Phase 1/2 Trial Integrating Allogeneic NK Cells in High-risk Advanced Stage III-IV Nasopharyngeal Cancer Patients
Phase: PHASE1
Conditions: Nasopharyngeal Carcinoma (NPC), Minimal Residual Disease, Recurrent Nasopharyngeal Carcinoma, Maximum Tolerated Dose
Sponsor: Singapore General Hospital

 
Version 6, Dated <16/January/2026> 
Page 1 of 43 
Restricted, Sensitive (Normal) 
 
 
 
 
 
CLINICAL TRIAL PROTOCOL 
 
PROTOCOL TITLE:
Phase 1/2 Trial integrating allogeneic NK cells in high-risk advanced Stage III–IV 
nasopharyngeal cancer patients  
 
PROTOCOL NUMBER: 
001
 
NCT NUMBER: NCT07247201 
PROTOCOL VERSION: 
Version 6
PROTOCOL DATE: 
16 January 2026
PRINCIPAL INVESTIGATOR: 
Lim Chwee Ming 
SITE PRINCIPAL INVESTIGATOR: 
Lim Chwee Ming, Senior Consultant, SGH
 
 
 
 

 
Version 6, Dated <16/January/2026> 
Page 2 of 43 
Restricted, Sensitive (Normal) 
1. BACKGROUND AND RATIONALE........................................................................................ 4 
1.1 GENERAL INTRODUCTION ........................................................................................... 4 
1.2 RATIONALE AND JUSTIFICATION FOR THE STUDY .................................................... 5 
1.3 RATIONALE FOR THE STUDY PURPOSE ..................................................................... 6 
1.4 RATIONALE FOR DOSES SELECTED ........................................................................... 6 
1.5 RATIONALE FOR STUDY POPULATION ....................................................................... 6 
1.6 RATIONALE FOR STUDY DESIGN ................................................................................. 6 
2. HYPOTHESIS AND OBJECTIVES ......................................................................................... 7 
2.1 HYPOTHESIS ................................................................................................................. 7 
2.2 PRIMARY OBJECTIVES ................................................................................................. 7 
2.3 SECONDARY OBJECTIVES ........................................................................................... 7 
2.4 POTENTIAL RISKS AND BENEFITS ............................................................................... 7 
2.4.1 POTENTIAL RISKS ...................................................................................................... 7 
2.4.2 POTENTIAL BENEFITS ............................................................................................... 7 
3 STUDY POPULATION ............................................................................................................ 8 
3.1 LIST THE NUMBER AND NATURE OF SUBJECTS TO BE ENROLLED ......................... 8 
3.2 CRITERIA FOR RECRUITMENT AND RECRUITMENT PROCESS ................................ 8 
3.3 INCLUSION CRITERIA ................................................................................................... 8 
3.4 EXCLUSION CRITERIA .................................................................................................. 9 
3.5 SUBJECT REPLACEMENT ............................................................................................. 9 
4 STUDY DESIGN ...................................................................................................................... 9 
4.1 RANDOMISATION AND BLINDING ...............................................................................11 
4.2 CONTRACEPTION AND PREGNANCY TESTING .........................................................11 
4.3 STUDY VISITS AND PROCEDURES .............................................................................12 
4.4 SCREENING VISITS AND PROCEDURES ....................................................................14 
4.5 STUDY VISITS AND PROCEDURES .............................................................................15 
4.6 FINAL STUDY VISIT.......................................................................................................17 
4.7 POST STUDY FOLLOW UP AND PROCEDURES .........................................................17 
4.8 DISCONTINUATION/WITHDRAWAL .............................................................................18 
4.8.1 DISCONTINUATION CRITERIA ..................................................................................18 
4.8.2 DISCONTINUATION VISIT AND PROCEDURES ........................................................18 
5 TRIAL MATERIALS ............................................................................................................... 18 
5.1 TRIAL PRODUCT(S) AND OVERVIEW OF NK CELL MANUFACTURING .....................18 
5.2 MANUFACTURING METHODOLOGIES ........................................................................20 
5.3 STORAGE AND PRODUCT ACCOUNTABILITY ............................................................23 
6 TREATMENTS ...................................................................................................................... 25 
6.1 RATIONALE FOR SELECTION OF DOSE .....................................................................25 
6.2 STUDY DRUG FORMULATIONS ...................................................................................25 
6.3 STUDY DRUG ADMINISTRATION .................................................................................25 
6.4 SPECIFIC RESTRICTIONS / REQUIREMENTS .............................................................25 
6.5 BLINDING ......................................................................................................................25 
6.6 CONCOMITANT THERAPY ...........................................................................................26 
7 SAFETY MEASUREMENTS .................................................................................................. 26 
7.1 DEFINITIONS .................................................................................................................26 

 
Version 6, Dated <16/January/2026> 
Page 3 of 43 
Restricted, Sensitive (Normal) 
7.2 COLLECTING, RECORDING AND REPORTING OF SERIOUS ADVERSE EVENTS 
(SAES) TO CIRB ..................................................................................................................26 
7.3 COLLECTING, RECORDING AND REPORTING OF SERIOUS ADVERSE EVENTS 
(SAES) TO THE HEALTH SCIENCES AUTHORITY (HSA) ..................................................27 
7.4 KNOWN OR POTENTIAL ADVERSE EVENT RELATED TO POST-INFUSION OF IP 
(ALLONK1) ..........................................................................................................................30 
7.5 KNOWN OR POTENTIAL SERIOUS ADVERSE EVENT RELATED TO POST-INFUSION 
OF IP (ALLONK1) .................................................................................................................30 
7.6 MANAGEMENT OF AE AND SAE ..................................................................................30 
7.7 SAFETY MONITORING PLAN .......................................................................................31 
B) 
DATA AND SAFETY MONITORING .............................................................................32 
7.8 COMPLAINT HANDLING ...............................................................................................32 
8 DATA ANALYSIS .................................................................................................................. 32 
8.1 DATA QUALITY ASSURANCE .......................................................................................32 
8.2 DATA ENTRY AND STORAGE ......................................................................................32 
9 SAMPLE SIZE AND STATISTICAL METHODS .................................................................... 33 
9.1 DETERMINATION OF SAMPLE SIZE ............................................................................33 
9.2 STATISTICAL AND ANALYTICAL PLANS ......................................................................33 
10 DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ....................................................... 38 
11 QUALITY CONTROL AND QUALITY ASSURANCE .......................................................... 38 
12 ETHICAL CONSIDERATIONS ............................................................................................ 38 
12.1 INFORMED CONSENT ................................................................................................38 
12.2 CONFIDENTIALITY OF DATA AND PATIENT RECORDS ...........................................39 
13 PUBLICATIONS .................................................................................................................. 39 
14 RETENTIONS OF TRIAL DOCUMENTS ............................................................................. 39 
15 FUNDING AND INSURANCE .............................................................................................. 39 
16 LIST OF ATTACHMENTS ................................................................................................... 40 
17  REFERENCES ................................................................................................................... 42 
 
 
 

 
Version 6, Dated <16/January/2026> 
Page 4 of 43 
Restricted, Sensitive (Normal) 
1. BACKGROUND AND RATIONALE 
Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer, primarily 
endemic in Singapore, Southeast Asia, Southern China and North Africa. A large majority 
(~70%) of NPC patients present with locally advanced disease at diagnosis and carry a 
high risk of recurrence (~30%) following definitive chemoradiotherapy (CRT). These 
relapses are highly lethal, and current chemotherapies can prolong progressive-free 
survival by 6-8 months only. To reduce NPC-related mortality, preventive strategies aiming 
to reduce the risk of relapses with treatment intensification upfront should be more effective 
than delivering treatments after relapses have occurred.  
 
Intensifying treatment in locally advanced NPC patients using adjuvant chemotherapy 
following conventional CRT is often not well-tolerated, given that these patients are still 
recuperating from the acute CRT toxicities. Conversely, immunotherapy options, such as 
cell-based therapy and immune checkpoint inhibitor, are good candidates of adjuvant 
treatments because they are very well-tolerated even when combined with chemotherapy.  
 
In line with this notion, our preliminary results have shown that the addition of functional 
NK cells to standard chemotherapy improved the prognostic outcome from chemotherapy 
alone based on the decrease in tumour growth rate and the final tumour volume. Therefore, 
this clinical trial is to validate if addition of Natural Killer (NK) infusion to NPC standard of 
care can reduce relapses and metastasis. Since NPC is ubiquitously associated with the 
Epstein Barr Virus (EBV) infection, detectable post-treatment circulating EBV-DNA is well-
documented to represent minimal residue disease (MRD), which portends a worse 
prognosis. Therefore, complete eradication of remaining circulating EBV-DNA NPC cells 
would eliminate MRD following treatment, thereby reducing the risk of future relapses.  
 
With these rationale in mind, our aim is to perform a dose escalating clinical trial to identify 
the maximum tolerate dose of allogeneic “off the shelf” NK cells integrated into the current 
standard of care treatment of advanced NPC patients; and investigate if addition of  
allogeneic “off the shelf” NK cells in the concurrent and adjuvant setting can reduce the 
probability of having detectable plasma EBV-DNA after definitive treatment.  
 
1.1 General Introduction 
NK cell is a member of the innate lymphoid cells (ILC) that is able to recognize and 
eradicate tumour cells without engaging MHC class I [1, 2]. A critical component of our 
innate immune system, NK cells is our first line of defence against carcinogenesis by their 
constant surveillance and elimination of circulating tumour cells [3, 4]. For the past decade, 
NK cells are constantly being explored as a new cell therapy in cancer treatment [5, 6]. 
The success in using NK cells to eliminate leukaemia has render much promise and merits 
to extend its use to treat solid tumour. In the past five years, several Phase I studies were 
being reported demonstrating good tolerance to the dosage of 0.5 to 3 billion allogeneic 
NK cells (derived from in-vitro expansion from PBMC) in cancer patients with no significant 
GVHD symptoms [7-10]. In addition, a study two years ago documenting the success in 
eliminating brain metastasis from NPC after failed chemotherapy using periodic infusion of 
2 billion of allogeneic NK cells  [11]. 

 
Version 6, Dated <16/January/2026> 
Page 5 of 43 
Restricted, Sensitive (Normal) 
1.2 Rationale and Justification for the Study 
This is a phase 1 leading to a Phase 2 clinical trial investigating addition of allogeneic NK 
cells to conventional CRT for locally advanced NPC patients. We hypothesis that allogeneic 
NK cells are well-tolerated and safe when used in combination with conventional CRT in 
treating locally advanced NPC and lead to elimination of post-treatment circulating EBV-
DNA from the current 25-30% to 10% among locally advanced NPC patients.  
 
From a mechanistic perspective, during concurrent CRT, the NPC tumour will be killed, and 
fragments of the EBV positive NPC tumour cells will be released into the circulation. In usual 
instance, the host circulating NK cells will be responsible to “mop” up these circulating NPC 
cells that may be still viable. However, due to the effects of immune suppression from 
chemoradiotherapy, some of these circulating NPC cells may not be adequately eliminated, 
resulting in missed opportunity to completely eradicate these circulating NPC cells. These 
viable cells may enter into dormancy and only to re-emerge subsequently as clinically 
evident cancer relapses. Therefore, in our trial design, we have purposefully integrated the 
use of allogeneic NK cells during the concurrent (2 doses) and adjuvant (4 doses) phase.  
 
We hypothesis that by supplementing NK cells in these critical phases, the circulating NPC 
cells can be effectively killed and eliminated. Additionally, we also have highly sensitive 
plasma EBV-DNA assays that will allow us to measure and track the circulating NPC cells 
during this trial.  A summary of our mechanistic explanation of our study is presented in 
Figure 1. 
 
