A Single-center, Open-label, Exploratory Trial of Autologous Immunotherapy for Hepatocellular Carcinoma (HCC) With Microvascular Invasion (MVI) After Radical Resection
Phase: PHASE2
Conditions: Hepatocellular Carcinoma, Malignant Neoplasm
Sponsor: Cancer Institute and Hospital, Chinese Academy of Medical Sciences

NCT03575806 
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A single-center, open-label, exploratory trial of autologous 
immunotherapy for Hepatocellular carcinoma (HCC) with 
microvascular invasion (MVI) after radical resection 
 
 
 
 
Sponsor: Cancer Institute and Hospital, Chinese Academy of Medical Sciences 
Collaborators: Newish Technology (Beijing)Co.,Ltd. 
Investigator Name: Hong Zhao 
ClinicalTrials.gov ID: NCT03575806 
Document date: 31 October 2019 
 
 
 

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Background 
Hepatocellular carcinoma (HCC) is one of the common cancer worldwide, which is the third cause 
of cancer related deaths. Radical hepatic resection remains the main treatment for hepatocellular 
carcinoma, the 5-year survival rate of HCC after surgery was 60-70%. In east Asian countries, the 
mortality rate for HCC is estimated at 360,000 pre year. Unfortunately, HCC is prone to 
postoperative recurrence that more than 50% of patients relapse within 2 years, which has become 
the key to restrict the therapeutic effect of HCC.  
Multi-factor analysis showed that vascular invasion, tumor number and size, preoperative AST 
increase, tumor resection margin and liver capsule invasion were the main factors influencing 
prognosis, among which vascular invasion was the most closely related variable to postoperative 
recurrence of HCC. 
Autologous cell immunotherapy is to collect patient's own immune cells and then given back to 
the patient after amplified in vitro that can improve the anti-tumor immune response and is effective 
treatment of the cancer. Tcm (central memory T cells) are effective anti-tumor immune cells that 
exhibit the long-term survival and self-renewal capacity in vivo. Autologous Tcm immunotherapy 
combining chemotherapy, surgery or radiotherapy would effectively prolong survival period, 
prevent tumor recurrence and metastasis, then improve quality of life in patients. 
 
Methods/design 
Study setting 
This trial is conducted at Cancer Institute and Hospital, Chinese Academy of Medical Sciences. 
 
Objective 
To investigate the clinical efficacy and safety of Tcm immunotherapy in hepatocellular carcinoma 
with microvascular invasion after radical resection. 
 
Trail design 
Single-center, open-label, exploratory trial. 
 

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Eligibility 
Inclusion criteria 
1. Be willing and able to provide written informed consent for the study. 
2. Subject has accepted radical hepatic resection, and preoperative imaging is no vascular 
invasion. 
3. Postoperative pathology confirmed Hepatocellular carcinoma with negative margin and 
microvascular invasion (MVI). 
4. Age between 18-75 years old. 
5. Radiology confirmed complete response (CR) after radical surgery. 
6. Child-Pugh A. 
7. Eastern Cooperative Oncology Group(ECOG) body condition score 0. 
8. Adequate hepatic and renal function:  
  Hemoglobin ≥ 9.0g/dl 
Absolute neutrophil count (ANC) > 1,500/mm3 
platelets ≥ 50,000/ul 
Total bilirubin (TBIL) ≤ 2mg/dl 
Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) ≤ 5 the upper 
limit of normal (ULN) for the institution 
Alkaline phosphatase (ALP) ≤ 4 the upper limit of ULN 
Prothrombin time (PT) > 50% or prothrombin time-international normalized ratio (PT-
INR) < 2.3 
Serum creatinine (CREA) ≤ 1.5 the upper limit of ULN. 
9. Female subjects have had a negative blood pregnancy test within 2 week,  
10. Subjects be willing to use appropriate contraception during the trial and 2 weeks after 
the last administration of immunotherapy. 
11. Radiology such as CT and MRI were performed in 4 weeks before the study.  

