrahepatic cholangiocarcinoma(CC) with - out any metastasis; (3) the existence of tumor thrombus in portal venous branch should be confirmed by histo - pathology or visible to the naked eye during the surgery. Key exclusion criteria included: (1) Patients with HIV infection, HCV infection, serious coronary artery disease or other diseases that the researchers consider not suit - able to be included in this study; (2) Patients with history of bone marrow transplantation or organ transplantation; (3) Patients with any form of immunodeficiency or his - tory of autoimmune disease; (4) Patients received prior treatment with any other immunotherapy within 1 month or have fewer than five identified actionable neoepitopes. After diagnosed with HCC, all enrolled patients firstly received radical surgical resection and then underwent prophylactic TACE within 2 months. Subsequently, personalized neoantigen vaccines were produced and administered subcutaneously at a prime-boost schedule. When the patient occurs HCC recurrence confirmed by imaging, he will reach the end of the clinical trial and receive standard clinical treatment. Physical examination, routine blood and biochemical tests and electrocardio - gram monitoring were used to assess the safety of each neoantigen vaccination. Meanwhile, we included 20 HCC patients who did not receive neoantigen vaccine as patient-controls though propensity scores matching (PSM). All PSM-control patients have similar clinical parameters with those 10 enrolled patients, such as clinical treatment (laparos - copy plus prophylactic TACE treatment), sex, age, vas - cular invasion, hemoglobin level (≥100 g/dl) and platelet level(≥80/l). Individuals were subsequently matched using a 1:2 nearest-neighbor matching algorithm within a caliper of 0.2 in PSM. All potential confounding factors in Supplementary Table S1 as covariates.HCC and peritumor tissues were collected at the time of hepatectomy. Serial blood samples, including plasma and peripheral blood mononuclear cells (PBMCs), were collected during whole clinical course, including pre - operative, postoperative, neoantigen vaccination time points and follow-up time points. Clinical response assessment (including imaging examination, ctDNA sequencing) and immune responses (including tissue sequencing, immunohistochemical staining, ELISpot, TCR sequencing, peripheral blood T lymphocyte subsets and cytokine assay) were conducted during neoantigen vaccination and follow up. Those detailed methods were shown in Supplementary Method. Tissue sequencing and epitope prediction To identify potential neoantigen profiling, HCC and matched peritumor tissues were used for whole exome sequencing with coverage depths of 200× and tran - scriptomic sequencing. Briefly, after DNA/RNA extrac - tion from HCC and matched peritumor tissue of each patient, exome and transcriptome libraries were further constructed according to the manufacturer’s instruc - tions, respectively. Then those libraries were sequenced by Fulgent. Co., Ltd. on Illumina Novaseq 6000 platform (paired end, 150 bp). The four-digit genotype of classical HLA class I genes (HLA-A, HLA-B and HLA-C) were assessed by Opti - Type [ 18] based on RNA-seq data, while HLA class II alleles (HLA-DRB1) were assessed by Seq2HLA. Somatic mutations were called using exome sequencing data by Mutect2 and Somaticsniper, and consistent somatic mutations were further confirmed by DeepSNV. All the somatic mutations were further validated at RNA level and somatic mutations with > = 20× depth and variant allele frequency (VAF) > = 0.05 in tumor RNA-seq data were kept. Immunogenicity of all remaining mutations were then evaluated using pVAC-Seq pipeline [19]. For each missense mutation, the binding affinity of 8 ~ 11 mer peptide containing mutated amino acids to patient’ s HLA class I alleles were predicted by NetMHCpan, NetMHC NetMHCcons, PickPocket, MHCflurry, while the binding affinity of corresponding 15-mer peptide to HLA class II alleles was predicted by NetMHCpanII. Mutations that produce mutant peptides with median IC50 < 500 nM of HLA class I or IC50 < 500 nM HLA class II alleles were considered as predicted neoantigens. All the predicted neoantigens were adopted to prepare long peptides (27mer) if the number was less than 30. If pre - dicted neoantigens greater than 30, epitopes were chosen in priority with the following rank order: (1) with both strong binding affinity (IC50 < 150 nM) to HLA class I and HLA class II alleles; (2) with either strong binding affinity to HLA class I and HLA class II alleles; (3) with Page 4 of 13 Cai et al. Molecular Cancer (2021) 20:164 higher VAF at RNA level; (4) with modest binding affin - ity (150 < IC50≦500) to both HLA class I or HLA class II alleles. Personalized neoantigen long‑peptide vaccine synthesis and vaccination To generate personalized neoantigen vaccines, up to 30 potential neoantigen mutations were selected and pri - oritized for clinical-grade long peptides synthesis (27 amino acids in length) by the standard solid-phase syn - thetic peptide chemistry with GMP-like standard (> 98% purity, endotoxin concentration was less than 0.01 EU/g). Among enrolled patients, 6~20 of neoantigen peptides were successfully synthesized in time for neoantigen vac - cine preparation. All such peptides were grouped into 2 ~ 4 pools (designated as pools 1 ~ 4, each pool con - tained 3 ~ 5 peptides, 0.3 mg/peptide) and received strict quality control, including sterility test, pyrogen detec - tion and abnormal toxicity test. Then neoantigen vaccine pools were further mixed with 0.5 mg poly:IC (polyi - nosinic-polycytidylic acid injection, Guangdong South China Pharmaceutical. Co) as adjuvant and then subcu - taneously (s.c.) injected at bilateral underarm and groin area, circularly. For neoantigen vaccination, patients were received neoantigen vaccine on days 1, 4, 8, 15 and 22 as the prime phase and on days 90 and 140 as the boost phase. Vaccination time is allowed within 15 days of the scheduled vaccination time in the boost phase. Statistical analysis Data from all enrolled patients received at least 5 planned prime phase vaccinations were included in the safety and clinical outcome evaluation. Descriptive statistics was used to determine safety of neoantigen vaccine. The RFS curves, immune response curves and ctDNA dynamic curves were plotted with GraphPad Prism 6 or R software. Results Study design and patient characteristics In this single-arm prospective clinical study, personal - ized neoantigen profiling was firstly performed for each newly diagnosed HCC patient with vascular invasion based on whole exome sequencing data and transcrip - tomic sequencing data of surgically resected tumor tis - sue and matched peritumor tissue (Fig. 1A). For each patient, 6 ~ 20 personalized neoantigen long peptides (27 aa) derived from somatic point mutations or RNA edit - ing events were synthesized for vaccine manufacture and were divided into 1~4 pools (termed as pools 1~4, each pool contained 3~5 long peptides). Following strict qual - ity control, the neoantigen vaccines together with poly:IC were administered at bilateral underarm and groin of HCC patient in a prime-boost schedule (days 1, 4, 8, 15 and 22 as the prime phase, days 90 and 140 as the boost phase) after radical surgery and prophylactic TACE. According to the investigator’s assessment, 10 patients with resectable HCC were included for evaluating the safety and efficacy of neoantigen vaccines. As shown in Fig. 1B, among 10 enrolled patients, pathological diag - nosis confirmed that 5 of them were HCC and other 5 patients were diagnosed as combined HCC and intra - hepatic cholangiocarcinoma (cHCC-CC). Nine patients diagnosed with HBV infection and 8 of them had con - comitant liver cirrhosis. The average tumor diameter of 10 patients was 7.7 cm (range, 2.8~11 cm). In total, 780 nonsynonymous somatic single-nucleotide variants (SNVs) were identified, with an average of 78 mutations (range, 7~148) in each patient using whole- exome sequencing of tumor tissue and matched peritu - mor tissue (Fig. 1B). Meanwhile, 33.5% (range, 0~66.7%) of these somatic mutations were further confirmed for expression by transcriptomic sequencing analysis (variant allele frequency > = 0.05) and identified as neoantigens by epitope prediction simultaneously (IC50 < 500 nM for HLA class I or II binding). The detailed HLA type and neoantigen sequence for neoantigen vaccine preparation of each enrolled patients were shown in Supplementary Table S2. As shown in Fig. 1B, HCC frequently missense mutated genes in protein coding region, such as TP53 and KALRN were observed in 50% and 40% of enrolled patients, respectively. As expected, among all identified neoantigens, neoantigens originated from TP53 muta - tions were observed in 40% (4/10) patients. Noteworthily, in one enrolled patient N18, no nonsynonymous somatic SNVs were identified in DNA level. Fortunately, 20 tumor specific nonsynonymous RNA editing sites were identi - fied and 6 of them were identified as neoantigens (Sup - plementary Fig. S1). Presently, the immunogenicity of neoantigen derived from RNA editing sites has been proved to have potential immunogenicity [15]. After fully informing potential treatment benefits and risks and obtaining informed consent from patient N18, we pre - pared a personalized neoantigen vaccine based on RNA editing sites for patient vaccination in a prime-boost schedule. The safety of neoantigen vaccination The median duration from hepatectomy to first vacci - nation was 86 days, ranging from 59 to 159 days. All 10 enrolled patients received 5 planned prime phase vacci - nations and 7 of them underwent another 2 boost vac - cinations. No obvious treatment related adverse events were observed and routine blood/biochemical tests also showed no obvious abnormalities during vaccinations Page 5 of 13 Cai et al. Molecular Cancer (2021) 20:164 (Supplementary Tables S3 and S4). Only some minor side effects (Grade 1), such as injection site reaction and fatigue, were reported after the vaccine injection but quickly disappeared without additional treatments. These results supported the high safety of neoantigen vaccines.Clinical outcome and immune response monitoring during vaccination and follow up The detailed timeline presentation of clinical treat - ments, vaccinations and clinical outcomes were shown in Fig. 2A. From November 26, 2018 to June 30, 2021, Fig. 1 The overview of study design, patient characteristics, and neoantigen profiles. A The infusion program of personalized neoantigen vaccine for HCC patients with vascular invasion. B Number of somatic mutations and neoantigens detected in each patient’s HCC tissue and corresponding clinicopathologic information in 10 enrolled patients. The percentage shows the proportion of the number of neoantigen mutations to the number of total somatic mutations. Heatmap summarized the somatic/neoantigenic hotspot gene profile in enrolled HCC patients. The neoantigen mutations of patient N06 and N09 all come from personalized gene mutations rather than hot gene mutations Page 6 of 13 Cai et al. Molecular Cancer (2021) 20:164 the median follow-up time of these 10 enrolled patients was 20.1 months (range, 10.9~32.7 months). Eight out of ten patients were confirmed with clinical relapse by MRI/CT imaging, with the median relapsed-free sur - vival (RFS) of 8.3 months after radical surgery; the other 2 patients remained relapsed-free (mean follow-up time Fig. 2 Clinical outcome and immune response monitoring in enrolled HCC patients during vaccination and follow up. A The detailed timeline presentation of clinical treatments, vaccinations and clinical outcomes for 10 enrolled HCC patients from surgery treatment until the deadline of clinical trial. B The Kaplan-Meier survival curve of RFS after receiving first neoantigen vaccination in enrolled 10 patients. C The Ex vivo IFN-γ ELISpot responses for PBMCs stimulated by personalized neoantigen pools between pre- and post-neoantigen vaccinations in 6 patients. D The Kaplan-Meier survival curve of RFS after radical surgery among patients with responsive neoantigens, patients without responsive neoantigens or only with prime vaccination and propensity scores matching control patients. E Long-term monitoring of Ex vivo IFN-γ ELISpot response for PBMCs stimulated by personalized neoantigen pools in 4 patients. PC indicated as positive control and NC indicated as negative control Page 7 of 13 Cai et al. Molecular Cancer (2021) 20:164 of 21.4 months). Kaplan-Meier survival analysis showed that the median RFS of enrolled patients after receiving first neoantigen vaccination was 7.4 months (Fig. 2 B). Generally, neoantigen vaccines need to be fully admin - istered by primer and boost vaccinations, and take 2 ~ 5 months to build up robust neoantigen-specific anti- tumor immune response in vivo. However, 3 patients (N24, N25 and N31) were relapsed within 2 weeks after prime phase vaccination and thus not received boost vac - cinations (Fig. 2A), which might leave insufficient time to induce enough anti-tumor immune response. Therefore, in vitro IFN-γ ELISpot assay was performed to monitor immune response of other 7 patients who received all boost vaccines by using autologous PBMCs stimulated by neoantigen pool-pulsed autologous DC cells. As shown in Supplementary Fig. S2, patient N06 did not generate obviously IFN-γ responses during vaccinations. How - ever, in 3 patients (N09, N22 and N27), the neoantigen specific reactivity of PBMCs against neoantigen peptide pools corresponding to neoantigen vaccines was consid - erably generated after all vaccinations when compared with corresponding negative control and pre-vaccina - tion sample (Fig. 2C). Meanwhile, early PBMC reactivity against neoantigen peptide pools before vaccination was observed in 3 patients (N13, N18 and N30), which may probably due to releasing tumor neoantigens of MRD by the prophylactic TACE treatment to further activate spe - cific T cells. However, during vaccinations, we also found that the neoantigen immune response of patient N13 decayed rapidly, and could not detect after boost vaccina - tion (Supplementary Fig. S3). This mean that neoantigen vaccine could not successfully activate the neoantigen- induced T cell responses in patient N13. In contrary, after all vaccinations, the neoantigen specific reactiv - ity in patient N18 was considerably stronger than pre- vaccination; patient N30 maintained a similar immune response intensity as before vaccination, which may be induced by neoantigen vaccines and prophylactic TACE, or that patient’s immune system was sensitive to neoan - tigens. Meanwhile, the neoantigen specific reactivity of PBMCs against each neoantigen individually was further confirmed that 36 out of 51(70.6%) individual long pep - tides could significantly induced measurable peptide- specific immune response in 5 patients (N09, N18, N22, N27and N30) and other 2 patents (N06 and N13) did not have responsive neoantigens after completed neoantigen vaccination (Supplementary Fig. S4). Interestingly, we found that the 6 neoantigen peptides derived from RNA editing in N18 patients showed 100% immune response rate (Supplementary Fig. S4), suggesting RNA edit - ing sites could indeed serve as neoantigen candidates in patients with low tumor mutation burden. Accordingly, 5 patients (N09, N18, N22, N27 and N30) were identified with responsive neoantigens after vaccinations. Analysis combining clinical data further indicated that patients with responsive neoantigens after fully vaccinations had significantly longer RFS after radical surgery than those without responsive neoantigens or only with prime vac - cination and propensity scores matching control patients enrolled in study (median RFS: 19.3 vs 6.7 vs 4.8 months, P = 0.035, Fig. 2D). Meanwhile, we also investigated the dynamic change in proportions of peripheral blood T lymphocyte subsets and serum levels of 6 cytokines (IL- 2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) in 10 enrolled patients during neoantigen vaccinations (Supplementary Table S5), but did not find a significant correlation with patients’ neoantigen response or RFS (data no shown). These results indicated that neoantigen-induced specific IFN-γ response can be used as an indicator for the effec - tiveness of neoantigen vaccine. Then, we further evaluated the durability of the neo - antigen-induced immune response in 4 patients (N09, N18, N22 and N27) who had follow-up PBMC samples after neoantigen vaccination. As shown in Fig. 2E, dur - ing follow up, neoantigen-specific immune response was still maintained relative strong at the 10 month after vac - cination in 2 patients (N18: average 67.8 spots in 2 neo - antigen pools; N27: average 97.3 spots in 2 neoantigen pools), but already relatively week at the 8 month after vaccination in patient N09 (average 17.8 spots in 3 neo - antigen pools) and patient N22 (average 24.5 spots in 3 neoantigen pools). Accordingly, the RFS of N18 and N27 patients is indeed longer than that of N09 and N22 patients (Fig. 2A). Overall, these results in our cohort suggested that personalized neoantigen vaccine could serve as an effective strategy to induce robust immune response in HCC patients. Immune microenvironment dynamics after neoantigen vaccination To explore the dynamics of immune microenvironment during neoantigen vaccination, we performed a toughly evaluation of patient N22 who had recurrent tumor tis - sue collected after neoantigen vaccination treatment. Patient N22 is a 46-year-old male, firstly diagnosed with HBV related HCC (MRI scan: 5.0 × 4.5 × 4.0 cm, at seg - ment 5 of liver) at Mengchao Hepatobiliary Hospital of Fujian Medical University. Then this patient received sur - gical resection and underwent prophylactic TACE after 1 month. Pathological examination showed that his tumor was an invasive cHCC-CC with stem cell characteristics. His local lesions showed visible vascular tumor thrombi and some circulating tumor cells, indicating high risk of recurrence after radical operation. Sequencing data pre - sented 44 potential neoantigens from 85 nonsynonymous somatic mutations and 13 of them were successfully Page 8 of 13 Cai et al. Molecular Cancer (2021) 20:164 synthesized in time and used for personalized neoan - tigen vaccine preparation. Three months after surgery, he started to receive neoantigen vaccination in planned prime-boost schedule (Fig. 3A). One years after surgical operation, MRI scan revealed a 1.4 cm recurrent lesion in primary HCC origin and he was then treated with second hepatectomy. Immunohistochemical staining revealed that the infiltration of CD8 positive T cells and granzyme B secretion in recurrent tumor tissues were obviously increased compared with the primary tumor (Fig. 3B). The recurrent tumor was further subjected to whole-exome sequencing and transcriptomic sequenc - ing to assess the influence of neoantigen vaccination on tumor immune microenvironment. Analysis of sequenc - ing data showed that the mean mutation allele frequen - cies for 13 neoantigen mutations identified in primary HCC were both obviously decreased by 89% at DNA level and 85% at RNA level in recurrent tumor, respectively (Fig. 3C); while the mean frequencies of other 72 somatic mutations in recurrent tumor were also correspondingly declined (72% in DNA level and 67% in RNA level, Sup - plementary Fig. S5). Meanwhile, analysis of clonal struc - ture dynamics between primary tumor and recurrent tumor revealed that 8 neoantigen mutations (other 5 neoantigen mutations were excluded due to copy number variation) are mainly located in cluster 2 and 3, which are significantly shrunk in recurrent tumors; and new clone (cluster 4) was indeed identified in recurrent tumors, sug - gesting tumor evolved under the pressure of neoantigen based immunotherapy to achieve tumor immune escape (Fig. 3D). Meanwhile, 9 new neoantigen mutations were also found in recurrent tumor (Supplementary Table S6). These results suggested that the proportion of tumor cells carrying these neoantigens in recurrent tumor sig - nificantly shrunk under neoantigen based immunother - apy. Furthermore, the heatmap analysis of immune cell infiltration by transcriptomic data showed that immune effector cells were significantly accumulated in recur - rent tumor including activated CD4+ T cells, activated CD8+ T cells, natural killer cells, and immature dendritic cells (Supplementary Fig. S6). Immunophenoscore dia - grams between primary HCC and recurrent tumor also indicated that the immune microenvironment in the recurrent tumor turn into “hot” with enhanced antigen presentation ability through MHC molecules, increased number of effector T cells, decreased expression of immune checkpoints in T cells (Fig. 3E). Moreover, we performed T cell receptor (TCR) sequencing in primary and recurrent tumor tissues to identify potential neoan - tigen associated TCR clone after vaccination. Two new TCR clones (CASSESPLYEQYF and CASTTSGSYEQYF) were detected in recurrent tumor, suggesting the pres - ence of neoantigen-induced specific T cells (Fig. 3F). Overall, these results indicated that the neoantigen vac - cine successfully activated the anti-tumor activity of T cells and could invade into the tumor site. The performance of ctDNA for evaluation of immune and clinical responses Real-time monitoring the immune and clinical responses is vital for assessing the responsiveness of neoantigen vaccination and predicting its efficacy. In this study, we monitored the clinical response of 6 enrolled patients with fully neoantigen vaccinations (except for N18 since his neoantigen derived from RNA editing) by tracking the dynamics of their personalized nonsynonymous somatic mutations in ctDNA. As shown in Fig. 4A and Sup - plementary Fig. S7, an average of 74 and 78.9% positive rate for neoantigen sites (15.4~100%) and other somatic mutations (29~100%) were observed in preoperative plasma. However, in patient N27 and N30, most neoan - tigen and other mutation sites were not well detected at ctDNA levels, resulting in inability to track tumor burden during clinical course. In other 4 patients, the dynamic change of neoantigens and other mutations in ctDNA were both well consistent with tumor burden evaluated by CT/MRI images during neoantigen vaccination and follow up (Fig. 4B-C and Supplementary Fig. S8A-B). Meanwhile, we also found postoperative ctDNA remained at a detectable level for those 4 patients before neoantigen vaccination, suggesting the existence of MRD even after radical surgery and following prophylactic TACE. The detailed ctDNA frequency of neoantigen mutations and other somatic mutations in each patient was shown in Supplementary Table S7. In 2 patients identified with neoantigen responses (N09 and N22), the mean frequency of overall ctDNA (treated neoantigen mutations and other mutations) was rising during first month of receiving five prime vaccinations, suggesting that MRD is progressing (Fig. 4B-C). More interestingly, Fig. 3 Clinical response and immune microenvironment dynamics for HCC patients during neoantigen vaccination and follow up. A Clinical event timeline and corresponding imaging of patient N22. Take the first dose of neoantigen vaccine as day 1. B Hematoxylin-eosin staining and Immunohistochemical staining of CD8 and granzyme B in primary tumor and recurrent tumor. C The mutation allele frequencies of treated neoantigen mutations in DNA level and RNA level between primary tumor and recurrent tumor. D The clonal structure dynamics between primary tumor and recurrent tumor. The asterisk indicated as neoantigen mutation. E Immunophenoscore diagrams between primary tumor and recurrent tumor. F TCR clone dynamics between primary tumor and recurrent tumor. VAF: variant allele frequency(See figure on next page.) Page 9 of 13 Cai et al. Molecular Cancer (2021) 20:164 Fig. 3 (See legend on previous page.) Page 10 of 13 Cai et al. Molecular Cancer (2021) 20:164 we then observed different ctDNA dynamic patterns of these 2 patients in the following plasma. From day 30 to day 90 during neoantigen vaccination, patient N09 showed both decrease in the mean frequency of treated neoantigen ctDNA and other ctDNA; while in N22 patients, only treated neoantigen ctDNA showed an Fig. 4 Tracking personalized nonsynonymous somatic mutations in ctDNA during the whole clinical course. A The profiling of neoantigen mutations and other somatic mutations detected in HCC and matched preoperative plasma. The time-course demonstration of quantified levels of treated neoantigen mutations and other somatic mutations in patient N09 ( B) and N22 (C), respectively. The small picture displays the dynamic of mean mutation allele frequencies in treated neoantigen mutations and other somatic mutations during neoantigen vaccination and follow up Page 11 of 13 Cai et al. Molecular Cancer (2021) 20:164 obviously decline and the frequency of other mutations in ctDNA is still slowly rising. After that, the frequency of overall ctDNA in patient N09 were increased from day 90 to day 140 but then decreased from day 140 to day 278 after receiving 2 booster vaccines; afterwards, his overall ctDNA frequency was increased again until recurrence happening. However, in the similar period, the over - all ctDNA frequency of patient N22 was continuously increasing even after receiving the two booster vaccines until recurrence happening at the day 331. This might be due to that the immune response induced by the neoanti - gen vaccine was insufficient to inhibit tumor progression in the boost phase or the HCC tumor occurred immune escape under the pressure of neoantigen immunotherapy. Significantly, the mean frequency dynamics of neoanti - gen mutations and other mutations observed in series of patient N22 ctDNA samples was well consistent with the results found in recurrent tumor, that is, the mean muta - tion frequency of other mutations is higher than that of treated neoantigens, implying that the immune response induced by neoantigen vaccine preferentially killed tumor cells carrying those neoantigens while other cells gained growth superiority. In other two patients (N13 and N06) without obvious neoantigen response detected in PBMCs after fully vaccinations, the overall ctDNA frequency did not show a similar pattern like in N09 or N22 (Supplementary Fig. S8). In summary, these results proved that real-time evaluation of the immune and clinical responses in HCC could be achieved by track - ing personalized neoantigen mutations in ctDNA during neoantigen vaccination. Discussion The high recurrence rate of HCC after radical surgery is the main reason for poor prognosis of patients. In China, the prophylactic TACE is the main anti-recurrence strat - egy for HCC but remains with controversies. Some clini - cal studies have shown that HCC patients with PVTT receiving prophylactic TACE after radical surgery could effectively prolong the recurrence-free survival time of about 2.4 months when compared with those without prophylactic TACE treatment, but other studies indicated that this treatment had poor anti-recurrence effect and considerable toxicity [20–23]. Therefore, it is extremely necessary to develop novel effective and safe anti-recur - rence strategies. Neoantigen vaccines, due to their high specificity, low side effects, and easy preparation, can be used as a potential strategy for anti-recurrence in solid tumors. More importantly, since sufficient tumor tissue and para-cancerous tissue can be likely obtained through surgery, we can comprehensively and accurately identify personalized neoantigen profiling by high-throughput sequencing; meanwhile, postoperative patients are not as prone to rapidly disease progression as terminal patients, sufficient time and resource are available for the prepa - ration of neoantigen vaccines. In this clinical study, 10 enrolled patients successfully received personalized neo - antigen vaccination with no obvious adverse events after radical surgery and prophylactic TACE. Their median RFS time reached 11.3 months after surgery. Interestingly, we also found that 5 patients who successfully induced a strong neoantigen response after fully vaccination had longer RFS than those without responsive neoantigens or only with prime vaccinations and PSM control patients. Although, due to the small sample size, it is necessary to further expand the sample size for verification. Therefore, neoantigen vaccination might be a potential strategy for developing anti-recurrence treatments in HCC. In addition, real-time monitoring the clinical response of neoantigen vaccination is crucial to provide essen - tial assistance for doctors’ clinical decisions. Due to the shortcomings of sensitivity and specificity, clinical protein markers or imaging scans could not effectively monitor the clinical response of neoantigen based immu - notherapy in patients with invisible tumor burden. In this clinical trial, we firstly try to real-time monitor treat - ment outcome of neoantigen vaccination by tracking the patient’s personalized neoantigen mutations and other mutations in plasma ctDNA. Significantly, we observed two different dynamic patterns of neoantigens and other mutations in ctDNA: one is that the dynamic change pat - tern of neoantigen mutations is similar with that of other mutations during neoantigen vaccinations, implying that patient may have low tumor heterogeneity, that is, most tumor cells contain certain neoantigen mutations and the immune response induced by neoantigen vac - cination could specifically kill tumor cells carrying these neoantigens. In such situation, the neoantigen mutations and other mutations in ctDNA might undergo similar changes. Another is that the dynamic change pattern of neoantigen mutations is not consistent with the patterns of other mutations, suggesting that patient may have high tumor heterogeneity, and only a part of the tumor cells contain neoantigen mutations. These results gave solid evidence for judging clinical response and maintenance time of neoantigen based immunotherapy, which would in-time help doctors to adjust therapeutic strategy. Meanwhile, based on the results of ctDNA, we also found that the duration of anti-tumor immune response induced by neoantigen vaccination is still limited, which is not enough to completely inhibit MRD progression. There are two possible reasons: on the one hand, high numbers of neoantigens could be identified in some patients, but only up to 20 neoantigens could be success - fully synthesized for neoantigen vaccine preparation due to limited time and cost effectiveness. On the other hand, Page 12 of 13 Cai et al. Molecular Cancer (2021) 20:164 with the high heterogeneity in HCC, some tumor cells might not carry such neoantigens and thus could not be well recognized by activated T cells, resulting in growth superiority during neoantigen vaccination. Therefore, it is necessary to further strengthen the anti-recurrence efficacy and immune durability of neoantigen vaccines by optimizing the neoantigen vaccination strategy and/ or combining with other treatment methods such as immune checkpoint inhibitors and tyrosine kinase inhib - itors [24]. Recently, some reports have shown that neo - antigen vaccine combined with PD-1 inhibitor in solid tumors such as melanoma and non-small cell carcinoma can further improve its anti-tumor efficacy [25], but its outcome in HCC remains to be further confirmed. In summary, neoantigen vaccination is a safe, fea - sible and effective strategy for HCC anti-recurrence after radical surgery. Moreover, tracking personalized neoantigen mutations in ctDNA could provide compre - hensive information for clinical response monitoring of neoantigen vaccination with high sensitivity and speci - ficity, and thus help the clinical application for indi - vidualized medicine. However, this study still has the following shortcomings: firstly, due to time and funding constraints, personalized neoantigen tetramers were not custom-made for neoantigen-specific T cell analy - sis; secondly, due to limited sample size enrolled in this study, the corresponding findings will still need to be validated in large-scale clinical trials. Abbreviations HCC: Hepatocellular carcinoma; TACE: Transcatheter Arterial Chemoemboliza- tion; MHC: Major histocompatibility complex; MRD: Minimal residual disease; ctDNA: Circulating tumor DNA; RFS: Recurrence free survival; PBMCs: Periph- eral blood mononuclear cells; TCR : T cell receptor; VAF: Variant allele frequency. Supplementary Information The online version contains supplementary material available at https:// doi.

