In our recent commentary [1], we were delighted to discuss your work [2] that represents a further step in the development of T cell therapy against hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). The study elegantly confirmed “the potential of HBV-T cell receptor (TCR) T cell immunotherapy for HBV-HCC”, an important point considering the scant number of HBV-HCC patients treated with HBV-TCR T cell therapy globally. To help the readers appreciate the significance of your new data, we provided a historical view of the different T-cell immunotherapy strategies that have been developed.

This is why we discussed not only your data obtained with lentiviral transduced HBV-TCR T cells (stably expressing HBV-TCR), but also the pros and cons of other approaches (like mRNA electroporated HBV-TCR T cells, transiently expressing HBV-TCR). We thought we provided a balanced view of the experimental and clinical results obtained thus far.

We were and remain convinced that the results are insufficient to decide which is the best approach (overall, no more than 20 patients have been treated and properly studied). We maintain that good quality data, like the one that you provided, is required to understand whether clinical efficacy against HBV-HCC will be better achieved using a treatment strategy obtained with T cells stably expressing HBV-TCR or with a “hit and run” strategy performed by T cells with limited functional lifespan (i.e., transient HBVTCR expression). Succinctly, we think that both strategies have shown good potential but clearly need to be improved to have robust clinical significance.

We were therefore surprised to receive this letter [3]. By repeating arguments that we have discussed in our commentary (e.g., mRNA TCRs have only a limited life span) and by suggesting some hypothetical induction of an anti-mRNA TCR immune response, the letter denigrates the mRNATCR approach and concludes that the only “suitable” therapy for HBV-HCC should be based on lentiviral-transduced stably expressing HBV-TCR T cells.

We understand that you are passionate about your approach. Nonetheless, in order to right the unbalanced claims listed in your letter, we are now compelled to provide detailed answers.

Firstly, we are delighted that there are groups that actively work to develop a therapy against HBV-HCC using T cells engineered with lentiviral vectors that achieve stable TCR expression. Indeed, differently to what you wrote (page 3, line 10), we not only pioneered the mRNA-TCR T cell approach [4] but we were also the first group to demonstrate that T cells stably expressing an HBV-TCR could target HBsAg-expressing HCC in vitro [5] and then in vivo [6]. Indeed, by performing the first-in-man treatment of a patient with extrahepatic recurrence of HCC expressing HBsAg using stably viral transduced HBV-TCR T cells [6], we provided the important proof-of-concept of the approach that you are now developing.

As you correctly pointed out, we then choose to pursue the mRNA electroporation approach for HBV-TCR T cells engineering [4,7], since we are of the opinion that there are associated advantages over viral transduction. We preferred to use the mRNA-TCR T cells clearly for safety reasons (no genetic modification, better control of off-target and off-tumour effects) [7], but also because there are caveats and limitations in the narrative of the ability of stably genetically modified TCR-T cells to establish a memory T cell response.

While it is nice to think that the adoptively transferred stably engineered HBV-TCR T cells will evolve into stem-like T cells that will not only remain in the body of the patients, but also establish, as you wrote, “a multipotent T cell population able to reconstitute the entire spectrum of memory and effector subset”, the reality in patients with solid tumor is a far cry from this rosy scenario. In particular, T cells become particularly compromised when they repetitively recognise foreign antigens in the liver. You should be aware of the liver tolerogenic features and how T cells specific for antigens expressed by the hepatocytes (i.e., HBV antigens) do not establish any stem-like memory T cell in chronic HBV infection [8]. In addition, a recent Nature publication coauthored by you, demonstrated additional mechanisms that inhibit the function of CD8 T cells targeting antigens in the liver through the action of liver endothelial cells [9]. It is rather surprising that the implications of this important discovery was not even mentioned in your arguments. Can lentiviral stably genetically modified HBV-TCR T cells selectively escape such liver-related tolerogenic/suppressive features?

Secondly, while you were arguing for T cell persistence being indispensable for HBV-TCR T cell treatment efficacy, the data presented in your manuscript [2] suggest that the stably genetically modified HBV-TCR T cells might persist but they do not seem to maintain their long term functionality. For the entire duration of almost 160 days, vector copy number data shows the persistence of their adoptively transferred HBV-TCR T cells with the development of the “ideal” stem-like phenotype. Even in the presence of these stem-like HBV-TCR T cells, AFP levels started to rise 60 days after the HBV-TCR T cell transfer (while it dropped between -7- and 28-days post infusion). It was even pointed out that in your AAV murine model, when HBV-TCR T cells were analyzed ex vivo 60 days after adoptive transfer, the TCR T cells were no longer activated by their cognate antigen. These results suggest a progressive dysfunction of the stably genetically modified HBV-TCR T cells in vivo, more than an establishment of “multipotent T cells”.