 
 
Figure 1: Mechanistic explanation to leverage on allogeneic NK cells to mop up circulating EBV-NPC 
cells following concurrent chemoradiotherapy. The lysed NPC cells following NK cell mediated 
cytotoxicity can further promote anti-NPC T cell responses from NK cell-dendritic cell cross 
presentation.  
 

 
Version 6, Dated <16/January/2026> 
Page 6 of 43 
Restricted, Sensitive (Normal) 
1.3 Rationale for the Study Purpose 
A large majority (~70%) of Nasopharyngeal Carcinoma (NPC) patients present with locally 
advanced disease at diagnosis carry a high risk of recurrence (~30%) following definitive 
chemoradiotherapy (CRT). Preventive strategies with treatment intensification upfront 
should be more effective than delivering treatments after relapses have occurred. 
However, intensifying conventional CRT as adjuvant treatment in locally advanced NPC 
patients is often not well-tolerated, given that these patients are still recuperating from 
acute CRT toxicities. Conversely, NK cell therapy has shown to be very well-tolerated when 
use as sole therapeutic in cancer patients. Together with their capacity to eliminate tumour 
cells without engaging MHC class I or prior antigenic engagement, allogeneic NK cell is 
expected to be an ideal adjuvant immunotherapy to be combined with chemotherapy. To 
date however, such combinatory therapeutic approach towards cancer treatment has not 
been investigated for NPC.  
In this clinical trial, we proposed a phase I study to determine the maximum Tolerated Dose 
(MTD) of allogeneic NK cells to be used in the trial, leading to a phase 2 study to evaluate 
the efficacy of such combined treatment approach on reducing cancer recurrence for NPC 
patients. 
1.4 Rationale for Doses Selected 
We anticipate the maximum tolerated dose (MTD) to be between 0.8x107 to 1.8x107 NK 
cells/kg (equivalent to approximately 0.5 to 1.0 billion NK cells) because this dosage range 
of allogeneic NK cells has been shown to be safe and feasible among several Phase I 
clinical trials [7-10]. Therefore, our team has adopted this in Phase I trial the same dosing 
regime in a carefully escalated manner.   
1.5 Rationale for Study Population 
 
We are selecting only locally advanced NPC patients who are at high risk of developing 
relapses in the future. Based on previous studies across many tertiary centres, locally 
advanced NPC patients (i.e.  Stage III-IVA/B, T1-4, N1-3, M0) are at higher risk of future 
cancer relapse following current standard of care treatment with concurrent CRT. With a 
larger tumour burden, we believe that addition of allogeneic NK cells can help to “mop” up 
the circulating EBV NPC cells during and after the standard of care CRT treatment.   
 
1.6 Rationale for Study Design 
 
In Phase 1 study, we will determine the MTD using the Bayesian Model Averaging 
continual reassessment method (BMA-CRM) study design [12, 13] where 5 doses 
(0.5x107, 0.8x107, 1.1x107, 1.4x107 and 1.8x 107cells/Kg) of NK cells will be evaluated. 
The primary goal of the BMA-CRM is to identify the maximum tolerated dose (MTD) of NK 
cells to be infused. An anticipated size of 6 patients will be accrued for the Phase 1 study. 
If the MTD is achieved earlier with smaller cohort of patients, we will proceed with phase 
2 portion without having to complete the projected 21 patient's accrual on the phase 1 
design. 
 

 
Version 6, Dated <16/January/2026> 
Page 7 of 43 
Restricted, Sensitive (Normal) 
The Phase 2 study is a single-arm trial derived from the anticipated proportion of locally 
advanced NPC patients (25-30%) who will continue to present with detectable circulating 
EBV-DNA following standard of care CRT paradigm. We hypothesize that in this single 
arm trial, adding allogeneic NK cells will completely eliminate the proportion of patients 
with detectable circulating EBV-DNA from 25-30% to 10%.  
 
Hence, applying the one proportion Z-score test, a sample size of 9 will achieve 80.5% 
power to detect a difference (P1-P0) of -0.2500% using a one-sided exact test with a 
significance level (alpha) of 0.05. Assume the withdrawal rate at 0.2 (20%), the total sample 
size is estimated at 12. In our phase 1 design, at least 6 patients will be treated at our MTD 
of allogenic NK cells, meaning that additional 25 patients will be accrued in the phase 2 
trial. 
 
2. HYPOTHESIS AND OBJECTIVES 
2.1 Hypothesis 
The hypothesis of this trial is allogeneic NK cells will be well-tolerated and safe when used 
in combination with conventional CRT in locally advanced NPC patients leading to the 
elimination of post-treatment circulating EBV-DNA from the current 30% to 10% in locally 
advanced NPC patients. 
2.2 Primary Objectives 
The primary objective of this trial is to determine the MTD of allogeneic NK cells that is 
deemed safe and tolerable when used with concurrent CRT in advanced NPC patients 
 
2.3 Secondary Objectives 
The secondary objective of this trial is to determine if adding allogeneic NK cells to NPC 
standard of care CRT treatment paradigm can decrease the percentage of NPC patients’ 
conversion to undetectable plasma EBV-DNA levels from 30 to 0%. The time to relapse 
will also be measured as an additional secondary endpoint.  
2.4 Potential Risks and Benefits 
2.4.1 Potential Risks 
Allogeneic NK cells are generally safe to administer with minimal toxicities reported. The 
risk of graft versus host disease (GVHD) are generally very low, in comparison with T cell 
therapy. As part of the release criteria of our NK cells, we will ensure that the minimum 
purity of NK cells to be at least 90%, before these cells are administered to patients. 
Once our MTD of NK cells is ascertained, this dose will be delivered in the phase 2 portion 
of the study. 
 
2.4.2 Potential Benefits 
Integrating NK cells into CRT can potentially reduce the risk of future cancer relapse of 
NPC patients receiving definitive treatment. This risk can be objectively measured using 
circulating plasma EBV-DNA post treatment. Therefore, in this trial, we will measure the 

 
Version 6, Dated <16/January/2026> 
Page 8 of 43 
Restricted, Sensitive (Normal) 
circulating plasma EBV-DNA longitudinally and ascertain if the addition of NK cells can 
reduce the risk of having detectable plasma EBV-DNA following current standard of care 
treatment of CRT.  
 
3 STUDY POPULATION 
3.1 List the Number and Nature of Subjects to be Enrolled 
We intend to accrue total of 31 locally advanced NPC patients for this Phase I leading to 
Phase II study. These patients are newly diagnosed advanced non-metastatic NPC 
patients who are at high risk of future cancer relapse.  
 
In addition, during the early Phase I portion, we aim to recruit 40 to 70 healthy blood donors 
from general public and relatives of NPC patients. The collected blood from these 
individuals will serve as starting source materials (PBMCs) for NK cell manufacturing. All 
donors must meet the study’s eligibility criteria and complete the required pre‑screening 
assessments prior to participation.  
 
3.2 Criteria for Recruitment and Recruitment Process 
We plan to enrol up to 31 patients (6 in Phase 1 and 25 in Phase 2) over a period of 24–
36 months from the following institutions: Singapore General Hospital, Sengkang General 
Hospital, Changi General Hospital, and the National Cancer Centre Singapore (NCCS). 
Newly diagnosed NPC patients with Stage III-IVA/B (T1-4, N1-3, M0)) disease and planned 
to start standard of care treatment will be identified and recruited. The detailed inclusion 
and exclusion criteria are outlined in Sections 3.3 and 3.4, respectively. Patients meeting 
the eligibility criteria will be approached to provide informed consent for participation in this 
clinical trial. 
 
In parallel, we plan to enrol approximately 40 to 70 healthy volunteers from the general 
public and relatives of NPC patients. Potential donors will undergo pre‑donation screening 
within seven days prior to the scheduled blood collection date. Individuals with a history of 
blood transfusion, major surgery, or any contagious disease within the past six months will 
be excluded. Donors must also abstain from taking any medications for at least 48 hours 
prior to blood collection. Only volunteers who meet all inclusion criteria and successfully 
pass the screening procedures will be invited to donate blood for the study. 
 
3.3 Inclusion Criteria 
a) Inclusion Criteria for study patient (NPC patient) as follows: 
 
Age 21-80 
 
NPC Stage III-IVA/B (T1-4, N1-3, M0) 
 
Able to tolerate conventional CRT 
 
Detectable EBV level at diagnosis  
 
Adequate organ function  
o ANC ≥ 1500/µL 
o Platelet count ≥ 100,000/µL 
o Creatinine clearance ≥60ml/minute 
o Total bilirubin ≤ 1.5 x upper limit normal (ULN) 
o AST ≤ 2 x upper limit normal 

 
Version 6, Dated <16/January/2026> 
Page 9 of 43 
Restricted, Sensitive (Normal) 
o  ALT ≤ 2 x upper limit normal 
 
ECOG performance status of 0-1 
 
b) Inclusion Criteria for Healthy Volunteers as follows: 
 
Age 21-50  
 
Body Mass Index (BMI): 19.0–30.0 kg/m² 
 
Haemoglobin level 
o Female: > 12.5 g/dL 
o Male: > 13g/dL. 
3.4 Exclusion Criteria 
a) Exclusion Criteria for study patient (NPC patient) as follows: 
 
Presence of Autoimmune Disease 
 
Any conditions resulting in Immunocompromised state (Drug induced) 
 
ECOG performance status  2 
 
Poor organ function 
 
Lactating or pregnant 
 
Serious concomitant disorders that would compromise the safety of the 
patient or compromise the patient's ability to complete the study. 
 
b) Exclusion Criteria for Healthy Volunteers as follows: 
 
Lactating or pregnant 
 
History of malignancy 
 
History of blood transfusion or major surgery within the past 6 months 
 
History of any contagious or transmissible disease within the past 6 
months 
 
Close contact with individuals diagnosed with any transmissible or 
contagious disease within the past 6 months 
3.5 Subject Replacement 
An accrued patient who fails to receive the first NK cell infusion during treatment will be 
considered non-evaluable and will be replaced. 
 
Similarly, a healthy volunteer whose screening results are positive for any listed contagious 
or transmissible disease, or who fails to meet the eligibility criteria, will not proceed to blood 
donation and will be replaced.   
 
4 STUDY DESIGN
This Clinical Trial comprised of an open label Phase 1 study leading to a single arm Phase 
2 study. 
 
Healthy volunteers will be recruited either through IRB‑approved brochures or, in cases 
where the volunteer is a relative of a study participant, the PI/Co‑I will communicate with 
them directly to explain the study details. All healthy volunteers will undergo screening for 
contagious or transmissible diseases prior to blood donation. Approximately 300–400 mL 

 
Version 6, Dated <16/January/2026> 
Page 10 of 43 
Restricted, Sensitive (Normal) 
of whole blood will be collected per donation using EDTA‑containing infusion bags, from 
volunteers who have been screened and confirmed eligible for blood donation. 
 
All collected blood units will be immediately transported to the designated GMP facility 
(ACTRIS) for processing and isolation of peripheral blood mononuclear cells (PBMCs). 
The isolated PBMCs will be aliquoted and subjected to quality testing, followed by 
cryopreservation using CS10 media. The cryopreserved PBMCs will be stored in liquid 
nitrogen at the ACTRIS facility. Once a sufficient PBMC cell yield is obtained, the cells will 
be processed for NK cell manufacturing. The final expanded NK cell product will be 
cryopreserved in liquid nitrogen and will only be reformulated on the day of infusion, after 
which it will undergo final quality control (QC) testing. The final formulated allogeneic NK 
cell product will then be administered to the NPC patient only. 
 