org/ 10. 1186/ s12943- 021- 01467-8. Additional file 1: Supplementary data. Additional file 2: Supplementary Figure S1. The profiling of DNA muta- tion and RNA editing identified in patient N18. Additional file 3: Supplementary Figure S2. The Ex vivo IFN-γ ELISPOT responses for PBMCs stimulated by personalized neoantigen pools of patient N06 during neoantigen vaccinations. Additional file 4: Supplementary Figure S3. The Ex vivo IFN-γ ELISPOT responses for PBMCs stimulated by personalized neoantigen pools of patient N13 during neoantigen vaccinations. Additional file 5: Supplementary Figure S4. The Ex vivo IFN-γ ELISPOT responses for PBMCs stimulated by individual neoantigen peptide after neoantigen vaccinations (5 months) in 7 patients. The peptide in blue font, orange font and green font indicated as pool1, pool2, and pool3, respectively.

Additional file 6: Supplementary Figure S5. The mutation allele fre - quencies of other mutations in DNA level and RNA level between primary tumor and recurrent tumor.Additional file 7: Supplementary Figure S6. The heatmap analysis of immune cell infiltration by transcriptomic data. Additional file 8: Supplementary Figure S7. The profiling of other mutations detected in matched preoperative plasma. Additional file 9: Supplementary Figure S8. The time-course demon- stration of quantified levels of treated neoantigen mutations and other somatic mutations in patient N06(A) and N13(B), respectively. The small picture displays the dynamics of the average of mutation allele frequen- cies in treated neoantigen mutations and other somatic mutations during neoantigen vaccination and follow up. Additional file 10: Supplementary Table S1. Baseline clinical character - istics between enrolled patients and PSM case-control patients. Additional file 11: Supplementary Table S2. The detailed HLA type and neoantigen sequence for neoantigen vaccine preparation of each enrolled patients. Additional file 12: Supplementary Table S3. Treatment-related adverse events in all enrolled patients. Additional file 13: Supplementary Table S4. The dynamics of routine blood and biochemical tests in all enrolled patients during neoantigen vaccinations. Additional file 14: Supplementary Table S5. The dynamic change in proportions of peripheral blood T lymphocyte subsets and serum levels of 6 cytokines in all enrolled patients during neoantigen vaccinations. Additional file 15: Supplementary Table S6. New neoantigen muta- tions identified in recurrent tumor. Additional file 16: Supplementary Table S7. The detailed ctDNA fre - quency of neoantigen mutations and other somatic mutations in enrolled patients during clinical course. Acknowledgments We appreciate the help of neoantigen screening and data analysis by the P&PMed Biotechnology Co.Ltd.(Shanghai, China). Conflict of interest No potential conflicts of interest were disclosed. Authors’ contributions ZC, XS, YZ, and XL conceived and designed the experiments. ZC, LQ, ZL, XL, XQ, FW, YZ and LL performed the experiments. XS, ZL, HC and YW analyzed the data. ZL, GC performed bioinformatic analyses. ZC, XS, YZ and XL wrote the manuscript. All authors have read and approved the final manuscript. Funding This work was supported by regional development project of Fujian Province [grant number 2019Y3001], the Scientific Foundation of the Fuzhou Health Commission [grant number 2019-S-wt3]. Availability of data and materials The data used to support the findings of this study are included within the article. The raw sequencing data reported in this article has been deposited at Big Data center (Nucleic Acids Res 2018), under the accession number of HRA000096 that are publicly accessible at http:// bigd. big.

ac.

cn/ gsa. Declarations Ethics approval and consent to participate This study was performed in agreement with the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice, and approved by the Institution Review Board of Mengchao Hepato - biliary Hospital of Fujian Medical University (Fujian, China). Consent for publication Written informed consent was obtained from the patients for publication of this letter. Page 13 of 13 Cai et al. Molecular Cancer (2021) 20:164 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissionsReady to submit y our researc h Ready to submit y our researc h ? Choose BMC and benefit fr om: ? Choose BMC and benefit fr om: Competing interests The authors have no conflicts of interest to declare. Author details 1 The United Innovation of Mengchao Hepatobiliary Technology Key Labora- tory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Xihong Road 312, Fuzhou 350025, Fujian Province, People’s Repub - lic of China. 2 The Liver Center of Fujian Province, Fujian Medical University, Fuzhou 350025, People’s Republic of China. 3 Mengchao Med- X Center, Fuzhou University, Fuzhou 350116, People’s Republic of China. 4 Department of Gastro - enterology, Second Affiliated Hospital of Wenzhou Medical University, Wen- zhou 325000, People’s Republic of China. 5 School of Basic Medicine, Wenzhou Medical University, Wenzhou 325000, People’s Republic of China. Received: 16 September 2021 Accepted: 22 November 2021 References 1. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statis- tics in China, 2015. CA Cancer J Clin. 2016;66:115–32. 2.