Of note, testing the functionality of adoptively transferred HBV-TCR T cells is no longer an impossible and insurmountable task. We recently developed a rapid and simple assay that measures ex vivo in the patient’s whole blood, the presence and functionality of TCR engineered T cells [10]. This assay is available to be used to resolve the issue of the functionality of the engineered TCR T cells persisting in the circulation of treated patients.

You are certainly correct to point out that the transient nature of mRNA-TCR T cells (T cell lose their exogenous TCR within 3–4 days after infusion) excludes the possibility of establishing a “long term tumor and virus control”. On the other hand, we cannot a priori exclude that T cell therapy might also act indirectly. A potent and transient tumor inflammation induced by mRNA-TCR T cells, might modify the tumor environment and allow induction of T cells of different tumor specificities. We are actively working to demonstrate that such a scenario does not represent only a hypothesis. At the same time, we completely refute the statement, present in point 2 of your letter, stating that mRNA HBV-TCR T cells treatment was not causing any “significant on target activity”. How did you reach this conclusion? Maybe the fact that one patient with advanced HBV-HCC had a treatment response significantly reducing the target lesion diameter (129 mm to 81 mm) with accompanying alanine aminotransferase flares [11], does not constitute “significant on-target activity”? How about the reduction of AFP and shrinkage of multiple lung lesions in a patient with HBV-HCC relapses in the lung? [12] We are eager to understand why direct measurements of tumour burden reduction and the lowering of indirect surrogates of tumour load represent a demonstration of “on target activity” only in the context of lentiviral transduced TCR T cells.

What was also not clear from your narrative is why it is perfectly acceptable to be treated over time with multiple infusions of drugs or antibodies but repetitive infusion of mRNA-TCR T cells is deemed unfeasible. Perhaps the answer to this question is provided in point 3 of your letter, where you raised the hypothesis of a phantomatic induction of anti mRNA-TCR T cell immunity. You suggested that repetitive infusion of engineered TCR T cells in the absence of lymphodepletion induces an autoreactive adaptive immune response capable of neutralizing mRNA-TCR T cells. You even suggested, with a rationale that we struggle to follow, that since mRNA is a potent inducer of immune response (that is true in a formulation used for example in vaccines - nothing to do with the engineering of mRNA electroporated T cells where the mRNA is not infused into the patient [4,7]), repetitive infusions of mRNA-TCR T cells will be particularly potent in the induction of such autoreactive T cells.

We are fascinated by the level of ingenuity and imagination contained in this hypothesis. We would like to reassure you that these events do not seem to occur in patients treated with repetitive doses of mRNA-TCR T cells. We not only never observed any “increased risk of adverse events” in these patients, but by starting to study longitudinally the ex vivo function of adoptive TCR-T cells, we started to gather data about the persistence of T cell functionality observed 3 days after infusion in a patient treated with 7 infusions of mRNA-TCR T cells (Fig. 1).

Clearly, in this patient with primary HBV-HCC who was never pre-conditioned before adoptive transfer, the repetitive infusion of mRNA HBV-TCR T cells did not result in any anti-TCR T or antibody response. since mRNA-TCR T cells retained their functionality. We will certainly have to confirm these data in other patients, but your narrative is not supported, for the moment, by any data.

Finally, the topic of cost is a big issue in T cell therapy field, but to make the simplistic claim that GMP grade viral vector production is cheaper than GMP mRNA production solely due to their “single infusion” approach is laughable. Due to the recent success of mRNA vaccination, methods of mRNA production underwent a fast development with a considerable reduction of price. Empirical evidence supporting the cheaper mRNA production approach can also be found in the gene therapy field where viral vectors were used to made genetic modifications [13]. The suggestion that lentiviral TCR-T cells production requires less time and is at a lower cost can be labelled as propaganda in the absence of any data.

We wish you all the best on your clinical trial and we also hope your work will bring some innovations to our field.