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Exclusion Criteria: 
1. Recurrent HCC. 
2. Portal vein embolus 
3. Cardiovascular disease: 
(1) Evidence of NYHA functional class III or IV heart disease. 
(2) Unstable coronary artery disease (CAD) is not allowed, while Myocardial 
Infarction (MI) 6 months of starting study is allowed.  
(3) Cardiac arrhythmias requiring antiarrhythmic drugs except β-blockers or digoxin 
are not allowed.  
(4) Uncontrolled hypertension.  
4. History of Human Immunodeficiency Virus (HIV) or syphilis infection.  
5. Severe inflammation, NCI CTCAE Version 3.0 grade > 2. 
6. Epilepsy requiring steroid or antiepileptic drugs.  
7. History of allotransplantation.  
8. History or any evidence of hemorrhage.  
9. patients undergoing renal dialysis.  
10. Pregnancy or breast-feeding. 
11. Prior or undergoing cancers that primary sites are different from the carcinoma of this 
study. Exceptions to this are: 
  Cervical carcinoma in situ (CIS) 
Cured basal cell carcinoma 
Superficial bladder tumor 
Cured cancers over 3 years before the study 
12. Uncontrolled Ascites by diuretic treatment. 
13. History of encephalopathy. 

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14. Gastrointestinal hemorrhage in 30 days before the study. 
15. History of esophageal variceal hemorrhage and it is no effective treatment to prevent 
the recurrence of hemorrhage. 
16. Major surgery except radical hepatic resection was performed in 4 weeks before the 
study.  
17. Autologous bone marrow transplantation (ABMT) in 4 weeks before the study. 
18. Concurrent treatment on another clinical trial or treatment on another clinical trial in 4 
weeks before the study. 
19. Drug abuse, medical treatment, mental illness or social disorders that would interfere 
with subjects' participation, or confound the results of the trial. 
20. Any condition that would interfere with or endanger the safety and compliance of 
subjects. 
 
Arms and interventions 
Experimental arm (Tcm + TACE group) 
After radical resection, subjects will receive TACE in a month instead of chemotherapy or targeted 
drugs. Peripheral Blood Mononuclear Cell (PBMC) obtained from peripheral blood that is 
extracted within 2 to 4 days before TACE will be cultured in vitro for 14 days, and then subjects 
will be treated with auto-immune cell transfusion. This is done for two cycles at a one-month 
interval. 
 
Active comparator (TACE group) 
After radical resection, subjects will receive TACE in a month instead of chemotherapy or targeted 
drugs. 
 
Outcomes 
we will evaluate the clinical efficacy and safety of autologous Tcm immunotherapy for 
HCC with MVI after radical resection according to Recurrence-Free Survival (RFS)as the 

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primary outcome measure. 
The data showed that the median RFS time was 9.5 months in control group, and did not 
reach the cut-off date in Tcm group for that both 12-month RFS rate and 24-month RFS 
rate were still greater than 50%. Gehan-Breslow-Wilcoxon test showed the time of RFS in 
Tcm group were significantly better than the control group when follow-up duration was 
12 months (P=0.049; HR=0.40; 95%Cl: 0.16-0.99), but the difference of the time of RFS 
between two groups did not have statistical significance when follow-up duration was 24 
months (P=0.06; HR=0.49; 95%Cl: 0.21-1.12). Above all, Tcm combined with TACE can 
obviously extended the time of RFS in early period after hepatectomy, and this protective 
efficacy might last for 12 months. 
 
Figure 1: Comparison of RFS between Tcm group and control group at 12 months (P=0.049) and 
24 months (P=0.060). P value were calculated by Gehan-Breslow-Wilcoxon test. 
 
Sample size 
Overall number of participants：48 Subjects (23 in Tcm + TACE group and 25 in TACE 
group) 
 
Statistical analysis 
Kaplan-Meier curves and Gehan-Breslow-Wilcoxon test are used to analyze the RFS in 
the two group. 
 