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SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 1 CANCER RESEARCH UK Reporting Analysis Plan Protocol Number: CRUKD/17/003 Protocol Name: AST-VAC2 EudraCT Number: 2016- 002577- 35 RAP Fin al Version: 2.0 Date: 09Mar2023 Replaces Version: N/A Date: N/A Name: Date: Title: Clinical Study Manager (Sponsor) Signature: Name: Date: Title: Medical Advisor (Sponsor) Signature: Name: Date: Title: Chief Investigator Signature: SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 2 SUMMARY OF CHANGES TO PREVIOUS REPORTING ANALYSIS PLAN VERSION This summary of changes is intended to highlight the important revisions that were made during the most recent update to the reporting analysis plan (RAP) to generate the current version. This document has been written based on information contained in the study protocol and data management plan detailed in the table below. Protocol version DMP version RAP version Revised section of RAP Summary of changes Date updated 5.0 2.0 1.0 5.9, 6.1 & 6.2 Section 5.9 – note added about patients with data censored for survival, referen ce to CEA listing added, Table 14 updated to refer to ‘Date of Death or Last Contact’. Section 6.1 – CEA listing added Section 6.2 – RAP tables 4, 5 & 6 numbering for CSR revised 12Dec2022 Final review prior to database lock Protocol version DMP version RAP version Revised section of RAP Summary of changes Date updated Protocol amendments may be applicable during the study. The RAP will be reviewed against the amendments and updated where necessary. The summary of changes table above should record all changes to the RAP in light of protocol amendments however if no changes were required, this should be recorded also. CONTRIBUTORS Medical Writer: Clinical Data Manager: Clinical Data base Programmer: Translational Scientist: Biomarker Development Scientist: SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 4 Table of Contents Summary of changes to previous Reporting Analysis Plan version ........................................................................... 2 LIST of abbreviations and definition of terms .........................................................................................................

3 1.

Introduction ................................................................................................................................................... 6 2. Trial Objectives ............................................................................................................................................... 7 3. Trial Design .................................................................................................................................................... 9 4. Patient Population and endpoints ..................................................................................................................11 4.1 Patient Populations ..............................................................................................................................11 5. Data Conventions and general analysis ..........................................................................................................12 5.1 Patient disposition ...............................................................................................................................12 5.2 Baseline characteristics ........................................................................................................................12 5.3 Patient withdrawal ...............................................................................................................................14 5.4 Protocol deviations ..............................................................................................................................14 5.5 Treatment compliance .........................................................................................................................15 5.6 Safety ...................................................................................................................................................16 5.7 Adverse Events .....................................................................................................................................17 5.8 Laboratory results ................................................................................................................................18 5.8.1 Additional safety tables ................................................................................................................18 5.8.2 Stopping rule criteria ...................................................................................................................19 5.9 Efficacy ................................................................................................................................................19 5.10 General data conventions .....................................................................................................................20 5.11 Decimal places .....................................................................................................................................21 5.12 Statistical software ...............................................................................................................................21 5.13 Supplementary analysis – Pharmacodynamics (data collected outside of the clinical database) .............21 5.13.1 HLA Pre -screening ........................................................................................................................21 5.13.2 Secondary endpoints ...................................................................................................................22 5.13.3 Tertiary endpoints .......................................................................................................................22 5.14 Other statistical analysis .......................................................................................................................25 5.14.1 Stratification and covariate analysis .............................................................................................25 5.14.2 Multivariate analysis ....................................................................................................................25 5.14.3 Subgroup analysis ........................................................................................................................25 5.14.4 Interim analysis ............................................................................................................................25 5.14.5 Final analysis ................................................................................................................................25 6. Tables listings and figures ..............................................................................................................................26 6.1 LISTINGS ..............................................................................................................................................26 6.2 SUMMARY TABLES ...............................................................................................................................27 7. Presentation of Results in Clinicaltrials.gov Including Additional Tables ..........................................................28 7.1 Arms and Interventions ........................................................................................................................28 7.2 Participant Flow ...................................................................................................................................29 SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 5 7.3 Baseline Characteristics ........................................................................................................................31 7.4 Reported Adverse Events (clinicaltrials.gov template sections) .............................................................32 7.5 Outcome Measures ..............................................................................................................................34 7.5.1 Outcome Measure 1 – Frequency and causality of AEs and SAEs to AST -VAC2 (Safety Population) 37 7.5.2 Outcome Measure 2 - Number of participants experiencing ISRs by grade (Safety Population) .....38 7.5.3 Outcome Measure 3 – Number of participants showing a durable peripheral immune response (Secondary Immunogenicity Endpoint Population) .........................................................................................39 7.5.4 Outcome Measure 4 – Mean fold change over baseline by timepoint (Secondary Immunogenicity Endpoint Population) .....................................................................................................................................40 7.5.5 Outcome Measure 5 - Tumour response according to Immune -Related Response Criteria (irRC) post vaccination (Response population) .........................................................................................................41 7.5.6 Outcome Measure 6 – Overall survival at 2 years post first vaccination (Safety Population) ..........42 7.6 Statistical Analyses ...............................................................................................................................42 8. Reporting of Clinical trial results to patients and public ..................................................................................43 9. References ....................................................................................................................................................43 SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 6 1. INTRODUCTION This document explains in detail the reporting analyses that will be carried out for Phase I trial of AST-VAC2 vaccine in patients with non -small cell lung cancer . The analyses described in this RAP are based upon and supplement those described in the current study protocol.

To support reproducibility of the research, a clear and comprehensive account of pre -planned reporting (or statistical) analyses must be avail able. This RAP will establish the essential items to be considered for interim and/ or final reporting requirements.

SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 8 SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 9 3. TRIAL DESIGN Table 1: Trial Design A Study design This is a first in man, open label, multi -centre, Phase I trial to investigate the safety, immunogenicity and patient survival following administration of the AST -VAC2 vaccine . Only one target dose (1× 107 cells) will be explored.

A reduced dose/administration schedule may be considered if required, based on emerging s afety data. B Patient group Patients with advanced NSCLC. C Sample size Approximately eight patients will be entered into this trial. The final number will depend on emerging safety data, the number of patient replacements required and vaccine availability . D Study intervention Patients will receive six AST -VAC2 vaccinations, administered weekly over six weeks . They will attend an end of vaccination visit 30 days after their last AST-VAC2 vaccination , and attend follow -up visits up to 12 months after their first AST -VAC2 vaccination . After which, patients will be followed up for safety quarterly by telephone for five years (post first vaccination). Information on OS will be collected for up to 2 years after the first vaccination for al l patient s or until all patients have died, whichever event occurs first. E Study analysis Safety evaluations will be conducted after the first patient has received AST -VAC2 and then again after the second and third patients have been treated. Once open recruitment has been initiated, safety review will be performed at regular intervals (as requ ired). A safety review committee, consisting of all Investigators, representatives of the Sponsor and representatives of the drug company (observers only), will review emerging study data, with particular reference to ensuring that patients are able to com plete the vaccination schedule without AST -VAC2 toxicity -related delays. See protocol Section 3.3.2. An interim analysis will be conducted once all patients administered AST-VAC2 have withdrawn, died or completed their 2- year follow - up visit for survival. Once this condition is met, a data cut -off will be established. All patient visits occurring on or before this date will be analysed and summarised in the clinical study report, including safety data to date and assessment of OS at 2 years. Any data collected after this date will be summarised in a supplemental report for the final analysis to satisfy the safety reporting requirements for AST -VAC2 as an advanced therapy investigational medicinal product (ATIMP). The final analysis will be conduct ed after one of the following conditions is met: • The trial is terminated early, e.g. due to toxicity; • The End of Trial has been reached (when all patients have either withdrawn from the trial or died, or the last patient SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 10 has completed their final follow -up visit [whichever is the latter]). Once one of the se conditions is met, a data cut -off will be established. All patient visits occurring on or before this date will be analysed and summarised in a supplemental report. Due to the small sample size, there ar e no formal statistical analyses planned for this trial. F Dose escalation schedule There is no dose escalation schedule for this study, only one dose will be explored. Stopping rule criteria can be found in protocol Section 3.3.3.