For the Phase 1 study: 
Determination of the maximum tolerated dose (MTD) using Continual Reassessment 
Method (CRM). 
1. Doses to be evaluated (0.5x107, 0.8x107, 1.1x107, 1.4x107 and 1.8x 107cells/kg). 
2. The starting dose to be evaluated is 0.8x107 cells/kg. 
3. Final allogeneic NK cells would be reformulated in a single infusion bag containing 
Plasma-Lyte 148, Interlukin-2 (200 IU/ml), and 2% Human Serum Albumin (HSA). 
4. Dose Limiting Toxicity (DLT): Grade 3 to 5 toxicities (using Common Terminology 
Criteria for Adverse Events CTCAE). 
5. Target Toxicity Limit (TTL): maximum probability of DLT occurring before rejecting 
the dose is set at 30% (0.3). 
 
For every case of DLT encountered, the toxicity limit will be reassessed to ascertain 
whether it has reached the TTL. In the instance when the cohort size is reached and the 
toxicity limit ≤ TTL, the dose will be escalated for MTD evaluation. Otherwise, if the toxicity 
limit > TTL, current dose will be rejected as MTD and the dose will be de-escalated for 
MTD evaluation. When the toxicity limit is very low, the dose escalation for MTD evaluated 
can be accelerated. i.e. escalating from dose 2 to 4 as shown in Figure 2. When 6 patients 

 
Version 6, Dated <16/January/2026> 
Page 11 of 43 
Restricted, Sensitive (Normal) 
treated at our set MTD do not develop grade 3-5 toxicities, the phase 1 portion of our study 
will be deemed to be completed, and then the set MTD will be accepted. Every accrued 
patient involved in MTD evaluation will undergo the standard NPC treatment with 
the NK cells infusion dose set at the previously evaluated dose.  
 
For the Phase 2 study: 
 
1. Standard Chemoradiotherapy will be implemented to every accrued NPC patient. 
2. The dose of NK cells used will be set at MTD or the dose where the patient is 
subjected during Phase 1. 
3. NK cells infusion will be carried out 1 time/week at week 3, 6, 7, 8, 9 and 10. 
4. Final allogeneic NK cells would be reformulated in a single infusion bag containing 
Plasma-Lyte 148, Interlukin-2 (200 IU/ml), and 2% Human Serum Albumin (HSA).   
5. Primary endpoint is set at the percentage of patients with detectable post-
treatment circulating EBV-DNA has decreased from 30% to 10%. 
6. Secondary endpoint is in determining the change in patient survival and 
recurrence rate within 24-36 months post-treatment under this new treatment 
regimen when compare with historic cohorts under standard treatments. 
 
The 
treatment regimen for the single arm Phase 2 study is showed in Figure 3. The 6 doses of 
NK cells will be infused once a week at specific week of treatment into every accrued 
patient. IL-2 will be co-formulated with the allogeneic NK cells with Plasma-Lyte148 and 
2% HSA as a single ready for infusion product. At Week 9, patients who's circulating 
EBV‑DNA levels have decreased but remain detectable will have the option to receive an 
additional four doses of NK cell infusions, administered from Week 12 to Week 15. 
 
4.1 Randomisation and Blinding 
This is as Phase 1 leading to single arm Phase 2 clinical trial. Hence, there will not be any 
randomization or blinding involved.   
 
4.2 Contraception and Pregnancy Testing 
For this trial, pregnant woman is a criterion for exclusion. Pregnant patients are excluded 
from receiving chemoradiotherapy as the standard of care CRT treatment. All females of 
reproductive age will be undergoing a pregnancy test as standard clinical care prior to 
receive CRT. 
 

 
Version 6, Dated <16/January/2026> 
Page 12 of 43 
Restricted, Sensitive (Normal) 
4.3 Study Visits and Procedures  
(a) Study Visits and Procedures for Healthy Volunteers as follows:   
Healthy Volunteers who indicate interest in participating in the study will be approached 
and the information about the study would be provided. Potential donors will be invited for 
in person visit, during which written consent would be obtained before carrying out any 
study related activities. The potential donors would be requested to complete a 
questionnaire which covers on medical history and lifestyle risk assessment. Then 20ml of 
blood would be collected for screening test to test for infectious disease. The screening 
result will be reviewed by PI/study team and are valid for 7 days. Donors who meet the 
eligibility criteria and have been tested negative will return within the 7-window period for 
the blood donation visit.   
  
During the blood donation visit, eligibility will be reconfirmed, and vital signs and basic 
physical examination would be carried out prior to donation. Once 300-400 ml of blood is 
collected, the donors will be observed for 10-15 minutes to ensure that they are stable and 
safe to dismissed. 
 
(b) Study Visits and Procedures for Study patient (NPC patient) as follows:  
 
A table of scheduled study assessments for screening is provided in Table 1, Table 2 and 
Appendix 1. The Study will conclude ~ 4 months after accrual. Patients following treatment 
will be followed up as per normal standard of care by their treatment physician at both NCC 
and their respective treating ENT specialists. All assessments must be performed and 
documented in the medical record for each patient.  
  
Screening evaluations will be performed ≤ 28 days before enrolment. Patients who agree 
to participate will sign the ICF before undergoing any screening procedure. The screening 
period begins on the date the ICF is signed. Screening evaluations may be repeated as 
needed within the screening period; the investigator will assess patient eligibility according 
to the latest screening assessment results. Results of standard-of-care tests or 
examinations performed before obtaining informed consent and ≤ 28 days before 
enrolment may be used for the purposes of screening rather than repeating the standard-
of-care tests unless otherwise indicated. Voluntary, written informed consent for 
participation in the study must be obtained before any study-specific procedures are 
performed.  
 
ICFs for enrolled patients and for patients who are screened but not enrolled will be 
maintained at the study site. All screening evaluations must be completed and reviewed to 
confirm that patients meet all eligibility criteria before enrolment.  
 
The investigator will maintain a screening log to record details of all patients screened and 
to confirm eligibility or record reasons for screening failure, as applicable. 
 
During the Screening Visit, a complete physical examination will be conducted, including 
evaluations of 1) head, eyes, ears, nose, and throat, 2) cardiovascular, 3) 
dermatological,4) musculoskeletal, 5) respiratory, 6) gastrointestinal, and 7) neurological 

 
Version 6, Dated <16/January/2026> 
Page 13 of 43 
Restricted, Sensitive (Normal) 
systems. Any abnormality identified during screening will be graded according to NCI-
CTCAE v5.0 and recorded.  
 
At subsequent visits (and as clinically indicated), limited, symptom-directed physical 
examinations will be performed. New or worsened clinically significant abnormalities are 
to be recorded as AEs. 
 
The tissue biomarker sample collection schedule is described in Appendix 1 and in the 
Schedule of Assessments in each subprotocol (after screening). Archival tumour tissues 
should consist of FFPE blocks or approximately 15 freshly cut unstained slides. If no 
archival tumour tissues are available, a fresh tumour biopsy at baseline (during screening 
period) is mandatory. Blood samples will be taken predose on Day 1 to measure the 
circulating plasma EBV-DNA and other correlative markers as detailed.   
Table 1  
Assessment 
Screening a 
Notes 
Days 
-28 to ~ -1 
Informed consent 
x 
 
Inclusion/exclusion 
criteria
x 
 
Enrolment 
x 
 
Demographics/medical 
history/prior 
medications 
x 
Includes age or year of birth, gender, 
self-reported race/ethnicity, tobacco 
consumption (ie, former or current or 
never) and history of treatment for the 
primary diagnosis, including prior 
medication, locoregional treatment 
plan. 
Information on radiographic studies 
performed before study entry may be 
collected for review by the investigator. 
Preexisting AEs at baseline should be 
recorded as medical history. 
Vital signs/ height and 
weight 
x 
Vital signs include body temperature, 
pulse rate, and blood pressure 
(systolic and diastolic). Pulse rate and 
blood pressure will be collected.
Physical examination 
x 
A complete physical examination is 
required.
ECOG Performance 
Status
x 
 
Adverse events 
x 
After the informed consent form has 
been signed but before the first 
administration of any study drug, only 
SAEs should be recorded.
Concomitant 
medications
x 
 
Haematology 
xa 

 
Version 6, Dated <16/January/2026> 
Page 14 of 43 
Restricted, Sensitive (Normal) 
Serum chemistry 
xa 
Local laboratory assessments on 
haematology, serum chemistry, 
coagulation parameters, and urinalysis 
will be conducted, of which certain 
elements will be collected.
Assessment 
Screening a 
Notes 
Days 
-28 to ~ -1 
Fresh tumour tissue 
x 
Pre-treatment Tissue will be collected 
either at time of diagnosis or after 
diagnosis, prior to to starting 
treatment.
 
4.4 Screening Visits and Procedures 
Patient accrual will be in accordance with the following chart showed in Figure 3. Only NPC 
patients who have not previously received CRT treatment for NPC will be eligible for the 
clinical trial. In addition, they must meet all inclusion criteria and do not fall within the 
exclusion criteria will be invited to give consent to participate in this clinical trial. After consent 
is given, the NPC patients will be accrued to start the trial. 
 
Moreover, healthy volunteers will be screened within seven days prior to the schedule 
donation date. Only donors who have tested and confirmed negative for infectious disease 
list below within seven (7) days of donation would be allowed to donate blood. Individuals 
who have undergone blood transfusion or major surgery or who have had any contagious 
disease in the past 6 months will be excluded. Donors must also refrain from taking any 
medications for at least 48 hours prior to blood collection. 
 
20ml of potential donors’ blood would be collected and would be tested for HIV-1/2 antigen 
and antibody, Human T-lymphotropic virus types I/II (HTLV-I/II), Syphilis, Cytomegalovirus 
(CMV) IgM and IgG, Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Hepatitis E virus 
(HEV) and Vesicular stomatitis virus (VSV) IgG. 
 

 
Version 6, Dated <16/January/2026> 
Page 15 of 43 
Restricted, Sensitive (Normal) 
4.5 Study Visits and Procedures 
 
1. All patients undergoing the trial will undergo conventional CRT as the standard treatment 
for NPC.   
2. For patients who are accrued for the Phase 1 study, the dose of NK cells used in the 
treatment will be the same as the dose be evaluated in the determination of MTD. Once 
MTD is established, it will be used as the dose for all patients accrued subsequently. 
3. Under the standard of care NPC treatment, chemotherapy will be carried out on week 1 
and week 4. In addition, radiation treatment will be implemented daily Monday to Friday (2 
Gyc per day) on week 1, 2, 3, 4, 5 and 6. 
4. NK cells will be infused intravenously on week 3, 6, 7, 8, 9 and 10.  
5. Interlukin-2 (200U/ml) will be delivered together with the NK cells in saline during NK 
infusion. 
6. A tumour tissue biopsy will be taken at screening period (<28 days before starting 
treatment) and at 11.  
7. Blood (20 ml) will be taken at screening period (<28 days before starting treatment) before 
treatment starts (week 0) and at week 4, 7, 9 and 11.  
8. For patients who are undergoing supplementary NK treatment at the end of the trial, four 
additional doses of NK cells will be infused by their early dosage respectively once a week 
on week 12, 13, 14 and 15. 
9. For patients receiving supplementary NK cells treatment, an additional blood (20 ml) and 
tumour tissue biopsy will be taken on week 16. 
 

