FCF Funded Projects

Generation of T cells with specificity to FLC’s chimeric fusion

Status: Completed

Timeframe: 2016 – 2017

Goals: Determine if T cells can be generated that react to FLC’s characteristic fusion protein

Principal Investigator: Ephraim Fuchs, MD

Study overview: In FLC, the characteristic DNAJB1-PRKACA fusion creates a novel protein with a unique amino acid sequence occurring at the junction of these two proteins.  This unique amino acid sequence could function as a “neoantigen” that can be recognized as foreign by the immune system of the patient or even the immune system of a healthy donor such as a sibling or parent.  The goal of this project was to identify the novel amino acid sequence that occurs in patients with FLC and to generate a response to this sequence from T cells of the immune system.

If T cells that react to the chimeric fusion protein of FLC could be generated and expanded in tissue culture, such T cells may be capable of killing cells that express this protein.  One possibility is to raise T cells against the fusion protein from a healthy donor and infuse these T cells after partial liver transplantation and bone marrow transplantation from this donor.  Such an infusion would be expected to reduce the risk of tumor relapse.

Key elements of the effort included:

  1. Obtaining tumor tissue from a biopsy and determining the sequence of chimeric fusion protein.
  2. Create peptides that spanned the junction between the DNAJB1 and PRKACA proteins.
  3. Collecting blood from the patient and tissue-matched relatives and culturing those cells in presence of the peptide, interleukin-2 (IL-2) and IL-15
  4. Testing the T cells for specificity to the peptide.

Results: A biopsy specimen from an FLC patient was sent for whole genome sequencing and targeted mutation analysis, which identified the fusion gene and predicted amino acid sequence.  The team synthesized 14 overlapping peptides, each containing the fusion amino acids and overlapping 13 amino acids from its neighboring peptide.  These fusion peptides were used to stimulate peripheral blood cells of the recipient and tumor-infiltrating lymphocytes (TILs) of the recipient.  In parallel, relatives of the patient donated blood and their lymphocytes were stimulated with overlapping peptides encompassing the fusion region of the patients DNAJB1-PRKACA fusion protein. 

The study identified that it was possible to grow T cells specific for the fusion protein in both cases. Both the patient and the donors had T cells that recognized and proliferated in response to the unique protein presented by the patient’s FLC tumor. 

Implications: This preliminary data supports the possibility of generating and expanding T cells that recognize proteins that are uniquely expressed by FLC. This ability to grow T cells that recognize the DNAJB1-PRKACA from patients and healthy, related donors, PD-L1 expression on FLC cells, and the presence of tumor-infiltrating lymphocytes in FLC, indicate potential promise for new immunotherapy approaches to treating FLC.