Trial status  

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Study period: 1 November 2016 to 31 October 2019 
 
Ethics approval and consent to participate 
This study was approved by the Ethics Committee of Cancer Institute and Hospital, 
Chinese Academy of Medical Sciences (Approval Number: 16-134/1213). 
The physician in charge will provide a detailed oral explanation supported by a written 
document to the patient or their legal representative and will then ascertain the patient’s 
understanding of the study. When the patient or their legal representative consents to 
participate in the study, the physician providing the explanation will enter the date and 
their name and that of the patient (or their legal representative) and the physician will sign 
the form. The physician will hand a copy of the signed consent form to the patient or their 
representative and store a copy (in electronic image format) in the electronic health 
record. The principal investigator will keep the original signed document. Withdrawal of 
consent will not disadvantage the patient or affect patient care. 
Participation in this study is voluntary, and participants can withdraw from the study at 
any time during the study as they wish. Non-participation will not cause any disadvantage 
to patients. The patient (or their legal representative) will confirm intent to withdraw by 
signing a withdrawal form. The physician in charge will countersign the withdrawal form. 
 
 
 

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Informed Consent Form 
 
Research number: CH-IT-001 
Research institute: Cancer Institute and Hospital, Chinese Academy of Medical Sciences 
The physician in charge of the study: Hong Zhao 
 
Dear patient: 
you are welcome to volunteer for the clinical trial of autologous immunotherapy for 
Hepatocellular carcinoma (HCC) with microvascular invasion (MVI) after radical 
resection. Before deciding whether be willing to participate in, you need to understand 
this study about the purpose and treatment, advantages and disadvantages, and expect you 
to do things in the process of research as well as the rights of you as a volunteer. Please 
read it carefully. If you have any questions, please ask the researcher responsible for the 
study. 
Background 
Hepatocellular carcinoma (HCC) is one of the common cancer worldwide, which is the third 
cause of cancer related deaths. More than 75% of the cases occurred in the Asia-Pacific region, 
which is largely related to the hepatitis β virus infection. The mortality rate for HCC is estimated at 
360,000 pre year in the east Asian countries. Radical resection is still the major treatment for HCC, 
radical hepatic resection remains the main treatment for hepatocellular carcinoma, the 5-year 
survival rate of HCC after surgery was 60-70%. Unfortunately, HCC is prone to postoperative 
recurrence that more than 50% of patients relapse within 2 years, which has become the key to 
restrict the therapeutic effect of HCC. Multi-factor analysis showed that vascular invasion, tumor 
number and size, preoperative AST increase, tumor resection margin and liver capsule invasion were 
the main factors influencing prognosis, among which vascular invasion was the most closely related 
variable to postoperative recurrence of HCC. 
It is concluded that there is no proven beneficial adjuvant therapy for HCC according to the analysis 
about published open-label trails of the adjunctive therapy of HCC. Therefore, it has 
become a priority in the treatment of HCC that conduct clinical trials to evaluate the 

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efficacy of adjuvant therapy after radical treatment. 
Memory T cells which are divided into Tem (effector memory T cells) and Tcm (central 
memory T cells) according to the function. Tcm are specific and durable anti-tumor 
immune cells according the result of study that the long-term observation of 19 
fibroblastoma patients after receiving autologous cell immunotherapy. The autologous cell 
immunotherapy in this clinical trail is provided by Newish Technology (Beijing)Co.,Ltd.. 
The informed consent form has been approved by ethics committee.  
Research purpose 
The purpose of this study is to assess the efficacy and safety of autologous 
immunotherapy in HCC patients after radical resection, and provide the basis for further 
study. 
Eligibility 
Inclusion criteria 
1. Be willing and able to provide written informed consent for the study. 
2. Subject has accepted radical hepatic resection, and preoperative imaging is no vascular 
invasion. 
3. Postoperative pathology confirmed Hepatocellular carcinoma with negative margin and 
microvascular invasion (MVI). 
4. Age between 18-75 years old. 
5. Radiology confirmed complete response (CR) after radical surgery. 
6. Child-Pugh A. 
7. Eastern Cooperative Oncology Group(ECOG) body condition score 0. 
8. Adequate hepatic and renal function:  
  Hemoglobin ≥ 9.0g/dl 
Absolute neutrophil count (ANC) > 1,500/mm3 
platelets ≥ 50,000/ul 
Total bilirubin (TBIL) ≤ 2mg/dl 