 
Version 6, Dated <16/January/2026> 
Page 16 of 43 
Restricted, Sensitive (Normal) 
 
Table 2
Assessment Period
Assessment Procedures
Notes
-28 to -1 
      1.   Draw Blood (20ml) 
      2.   Tumour Tissue Biopsy 
     3.   EBV assay 
 
Week 1 
1. Chemo-drug Administration 
2. Radiation Therapy Administration 
(1 dose daily, 5 doses/week) 
 
Week 2 
1. Radiation Therapy Administration 
(1 dose daily, 5 doses/week) 
 
Week 3 
1. Radiation Therapy Administration 
(1 dose daily, 5 doses/week) 
2.
NK cells infusion 
 
Week 4 
1. Draw Blood (20ml) * 
2. EBV assay 
3. Chemo-drug Administration 
4. Radiation Therapy Administration 
(1 dose daily, 5 doses/week) 
*Draw blood before 
Chemo-drug 
administration but 
they may not be on 
the same day. 
Week 5
1.
Radiation Therapy Administration

 
Version 6, Dated <16/January/2026> 
Page 17 of 43 
Restricted, Sensitive (Normal) 
(1 dose daily, 5 doses/week) 
Week 6 
1. Radiation Therapy Administration 
(1 dose daily, 5 doses/week) 
2.
NK cells infusion 
 
Week 7 
1. Draw Blood (20ml) * 
2. EBV assay 
3. NK cells infusion 
*Draw blood before 
NK cells infusion 
but they may not be 
on the same day.
Week 8 
1.
NK cells infusion
 
Week 9 
1. Draw Blood (20ml) * 
2. EBV assay 
3. NK cells infusion 
*Draw blood before 
NK cells infusion 
but they may not be 
on the same day.
Week 10
1.
NK cells infusion
Week 11 
1. Draw Blood (20ml) 
2. Tumour Tissue Biopsy 
3.
EBV assay
 
Week 12 (Optional) 
1. NK cells infusion  
 
Week 13 (Optional) 
1. NK cells infusion  
 
Week 14 (Optional) 
1. NK cells infusion  
 
Week 15 (Optional) 
1. NK cells infusion  
 
Week 16 (Optional) 
1. Draw Blood (20ml) 
2. Tumour Tissue Biopsy 
3. EBV assay 
 
 
4.6 Final Study Visit 
At the end of the trial, the patients will be reviewed for their tolerability and safety of the 
allogeneic NK cell infusion.  
 
4.7 Post Study Follow up and Procedures 
As part of standard of care NPC surveillance protocol, both clinical and radiological 
assessments will be performed as follow: 
a) Clinical Assessments: Physical examination (including flexible nasopharyngoscopy to 
examine the nasopharynx) will be performed monthly for the first year and bi-monthly 
for the second and third year as part of the routine NPC surveillance protocol.  
b) Radiological Assessments: Baseline radiological imaging for tumour assessment and 
staging will be performed. Post-treatment radiological imaging will also be performed at 
4-6 months post completion of treatment. Subsequent radiological studies will be 
performed as clinically indicated, determined by the treating physicians. The Response 
Evaluation Criteria in Solid Tumours (RECIST 1.1 criteria) will be used for assessment 
of response to therapy (from baseline to 4–6-month post treatment scans). 

 
Version 6, Dated <16/January/2026> 
Page 18 of 43 
Restricted, Sensitive (Normal) 
4.8 Discontinuation/Withdrawal 
4.8.1 Discontinuation Criteria 
Withdrawal Criteria: 
1. 
Documented disease progression 
2. 
Death 
3. 
Development of an unacceptable toxicity, which in the opinion of the PIs, would render 
continued participation harmful.  
4. 
Research subject or LAR withdraws consent from the study.  
5. 
Investigator withdraws the subject from the study, in subject’s best interest.  
6. 
Non-compliance with protocol medications/administrations and/or required follow-up as 
judged by the PI. 
7. 
Unable to be contacted and/or effectively monitored by the PIs and/or designees for 
follow-up (lost to follow-up). 
8. 
Unable to receive the study infusion due to development of a significant health disorder, 
which in the judgment of the PI, would make proceeding with this study intervention to 
be not in the best medical interest of the participant.  
9. 
Receipt of any therapy specifically for treatment of primary disease not defined in this 
protocol. 
 
4.8.2 Discontinuation Visit and Procedures 
Subjects may also withdraw voluntarily from receiving the study intervention for any reason 
during the study and they will continue to receive standard of care treatment for their cancer 
and follow up.  Patients who withdraw from the study can give consent for their archival tissue 
samples to be used for correlative analyses with those who have completed the trial.  
 
5 TRIAL MATERIALS 
5.1 Trial Product(s) and Overview of NK Cell Manufacturing  
The source of the allogeneic NK cells includes: 
1. Healthy Donor from the public 
2. Donor from Patients healthy relatives  
3. GMP graded leukopaks. 
 
A) Collection and Handling Donated Blood 
 
Approximately 300–400 mL of whole blood will be collected per donation using EDTA-
containing infusion bags. All collected units will be transported immediately to the designated 
GMP facility for isolation of peripheral blood mononuclear cells (PBMCs). The isolated 
PBMCs will be aliquoted, and quality tested before freezing in CS10 media and store in Liquid 
Nitrogen.  
 
B) Manufacturing of Allogeneic NK cells 
 
Manufacturing of the Allogenic NK cells will be performed by ACTRIS- our national cell 
manufacturing facility. 

 
Version 6, Dated <16/January/2026> 
Page 19 of 43 
Restricted, Sensitive (Normal) 
 
This process includes: 
 
Isolation of PBMC from whole blood 
 
Enrichment and Expansion of NK cells from the isolated PBMCs 
 
Preparation and formulation of allogenic NK cells  
 
C) Overview of NK cell manufacturing  
 
The NK cell Manufacturing workflow is shown in the flow diagram below. 
 
1. 
Blood is obtained from: 
A) consented healthy relative of cancer patients (300 ml - 400ml) 
B) consented healthy public donor (300-400ml) 
C) Commercial GMP leukopaks. 
2. 
PBMC will be isolated from whole blood via SepMate 50. 
3. 
The isolated PBMC will be analysed for Sterility test (QC1). 
4. 
PBMC will be aliquoted and frozen at 5x106 cells/cryovial. 
5. 
For batches of PBMC that did not clear the sterility test will be disposed in compliance 
with the Institutional guidance.  
6. 
When sufficient PBMC cells are accumulated, they will be thawed and undergo NK 
cells enrichment using the automated CliniMACS Prodigy system. 
7. 
The NK cells enrichment is achieved using CliniMACS CD3/CD56 complete kit used 
in conjunction with the automated CliniMACS Prodigy system. 
8. 
After enrichment, a sample of the purified NK cells will undergo the Viability and 
Composition test (QC2). 
9. 
The purified NK cells will be immediately used for in-vitro NK cells expansion. 
10. Expansion of NK cells will be carried out in G-Rex100M open system Bioreactors. 
11. Proliferation of NK cells is stimulated by cytokine cocktail (IL-15(50ng/ml) and IL-
18(250ng/ml)) + MICA (50ng/ml) 
12.
Cell count will be determined on a regular basis (every 3-4 days).

 
Version 6, Dated <16/January/2026> 
Page 20 of 43 
Restricted, Sensitive (Normal) 
13. When the cell density is ≥ 1x106 cells/ml NK cells culture will be distributed to 5 G-
Rex100M Bioreactors to enable further expansion. 
14. Three days later, 300ml of NK MACS media suppled with 5% Human AB serum and 
IL-2 (200IU/ml) are added to each G-Rex100M bioreactor. 
15. Two days later, 500ml of NK MACS media suppled with 5% Human AB serum and 
IL-2 (200IU/ml) are added to each G-Rex100M bioreactor to a volume of 1000ml. 
16. Cell count is performed every 3-4 days.  
17. After another 10-14 days when the cell density is ≥ 2.5 x106 cells/ml, expanded NK 
cells will be harvested for QC testing. 
18. Spent medium (40mL) will be sent for sterility test.  
19. Also, samples of cells will be tested for Purity and Viability (QC3) as well as a cell 
count. 
20. Then, expanded NK cells will be aliquoted and freeze at 5x108 cells/ml in 20 ml of 
CS10 per freezing bag. 
21. On the day of NK cells infusion, sufficient bags of cryo-preserved expanded NK cells 
will be thawed and dilute 10 volumes of 0.9% saline supplemented with 5% Human 
serum albumin (HSA). 
22. Centrifuge the thawed NK cells (500xg, 10 mins, 4oC) to pelletize the cells. Then, the 
supernatant is removed. 
23. A small sample of the thawed NK cells will undergo test for purity and Viability (QC4). 
24. Resuspend the rest of the NK cells into infusion media (Plasmalyte 148, HSA (2%), 
IL-2 (200IU/ml)). 
25. Store the cells at 4oC (no more than 6 hours). 
26. If the viability of the NK cells <70%, NK cells viability can be increased via counterflow 
centrifugation using the Rotea system. 
27. Any excess expanded NK cells will be passed to the research lab.  
 
5.2 Manufacturing Methodologies 
 
A) Isolation of PBMC from blood followed by NK cells enrichment: 
Materials: 
 
The source of Blood will be from consented healthy relative of cancer patients 
(300 - 400 ml); 
 
Consented healthy public donor (300-400ml) or Commercial GMP leukopaks.  
 
SepMate 50 (StemCell Technologies, cat no: 86450). 
 
Ficoll-Paque Premium density gradient medium (Cytiva, cat no: 17544202) 
 
CryoStor CS10 freezing medium (Biolife solutions, cat no: 210102) 
 
TheraPEAK PBS buffer (Lonza Bioscience, cat no: BEBP17-516Q) 
 
Human Serum Albumin  
Methods:  
1. Add 15 ml of Ficoll-Paque into the SepMateTM tube by carefully pipetting it through 
the central hole of the SepMateTM insert. The top of the density gradient medium 
will be above the insert. 
2. Dilute the whole blood with an equal volume of PBS + 2% human serum albumin 
and mix gently. 
3. Keeping the SepMateTM tube vertical, add the diluted whole blood by pipetting it 
down the side of the tube. The diluted whole blood will mix with the lymphoprep 
above the insert. 

 
Version 6, Dated <16/January/2026> 
Page 21 of 43 
Restricted, Sensitive (Normal) 
4. Centrifuge at 1200 x g for 10 minutes at room temperature, with the brake on.  
5. Pour off the top layer, which contains the enriched PBMCs, into a new tube. Do 
not hold the SepMateTM tube in the inverted position for longer than 2 seconds. 
6. Wash the PBMC layer with 4 volumes of PBS supplemented with 2% human 
serum albumin. 
7. Pelletize the PBMC (600xg, 10 min, RT). 
8. Resuspend in NK MACS medium. 
9. Draw a sample to perform a viable cell count followed by pelletization of the 
PBMC (600xg, 10 min, RT). 
10. Remove the supernatant for sterility test. 
11. Resuspend in CS10, aliquot and freeze the PBMC at 1x108 cells/ml/cryovial. 
 
