Perspectives on FLC immunotherapy

In March 2024, a six-institution team led by Paul Thomas (St. Jude Children’s Research Hospital) and Scott Strome (University of Tennessee) published an article in Cell Reports Medicine about the isolation of T-cell receptors specific for the fusion protein driver of fibrolamellar carcinoma (FLC). (Click here to read the publication). Below is a discussion of the FCF-funded study’s results and the implications for future FLC immunotherapy treatments as summarized by Mark Furth, Chief Science Officer of FCF.


T-cell receptor (purple). The antigen (light green) is a peptide from a tumor cell.

Therapies that enhance the body’s immune response against tumor cells are dramatically improving outcomes for patients with many cancers. Immunotherapy harnesses the ability of certain white blood cells to hunt down and destroy cancer cells. For such search and destroy missions, “killer T cells” (a type of lymphocyte) recognize specific chemical features, called antigens, that mark abnormal cells. To do so, each killer T cell produces on its surface a receptor protein (called a T-cell receptor or TCR) that binds specifically to a unique antigen, like a key matches a lock. These TCRs recognize peptides, which are fragments of proteins from 8 to 24 amino acids long. All cells throughout the body are constantly chopping up their internal proteins and displaying the resulting peptides on their surfaces. Individual cells are constantly “interrogated” by passing T cells looking for surface peptides that are abnormal. T cells with a matching TCR have an extraordinary ability to find a rare abnormal peptide in a haystack of normal ones, and to selectively kill cells that include that abnormal peptide.

To comprehensively find cancerous cells and cells infected by viruses, the pool of T cells in a human body includes enough distinct TCRs to recognize billions of possible antigens.

The ability of tumor cells to provoke a strong immune response is often related to the number of genetic mutations. As tumor cells proliferate, an increased frequency of mistakes in copying their DNA causes the accumulation of a variety of proteins with random substitutions, deletions, or rearrangements in their amino acid sequences. These mutant proteins generate peptides that, when displayed on the cancer cell surface, may be recognized as new antigens (neoantigens) and trigger attack by a pack of killer T cells. However, to evade attack, many potentially susceptible cancers co-opt a system of “immune checkpoints” that signal T cells to back off and not kill their targets. To overcome this problem, a class of drug called immune checkpoint inhibitors (ICIs) was developed and approved for medical use in 2016. ICIs are especially effective against tumors that accumulate many random mutations, presumably because the presence of more neoantigens can activate a larger set of killer T cells that can attack the cancer once any protective immune checkpoints are disabled.

How does this apply to fibrolamellar carcinoma (FLC)?

On the scale of mutation frequency, FLC falls at the low end of cancers, and so generates relatively few neoantigens. Perhaps for this reason, ICIs generally have not proven highly beneficial against FLC. However, a few cases of exceptionally strong clinical responses to ICIs, when given in combination with other types of anticancer drugs, provide encouragement that highly effective immunotherapy for FLC may be achievable.

What did the St. Jude study team look for?

Paul Thomas, PhD of the St. Jude Children’s Research Hospital

The six-institution study team led by Paul Thomas (St. Jude Children’s Research Hospital) and Scott Strome (University of Tennessee) explored a novel strategy towards this goal. In the published paper, they report “proof-of-concept” for a potentially universal immunotherapy for FLC, directed against a single neoantigen, the fusion protein that drives this cancer. (Allison Kirk, et al., Cell Reports Medicine, March 19, 2024)

The investigators leveraged a signature feature of FLC. As discovered by Sandy Simon (Rockefeller University) and colleagues, and reported in 2014, nearly 100% of FLC tumors arise after a loss of DNA on human chromosome 19 occurs in a liver cell, permanently joining two nearby genes to form a fused gene designated DNAJB1-PRKACA. The fusion changes and “activates” an important cellular enzyme, protein kinase A (PKA), making it cancer-causing. The gene fusion turns PKA it into a chimeric protein (abbreviated as “DP”) in which a small segment of the “PKAc” amino acid chain (the enzyme’s “catalytic subunit”) is replaced by a segment from the unrelated DNAJB1 protein. Normal PKA plays important roles in regulating cell metabolism and proliferation. However, production of the abnormal DP version of PKA, usually at elevated levels, drives the development of FLC cancers. In addition, DP remains essential for the survival and proliferation of the cancer cells as the disease progresses.

Remarkably, the joining of “D” and “P” portions of the DP chimera always occurs at precisely the same place on the two original amino acid chains. Therefore, when fragments of the cancer driving protein are displayed on the surface of FLC cells, the set of peptides spanning this junction should be unique to the cancer cells. The study team, therefore, asked if T cells that recognize DP junctional peptides can be found in FLC patients. They reasoned that such anti-DP immune cells might be enriched among “tumor-infiltrating lymphocytes” (TILS), immune cells that have migrated into tumor tissue. Furthermore, if a patient’s immune system was mounting an effective response against DP, they expected to see large numbers of T cells having TCRs specific to a junction-spanning peptide.

What did they find?

Examination of tumor samples from two dozen FLC patients did reveal the presence of TILs, although only in small numbers. About 20% of these immune cells appeared to be killer T cells. However, disappointingly, the investigators did not observe expanded clusters of killer cells bearing a selected set of TCRs, as they would have expected to see if patients were mounting very strong immune responses against their FLC cells.

Could any DP-specific killer T cells be found?

The team searched intensively for such cells in the TIL populations obtained from four additional FLC cases. From one patient, they struck gold! When they challenged this patient’s T cells with seven different peptides from the fusion junction that were predicted to be DP-specific neoantigens, a few of the patient’s T cells bound one of these peptides tightly. Furthermore, this peptide evoked a strong response from the T cells – the release of immune factors (e.g., interferon) known to be crucial in the attack of killer T cells against cancer cells. The team next isolated from these T cells the gene that encoded its unique TCR. When transferred into “naïve” T cells, this TCR gene conferred the ability to bind and respond to the correct DP junction peptide. Most importantly, the engineered T cells now recognized and killed target cells that expressed the intact DP driver protein.

Analysis of the peptide bound by the cloned TCR highlights the amazing ability of the immune system to recognize and respond to a cancer neoantigen. This peptide is 10 amino acids long. Its sequence comprises the 9 amino acids derived from the “D” protein to the left of the DP junction, plus a single adjacent amino acid from the “P” side (valine). When that “P”-derived residue was replaced with the corresponding amino acid from the normal “D” protein (glycine) the TCR’s ability to bind the peptide and to enable killing of target cells were dramatically diminished.

How could these findings enable “precision immunotherapy” for FLC patients?

These findings point to two approaches towards immunotherapy for this cancer that are under active investigation:

  • Vaccination
    While it appears that few, if any, FLC patients spontaneously mount a strong immune response against the cancer’s DP driver protein, it may be possible to enhance anticancer immunity with a vaccine. Two independent research groups, one at the University of Tuebingen in Germany, another at Johns Hopkins University (Baltimore, MD), have tested this idea by vaccinating patients with a peptide corresponding to the fusion junction region of the FLC driver protein DP. Both groups used a 24mer peptide, with 12 amino acids from the D and P sides of the junction.

    In 2022 the Tuebingen team, led by Juliane Walz, reported encouraging results in an initial test of their anti-DP vaccine in a single FLC patient. The patient previously had suffered recurrent relapses at short time intervals after the cancer responded to chemotherapy. When the patient received the peptide vaccine, in addition to the same chemotherapy, it evoked a strong immune response. In contrast to the earlier treatments with chemotherapy alone, this time the cancer did not relapse quickly. The patient has remained apparently disease-free for more than three years. A clinical trial of that peptide vaccine (Fusion-VAC-XS15) in combination with an ICI is now underway in FLC patients ( ID NCT05937295).

    In 2020 the Johns Hopkins group, led by Mark Yarchoan and Marina Baretti, initiated a pilot study in FLC patients of a similar peptide vaccine in combination with two ICIs ( ID NCT04248569). At a recent cancer immunotherapy conference (November 2023) they reported results on the first 12 evaluable trial subjects. A majority of these patients (75%) developed strong immune responses to the immunizing peptide, which in most cases was associated with clinical benefit, i.e., disease stabilization or regression. Three of these patients experienced dramatic, sustained reductions in tumor burden, and two of them, who had surgery to remove small areas of possible residual disease, now appear cancer-free.
Marina Baretti, MD of Johns Hopkins University
  • Cell-based immunotherapy
    The Johns Hopkins and St. Jude teams have been collaborating closely in studies of immune responses against the DP fusion protein. A current shared goal is to isolate TCR genes from patients immunized with the peptide vaccine for the fusion junction.

    These anti-DP TCR genes could eventually be used for precision immunotherapy by “adoptive cell therapy.” In that potential future therapy, T cells would be taken from a patient diagnosed with FLC and genetically modified to produce a known, strong anti-DP TCR that had already been identified and cloned from an immune donor. The engineered cells would be expanded in laboratory culture and large numbers would be infused into the patient to provide an overwhelming force of T cells that are armed to hunt down and destroy FLC cancer cells. Moreover, “memory” immune cells can remain in the body throughout an individual’s lifespan. Their continuing presence could provide a perpetual defense against any surviving cancer cells that may lurk silently, only to reawaken, proliferate, and grow into recurrent tumors.

    While potentially very promising, immunotherapy treatments based on TCRs have significant complexity because of how T cells recognize abnormal peptides. The peptides processed in a cell are bound into grooves of cell surface proteins of the HLA (human leukocyte antigen) system for “inspection” by surveilling T cells. Immunologists have learned that a TCR that has been cloned and transferred into another patient’s T cells can only recognize a peptide antigen if that peptide is displayed by a HLA protein that exactly matches the HLA protein used by the original patient. There are many variants of HLA proteins distributed across the human population. Nevertheless, it should be feasible to assemble large enough collection of DP-specific TCRs matched to different HLA variants, especially those that are relatively common in the population, to enable “off the shelf” engineering of T cells for a large majority of FLC patients.

    Another important area of research for this type of therapy is to determine whether killer T cells, identified by a marker called CD8, are indeed the most critical actors in immunotherapy of FLC, as has been generally assumed. However, both groups studying peptide vaccines for DP have found evidence that a different class of T cells called “helpers,” marked by CD4, may play a critical role in the immune response to the fusion protein. Efforts to isolate and characterize anti-DP TCRs from both CD8+ and CD4+ T cells are underway, and may provide further insights and tools to drive the development of effective immunotherapy of FLC.