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Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) ≤ 5 the upper 
limit of normal (ULN) for the institution 
Alkaline phosphatase (ALP) ≤ 4 the upper limit of ULN 
Prothrombin time (PT) > 50% or prothrombin time-international normalized ratio (PT-
INR) < 2.3 
Serum creatinine (CREA) ≤ 1.5 the upper limit of ULN. 
9. Female subjects have had a negative blood pregnancy test within 2 week,  
10. Subjects be willing to use appropriate contraception during the trial and 2 weeks after 
the last administration of immunotherapy. 
11. Radiology such as CT and MRI were performed in 4 weeks before the study.  
Exclusion Criteria: 
1. Recurrent HCC. 
2. Portal vein embolus. 
3. Cardiovascular disease: 
(1) Evidence of NYHA functional class III or IV heart disease. 
(2) Unstable coronary artery disease (CAD) is not allowed, while Myocardial 
Infarction (MI) 6 months of starting study is allowed.  
(3) Cardiac arrhythmias requiring antiarrhythmic drugs except β-blockers or digoxin 
are not allowed.  
(4) Uncontrolled hypertension.  
4. History of Human Immunodeficiency Virus (HIV) or syphilis infection.  
5. Severe inflammation, NCI CTCAE Version 3.0 grade > 2. 
6. Epilepsy requiring steroid or antiepileptic drugs.  
7. History of allotransplantation.  
8. History or any evidence of hemorrhage.  
9. patients undergoing renal dialysis.  

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10. Pregnancy or breast-feeding. 
11. Prior or undergoing cancers that primary sites are different from the carcinoma of this 
study. Exceptions to this are: 
  Cervical carcinoma in situ (CIS) 
Cured basal cell carcinoma 
Superficial bladder tumor 
Cured cancers over 3 years before the study 
12. Uncontrolled Ascites by diuretic treatment. 
13. History of encephalopathy. 
14. Gastrointestinal hemorrhage in 30 days before the study. 
15. History of esophageal variceal hemorrhage and it is no effective treatment to prevent 
the recurrence of hemorrhage. 
16. Major surgery except radical hepatic resection was performed in 4 weeks before the 
study.  
17. Autologous bone marrow transplantation (ABMT) in 4 weeks before the study. 
18. Concurrent treatment on another clinical trial or treatment on another clinical trial in 4 
weeks before the study. 
19. Drug abuse, medical treatment, mental illness or social disorders that would interfere 
with subjects' participation, or confound the results of the trial. 
20. Any condition that would interfere with or endanger the safety and compliance of 
subjects. 
Study methods 
Experimental arm (Tcm + TACE group): After radical resection, subjects will receive 
TACE in a month instead of chemotherapy or targeted drugs. Peripheral Blood 
Mononuclear Cell (PBMC) obtained from peripheral blood that is extracted within 2 to 4 
days before TACE will be cultured in vitro for 14 days, and then subjects will be treated 
with auto-immune cell transfusion. This is done for two cycles at a one-month interval. 

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Active comparator (TACE group): After radical resection, subjects will receive TACE in 
a month instead of chemotherapy or targeted drugs. 
Withdrawal from the study 
In the following cases, the researcher could suspend the test without your consent:  
1. When researcher decides that continue test is not good for you. 
2. When you happened severe adverse events in the process of trial. 
3. When you fail to take the medicine according to the doctor's instructions. 
Research process 
The subjects had to sign a consent form before participating in this trial, you will 
participate in this trail into the treatment group or control group as you meet the criteria. 
According to the principle of random grouping and into experimental arm (Tcm + TACE 
group) or active comparator (TACE group). Requirements as follows: 
experimental arm (Tcm + TACE group): After radical resection, you must be in 
accordance with the requirements of the trail and the researcher during the trail. You are 
informed to sign the informed consent after meeting criteria by the researcher screening 
your conditions. twenty days later you would return to research institute and 80-100 ml 
blood is extracted that will be sent to the laboratory for amplification of 14 days. It will be 
two days in advance to inform you that you are returning to research institute and will be 
treated with auto-immune cell transfusion intravenously. you would be treated with 
TACE one month later. This is done for two cycles at a one-month interval. During this 
trail, comprehensive interrogation, physical examinations, laboratory examinations and 
imaging examinations are required before the first auto-immune cell transfusion and 
every three months postoperatively, the observation time is 24 months. 
active comparator (TACE group): you would be treated with TACE one month after 
radical resection, this would be done for two cycles at a one-month interval. During this 
trail, comprehensive interrogation, physical examinations, laboratory examinations and 
imaging examinations are required before the first auto-immune cell transfusion and 
every three months postoperatively, the observation time is 24 months. 