B) NK cells Enrichment and Expansion: 
Materials: 
 
NK MACS GMP Medium (Mltenyi Biotec, catalog no. 170-076-356)  
 
CliniMACS CD3/CD56 complete kit (Miltenyi Biotec, cat no: 200-074-004) 
 
Human Serum Albumin  
 
IgG solution (SK Plasma) 
 
Sterile water for injection (B. Braun Medical Inc) 
 
GMP Human AB Serum (Akron Bio, catalog no: AR1010-0100)  
 
GMP IL-2, IL-15 and IL-18  
 
Soluable MICA protein (SinoBiological, cat no: 12302-H08H)  
 
G-Rex 100M open system Bioreactors (Wilson Wolf)  
 
Single use disposable Heamocytometer (Incyto, cat no: DHC-N01) 
 
Trypan blue solution, 0.4% (Bio-Rad, cat no. 1450021) 
 
CryoStor CS10 freezing medium (Biolife solutions, cat no: 210102) 
 
T175 culture flasks (Thermofisher Scientific, cat no: 159910) 
 
CryoMACS freezing bag 50 (Miltenyi Biotec, cat no: 200-074-400) 
 
Plasmalyte 148 solution  
 
Method: 
 
Steps 
 
Day 0 
1. Frozen 15x109 PBMC cells are thawed. 
2. Pooled the PBMC cells in NK MACS media supplemented 
with 10% human serum albumin (HSA). 
3. Wash once with PBS supplemented with 2% HSA. 
4. Re-suspend PBMC in NK MACS media supplemented with 
2% HSA before transferring into a 600ml transfer bag. 
5. Collect a sample of total NK cells for Purity and Viability 
determination using Flow analysis.  
6. Also, the purified NK cells will undergo a viable cell count to 
ascertain the total number of NK cells. 
7. Introduce the PBMC cells into the CliniMACS Prodigy system 
for automated NK cells enrichment using the CliniMACS 
CD3/CD56 complete kit. 

 
Version 6, Dated <16/January/2026> 
Page 22 of 43 
Restricted, Sensitive (Normal) 
8. After enrichment, 200x106 purified NK cells are pelletized by 
centrifugation (300xg, 5 min, RT) follow by resuspension in 
200mL of NK Growth Medium supplemented with Human AB 
Serum (5%), IL-2 (200 IU/ml), MICA (50ng/ml) and cytokine 
cocktail (IL-15 (50ng/ml) and IL-18(250ng/ml)). 
9. The re-suspended NK cells are introduced into the G-
Rex100M bioreactor before placing the bioreactor into an CO2 
incubator. 
10. The culture condition is to be maintained at 37°C, 5% CO2. 
11. Growing NK cells will settle at the bottom of the bioreactor to 
undergo proliferation. 
 
Day 3 
1. Perform a viable NK cell count. 
2. Add 800mL of fresh NK MACS media supplemented with 5% 
human AB serum and 200IU/ml IL-2.  
3. After the addition, the total culture volume is 1000mL.  
 
When NK 
cells density 
is ≥ 1 × 106 
cells/ml. 
(typically 
between 
Days 9-12 
1. NK cell count will be performed once every 3-4 days. 
2. To perform a viable NK cell count, 800mL of the spent medium 
in the G-Rex100M Bioreactor are remove first. 
3. NK cells at the bottom will be mixed in the residual 200mL of 
medium.  
4. A sample is then taken for cell count.  
5. The cell number is estimated by trypan blue exclusion method 
with a haemocytometer.  
6. The 800mL removed in Step 2 will be put back into the G-Rex 
Bioreactor to enable further culture.  
7. When NK cells density is ≥ 1 × 106 cells/ml, the 1000ml NK 
cell culture is equally distributed into five G-Rex100M 
bioreactors. 
8. Additional MICA is added to a final concentration of 50ng/ml. 
9. Additional IL-2 is added to a final concentration of 200IU/ml. 
 
Three days 
later 
 
1. A viable NK cells cell count is performed as outlined before. 
2. Add 300ml of NK MACS media supplemented with 5% 
human AB serum and IL-2 (200IU/ml) to each G-Rex100M 
bioreactor. 
Two days 
later 
1. Perform a viable cell count as outlined before. 
2. Add another 500ml of NK MACS media supplemented with 
5% human AB serum and IL-2 (200IU/ml) to each G-Rex100M 
bioreactor to a volume of 1000ml. 
 
After 
another 10-
14 days 
when the 
1. Perform a viable NK cell count as outlined before. 
2. The expanded NK cells are harvested.  
3. During NK cells harvesting, 40mL of spend medium is 
collected for Sterility test. 

 
Version 6, Dated <16/January/2026> 
Page 23 of 43 
Restricted, Sensitive (Normal) 
cell density 
is ≥ 2.5 × 
106 cells/ml 
4. A cell sample is collected for purity and viability determination 
by flow cytometry. 
5. Another cell sample is collected for cytotoxicity assay to be 
performed in our research lab. 
6. The rest of the NK cells will be labelled, aliquoted and frozen 
in CS10 at 5x108 cells/20mL in each 20ml CryoMACS freezing 
bag 
7. The bags are cryopreserved and stored, ready to be use. 
 
C) NK cells preparation for infusion: 
 
Day of 
infusion 
1. Thawed sufficient cryo-preserved NK cells for infusion. 
2. Dilute the thawed NK cells with 10x volume of washing buffer 
(Saline solution, HSA (5%)) 
3. Centrifuge (500xg, 10min,4oC) to pelletize the NK cells.  
4. Remove the supernatant containing DMSO. 
5. Resuspend the NK cells in infusion media (Plasma-Lyte148, 
HSA (2%), IL-2(200IU/ml)).  
6. Take a sample to analyse the purity and viability. 
7. As a risk minimizing step, if the viability falls below the range of 
40% - 70%, the viability is increase through counterflow 
centrifugation via the Rotea system.  
8. Keep the NK cells in infusion bag at 4oC (no more than 6 hr). 
 
 
D) Sterility Test: 
The cell culture Sterility assay will be outsourced to TUV Ltd. In this assay, the cell culture 
media is to be determined for the absence of the following: 
 
Mycoplasma  
 
Endotoxin  
 
Sterility 
 
 
E) Purity and Viability Test: 
The purity and viability of the expanded NK cells is determined by Flow Cytometry 
Analysis. In brief, approximately 1x106 NK cells from the expansion will be stained for 
CD56 (Miltenyi Biotec, cat no: 130-114-549), CD3 (Miltenyi Biotec, cat no: 130-114-710, 
and 7AAD Viability Dye (Miltenyi Biotec, cat no: 130-111-568). After staining, the purity 
and viability of NK cells can be calculated 
 
5.3 Storage and product accountability 
 
A. Storage: 
 
 
Allogeneic NK cells must be stored in a cryopreserved state at ≤ -150°C (e.g., 
vapor phase of liquid nitrogen) to maintain viability and functionality.

 
Version 6, Dated <16/January/2026> 
Page 24 of 43 
Restricted, Sensitive (Normal) 
 
Upon thawing, cells should be used immediately or within a specified short 
window (typically within 6 hours at 2–8°C) to preserve cell quality. 
 
Avoid repeated freeze-thaw cycles to prevent loss of viability. 
 
B. Label Elements on the Investigator Product 
 
 
Product Identification: AlloNK1 – Allogenic NK cells 
 
Batch/Lot number: Unique identifier for traceability (each manufacturing run). 
 
Study Protocol Number/Code: 001  
 
Statement “For Investigational Use only” 
 
Route of Administration: For intravenous infusion only 
 
Storage Conditions: Store at 2–8 °C. Do not freeze and protect from light. 
 
Date of Manufacture / Expiration: Date of Manufacture, Date of Expiration 
 
Quantity/ Volume/ Cell Dose: Total cell number, total volume (ml) and the 
dosage 
 
Sponsor Name & Contact:  Dr Lim Chwee Ming, (65) 98247996 
 
Handling/ Special Precautions: Handle using aseptic technique and for 
allogenic use only 
 
Instruction of Use: See Investigator’s Brochure for administration instructions 
 
C. Shipping: 
 
Cryopreserved NK cell products must be shipped in validated dry vapor shippers 
that maintain temperatures at or below -150°C throughout transit. 
 
Shipping containers must be equipped with temperature monitoring devices to 
document temperature stability during transport. 
 
Shipping must be coordinated to ensure timely delivery and immediate transfer 
to appropriate storage upon receipt. 
 
Shipping documentation must include product identification, batch number, 
storage instructions, and emergency contact information. 
 
D. Disposal of Investigational Product on site 
 
Any unused, or nonconforming NK cell products must be disposed of following 
institutional and regulatory biosafety guidelines for biohazardous materials. 
 
Disposal procedures should ensure inactivation of viable cells through validated 
methods (e.g., autoclaving, chemical disinfection). 
 
All disposal activities must be documented, and records maintained in 
accordance with applicable regulations. 
E. Disposal of Expired Investigational Product  

 
Version 6, Dated <16/January/2026> 
Page 25 of 43 
Restricted, Sensitive (Normal) 
 
Any expired NK cell products will be transferred to our research lab to be used for 
preclinical investigation. 
 
6 TREATMENTS 
6.1 Rationale for Selection of Dose 
In this trial, allogeneic NK cells are being used to add onto the standard chemoradiotherapy 
for NPC. The Maximum Tolerated Dose (MTD) will be between 0.5x107 to 1.8x107 NK 
cells/Kg (equivalent to approximately 0.5 to 1.0 billion NK cells). This dosage range of 
allogeneic NK cells is selected based on several Phase I cancer studies as a guide [7-10].  
 
In these Phase I studies using allogeneic NK cells as the sole therapeutics, the dosage of 
allogeneic NK cells in the range of 1- 3 billions allogeneic NK cells exhibiting well tolerance. 
Therefore, in this Phase I trial with the infusion of allogeneic NK cells complementing 
standard chemoradiotherapy, we have proposed a lower dose of 0.5x107 to 1.8x107 NK 
cells/Kg (equivalent to approximately 0.5 to 1.0 billion NK cells) 
 
6.2 Study Drug Formulations 
The NK cells to be infused into NPC patients will undergo a media change to infusion media 
(Plasma-Lyte148, 2% human serum albumin, Interleukin-2 (200 IU/ml)) before transferred 
into in infusion bag and will be stored at 4°C (no more than 6 hr). 
 
6.3 Study Drug Administration 
Six doses of allogeneic NK cells at MTD will be administered for every accrued patient at 
during and after the radiotherapy via intravenous injection. Intravenous injection is selected 
because the intended target of the infused allogeneic NK cells is the small NPC cell clusters 
generated from the solid NPC tumour circulating in the blood during and after radiotherapy. 
The allogeneic NK cells infused into each patient will be derived from the PBMC of up to of 
healthy volunteers. 
6.4 Specific Restrictions / Requirements  
Routine pre-medication including steroid, antihistamine, and acetaminophen is not allowed 
prior to NK cell infusion.  
 
In the event of an infusion reaction, the patient may be treated accordingly although steroids 
should be avoided if possible (though not absolutely contraindicated). The use of agents such 
as H2 antagonists, antihistamines, acetaminophen, etc. is permitted for the treatment of 
infusion reactions. After appropriate treatment and resolution of the infusion reaction, NK cell 
infusion may be resumed at the discretion of the treating physician. 
6.5 Blinding 
 
This Study is an open label Phase 1 which does not require blinding  
 

 
Version 6, Dated <16/January/2026> 
Page 26 of 43 
Restricted, Sensitive (Normal) 
6.6 Concomitant therapy 
1. Radiotherapy will be given as per standard of care treatment.  
2. Chemotherapy:  
i) 
Platinum based chemotherapy regime is usually administered as the 
standard of care chemotherapy option Platinum based chemotherapy 
regime is usually administered as the standard of care chemotherapy option 
for NPC.  
ii) 
In patients who cannot receive chemotherapy or declined chemotherapy 
option, they will still be eligible to participate in this trial.    
iii) 
Patients who are prescribed other chemotherapy beside the platinum-based 
chemotherapy option due to adverse response to platinum chemotherapy 
are also eligible to participate in this trial.  
7 SAFETY MEASUREMENTS 
7.1 Definitions 
An adverse event (AE) is any untoward medical occurrence in a trial participant administered 
a cell, tissue and gene therapy products (CTGTP) and which does not necessarily have a 
causal relationship with this treatment. 
 
An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding, for example), symptom, or disease temporally associated with the NK cell 
product. 
 
A serious adverse event (SAE) is any untoward medical occurrence that at any dose: 
 
results in death 
 
is life-threatening 
 
requires inpatient hospitalisation or prolongation of existing hospitalisation 
 
results in persistent or significant disability/incapacity, or 
 
is a congenital anomaly/birth defect 
7.2 Collecting, Recording and Reporting of Serious Adverse Events (SAEs) to CIRB  
The reporting requirements will be in accordance with the reporting requirements published 
on CIRB website at the time when the event took place. 
 
Only related SAEs (definitely/ probably/ possibly) will be reported to CIRB. Related means 
there is a reasonable possibility that the event may have been caused by participation in the 
clinical trial.   
 
The investigator is responsible for informing CIRB after first knowledge that the case qualifies 
for reporting. Follow-up information will be actively sought and submitted as it becomes 
available.  
 
Related AEs will not be reported to CIRB.  However, the investigator is responsible to keep 
record of such AEs cases at the Study Site File. 

 
Version 6, Dated <16/January/2026> 
Page 27 of 43 
Restricted, Sensitive (Normal) 
7.3 Collecting, Recording and Reporting of Serious Adverse Events (SAEs) to the 
Health Sciences Authority (HSA) 
The investigator will assess the severity of each AE and SAE reported during the study. AEs 
and SAEs should be assessed and graded based upon NCI-CTCAE v5.0. 
Toxicities that are not specified in NCI-CTCAE will be defined as follows: 
• Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated 
• Grade 2: Moderate; minimal, local, or non-invasive intervention indicated; limiting 
age-appropriate instrumental activities of daily living (ADL) 
• Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
ADL 
• Grade 4: Life-threatening consequences; urgent intervention indicated 
• Grade 5: Death related to AE 
 
The following are not considered to be SAEs: 
• Hospitalization for elective treatment of a preexisting condition that did not worsen 
from baseline 
• Hospitalization for social/convenience considerations 
• Scheduled therapy for the target disease of the study, including admissions for 
transfusion support or convenience 
 
The assessment of study drug relationship to each AE will be reported on the appropriate 
source document (and SAE form, in the event of an SAE) by the Investigator or designee 
according to his or her best clinical judgment. The criteria listed in Table xx should be used 
to guide this assessment. Please note that not all criteria must be present to be indicative of 
a particular drug relationship.  
 
In this study, imAEs are of special interest. Potential imAEs are listed in Table 2 below:  
Table 2:  Adverse Event Causality Guidelines 
Body System Affected 
Events 
Skin (mild-common) 
pruritus or maculopapular rash; vitiligo 
Skin (moderate) 
follicular or urticarial dermatitis; 
erythematous/lichenoid rash; Sweet syndrome 
Skin (severe-rare) 
full-thickness necrolysis/Stevens-Johnson 
syndrome 
Gastrointestinal 
colitis (includes diarrhoea with abdominal pain or 
endoscopic/radiographic evidence of 

 
Version 6, Dated <16/January/2026> 
Page 28 of 43 
Restricted, Sensitive (Normal) 
inflammation); pancreatitis; hepatitis; ALT/AST 
elevation; bowel perforation 
Endocrine 
thyroiditis, hypothyroidism, hyperthyroidism; 
hypophysitis with features of hypopituitarism, eg, 
fatigue, weakness, weight gain; insulin-
dependent diabetes mellitus; diabetic 
ketoacidosis; adrenal insufficiency 
Respiratory 
pneumonitis/diffuse alveolitis 
Eye 
episcleritis; conjunctivitis; iritis/uveitis 
Musculoskeletal 
arthritis; arthralgia; myalgia; myasthenic 
syndrome/myasthenia gravis; myositis 
Blood 
anemia; leukopenia; thrombocytopenia 
Renal 
interstitial nephritis; glomerulonephritis; acute 
renal failure 
Cardiac 
pericarditis; myocarditis; heart failure 
Neurologic 
encephalitis; Guillain-Barre syndrome; 
meningitis; meningoradiculitis; 
meningoencephalitis; neuropathy 
  
Classification 
Definition 
Likely Related 
There is a clear temporal association between 
the event and the administration of study drug, 
a plausible mechanism for the event to be 
related to the study drug and causes other 
than the study drug has been ruled out, and/or 
the event re-appeared on re-exposure to the 
study drug.
Probably Related 
The event is a known or suspected effect of 
the study drug and follows a reasonable 
temporal sequence from the administration of 
study drug but cannot be readily explained by 
study participant or study factors, and/or the 
event 
ceases 
or 
diminishes 
with 
discontinuation of study medication 
Possibly Related 
There is an association between the event 
and the administration of the study drug and 
there is a plausible mechanism for the event 
to be related to study drug, but there may also 
be alternative etiology, such as characteristics 
of the patient's clinical status or underlying 
disease. 
Unlikely Related 
The event is unlikely/remotely possible 
/probably not related to the study drug and 

 
Version 6, Dated <16/January/2026> 
Page 29 of 43 
Restricted, Sensitive (Normal) 
likely to be related to factors other than the 
study drug. 
Not Related 
The event is related to an etiology other than 
the study drug (the alternative etiology must 
be documented in the study patient’s medical 
record). 
 
 

 
Version 6, Dated <16/January/2026> 
Page 30 of 43 
Restricted, Sensitive (Normal) 
7.4 Known or potential adverse event related to post-infusion of IP (AlloNK1) 
Events that are undesirable but usually manageable or mild/moderate, without causing life-
threatening risk, hospitalization, or permanent impairment such as the following: 
 
Infusion-related reactions (<5%): fever, chills, rigors, headache, mild hypotension. 
 
Mild Infections (<5%): uncomplicated bacterial/viral infection related to transient 
immunosuppression. 
 
Cytopenia (5-10%): neutropenia, anaemia, thrombocytopenia (usually CRT-
related). 
 
Mild Hepatic/renal toxicities (<5%): Transient enzyme elevations or creatinine rise. 
 
Mild Neurotoxicity (<1%): mild confusion without major sequelae. 
 
7.5 Known or potential serious adverse event related to post-infusion of IP (AlloNK1) 
Events that result in death, are life-threatening, require/prolong hospitalization, cause 
persistent disability, or are medically significant such as the following: 
 
Cytokine Release Syndrome (CRS):  even if rare and milder than CAR-T cells, if it 
causes hypotension requiring vasopressors, high-grade fever, or ICU-level care, it 
will be considered as an SAE. 
 
Severe infections: presence of sepsis, pneumonia, or other infections requiring IV 
antibiotics or hospitalization. 
 
Graft-versus-host disease (GVHD): any clinically significant GVHD is considered 
an SAE. 
 
Hepatic/renal toxicities – acute liver failure, grade 3–4 enzyme elevations, renal 
failure requiring dialysis. 
 
Neurotoxicity: seizures, encephalopathy, or any neurological event requiring 
intervention or hospitalization. 
 
Tumour lysis syndrome: especially if it leads to renal impairment, arrhythmia, or 
hospitalization. 
 
7.6 Management of AE and SAE 
Our team comprises of haematologists specializing in managing these cell therapy related 
complications indicated table 4: 
Table 4 
Events
Recommended Management
Infusion reaction (mild–
moderate) 
Pause/slow infusion, give antipyretics, antihistamines; 
monitor vitals closely. 
CRS (grade ≥2) 
Supportive care (IV fluids, oxygen), consider 
tocilizumab / corticosteroids as deemed appropriate by 
our haematologist. Escalate to HD or ICU care if 
hypotensive requiring vasopressors. 

 
Version 6, Dated <16/January/2026> 
Page 31 of 43 
Restricted, Sensitive (Normal) 
Infection 
Prompt cultures, broad-spectrum antibiotics; 
prophylactic antimicrobials per institutional guidelines 
during neutropenia.
Cytopenia 
Growth factor support (G-CSF), transfusions as 
indicated.
GVHD (if suspected) 
Standard immunosuppressive therapy (high-dose 
steroids) and consult transplant/immune specialists.
Neurotoxicity 
Supportive management, consider corticosteroids, 
seizure prophylaxis if indicated.
Tumour lysis / electrolyte 
abnormalities 
 
Hydration, monitor electrolytes, rasburicase/allopurinol 
as per protocol. 
 
7.7 Safety Monitoring Plan 
The principal investigator will be responsible for the safety and well-being of subjects, and 
integrity of data collected.  
 
a) Monitoring Requirement for NPC patient receiving IP: 
 
Parameter 
Timing/Frequency 
Notes 
Vital signs (temperature, 
blood pressure, heart rate, 
respiratory rate, oxygen 
saturation) 
Before, during, and after 
each NK cell infusion (e.g., 
baseline, 15 min, 30 min, 1 h, 
2 h post-infusion) 
Monitor for infusion 
allergic reactions, 
hypotension, 
fever/chills. 
Complete blood count 
(CBC) with differential & 
platelets 
Baseline, then at least twice 
weekly during 
chemoradiotherapy, and prior 
to each NK cell infusion 
 
Detect cytopenia 
from CRT 
Comprehensive metabolic 
panel (liver enzymes, renal 
function, electrolytes) 
Baseline, weekly or more 
frequently if abnormal 
Monitor for 
hepatic/renal 
toxicity. 
Coagulation profile (PT, 
aPTT, fibrinogen) 
Baseline and as clinically 
indicated 
Evaluate for 
coagulopathy and 
possible CRS or 
infection. 
Infection screening (blood 
cultures, CRP/procalcitonin 
if febrile) 
 
Baseline and as clinically 
indicated 
 
Example for rare 
rick of neutropenic 
infection.
Immune parameters (NK 
cell counts, cytokine panels, 
EBV DNA load) 
 
Baseline, then specified time 
points post-infusion 
Monitor NK 
persistence, 
immune 
activation, EBV 
viral load in NPC 
context.

 
Version 6, Dated <16/January/2026> 
Page 32 of 43 
Restricted, Sensitive (Normal) 
Cardiac monitoring (ECG, 
troponin, echocardiogram 
Baseline and if any 
symptoms 
Recommended if 
CRT includes 
cardiotoxic agents 
or for severe CRS
 
Neurological checks 
Daily during hospitalization; 
at each visit outpatient 
Watch for 
neurotoxicity (rare).
 
b) Data and Safety Monitoring 
 
A safety review committee (SRC) will monitor the safety data during Phase 1 of the study 
on a periodic basis to help ensure the ongoing safety of study patients. The SRC will 
consist of at least 3 investigators of the study, a pharmacist and biostatistician. The SRC 
will meet regularly during the Phase 1 dose escalation portion of the study to review all 
accumulated available data, including safety and preliminary efficacy to make 
recommendations on continuation, modification, or suspension of the study. The SRC 
will make recommendations concerning dose escalation/de-escalation or any changes 
to the dosing paradigm based on review of available data prior to proceeding to the next 
dose level. The SRC will also make recommendations about the dose selected for Phase 
2 and overall study conduct. 
7.8 Complaint Handling  
Any compliant will be submitted to the SRC for their review process. A response will be 
provided within 21 days. 
8 DATA ANALYSIS 
8.1 Data Quality Assurance 
 
Data will be collected and recorded by a clinical research coordinator. The investigator will 
verify that all data entries are accurate and correct, including verification that the subject fulfils 
the criteria for entry into the study before study treatment commences. 
8.2 Data Entry and Storage 
All data obtained in the study described in this protocol will be recorded on paper case report 
forms (CRFs). The CRF for each subject will be presented in a folder. The CRF will be 
completed chronologically and updated regularly in order to reflect the most recent data on 
the patient included in the study. 
 
Each CRF must be neatly filled in with a black-inked pen. For each page on which information 
is entered, the subject number must be recorded. CRFs will be dated and signed by an 
investigator. 
 
