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Page 4 of 7 Hafezi et al. Hepatoma Res 2020;6:23 I http://dx.doi.org/10.20517/2394-5079.2020.02

ABILITY OF HBV-SPECIFIC TCR REDIRECTED T CELLS TO RECOGNIZE HCC CELLS WITH
SHORT HBV-DNA INTEGRATION
As described above, the feasibility of T-cell immunotherapy was first demonstrated in the scenario wherein
HBsAg was expressed and secreted only by the HCC metastases and not by the transplanted liver. However,
unlike that patient, HBV-DNA integrations in HCC cells do not always involve the complete open reading
frame of HBV antigens. Although HBV-DNA integration could be seen in 60%-80% of HCC cases, most
HCC cells appear HBsAg negative when tested with antibodies targeting the whole HBsAg [22,23] . This is easily
explained by the detection of HBV-DNA integration that is often incomplete in normal and transformed
hepatocytes and hence generate HBV-human chimeric protein [24-26] and not whole HBV proteins.

Antibody-based techniques, which depend on the recognition of conformational epitopes, are unable
to detect these chimeric proteins and therefore these HBV-HCC tumours are often negative for HBV
antigens [22,27] . This explains why utilizing HBV-related antigens as a tumor-specific antigen was highly
controversial and thought to be applicable only for a minority of HCC patients presenting whole HBV
antigens in their HCC relapses. However, unlike antibodies, TCRs detect short linear sequences of
HBV antigens (9-10 amino acid long) that can be derived from HBV-human chimeras present in HBV
serologically negative HCC cells. We have recently tested this possibility in vitro utilizing HCC cells negative
[12]
for HBV antigens when assayed using antibody based techniques . We utilized HBV-specific CD8 T cells
and antibody specific for HLA-class I/HBV epitopes and were able to demonstrate that the production and
presentation of HBV-specific CD8 T cell epitopes can take place in naturally HBV serologically negative
[12]
HCC cells with short HBV-DNA integration . This in vitro demonstration showed the possibility of
utilizing short sequences of HBV-DNA integration present in the majority of HBV-related HCC as a robust
tumor-associated antigen which can then be applied for HBV-specific T cell engineering. Furthermore,
despite the high genetic diversity between HCC cells in the same patient [28-30] , single-cell genome sequencing
[31]
of HBV-HCC cells showed remarkable homology of HBV integration across multiple single tumor cells .
This suggests that TCR-redirected T cells specific for single epitopes should be able to target the majority of
HCC cells present in a patient.

To show feasibility of this strategy, HBV integration profiles of HCC metastases from two LT patients with
[12]
undetectable HBsAg in the serum were analyzed . HBV-specific TCR-redirected T cells targeting the
epitope encoded by the detected HBV integrations were engineered. Following adoptive T cell therapy,
significant volume reduction of several pulmonary metastases has been seen in one patient.

Importantly, in these patients, TCR-T cells were engineered through mRNA electroporation and not virally
transduced. This approach offers inherent safety features due to the labile nature of mRNA [Figure 1].
Unlike viral DNA, mRNA transfection has no risk of causing random integration in the human genome.
More importantly, controlled pharmacokinetics of the therapy allows for dose escalation by multiple repeat
injections which can be personalized for each patient. Multiple infusion of mRNA TCR-T cells (~up to
600 million TCR-T cells) showed no significant increase in serum cytokine levels as well as no evidence
[12]
of graft inflammation in both patients after receiving this therapy . Although this therapy was not able
to completely eliminate HCC relapses in both patients, one had a dramatic reduction in size of multiple
metastatic lesions in the lung without the detection of new lesions and he was clinically well for almost 2 years
[12]
during treatment . Unfortunately, abdominal and retroperitoneal HCC relapses that were already present
in the patient at the time of TCR therapy did not show any response, progressively expanded and ultimately
led to the patient’s death (Personal communication). Even though we were unable to investigate the real
cause of the unresponsiveness of HCC relapses in different anatomical locations, such data suggests that
a tumor’s anatomical location together with its inflammatory status, could have a negative impact on
TCR-T therapy outcome.