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Bourinbaiar et al. Hepatoma Res 2020;6:2 I http://dx.doi.org/10.20517/2394-5079.2019.25 Page 3 of 8

ablation, percutaneous ethanol injection, or their combinations. At the presentation, all patients were
either under palliative care or pronounced incurable without available treatment options - underlining the
disease gravity. As the overwhelming majority of our HCC patients were in terminal stage of the disease,
they often presented with a variety of symptoms related to decompensated cirrhosis including ascites, edema,
variceal bleeding, portal thrombi, and hepatic encephalopathy. Besides these symptoms, abdominal discomfort
and less frequently pain were common. Other encountered complaints were fatigue or weakness, cachexia,
anorexia, skin itch, bleeding from gums and nose, vomiting, and jaundice. As no other intervention options
except palliative care were available, patients consented to receive a once-per-day pill of Hepko-V5 (n =14) or
identically appearing placebo pill (n =16) with follow-up lab tests at Months 1 and 2. Patients were instructed
to take one pill before bedtime and come back after running out of pills after 30 days. Patients had
baseline measurement for alpha-fetoprotein and standard liver function tests such as alanine transaminase
(ALT), aspartate transaminase (AST), alkaline phosphatase, and total bilirubin. Additional tests included
creatinine and prothrombin time. In such a way, we had a cohort representative of the real-life situation in
Mongolia, where almost every patient is diagnosed only after symptoms of the disease become apparent or
when tumors recur.

V5
V5 is derived from pooled blood of patients with HCC who are often viral hepatitis C, hepatitis B, and
Delta virus carriers by employing proprietary technology developed by us. The process of manufacturing
has been described in detail previously: it involves heat and chemical inactivation with subsequent
[9]
formulation into a tablet capable of withstanding digestive degradation in the stomach . The principle
for mechanism of action of V5 is not much different from established principles with old-fashioned killed
vaccines, e.g., hepatitis B vaccine made from pooled plasma. V5 has been approved in Mongolia as a
biologically active product since 2008. V5 Immunitor is presented as an 850 mg coated pink pill, with 30
pills per one package. The recommended dose is one to two pills per day, and the dose being used in this
study is one pill per day. The preparation is stable at ambient room temperature for at least three years.

Flow cytometry analysis
Peripheral blood mononuclear cells (PBMCs) from the blood of healthy donors were obtained by density
gradient centrifugation. Lymphocytes were isolated from PBMCs using anti-human CD4 or CD8 magnetic
MACS microbeads following the manufacturer’s protocol (Miltenyi Biotec, Somerville, MA 02143, USA).
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Obtained cells were incubated with a crushed pill of V5 diluted one million-fold (10 ) in saline for 48 h and
then subjected to fluorescence-activated cell sorter (FACS) analysis using a FACSCalibur flow cytometer
(BD Biosciences, San Jose, CA). V5-exposed and control cells were stained with antibodies against CD3,
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CD4, CD8, IFN-γ, TNF-α, and IL-2 obtained from BD Biosciences. Briefly, cells (1 × 10 ) were stained either
with anti-CD4 or -CD8 and anti-CD69 or -Ki67 (BD Pharmingen). Labeled cells were fixed with 0.5%
paraformaldehyde and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Statistical analysis
The primary endpoint for this study was the effect of Hepko-V5 on serum AFP levels as a surrogate marker
of tumor burden changes. Secondary objectives were safety, adverse effects, and changes in liver function
parameters, creatine levels, and pro-thrombin time. The difference between pre- and post-treatment
parametric values was assessed by paired Student’s t-test and linear regression analysis. Wilcoxon matched
pairs ranking test and contingency table analysis were employed for categorical data using the statistical
calculator GraphPad, which is freely available online (GraphPad Software, 2365 Northside Dr., San Diego,
CA 92108, USA). The significance level for all tests was set at P ≤ 0.05.

Ethics approval and written consent
All patients were explained benefits and risks of the intervention and provided informed written consent
agreeing to participate. The study was approved by the Institutional Review Board of Immunitor LLC