Errors must be corrected by drawing a single line through the incorrect entry and by writing 
the new value as close as possible to the original. The correction must then be initialed and 
dated by an authorized study team member. 
 

 
Version 6, Dated <16/January/2026> 
Page 33 of 43 
Restricted, Sensitive (Normal) 
CRFs and Investigator Site Folder will be kept in a secure cabinet, under lock and key. Only 
members of the study team can have access to the data.  
9 SAMPLE SIZE AND STATISTICAL METHODS 
9.1 Determination of Sample Size  
 
In the Phase I study, we will be using the Bayesian Model Averaging continual reassessment 
method (BMA-CRM) study design [12, 13]. The primary goal of the BMA-CRM is to identify 
the maximum tolerated dose (MTD) of NK cells to be infused. In this Phase I study, minimum 
of 21 patients will be accrued.  Five doses of NK cells will be used (0.5x107, 0.8x107, 1.1x107, 
1.4x107 and 1.8x 107cells/Kg). We will begin with the dose 0.8x107cells/kg and the dose 
escalation is restricted to a single dose level. Once there is an occurrence of 1 toxicity, the 
CRM will be implemented. The CRM method estimates toxicity at each dose using Bayesian 
methodology to update a dose-toxicity curve for each cohort of 3 patients. We assume the 
prior toxicity probability estimates at 10%, 15%, 20%, and 25% at each level. To avoid 
subjectivity in setting priors, the BMA-CRM specifies multiple sets of priors and takes the 
average across the CRM models to avoid the potential estimation bias caused by 
misspecification. We shall terminate the phase at 21 patients or when CRM dictate that 6 
patients be treated at a particular dose, at which point the current dose estimate is declared 
the MTD.  
 
Phase 2 is a single-arm trial; the one-sample proportion test will be used to test the proportion 
deduction based on the binomial distribution. A sample size of 5 achieves 80.549% power to 
detect a difference (P1-P0) of -0.2500 using a one-sided exact test with a significance level 
(alpha) of 0.05. These results assume that the population proportion under the null 
hypothesis (P0) is 1.00. Assume the withdrawal rate at 0.2 (20%), the total sample size is 
estimated at 6. From phase 1a, for those patients treated at MTD, these patients will be 
followed up and included in the phase 2 study. As likely there will be around six patients 
treated at MTD (at least two cohorts), thus only an additional one patient will be included for 
the phase 2 trial.  
 
9.2 Statistical and Analytical Plans 
a. General Considerations 
 
The phase 1 study will be an open-label, dose-escalation trial, with a maximum of 21 NPC 
patients to be accrued for NK cells treatment. The primary endpoint is the occurrence of 
any of the toxicities during 4 weeks of treatment as assessed by AEs attributed (see session 
7.3). Five doses of NK cells will be used for the treatment (d1 = 0.5x107, d2= 0.8x107, d3 = 
1.1x107, d4 = 1.4x107, and d5 = 1.8x107 cells/kg). The maximum tolerated dose (MTD) will 
be estimated as the highest dose with an estimated probability of a dose limiting toxicity 
(DLT) of less than 30%.  
 
We will apply 2-stage continual reassessment method (CRM) design (Cheung 2005 & 2011; 
Goodman 1995). First, we specify a stage 1 design as a predetermined nondecreasing dose 
sequence.  Because the 3+3 algorithm is familiar, we consider the “group-of-three” stage 1 
design whereas escalation takes place after every group of three nontoxic outcomes. The 
stage 1 design is in effect until the first observed toxicity. Secondly, the trial turns to the 
model-based CRM for dose assignment. CRM method generally has a superior operating 

 
Version 6, Dated <16/January/2026> 
Page 34 of 43 
Restricted, Sensitive (Normal) 
performance as it bases estimation on all prior dose data rather than just the last 3 patients; 
this usually resulted in more accurate estimation of the MTD, fewer toxicities and more 
patients treated at assumed efficacious doses close to the MTD.  
 
Specifically, for stage 1:  
The first three patients (xi,0) will be recruited at dose level d2. Starting at d2 is because that 
NK cell treatments are given at established doses which is thought to be non-toxic. 
 
If no DLT occurs, subsequent three patients will be recruited at one dose higher (unless 
current dose is D6).  
 
x1,0 = x2,0  = x3,0  = d2, x4,0 = x5,0  = x6,0  = d3,. . . 
 
The MTD will be estimated as the last dose (d5). 
 
If a DLT occurs, the trial will progress to stage 2 using a CRM to assign the dose (see 
below).  
 
For stage 2:  
 
The study will progress to this stage if a DLT occurs in stage 1. We will assign 3 patients at 
a time to each does and update the model-based estimates between every group of 3 
patients, using information of all patients with DLTs known by the next patient recruitment 
time. Escalation by more than 1 level will not be allowed. If the number of consecutive 
patients on the current dose is 6 or 21 patients in total, then the trial will stop and the MTD 
will be estimated based on a CRM of the recruited patients.  
 
We use maximum likelihood estimation in conjunction with the CRM. The idea is analogous 
to the Bayesian CRM, with the data observed in the first i −1 patients. The dose for the next 
subject is computed based on the maximum likelihood estimates of coefficients for given 
data. 
 
A CRM will be implemented via the function implemented in R package “dfcrm” [15]. The 
prior toxicity estimates are set at 2.5%, 5%, 10%, 20%, 30% for the dose levels from D1 to 
D6. We shall terminate the phase at 21 patients or when a dose level is selected 6 times at 
which point the current dose estimate is declared the MTD. 
 
The properties of the CRM procedure are assessed using the CMR Suite v1.00 using 
simulated data [12, 13]. We assume patients recruited randomly, with average arrival of 
1.25 per month, toxicity assessment period is within 30 days, and target toxicity limit (TTL) 
probability is 30%.  The simulation is applied to 5 scenarios from true low to very high toxicity 
rates using 1000 replicates for each scenario.  The table below compares the performance 
in each circumstance in terms of the total and an average number of DLT, the proportion of 
times each dose level is selected as MTD, the proportion of total patients treated at each 
dose level, and the duration of trial time. If the true toxicity is low, around 19.8 months are 
expected to complete the trial, and the toxicity per trial is low at 2.7, and MTD is estimated 
at the dose level 5. 
 
Model 
Target Toxicity Probability 
0.300

 
Version 6, Dated <16/January/2026> 
Page 35 of 43 
Restricted, Sensitive (Normal) 
Safety Stopping Probability
(Upper Limit Pr(tox) at Lowest 
Dose) 
0.700
Maximum Sample Size
21
Cohort Size 
3
Starting Dose Level
2
Toxicity Assessment Period 
(Days)
30
 
Prior Median Probabilities of Toxicity at Each Dose Level: 
Dose
Prob.
1 
0.025 
2
0.050
3 
0.100 
4
0.200
5
0.300
 
Scenarios and Results 
 
Scenario: Very High Toxicity 
Dos
e 
Leve
l 
True 
Prob(tox) 
Selection 
Probability  
# of Subjects 
Treated 
# of 
Toxicities 
1
0.200
0.11
1.7
0.4
2
0.300
0.24
7.3
2.2
3
0.400
0.22
4.6
1.8
4
0.500
0.07
1.5
0.7
5
0.600
0.01
0.2
0.1
Probability of Early Termination: 0.35
Toxicities per Trial: 5.2 
Total Trial Time (Months): 15.65
 
Scenario: High Toxicity 
Dose 
Level 
True 
Prob(tox) 
Selection 
Probability  
# of Subjects 
Treated 
# of 
Toxicities 
1
0.100
0.03
0.8
0.1
2
0.200
0.17
6.7
1.4
3
0.300
0.35
6.5
1.9

 
Version 6, Dated <16/January/2026> 
Page 36 of 43 
Restricted, Sensitive (Normal) 
4
0.400
0.25
3.6
1.4
5
0.500
0.05
0.8
0.4
Probability of Early Termination: 0.16 
Toxicities per Trial: 5.2
Total Trial Time (Months): 19.01
 
Scenario: Medium Toxicity 
Dose 
Level 
True 
Prob(tox) 
Selection 
Probability  
# of Subjects 
Treated 
# of 
Toxicities 
1
0.050
0.00
0.1
0.0
2
0.100
0.03
4.6
0.5
4
0.250
0.32
5.2
1.3
5
0.300
0.41
4.2
1.3
Probability of Early Termination: 0.04
Toxicities per Trial: 4.2 
Total Trial Time (Months): 20.55
 
Scenario: Low Toxicity 
Dose 
Level
True 
Prob(tox)
Selection 
Probability
# of Subjects 
Treated
# of 
Toxicities
1
0.000
0.00
0.0
0.0
2
0.025
0.00
3.2
0.1
3
0.050
0.00
3.5
0.2
4
0.100
0.06
4.3
0.4
5
0.200
0.93
9.9
2.0
Probability of Early Termination: 0.01
Toxicities per Trial: 2.7
Total Trial Time (Months): 19.85 
 
 
 
 
Phase 2: Statistical Analysis and Power calculation (new to 10%) 
The reference we use in comparison with our single arm trial finding is derived from the 
anticipated proportion of locally advanced NPC patients (25-30%) who will continue to 
present with detectable circulating EBV-DNA following standard of care CRT paradigm. 
We hypothesize that in this single arm trial, adding allogeneic NK cells will completely 
eliminate the proportion of patients with detectable circulating EBV-DNA from 25-30% to 
10%. Hence, applying the one proportion Z-score test, a sample size of 26 will achieve 
81.2% power to detect a difference (P1-P0) of 20% using a one-sided exact test with a 
significance level (alpha) of 0.05. Assuming the withdrawal rate at 0.2 (20%), the total 

 
Version 6, Dated <16/January/2026> 
Page 37 of 43 
Restricted, Sensitive (Normal) 
sample size is estimated at 31. In our phase 1 design, at least 6 patients will be treated 
at our MTD of allogenic NK cells, meaning that additional 25 patients will be accrued in 
the phase 2 trial. 
The proportion estimate and its confidence interval will also be reported. We will also 
perform sensitivity analyses to assess the impact of missing data and potential 
confounding variables. 
 
The secondary endpoint of the study is to determine the change in patient survival and 
recurrence rate within 24-36 months post-treatment when compared with historic cohorts 
under standard treatments. This is an exploratory aim due to small sample size. We will 
apply Kaplan-Meier curves to plot the survival and recurrence rate with time. We will 
estimate the median and mean survival time, and the confidence intervals. The median 
survival time will be estimated. We will compare the survival and recurrence rates 
between the study cohort and the historical control group using log-rank tests.  
 
b. Safety Analyses 
The phase 1 study is safety-oriented design. Adverse events will be monitored and 
recorded throughout the study. Dose toxicity evaluation after receiving the treatment will 
be conducted. Dose escalation would occur only if no toxicity occurred or as suggested 
by CRM. Dose-limiting toxicities will be defined and recorded. We will also examine the 
proportion of patients who experience treatment-related adverse events and report them 
separately. 
 
9.3 Interim Analyses 
 
The dose-escalation design CRM foresees that decisions based on the current data are 
taken before the end of the study. More precisely, after each cohort in the dose-escalation 
part, the next dose will be chosen depending on the observed data. The study may be 
stopped early if predefined stopping rules are met.  
 
At the mid-point of Phase 2, we will access the progress of our primary endpoint by the 
determination of the percentage of patients achieving undetectable EBV-DNA levels and 
secondary endpoint of recurrent rate among all the accrual for the trial thus far. 
9.4 Describe the types of statistical interim analyses, including their timing 
 
1. See the analysis method session for CRM methods.  
2. A statistical two tailed test will be performed to examine the decrease in the percentage 
of patients with undetectable EBV-DNA levels, 
3. Similar statistical analysis will be carried out to analyse the NPC recurrence rate among 
the accruals for the trial at the point of analysis.  