For full details of the trial design, background and rationale for the study, please refer to the current study protocol. SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 11 4. PATIENT POPULATION AND ENDPOINTS Patients must fulfil all the inclusion/exclusion criteria to be eligible for entry to the trial. Refer to the study protocol for the complete list of inclusion and exclusion criteria. 4.1 Patient Populations The analysis sets are defined as follows: Full Analysis Population (FAP): All patients who enrolled onto the main trial. Patients who are enrolled in error onto the main trial (due to ineligibility/administrative error), prior to receiving any AST -VAC2 vaccinations , will be excluded from the FAP. Safety Population: All enrolled patients who received at least one AST -VAC2 vaccination . This population will also be used for overall survival at 2 years post first AST -VAC2 vaccination. Patients are evaluable for survival regardless of whether they go on to receive another anti -cancer therapy. HLA Population: All patients who had a sample t aken for HLA analysis. Secondary Immunogenicity Endpoint Population: All eligible patients who received at least three AST-VAC2 vaccinations and have had a baseline blood sample and at least two post -vaccination blood sample s taken . Response Population: All eligible patients who have received at least one AST -VAC2 vaccination and have had a baseline assessment of disease and at least one repeat disease assessment measured according to irRC. For details of irRC, refer to Appendix 3 of the study protocol. If rapid tumour progression occurs before completion of the scheduled six AST -VAC2 vaccinations, the patient will be classified as having early progression. Patients with early progression will be included in the response population. SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 12 5. DATA CONVENTIONS AND GENERAL ANALYSIS 5.1 Patient disposition Patients excluded from any population (defined in Section 4.1) will be detaile d. The accrual and trial discontinuation details will be presented descriptively. This should include details of: • Screening failure patients o Pre-screening – patients were pre -screened for human leucocyte antigen ( HLA) status as part of the study. The number of patients who underwent HLA screening and the number of patients HLA A*02:01 positive and negative will be summarised . o Overall screening failure information is available via the e-screening logs and will be described descriptively . • Information on ineligible patients who were enrolled and/or received AST -VAC2 . • Reasons for vaccination discontinuation by number of vaccinations received will be described by counts and percentages. Reasons for vaccination discontinuation other than disease progression will be detailed and summarised separately (Section 5.3). 5.2 Baseline characteristics Demographics and baseline characteristics will be summarised for all enrolled patients. Table 2: Summary of Patient Demography (Full Analysis Population) All Patients N=X Patients Sex Male N (%) Female Age (Years) Mean Median Min Max WHO performance status 0 N (%) 1 2 3 4 *Footnote: withdrew from the study prior to first v accination SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 13 In case of pre -treatment characteristics with multiple measurements per patient before the start of treatment (laboratory assessments, vital signs), the baseline measurement will be considered the last value prior to or on the first day of vaccination . Baseline performance status assessments will be summarised with frequency counts. For the cancer history, histologic diagnosis, number of baseline lesions, and involvement in the different sites will be summarised. If incomplete dates are recorded, the rules descri bed in Section 5.10 will be used for imputation. The primary tumour sites and baseline lesions will be recorded in order to categorise them accurately in the analysis. A frequency tabulation of the different types of previous oncologic surgery (excluding only diagnostic or palliative procedures), radiotherapy, or anti -cancer systemic therapy will be given. Table 3: Primary Diagnosis at Baseline (Full Analysis Population) No. of Patients (N=XX) Primary tumour type N (%) Stage at study entry Stage I Stage II Stage III Stage IV Not applicable Not known Table 3 will be manually generated for the Clinical Study Report (CSR) based on the programmed table from the clinical trial database to allow for better grouping by primary tumour type. Table 4: Summary of Prior Treatment for M alignant Disease (Full Analysis Population) No. of Patients (N=XX) Prior treatment Surgery N (%) Radiotherapy Chemotherapy/other SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 14 Table 5: Summary of Prior L ines of C hemotherapy/ Other Therapy (Full Analysis Population) No. of Patients (N=XX) Lines of chemotherapy/other 1 N (%) 2 3 4 5, etc Median (range) X (X-X) Table 5 will be manually generated for the Clinical Study Report based on the ‘Previous Treatment for Malignant Disease – Chemotherapy/Other Therapy’ listing . Table 6: Summary of B aseline Disease Sites (Full Analysis Population) Baseline disease type No. of Patients (N=XX) Patient Numbers Primary tumour N (%) XX/XXX, XX/XXX Local recurrence Regional nodes Metastatic nodes Lung metastases Liver metastases Etc 5.3 Patient withdrawal Reasons for patient withdrawal from vaccination will be provided as the data are available and will be presented descriptively . 5.4 Protocol deviations A protocol deviation is defined as any departure from what is described in the protocol of a clinical trial approved by an Independent Ethics Committee and Competent Authorities.

Therefore, it applies to deviations related to patient inclusion and clinical procedures (e.g. assessments to be conducted or parameters to be determined), and also to other procedures described in the protocol that concern the Good Clinical Practice (GCP) guidelines or ethical issues (e.g. issues related to obtaining the patients’ Informed Consent, data reporting, the responsibilities of the Investigator, etc.). Protocol deviations are captured throughout the trial open phase on the Sponsor’s central tracker and can be filtered by study and by deviation category or on the study specific pharmacokinetic/pharmacodynamic ( PK/PD ) deviations tracker (and the PK/PD tab [version 1] of the standard study tracker for deviations prior to set up of PK/PD deviations tracker) . Standard deviation categories have been defined by the Sponsor and are further defined by those which are deemed reportable (important deviations) in the CSR. Those deviations which have been coded as CSR reportable SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 15 will be summarised for all patients, according to the categories allocated at identification. Deviations are reviewed manually as per Sponsor standard operating procedures ( SOPs ). A summary ta ble with the number of patients with deviations will be presented per criterion. Deviations with no effects on the risk/benefit ratio of the clinical trial (such as minimal delays in assessments or visits) will be distinguished from those that might have a n effect on this risk/benefit ratio. The following are pre -defined protocol deviations with a direct bearing on the primary outcome and therefore will be reported in the CSR. A summary including but not necessarily restricted to the following categories w ill be presented: • Ineligible patients as per protocol. • Patient not withdrawn as per protocol. • Excluded concomitant medication. • Incorrect investigational medicinal product ( IMP) dose or schedule . Table 7: Protocol Deviations per C riterion Deviation criteria Number of Patients Patient Numbers Contraindicated medication Dosing error Eligibility criteria IMP (administration of expired/quarantined IMP) Missed visit or investigation PK/PD samples and endpoints Visit o r investigation conducted outside of window Imaging Other (serious breach/urgent safety measure not reported in required timelines) Deviations relating to all the other PD samples /endpoints will be documented in the individual PD reports and not in this summary table . 5.5 Treatment compliance Patients are expected to re ceive six AST-VAC2 vaccinations over a period of six weeks with an interval of 7 days (+/ -1 day) between each of the vaccination days . Dose reductions (i.e.

, reduced dose or modified number of vaccinations) are not expected but would be considered by the Sponsor an d Chief Investigator based on vaccine availability or if plateaued immune responses were observed with fewer vaccinations in those patients already treated . SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 17 5.7 Adverse Events Pre-treatment AEs will be defined as those where “Did this AE start prior to first vaccination ?” is ticked. Treatment Emergent AEs (TEAEs) will be defined as those where “Did this AE start prior to first vaccination ?” is not ticked. Related AEs are those where causality to IMP is considered to be Possible, Probable or Highly Probable. The frequency of adverse events will be summarised overall . A patient can be counted multiple times per row for the No. of Episodes but will only be counted once per row for the No.

of Patients. Table 8: Frequency of All Adverse Events (Safety Population) SYSTEM ORGAN CLASS Preferred Term No. of Episodes Reported No. of Patients N=XX All AEs N N (%) BLOOD AND LYMPHATIC… Anemia CARDIAC DISORDERS Sinus tachycardia Etc There will be additional versions (as required) of the above table based on pre -treatment AEs, TEAEs , treatment emergent SAEs (any AE that is considered Serious) and related TEAEs. Overview of TEAEs will be presented for all patients. An AE is considered to have led to withdrawal if an adverse event with “Did the AE cause the subject to be discontinued from the study?” is recorded as yes. Table 9: Overview of Treatment Emergent Adverse Events (Safety Population) Patients with TEAEs Overall No. of Patients N=XX Patients with >=1 TEAE N (%) Patients with >= 1 TE SAE Patients with >= 1 related TEAE Patients with >= 1 CTCAE Grade 3, 4 or 5 TEAE Patients with >= 1 AE leading to withdrawal * Patients who died due to TEAE *Footnote: AEs that lead to withdrawal of vaccination ; patients remained on study for assessmen t of response, safety and overall survival. TEAEs leading to withdrawal of vaccination will be summarised descriptively . Patients will be included if an AE has “Action taken” recorded as “Drug Withdrawn” or “Did the AE cause the subject to be discontinued from the study?” is recorded as yes. All TEAEs and related TEAEs by worst CTCAE grade within an episode will be presented by number of TEAEs and by numbe r of patients (see Table 10 and Table 11, respectively ). SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 18 Table 10: Frequency of Treatment Emergent A dverse Event s and Related Treatment Emergent Adverse Events by Worst CTCAE Grade within an Episode (Safety Population) SYSTEM ORGAN CLASS Preferred Term All TEAEs AST-VAC2 Related TEAEs _______Grade______ _______Grade______ Total 1 2 3 4 5 Total 1 2 3 4 5 Overall total N N N N N N N N N N N N BLOOD AND LYMPHATIC… Anaemia CARDIAC DISORDERS Sinus tachycardia Etc Table 11: Frequency of Patients with Treatment Emergent A dverse Event s and Related Treatment Emergent Adverse Events by Worst CTCAE Grade within an Episode (Safety Population) SYSTEM ORGAN CLASS Preferred Term All TEAEs AST-VAC2 Related TEAEs No. of patients with > =1 AE (N=XX) No. of patients with >=1 AE (N=XX) _______Grade______ _______Grade______ Total 1 2 3 4 5 Total 1 2 3 4 5 Overall total N (%) N N N N N N (%) N N N N N BLOOD AND LYMPHATIC… Anaemia CARDIAC DISORDERS Sinus tachycardia Etc 5.8 Laboratory results Laboratory results (haematology, biochemistry and urinalysis) will be presented in data listings as an appendix to the CSR. Any significant out of range values are captured as AEs and will be presented in the summary AE tables (see Section 5.7). 5.8.1 Additional safety tables A table of ISR grade by vaccination number will be produced. SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 19 Table 1 2: Summary of I njection Site Reactions by Grade and Vaccination Number (Safety Population) ISR Grade Vaccination number at onset Total 1 2 3 4 5 6 1 N N N N N N N 2 3 4 5 5.8.2 Stopping rule criteria Any AEs that meet the stopping rule criteria will be presented descriptively. 5.9 Efficacy Documentation of tumour response is a secondary o bjective of this trial. A nti-tumour activity will be measured according to the modified irRC; (see Protocol Appendix 3 ) at 30 days (+/- 5 days) after the last AST-VAC2 vaccination in all patients . The overall response will be presented for the ‘Response Population’ (see Table 13). The response rate is defined as the ratio of patients with any response ( immune -related complete response [ irCR] or immune -related partial response [ irPR]) to the total number of patients included in the efficacy population. Overall survival will be assessed in the ‘Safety Po pulation’ and is defined as the time from the first AST-VAC2 vaccination to the date of death (regardless of whether the patient went on to receive other anti-cancer treatments) or date of last contact (if patient is still alive or lost to follow -up). Survival will be summarised for each patient as shown in Table 14.

Patients with data censored for survival will be indicated with a ‘+’ after the number of days. CEA data will be listed per patient. Table 1 3: Overall R esponse (Response Population) Overall Tumour Response No. of Patients N=XX irCR X (XX.X%) irPR irSD irPD NE Response rate (irCR + irPR) SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 20 Table 1 4: Summary of Survival (Safety Population) Patient Time to Death (or Last Contact) (days) XX/XXX N XX/XXX XX/XXX XX/XXX Median (range) X (X-X) Table 1 5: Two Year Survival (Safety Population) Patient s alive Patients deceased N (%) [ XX/XXX , XX/XXX …] N (%) [XX/XXX, XX/XXX… ] 5.10 General data conventions There is only one dose level for this study, as specified in the study protocol. Patients who deviate from the assigned dose and/or schedule will be clearly described in the CSR with regards to their treatment modification and if applicable in the list of protocol deviations ( Section 5.4).

