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Hafezi et al. Hepatoma Res 2018;4:16 I http://dx.doi.org/10.20517/2394-5079.2018.55 Page 5 of 8

hepatocytes have been removed, reducing the risk of overt destruction of functional hepatocytes. This risk
is further lowered by confirming the absence of HBcAg, HBsAg and HBV DNA from a biopsy sample of
his transplanted liver. Second, immunohistochemistry analysis of his metastatic tumour nodules shows the
expression of HBsAg. This not only suggest that the tumour cells can be recognized by HBV-specific TCR-T
cells, it also means that the serum levels of HBsAg could be used as a surrogate to monitor the efficacy of
4
therapy as HBsAg is only produced by the tumour cells. With a single infusion of small numbers (10 HBV-
specific TCR-T cells/kg) of retroviral transduced TCR-T cells, the cells expanded efficiently in vivo (~2% of
CD8 T cells), and a reduction of over 90% of the serum HBsAg levels was achieved within 30 days without
exacerbation of liver inflammation or any detectable on/off-target toxicities. This was not observed over the
duration of one year when multiple radiotherapy and surgical resections of the tumours were performed.
Unfortunately, the patient was treated at a very late stage and he succumbed to his disease after 8 weeks
of monitoring. Nonetheless, the promising results obtained from this proof-of-concept therapy warranted
further development of this treatment approach.

PRACTICAL CONSIDERATIONS FOR SAFE AND EFFECTIVE T CELL IMMUNOTHERAPY OF
HBV-HCC
Despite the encouraging data obtained from the proof-of-concept therapy described above, additional
considerations will have to be addressed in order to develop a safe and effective TCR-T cell immunotherapy
for HBV-HCC. The first consideration is the issue of safety associated with the use of viral vectors for the
delivery of the TCR gene construct. The oncogenic effect mediated by the insertion of the TCR gene into
the host genome is a potential concern. More importantly, viral transduction generates T cells that stably
expresses the HBV-specific TCR, allowing them to expand in vivo. This in vivo persistence may be beneficial
for tumour eradication, but it would also pose a safety concern as the quantity and function of the modified
[32]
T cells could not be easily controlled if a treatment related adverse event were to occur . An alternative is to
[41]
introduce the TCR gene via the electroporation of in vitro transcribed functional mRNA [Figure 1] . This
approach will not result in insertional mutagenesis and the expression of the introduced TCR is transient,
while maintaining the anti-tumour effects. Not only will you have better control of the TCR-T cell function,
the transient expression also allows clinical trials to be designed with an intra-patient dose-escalation
protocol and thereby improving the safety. At the moment, HBV-specific TCR-T cells modified through
mRNA electroporation have been extensively characterized in vitro and in in vivo pre-clinical models and
[42]
is currently utilized in clinical trials for the treatment of HBV-HCC in liver transplanted patients . In
addition, the transient function of mRNA electroporated T cells is ideal for the treatment of the majority of
HBV-HCC patients who have not undergone liver transplantation, where the risk of on-target off-tumour
lysis of functional but HBV infected hepatocytes is high.

Patient selection is also a critical issue that needs to be addressed. Barring inclusion and exclusion criteria
associated with clinical parameters, at the moment, patient eligibility is dictated solely by the HLA haplotype
[29]
of the patient . In which case, the patient is suitable for therapy if he/she expresses the appropriate HLA
molecule capable of presenting the T-cell epitope recognized by the TCR-T cells. This simplistic criteria only
[29]
takes into consideration the HLA component of the complex recognized by the TCRs For the therapy to
be effective, one has to be able to account for the presence or absence of the T cell epitope on the HCC cells.
Ideally, this can be achieved using TCR-like antibodies specific for every HLA/HBV-epitope complex [43,44]
[45]
but the diversity of complexes makes this approach unfeasible . Peptide elution and mass spectrometry
[46]
strategies might seem possible, but such techniques is highly specialized and complex, and at the moment
restricted primarily to academic research and not clinical application. As a compromise, the detection of
HBV proteins, DNA or mRNA would suffice with the assumption that antigen processing and epitope
presentation occurs as expected. This is simpler in the situation where the complete open reading frame
of a HBV antigen is integrated in the HCC cells. Detection of HBV antigens by serological means through
immunohistochemistry analysis of tumour tissues would be sufficient. However, a recent study demonstrated