 
Version 6, Dated <16/January/2026> 
Page 38 of 43 
Restricted, Sensitive (Normal) 
10 DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
The investigator(s)/institution(s) will permit study-related monitoring, audits and/or IRB review 
and regulatory inspection(s), providing direct access to source data/document. 
In accordance with ICH GCP guidelines, the study monitor must have direct access to the 
investigator’s source documentation in order to verify the data recorded in the investigator 
file for consistency. The monitor is responsible for routine review of the investigator file at 
regular intervals throughout the study to verify adherence to the protocol and the 
completeness, consistency, and accuracy of the data being entered on them. The 
investigator agrees to cooperate with the monitor to ensure that any problems detected during 
these monitoring visits are resolved. 
 
11 QUALITY CONTROL AND QUALITY ASSURANCE 
Data will be collected and recorded by a clinical research coordinator. The investigator will 
verify that all data entries are accurate and correct, including verification that the subject fulfils 
the criteria for entry into the study before study treatment commences. 
12 ETHICAL CONSIDERATIONS 
This study will be conducted in accordance with the ethical principles that have their origin in 
the Declaration of Helsinki and that are consistent with the Good Clinical Practice and the 
applicable regulatory requirements. 
 
This final Clinical Trial Protocol, including the final version of the Participant Information Sheet 
and Consent Form, must be approved in writing by the Centralised Institutional Review Board 
(CIRB) and regulatory approval from Health Sciences Authority (HSA), prior to enrolment of 
any patient into the study. 
 
The principal investigator is responsible for informing the CIRB and HSA of any amendments 
to the protocol or other study-related documents, as per local requirement. 
12.1 Informed Consent 
 
The study will be conducted in accordance with the protocol, International Conference on 
Harmonization (ICH) GCP guidelines, applicable regulations and guidelines governing 
clinical study conduct and ethical principles that have their origin in the Declaration of 
Helsinki. The investigator will ensure that the study is conducted in accordance with the 
provisions as stated in the ICH regulations and complies with prevailing local laws and 
customs. Prior to the initiation of any screening or study-specific procedures, the investigator 
and/or his/her representative will explain the nature of the study to the subject and answer 
all questions regarding this study. Each informed consent will be reviewed, signed and dated 
by the subject and the person who administered the informed consent. A copy of each 
informed consent will be given to the subject, and each original will be placed in the 
Investigator Site Manual.  

 
Version 6, Dated <16/January/2026> 
Page 39 of 43 
Restricted, Sensitive (Normal) 
12.2 Confidentiality of Data and Patient Records 
Information collected for this study will be kept confidential. This confidentiality is extended 
to cover testing of any biological samples and genetic tests in addition to the clinical 
information relating to the participating subjects. Patient records, to the extent of the 
applicable laws and regulations, will not be made publicly available. Data collected and 
entered into the Case Report Forms are the property of SGH. In the event of any publication 
regarding this study, patient identity will remain confidential. 
13 PUBLICATIONS 
 
The investigators have the right to publish the results of the study, but with due regard to the 
protection of confidential information. 
 
14 RETENTIONS OF TRIAL DOCUMENTS 
 
Records for all participants, including CRFs, all source documentation (containing evidence 
to study eligibility, history and physical findings, laboratory data, results of consultations, etc) 
as well as DSRB records and other regulatory documentation will be retained by the PI in a 
secure storage facility for up to 10 years from time of study termination. The records will be 
accessible for inspection and copying by authorized authorities. 
15 FUNDING AND INSURANCE 
 
The Insurance is covered under the Singhealth Insurance policy for clinical trial in its terms 
and provisions, its legal liability for injuries caused to participating persons and arising out of 
this research performed strictly in accordance with the scientific protocol as well as with 
applicable law and professional standards. Since this is a first in man clinical trial, additional 
insurance will be acquired. 
 
The funding is supported under the PI’s National Medical Research Council. Clinician 
Scientist Award (CSA) and Clinical Trial Grant – Investigator Initiated trial (CTG-IIT). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Version 6, Dated <16/January/2026> 
Page 40 of 43 
Restricted, Sensitive (Normal) 
16 LIST OF ATTACHMENTS 
 
 
A) Appendix 1 Study Schedule 
 
Assessment Period
Assessment Procedures
Notes
-28 to -1 
      1.   Draw Blood (20ml) 
      2.   Tumour Tissue Biopsy 
      3.   EBV assay  
 
Week 1 
1. Chemo-drug Administration 
2. Radiation Therapy 
Administration 
(1 dose daily, 5 doses/week) 
 
Week 2 
1. Radiation Therapy 
Administration 
(1 dose daily, 5 doses/week) 
 
Week 3 
1. Radiation Therapy 
Administration 
(1 dose daily, 5 doses/week) 
2. NK cells infusion 
 
Week 4 
1. Draw Blood (20ml)* 
2. EBV assay 
3. Chemo-drug Administration 
4. Radiation Therapy 
Administration 
*Draw blood before 
Chemo-drug 
administration 

 
Version 6, Dated <16/January/2026> 
Page 41 of 43 
Restricted, Sensitive (Normal) 
(1 dose daily, 5 doses/week) 
Week 5 
1. Radiation Therapy 
Administration 
(1 dose daily, 5 doses/week) 
 
Week 6 
1. Radiation Therapy 
Administration 
(1 dose daily, 5 doses/week) 
2. NK cells infusion 
 
Week 7 
1. Draw Blood (20ml) * 
2. EBV assay 
3. NK cells infusion 
*Draw blood before NK 
cells infusion 
Week 8 
1. NK cells infusion 
 
Week 9 
1. NK cells infusion 
 
Week 10 
1. NK cells infusion 
 
Week 11 
1. Draw Blood (20ml) 
2. Tumour Tissue Biopsy 
3.
EBV assay
 
Week 12 (Optional) 
1. NK cells infusion 
 
Week 13 (Optional) 
1. NK cells infusion 
 
Week 14 (Optional) 
1. NK cells infusion 
 
Week 15 (Optional) 
1. NK cells infusion 
 
Week 16 (Optional) 
1. Draw Blood (20ml) 
2. Tumour Tissue Biopsy 
3. EBV assay 
 
B) NK cell infusion 
 
1. 6 doses will be administered during the standard of care treatment of concurrent 
chemoradiotherapy (2 doses as concurrent and 4 doses as adjuvant therapy). 
2. The first 3 patients admitted for the administration of the first dose of NK cells will be 
monitored overnight. The expected duration of NK cell infusion is approximately 30-
60 mins over a slow infusion. If there are no acute reactions to NK cell infusion for 
the first 3 patients, subsequent doses to the 3 patients can be treated as outpatient 
with a monitoring of 4-6 hours following infusion. 
3. Without acute reaction, NK cells infusion for the rest of the patients accrued will be 
treated as outpatient with a monitoring of 4-6 hours following infusion 
4. Patients who have persistently detectable circulating plasma EBV-DNA copies after 
the 6 doses of NK cell infusions will have the option of receiving an additional 4 
weekly cycles of NK cells infusion.  
 
C) Radiation Therapy 
Conventional radiotherapy will be given as per standard of care treatment of NPC 

 
Version 6, Dated <16/January/2026> 
Page 42 of 43 
Restricted, Sensitive (Normal) 
 
D) Chemotherapy 
Platinum based chemotherapy regime is usually administered as the standard of care 
chemotherapy option for NPC. In patients who cannot receive chemotherapy or declined 
chemotherapy option, they will still be eligible to participate in this trial. Patients who are 
prescribed other chemotherapy beside the platinum-based chemotherapy option due to 
adverse response to platinum chemotherapy are also eligible to participate in this trial.  
 
E) Correlative tissue Biopsy and blood analyses 
 
1. A total of 2 tumour biopsies will be performed in this trial (once prior to treatment and 
the other after the completion of the standard treatment and 6 doses of NK cells 
infusion).  
2. Blood (approximately 20 ml) will be drawn 5 times (prior to treatment, week 4, 7, 9 and 
11) during the trial. Sufficient plasma will be sent for plasma EBV detection, and the 
rest will be frozen for analysis at a later stage. 
3. PBMC will be isolated and stored for correlative analyses. 
 
17  REFERENCES  
 
1. 
Del Zotto, G., et al., Markers and function of human NK cells in normal and pathological 
conditions. Cytometry B Clin Cytom, 2017. 92(2): p. 100-114. 
2. 
Wu, J. and L.L. Lanier, Natural killer cells and cancer. Adv Cancer Res, 2003. 90: p. 
127-56. 
3. 
Dianat-Moghadam, H., et al., NK cells-directed therapies target circulating tumor cells 
and metastasis. Cancer Lett, 2021. 497: p. 41-53. 
4. 
Han, B., et al., Altered NKp30, NKp46, NKG2D, and DNAM-1 Expression on Circulating 
NK Cells Is Associated with Tumor Progression in Human Gastric Cancer. J Immunol 
Res, 2018. 2018: p. 6248590. 
5. 
Myers, J.A. and J.S. Miller, Exploring the NK cell platform for cancer immunotherapy. 
Nat Rev Clin Oncol, 2021. 18(2): p. 85-100. 
6. 
Shimasaki, N., A. Jain, and D. Campana, NK cells for cancer immunotherapy. Nat Rev 
Drug Discov, 2020. 19(3): p. 200-218. 
7. 
Ciurea, S.O., et al., Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived 
NK cells after haploidentical transplantation. Blood, 2017. 130(16): p. 1857-1868. 
8. 
Hoogstad-van Evert, J., et al., Intraperitoneal infusion of ex vivo-cultured allogeneic NK 
cells in recurrent ovarian carcinoma patients (a phase I study). Medicine (Baltimore), 
2019. 98(5): p. e14290. 
9. 
Ishikawa, T., et al., Phase I clinical trial of adoptive transfer of expanded natural killer 
cells in combination with IgG1 antibody in patients with gastric or colorectal cancer. Int J 
Cancer, 2018. 142(12): p. 2599-2609. 
10. 
Lin, M., et al., Pembrolizumab plus allogeneic NK cells in advanced non-small cell lung 
cancer patients. J Clin Invest, 2020. 130(5): p. 2560-2569. 
11. 
Jin, Y.Y., et al., Chemoradiotherapy combined with NK cell transfer in a patient with 
recurrent and metastatic nasopharyngeal carcinoma inducing long-term tumor control: A 
case report. Medicine (Baltimore), 2020. 99(43): p. e22785. 
12. 
O'Quigley, J., M. Pepe, and L. Fisher, Continual reassessment method: a practical 
design for phase 1 clinical trials in cancer. Biometrics, 1990. 46(1): p. 33-48. 

 
Version 6, Dated <16/January/2026> 
Page 43 of 43 
Restricted, Sensitive (Normal) 
13. 
Yin, G. and Y. Yuan, Bayesian model averaging continual reassessment method in 
phase I clinical trials. Journal of the American Statistical Association, 2009. 104(487): p. 
954-968. 
14. 
cheung, Y.K., Coherence principles in dose-finding studies. Biometrika, 2005. 92: p. 10. 
15. 
Cheung, Y.K., Dose FInding by the Continual Reassessment Method. 2011: Chapman & 
Hall/CRC press. 
16. 
Goodman, S.N., M.L. Zahurak, and S. Piantadosi, Some practical improvements in the 
continual reassessment method for phase I studies. Stat Med, 1995. 14(11): p. 1149-61.