Continuous variables will be summarised and presented with summary statistics.

The median OS will be presented. Categorical variables will be summarised in frequency tables. Percentages in the summary tables will be rounded and may therefore not always add up to exactly 100%. The convention in Rave is that an unknown day resolves to 1st of the month and an unknown month resolves to January. Dates may be ordered by this ; however , CRUK do not perform calculations on unknown dates except as stated below. Durations of AEs: the start date of an AE is considered as Day 1 of the event and should be included in all duration calculations (i.e. if an AE starts and stops on same day, the duration should be reported as one day). - For unrelated AEs, those with missing or partially completed end dates will not be excluded from analysis and the duration of the AE will be calculated from the first day of the month (if unknown) or first day of the year (based on RAVE conventions) . - For related AEs, prior to final data lock, an end date will be either i) confirmed AE end date or ii) stabilisation of AE. Cases where this is not possible are if the patient was lost to follow up . If lost to follow -up then the AE would stay as not recovered/not resolved. In this case duration would not be calculated. Time to onset of AEs from IMP administration: the time to onset should be calculated from the date of the first AST -VAC2 vaccination and AEs occurring within a specific vaccination window (e.g. start date from date of 2 nd vaccination to before date of 3rd vaccination) should also have times to onset calculated from the date of the last vaccination administered in that window (e.g. Vaccination 2) . Onset time will be SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 21 calculated as 0 if the AE occur s on the same day as the dose ; however , if the start time is missing, onset time will not be calculated (only applicable when the drug admin has a start time entered) . AE assignment: AE is assigned to the vaccination number it begins in .

This is regardless of whether the AE start time shows the AE started before the dose that day. The only exception is for the first vaccination ; if ‘Did AE occur prior to first dose’ is checked then no vaccination number will be assigned. Duration of treatment : this is from the day of the first vaccination until the day of the final vaccination . Duration of treatment will be described using summary statistics. Completed vaccination : AST-VAC2 vaccinations are to be given once a week for 6 weeks. A patient is considered to have completed vaccination when vaccination data entered is for each of those expected weekly visits (despite dose modifications) . Treated patients: If the drug administration form in the eCRF has the ‘date of vaccination ’ completed, this constitutes a treated patient. 5.11 Decimal places When data are used in calculations it important that rounding is only conducted when the final test result is obtained (to avoid accumulation of errors).

All percentages should be presented to 1 decimal place. If a percentage value is less than 0.1% on rounding, then use ‘<0.1%’.

Days to be presented to 0 decimal places. 5.12 Statistical software Medidata Rave will be used as the Electronic Data Capture (EDC) system for the trial. SAS 9.4 will be used to generate data listings and summary tables/graphs. 5.13 Supplementary analysis – Pharmacodynamics (data collected outside of the clinical database) Pharmacodynamic anal yses will be described in supplementary lab report s . There are no primary endpoint pharmacodynamic assays for this study. 5.13.1 HLA Pre-screening DNA extracted from whole blood taken at pre -screening will be analysed by Luminex (or similar method) for HLA-A*02:01 allelic string (HLA population) . The whole A* allele will be reported for each patient. Results are reported per patient with in 14 days of receipt of the sample . The HLA-A*02:01 status (positive or negative) is also captured in the clinical database on the enrolment eCRF. SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 22 The final report is expected no more than two months after completion .

No formal statistical analysis is planned.

The data will be summarised in the CSR and the final report included as an appendix . 5.13.2 Secondary endpoint s 5.13.2.1 Identification of peptide specific T cells (Secondary Immunogenicity Endpoint Population) Immunological response will be measured in whole blood (PBMCs) by hTERT specific T cells as measured by ELISPOT analysis at the following time points: • baseline, • vaccination weeks 3, 4 and 6, • 2 weeks post last vaccination and • 3, 6 and 12 months post firs t vaccination. Vaccine induced ELISPOT response in blood will be reported longitudinally for each patient. The total number of patients showing durable peripheral immune response, defined as a change in one validated assay at two time points after at least two vaccinations (where a change is defined as 2.5 fold change over baseline [ after removal of background ], assay control and >35 spots/10 6 cells) will be reported. Interim reports are expected at timepoints to be agreed between the Sponsor and the laboratory.

Interim reports will be provided within one month of completing the agreed patient analyses. A final report is expected no more than 10 weeks after the last sam ple is analysed. The data will be reported in tables and graphs/ figure s. No formal statistical analysis is planned. The results will be summarised in the CSR and the final report included as an appendix. 5.13.3 Tertiary endpoints SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 23 SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 24 SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 25 5.14 Other statistical analysis 5.14.1 Stratification and covariate analysis No stratification or covariate analysis is planned. 5.14.2 Multivariate analysis No multivariate analysis is planned. 5.14.3 Subgroup analysis Given the small number of patients to be vaccinated, no subgroup analyses are planned. 5.14.4 Interim analysis An interim analysis will be conducted once all patients administered AST -VAC2 have withdrawn, died or completed their 2- year follow -up visit for survival. Once this condition is met, a data cut -off will be established. All patient visits occurring on or before this date will be analysed and summarised in the clinical study report including safety data to date and assessment of OS at 2 years. Any data collected after this date will be summarised in a supplemental report produced for the final analysis to satisfy the safety reporting requirements for AST -VAC2 as an advanced therapy investigational medicinal product (ATIMP ). The relevant data will be uploaded to ClinicalTrials.gov after the CSR is approved. 5.14.5 Final analysis The final analysis will be conducted after one of the following conditions is met: • The trial is terminated early (e.g. due to toxicity) • The End of Trial has been reached (defined as the date when all patients have either withdrawn from the trial or died, or the last patient has completed their final follow- up visit [whichever is the latter]. The final follow -up visit is scheduled to take place 5 years after t he patient’s first vaccination) . Once one of the conditions is met, a data cut -off date will be established. All patient follow -up visits occurring on or before this date will be analysed and summarised in a supplemental report. SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 26 6. TABLES LISTINGS AND FIGURES 6.1 LISTING S CSR Appendix Subsection CSR Listing Number Listing New Listing Name for CSR 16.2.1 Discontinued Patients 16.2.1.1 Vaccine Received Vaccine Received 16.2.1.2 End of Vaccination Vaccination Completion 16.2.2 Protocol Deviations 16.2.2.1 To be provided from the central deviation tracker N/A 16.2.3 Patients excluded from Efficacy Analysis 16.2.3.1 Enrolment Enrolled Patients 16.2.4 Demographic and Baseline Data 16.2.4.1 Demographics Patient Demography 16.2.4.2 Diagnosis Diagnosis of Primary Disease 16.2.4.3 Medical History Medical History 16.2.4.4 Medical Procedures Medical Procedures 16.2.4.5 Prior and Concomitant Medication s Prior and Concomitant Medication s 16.2.4.6.1 Surgery Previous Treatment for Malignant Disease - Surgery 16.2.4.6.2 Chemotherapy/other therapy Previous Treatment for Malignant Disease – Chemotherapy/Other Therapy 16.2.4.6.3 Radiotherapy Previous Treatment for Malignant Disease - Radiotherapy 16.2.4. 7 Physical Examination Physical Examination 16.2.4. 8 Chest X-ray Results Chest X -ray 16.2.5 Compliance and Drug Administration 16.2.5.1 Drug Administration - Vaccination Drug Administration - Vaccination 16.2.6 Efficacy Response 16.2.6.1 Immune Response Index Lesions Immune Response Index Lesions 16.2.6.2 Immune Response Non-Index Lesions Immune Response Non -Index Lesions 16.2.6.3 Immune Related Response Criteria Immune Related Response 16.2.6.4 New Anti-Cancer Therapy New Anti -Cancer Therapy 16.2.6.5 Survival Survival 16.2.6.9 CEA Sample CEA Sample 16.2.7 Safety 16.2.7.1 Adverse Events Adverse Events SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 27 16.2.7.2 Adverse Events by SOC Adverse Events by SOC 16.2.7.3 Follow -Up Follow Up 16.2.7.4 WHO WHO Performance Status 16.2.7.5 Haematology Haematology : Haemoglobin, WBC, Neutrophils, Lymphocytes , Eosinophils, Platelets 16.2.7.6 Biochemistry Biochemistry : Sodium, Potassium, Adjusted calcium, Phosphate, Urea, Creatinine, Total Protein, Albumin, Bilirubin, ALP, ALT, AST, CRP 16.2.7.7 Urinalysis Urinalysis : Glucose, Protein, Blood, pH 16.2.7.8 Vital Signs Vital Signs 16.2.7.9 Pregnancy Pregnancy 16.2.7.10 ECG ECG 6.2 SUMMARY TABLES Table Number in RAP CSR Table Number Table Name Table 1 N/A Trial Design N/A: to be produced as part of lab reporting In-text table Human Leucocyte Antigen Status at Screening Table 2 14.1.1 Summary of Patient Demography (Full Analysis Population) Table 3 14.1.2 Primary Diagnosis at Baseline (Full Analysis Population) Table 6 14.1. 3 Summary of B aseline Disease Sites (Full Analysis Population) Table 4 14.1.

4 Summary of Prior T reatment for Malignant Disease (Full Analysis Population) Table 5 In-text table (will refer to listing) Summary of Prior Lines of Chemotherapy/Other Therapy (Full Analysis Population) Table 7 In-text table (will refer to listing) Protocol D eviations per C riterion Table 9 14.3.1 Overview of Treatment Emergent Adverse Events (Safety Population) Table 8 14.3.2 Frequency of All Adverse Events (Safety Population) N/A: based on Table 8 14.3.3 Frequency of Pre -Treatment Adverse Events (Safety Population) N/A: based on Table 8 14.3.4 Frequency of Treatment Emergent Adverse Events (Safety Population) N/A: based on Table 8 14.3.5 Frequency of Treatment Emergent Serious Adverse Events (Safety Population) SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 28 N/A: based on Table 8 14.3.6 Frequency of Related Treatment Emergent Adverse Events (Safety Population) Table 10 14.3.7 Frequency of Treatment Emergent A dverse Event s and Related Treatment Emergent Adverse Events by Worst CTCAE Grade within an Episode (Safety Population) Table 11 14.3.8 Frequency of Patients with Treatment Emergent Adverse Events and Related Treatment Emergent Adverse Events by Worst CTCAE Grade within an Episode (Safety Population) Table 1 2 14.3.9 Summary of Injection Site Reactions by Grade and Vaccination Number (Safety Population) Table 1 3 14.2.1 Overall R esponse (Response Population) Table 1 4 14.2.2 Summary of Survival (Safety Population) Table 1 5 14.2.3 Two Year Survival (Safety Population) 7. PRESENTATION OF RESULTS IN CLINICALTRIALS.GOV INCLUDING ADDITIONAL TABLES The following information will be used for presentation and upload of trial data on Clinical Trials.gov.

The data presented will be consistent with the final CSR but will be tabulated in a format that fits with the ClinicalT rials.gov requirements1. The tables programmed for C linicalT rials.gov will not form part of the Clinical Study Report (CSR). 7.1 Arms and Interventions The trial will be presented as a single group design .

Data will be presented for one arm. Treatment arm: Participants with advanced NSCLC, to receive AST -VAC2 Treatment Arm type : Experimental Arm Description : Participants will receive up to a maximum of six vaccinations over six weeks. Each weekly AST -VAC2 vaccination will be administered as a split dose via two intradermal injections at a target dose defined as 1 x 107 viable cells. There is no dose escalation planned during the study; only one dose level will be explor ed. Intervention type: Biological/Vaccine Intervention Name(s): AST-VAC2 Other Intervention Name(s) : N/A Intervention Description : Allogeneic dendritic cell vaccine. 1 ClinicalTrials.gov Results Data Element Definitions for Interventional and Observational Studies found here SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 29 7.2 Participant Flow The participant flow section of the results upload will include the following details. The programmed table output for reason not completed may be re -categori sed to ClinicalT rials.gov categories and reviewed by C linical Study Manager for approval. Period Title Overall Study Arm/Group Title Experimental: AST -VAC2 Arm/Group Description Participants will receive up to a maximum of six vaccinations over six weeks. Each weekly AST -VAC2 vaccination will be administered as a split dose via two intradermal injections at a target dose defined as 1 x 107 viable cells. There is no dose escalation planned during the study; only one dose level will be explored. Number of Participants Started (participants enrolled into main trial) * Complete d (6 vaccinations) Not completed Reason not completed Adverse Event Death Lack of Efficacy Lost to Follow -up Physician Decision Pregnancy Protocol Violation Withdrawal by Subject Disease Progression SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 30 Sponsor ’s decision to terminate the trial Started (participants entered follow -up period) Complete d (2-year OS follow up) Not completed Reason not completed Lost to follow -up Withdrawal of consent Death Declined to attend follow -up visit SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 31 7.3 Baseline Characteristics The baseline characteristics section of the results upload will include the following details. The programmed table output for reason not completed may be re-categori sed to C linicalT rials.gov categories and reviewed by C linical Study Manager for approval.

Note: CDD trials don’t rout inely collect data on race. Ethnicity information is collected for some trials, where ethnicity information is collected, the standard category of Race will be entered as “not collected” (or equivalent) on C linicalT rials.gov and Ethnicity information provided as an additional baseline measure. Arm/Group Title Experimental: AST -VAC2 Arm/Group Description Participants will receive up to a maximum of six vaccinations over six weeks. Each weekly AST -VAC2 vaccination will be administered as a split dose via two intradermal injections at a target dose defined as 1 x 107 viable cells. There is no dose escalation planned during the study; only one dose level will be explored. Overall Baseline Participants Number of Participants Baseline Analysis Population Description * Age, Number Analysed Age, Median (Full range) Sex, Number Analysed Male, number of participants Female, number of participants Ethnicity Not collected SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 32 7.4 Reported Adverse Events (clinicaltrials.gov template sections) AE Reporting Timeframe: Safety data will be collected from the time of informed consent until 2 years from the first dose of AST -VAC2 for this interim report . Patients are followed -up for 5 years; if there are any changes to information reported at end of trial, this will be updated.

The average (median and range) time from consent to the end of follow up will be calculated from the database presented in the AE tables. All-Cause Mortality will be presented as per the C linicalT rials.gov template as follows (based on a programmed table) : Note: CT.go v Definition of Total all cause m ortality: Total of all anticipated and unanticipated deaths due to any cause . Experimental: AST -VAC2 Affect/At Risk (%) # Events Total All Cause Mortality * Serious Adverse events will be presented as per the C linicalT rials.gov template as follows (based on a programmed table) : Experimental: AST -VAC2 Affect/At Risk (%) # Events Total <AE term> <AE term> <AE term> Other (non -Serious) Adverse events will be presented as per the clinicaltrials.gov template as follows (based on a programmed table) : Frequency Threshold Above Which Other Adverse Events are Reported: 0% Experimental: AST -VAC2 Affect/At Risk (%) # Events Total SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 33 <AE term> <AE term> <AE term> SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 36 were not evaluable at radiological disease assessment (computerised tomography and/or magnetic resonance imaging) at the End of Vaccination visit according to irRC (Appendix 3 of protocol) . Time Frame: Baseline to End of Vaccination visit ( 30 days post last vaccination ). Measure Type: Count of participants Measure of dispersion/precision: Not applicable To determine the 2 -year overall survival (OS) in patients receiving the AST -VAC2 vaccine. Measurement of overall survival at two years post first vaccination. Measure Title : Overall survival at 2 Years Post First Vaccination Measure Description Number of participants alive at 2 years post their first vaccination . Time Frame: From first AST -VAC2 vaccination to 2 years post first vaccination . Measure Type: Count of participants Measure of dispersion/precision: Not applicable SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 37 7.5.1 Outcome Measure 1 – Frequency and causality of AEs and SAEs to AST -VAC2 (Safety Population) The following table will be programmed to provide results for f requency and causality of AEs and SAEs to AST -VAC2 . Arm/Group Title Experimental: AST -VAC2 Frequency and causality of AEs , SAEs and Grade ≥3 AEs to AST - VAC2 Measure type: Number Unit of measure: not applicable Number of participants analysed <N> SAEs <data> Non-serious AEs <data> Grade ≥3 AEs <data> Related SAEs <data> Related non -serious SAEs <data> Related Grade ≥ 3 AEs <data> SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 38 7.5.2 Outcome Measure 2 - Number of participants experiencing ISRs by grade (Safety Population) The following table will be programmed to provide results for number of pa rticipants experiencing ISRs Grade 1 to 4. Arm/Group Title Experimental: AST -VAC2 Participants experiencing ISRs Grade 1 to 4 Measure type: Count of participants Unit of measure: participants Number of participants analysed <N> Grade 1 <data> Grade 2 <data> Grade 3 <data> Grade 4 <data> SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 39 7.5.3 Outcome Measure 3 – Number of participants showing a durable peripheral immune response (Secondary Immunogenicity Endpoint Population) The following table will be programmed to provide results for the number of pa rticipants with a durable periperal immune response . Arm/Group Title Experimental: AST -VAC2 Participants showing a durable peripheral immune response Measure type: Count of participants Unit of measure: participants Number of participants analysed <N> Durable response <data> Non-durable response <data> Undetermined (baseline sample did not pass acceptance criteria or insufficient timepoints ) <data> SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 40 7.5.4 Outcome Measure 4 – Mean fold change over baseline by timepoint (Secondary Immunogenicity Endpoint Population) The following table will be programmed to provide results for the mean fold change from baseline . Arm/Group Title Experimental: AST -VAC2 Overall Number of Participants Analysed <N> Mean fold change over baseline Measure type: Mean Unit of measure: full range Number of participants analysed <N> Week 3 <data> Number of participants analysed <N> Week 4 <data> Number of participants analysed <N> Week 6 <data> Number of participants analysed <N> 2 weeks post last vaccination <data> Number of participants analysed <N> 3 months post first vaccination <data> Number of participants analysed <N> 6 months post first vaccination <data> Number of participants analysed <N> 12 months post first vaccination <data> SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 41 7.5.5 Outcome Measure 5 - Tumour response according to Immune -Related Response Criteria (irRC) post vaccination (Response population) The following table will be programmed to provide results for tumour response . Arm/Group Title Experimental: AST -VAC2 Tumour response according to irRC post vaccination Measure type: Count of participants Unit of measure: participants Number of participants analysed <N> Complete response (irCR) <data> Partial response (irPR) <data> Stable disease (irSD) <data> Progressive disease (irPD) <data> Not evaluable (NE) <data> SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 42 7.5.6 Outcome Measure 6 – O verall survival at 2 years post first vaccination (Safety Population) The following table will be programmed to provide results for overall survival . Arm/Group Title Experimental: AST -VAC2 Overall survival at 2 years post first vaccination Measure type: Count of participants Unit of measure: participants Number of participants analysed <N> Alive <data> Deceased <data> 7.6 Statistical Analyses No statistical analyses are to be performed on the primary or secondary endpoints . SM.TEMP.013 version 5 CRUKD/17/003 RAP Version: 2.0 09Mar2023 FINAL 43 8.

REPORTING OF CLINICAL TRIAL RESULTS TO PATIENTS AND PUBLIC When considering preparation of information that is for patients and their carers/families. It is important to take into account the language used and to allow opportunity to tailor information depending on the patients’ experience/current situation: - Refer to Guidance on use of Plain Language and Readability checks in the PICD toolki t here At the End of the Trial At the end of the trial summary results in lay language will be provided on the CRUK website at the following link: A trial of a vaccine called AST -VAC2 in non small cell lung cancer . Additionally, a link to the CRUK lay summary of the results will be a dded to the HRA website . CRUK CDD will provide Investigator Sites with a .pdf copy of the results from the CRUK trials database for distribution to pa tients and their families (as appropriate and at the discretion of the investigator). 9.

REFERENCES • Guidelines for the C ontent of S tatistical Analysis Alans in Clinical Trials, JAMA December 2017, Volume 318, Number 23. Gamble et al.

Statistical Analysis Plan Study Code D419EC00001 Edition Number 4.0 Date 19Apr2023 Phase I/II, Open -Label, Multicenter Study to Evaluate the Safety , Tolerability and Preliminary Efficacy of Durvalumab Monotherapy or Durvalumab in Combination with Tremelimumab in Pediatric Patients with Advanced Solid T umors and Hematological Malignancies Statistical Analysis Plan Study Code D419EC00001 Edition Number 4.0 Date 19Apr2023 CONFIDENTIAL AND PROPRIETARY 2of 65TABLE OF CONTENTS TITLE PAGE ........................................................................................................................ 1 TABLE OF CONTENTS ......................................................................................................

2 LIST OF ABBR EVIATIONS ............................................................................................... 5 AMENDMENT HISTORY ................................................................................................... 8 1 STUDY DETAILS ........................................................................................... 10 1.1 Study objectives................................................................................................ 10 1.1.1 Primary object ives............................................................................................ 10 1.1.2 Secondary object ives........................................................................................ 11 1.1.3 Safety objectives............................................................................................... 12 1.1.4 Exploratory objectives ...................................................................................... 12 1.2 Study design ..................................................................................................... 12 1.3 Number o f patients ........................................................................................... 18 2 ANALYSIS SETS ............................................................................................ 20 2.1 Definit ion of analysis sets ................................................................................. 20 2.2 Violations and deviat ions.................................................................................. 22 3 PRIMARY AND SECONDAR Y VARIABLES ............................................... 27 3.1 Derivat ion of visit tumor responses ................................................................... 27 3.1.1 Derivat ion of RECIST 1.1 visit responses ......................................................... 27 3.1.1.1 Target l esions (TLs) –site Invest igator data ...................................................... 28 3.1.1.2 Non-target l esions (NTLs) and new lesio ns –site Investi gator data. .................. 33 3.1.1.3 Overall visit response –site Invest igator data .................................................... 34 3.2 Efficacy Variables ............................................................................................ 35 3.2.1 Object ive response rate (ORR) .......................................................................... 35 3.2.2 Progression -free survival (PFS) ........................................................................ 35 3.2.3 Overall Survival (OS) ....................................................................................... 37 3.2.4 Durati on of response (DoR) .............................................................................. 38 3.2.5 Best objective response (BoR) .......................................................................... 38 3.2.6 Disease control rate (DCR) ............................................................................... 39 3.2.7 Proporti on alive and progression free at 12 (APF12) and 18 (APF18) months ...39 3.2.8 Overall survival at 12 (OS12) and 24 (OS24) months ........................................ 39 3.3 Safety Variables................................................................................................

39 3.3.1 Adverse Events ................................................................................................. 40 3.3.2 Laboratory data ................................................................................................. 42 3.3.3 ECG data .......................................................................................................... 43 3.3.4 Vital signs ......................................................................................................... 43 3.3.5 Exposure and dose intensit y.............................................................................. 43 3.3.6 Dose Limit ing Toxi cities (DLTs) ...................................................................... 44 Statistical A nal ysis Pla n St u d y C o de D 4 1 9 E C 0 0 0 0 1 E diti o n N u m ber 4.

0 Date 1 9 A pr 2 0 2 3 C O N FI D E N TI A L A N D P R O P RI E T A R Y 4of 6 5 4.

2. 6 C o nc o mita nt a n d ot her treat me nt ................................ ...................................... 6 3 4. 2. 7 Bi o m ar ker data ................................ ................................

................................ . 6 3 4. 2.

7. 1 A nt i b o d y ti t er m eas ure me nts bef ore a n d after r o uti ne i m m u nizati o n ................. 6 3 4.

2. 7. 2 .......................................................... 6 4 5 I N T E RI M A N A L Y S E S ................................ .................................................... 6 4 6 C H A N G E S O F A N A L Y SI S F R O M P R O T O C O L ............................................ 6 4 7 R E F E R E N C E S ................................ ................................

................................

. 6 5 8 A P P E N DI X ( N O T A P P LI C A B L E) ................................ .................................. 6 5 LI S T O F T A B L E S Ta ble 1 Ke y c o nsi derat i o ns f o r D R C ................................ ......................................... 1 5 Ta ble 2 Mali g na nt s oli d t u m or c o h orts ................................ ...................................... 1 6 Ta ble 3 A nal ysis sets ................................ ................................

................................

. 2 0 Ta ble 4 S u m mar y o ut c o m e varia bles a n d a nal ysis sets ............................................... 2 1 Ta ble 5 Criteria f or I n vest i gat or assess me nts ............................................................. 2 7 Ta ble 6 T L Visit Res p o nses ( R E CI S T 1. 1) ................................ ................................ 2 9 Ta ble 7 N T L Visit Res p o nses ( R E CI S T 1. 1) ............................................................. 3 3 Ta ble 8 O verall visit res p o nses ................................ .................................................. 3 5 Ta ble 9 Pre -pla n ne d statistical a nal ysis t o be c o n d ucte d ............................................ 5 2 LI S T O F FI G U R E S Fi g ure 1 D ose le vels –D ose -fi n di n g p ha se .............................................................

1 4 Fi g ure 2 D ose -fi n di n g: de -escalat i o n sc he ma .......................................................... 1 6 C CI Statistical Analysis Plan Study Code D419EC00001 Edition Number 4.0 Date 19Apr2023 CONFIDENTIAL AND PROPRIETARY 5of 65LIST OF ABBREVIATION S Abbreviation or special termExplanation ADA Anti-drug antibody AE Adverse event AEPI Adverse event of possible interest AESI Adverse event of special interest ALT Alanine transaminase APF Alive and progression -free APF12/18 Proportio n of patients alive and progression -free at 12 / 18 months from first dose of study treatment AST Aspartate transaminase AUC Area under the curve BMI Body mass index BoR Best objective response CAR -T Chimeric antigen receptor T -cell therapy CI Confidence interval Cmax Maximum serum concentration Cmax ss Maximum plasma concentration at steady state Cmin Minimum serum concentration Cmin ss Minimum plasma concentration at steady state COVID -19 Coronavirus Disease 2019 CPA Clinical Pharmacology Alliance CR Complete response CSP Clinical study protocol CSR Clinical study report CT Computed tomography CTCAE Common Terminology Criteria for Adverse Event CTLA -4 Cytotoxic T-lymphocy te-associated antigen 4 CV Coefficient of variation DCR Disease control rate DCO Data cut off DLT Dose -limiting toxicity DoR Duration of response Statistical Analysis Plan Study Code D419EC0000 1 Edition Number 4.0 Date 19Apr2023 CONFIDENTIAL AND PROPRIETARY 6of 65Abbreviation or special termExplanation DRC Data Review Committee ECG Electrocardiogram eCRF electronic case report form FAS Fullanaly sis set GeoSD Geometric standard deviation Gmean Geometric mean HCV Hepatitis C virus HIV Human immunodeficiency virus HLT Higher -level terms ICs Immune cells IHC Immuno histochemistry imAE Immune -mediated adverse event INRC International Neuroblastoma Response Criteria irAE Immune -related adverse event irRECIST Immune -related Response Evaluation Criteria in Solid Tumors KM Kaplan -Meier LD Longest diameter LLOQ Lower limit of quantitation MedDRA Medical Dictionary for Regulatory Activities MR Minor response MRI Magnetic resonance imaging MTD Maximum tolerated dose NA Not applicable NB Neuroblastoma NC Not calculable NCI Natio nal Cancer Institute NE Not evaluable NED No evidence of disease NK Natural killer NQ Non-quantifiable NTL Non-target lesion ORR Objective response rate Statistical Analysis Plan Study Code D419EC00001 Edition Number 4.0 Date 19Apr2023 CONFIDENTIAL AND PROPRIETARY 7of 65Abbreviation or special termExplanation OS Overall survival OS12/24 Proportio n of patients alive at 12 / 24 months from first dose of study treatment PCR Polymerase chain reaction PD Progression of disease PD-L1 Programmed cell death ligand 1 PD-L2 Programmed cell death ligand 2 PFS Progression -free survival PK Pharmacokinetics PR Partial response PT Preferred term q4w Every 4 weeks RDI Relative dose intensity RECIST Response Evaluation Criteria in Solid Tumo rs RNA Ribonucleic acid RP2D Recommended Phase II dose SAE Serious adverse event SAP Statistical Analysis Plan SARC -1 Osteosarcoma and Ewing sarcoma SARC -2 Rhabdomyosarcoma, non -rhabdomyosarcoma, and other sarcomas SARCOMA Osteosarcoma and Ewing sarcoma; soft -tissue sarcomas: Rhabdomyosarcoma, non - rhabdomyosarcoma, and other sarcomas SAS Safety analysis set SD Stable disease SoA Schedule of activities SOC System organ class STD Standard deviation STO Other solid tumors TL Target lesion TSH Thyroid stimulating ho rmone ULN Upper limit of normal Statistical A nal ysis Pla n St u d y C o de D 4 1 9 E C 0 0 0 0 1 E diti o n N u m ber 4. 0 Date 1 9 A pr 2 0 2 3 C O N FI D E N TI A L A N D P R O P RI E T A R Y 8of 6 5 A M E N D M E N T HI S T O R Y D ate Brief descri pti o n of c h a n ge 0 8J U N 2 0 2 1 T here ha ve bee n 3 pr ot oc ol versi o ns si nce t he ori gi nal S A P was create d, u p dates as f oll o ws: Secti o n 1, u p date d t o ali g n o bjecti ves wit h pr ot oc ol versi o n 5.

St u d y desi g n a n d patie nt n u m bers u p date d t o reflect c o h orts re m o ve d a n d re vise d patie nt n u m bers i n pr ot oc ol versi o n 5. Secti o n 3. 1 u p date d t o reflect ne w t u m o ur assess me nt sc he d ule a n d t o re m o ve refere nces t o c o h orts re m o ve d i n pr ot oc ol versi o n 5. Secti o n 3. 3 re m o ve d refere nces t o E C G i nter val data as t his is n ot bei n g c ollecte d. Secti o n 3.

5. 1 a n d 3. 5. 2, refere nces t o t he H L c o h ort were re m o ve d. Secti o n 4. 2 la y o ut of o ut p uts f or d ose -fi n di n g part a me n de d t o i ncl u de se parate s u m maries b y wei g ht gr o u p. Secti o n 4.

2.

3. 2 clarifie d t hat d ose m o dificati o n i n A E o ut p uts mea ns i nterr u pti o n or disc o nti n uati o n, as i ncreases a n d decreases are n ot per mitte d. Secti o n 4.

2. 2, r e m o ve d refere nces t o c o h orts re m o ve d i n pr ot oc ol ver si o n 5. Clarifie d efficac y o ut p uts are f or d ose -e x pa nsi o n o nl y a n d acc or di n gl y re m o ve d refere nces t o I N R C w hic h is f or t he N B c o h ort e nr olle d i n d ose -fi n di n g o nl y . Secti o n 4.

2. 5, C O VI D -1 9 pa n de mic relate d o ut p uts ha ve bee n a d de d. Secti o n 4.

2.

3. 5 re m o ve d re fere nce t o listi n g of p h ysical e xa mi nati o n data, as a n y p h y sical e xa mi nati o n fi n di n gs are re p orte d as a d verse e ve nts. Secti o n 4.

2.

3. 6 b or derli ne E C G res ults a d de d t o re p orti n g as a se parate cate g or y. Secti o n 5, patie nt n u m bers f or t he D R C u p date d i n li ne wit h pr ot oc ol versi o n 5. 2 4J U N 2 0 2 1 Secti o n 2. 1, a me n de d t he E val ua ble f or res p o nse a nal ysis set a n d a d de d t he se nsiti vit y a nal y sis f or u nc o nfir me d O R R. Secti o n 3, re m o ve d I N R C refere nces t hr o u g h o ut . Secti o n 3.

2. 2, a me n de d t he misse d visits t o acc o u nt f or t he c ha n ge t o 1 6 wee kl y efficac y assess me nt bei n g o pti o nal a n d clarifie d a R E CI S T visit res p o nse of N E d oes n ot c o u nt as a misse d visit. Secti o n 3.