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Benefits 
By studying your case, it will provide the necessary advice for your treatment, or provide 
useful information for the study of the disease. 
Rights and interests of subjects 
1. You have the right to know the study about the purpose, methods, advantages and 
disadvantages, rights and interests by consulting the researcher. 
2. You may choose not to participate in this study, or at any time inform the researcher to 
request withdrawal from the study. Your data will not be included in the study results, 
and any medical treatment and benefits will not be affected. 
3. You have the right to privacy: see the confidentiality section of this text. 
Adverse reactions and safety measures  
Any research will have risk, discomfort and inconvenience. In preclinical trials, It is 
found no severe side effects about cells immunotherapy, but any kind of treatment may 
cause some adverse reactions. Due to individual difference, possible adverse reactions are 
infection, fever, etc., or any unanticipated adverse reactions that could be very serious 
even life threatening. If adverse reactions occur, the doctor will take timely corresponding 
treatment methods. During the treatment, the doctor will monitor you for possible adverse 
reactions strictly and may terminate your continued participation in the study if necessary. 
Subject responsibilities 
As a study subject, you have the following responsibilities: If you are willing to 
participate in the clinical trial, you should sign the informed consent after carefully 
reading the informed consent. keep appointments and see doctors regularly; provide true 
information about your medical history and current physical condition; Inform the study 
physician of any discomfort during the study period; Tell your research doctor if you have 
been involved in other studies recently or are currently involved in other studies. 
Costs 

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The study will no additional cost, cell immunotherapy are provided free of charge for you 
by Newish Technology (Beijing)Co.,Ltd., other fees shall be borne by you. 
Compensation 
If you have severe adverse events which caused by cell immunotherapy such as death, 
life-threatening and severe dysfunction confirmed by the study doctor in the study, please 
contact your study doctor, he/she will provide you with diagnosis and treatment timely, at 
the same time, you will get the appropriate economic compensation provided by Newish 
Technology (Beijing)Co..Ltd..  
Confidentiality 
if you decide to participate in this study, your personal data in and during the study are 
confidential. Your tissue specimen will be identified by a study number rather than your 
name. Information that identifies you will not be disclosed to anyone other than members 
of the research group unless your permission is obtained. All research members and 
research bidders are required to keep your identity confidential. Your file will be kept in a 
locked filing cabinet for researchers only. To ensure that the study is conducted in 
accordance with the regulations, if necessary, members of the government management 
department or the ethics review committee may refer to your personal data in the research 
unit as required. When the results of this study are published, no information about you 
will be disclosed. 
 
 

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Informed consent signature 
If you understand the content of this study and agree to participate in it, please sign the 
informed form. Each copy should be retained by the investigator and the subject or the 
client in duplicate. 
Declaration of consent:  
1. I confirm that I have read and understood the informed consent of this study, the 
possible problems in the process of study and solution have been explained to me, and I 
have the chance to make their own questions. 
2 I have been clear that participating in the study belongs to the voluntary, I can choose 
not to participate in this study and my rights and interests will not be affected.  
3. I agree to participate in this study after much consideration. 
Subject’s name:                     Date:                  
(note: if the subject is unable to sign the informed consent, please ask statutory agent to 
sign it.) 
statutory agent’s name:                    Relationship with the subject:                
Date:                
(note: if both the subject and statutory agent are unable to sign the informed consent, 
please ask independent witness to sign it.) 
Independent witness’s name:                  
Date:                
The following completed by the doctor who performs the process of informed consent: 
The researcher stated: I confirm that I have explained and discussed the purpose, 
requirement and potential risk of this study. Subjects volunteered for this study. I ensure 
that a signed informed consent is given to the subject or client for retention. 
Researcher’s name:                             Date: