Developing Novel Cellular Therapies for Cutaneous T Cell Lymphoma (CTCL): Combining Immunopeptidomics and Endogenous T Cell (ETC) Therapy Platforms to Identify and Target Shared CTCL-Associated Antigens
Authors: Bermack Casey , Pan Ke , Chiu Yulun , Malpica Luis , Iyer Swaminathan P , Flowers Christopher R. , Yee Cassian 
Published: December 2024

Cutaneous T cell lymphomas (CTCL) are rare, largely incurable lymphomas that begin in the skin and may progress to involve the lymph nodes, peripheral blood, and viscera. In late/advanced stage disease, prognosis is poor with median survival of 2.5 years. To date, the only curative treatment option is allogeneic bone marrow transplantation which offers limited 5-year survival rates of 38%. Although several studies support the role of cytotoxic T-lymphocytes (CTLs) in CTCL disease control, the tumor-associated peptide-major histocompatibility complex (pMHC) antigens recognized by tumor-reactive CTLs are unknown.

By applying the Yee lab's well-established pipelines for immunopeptidome prediction and validation, we have identified novel antigen targets for cellular therapy in CTCL. We analyzed RNA-sequencing data from 4 cell lines and 22 tumor samples to identify CTCL-associated genes which are significantly upregulated in CTCL cells relative to healthy tissues. Gametocyte Specific Factor 1 (GTSF1) emerged as a top candidate, as its ubiquitous over-expression across CTCL cells and highly selective expression in tumors and germinal tissues support the wide-reaching potential of this therapeutic target. Prior studies have reported an association between the increased expression of this cancer testis antigen with advanced stage disease and poor prognosis in CTCL patients. Furthermore, GTSF1 expression is also upregulated in other lymphoma subtypes (including diffuse large B cell lymphoma), leukemias, and myelomas. Utilizing our mass-spectrometry-verified tumor-associated antigen immunopeptidome collected from 800+ cell lines and tumor tissue samples covering 20+ tumor types, we identified a pMHC antigen from GTSF1 to serve as a novel cellular therapy target.

Endogenous T-cell (ETC) therapy involves isolating rare populations of endogenous tumor-reactive T-cells from peripheral blood and expanding these cell populations ex vivo to promote functional and regenerative phenotypes for durable responses. Using the Yee lab's ETC therapy platform, we have isolated and validated GTSF1-targeting CTLs from the leukapheresis products of three healthy donors. Donor-specific dendritic cells were generated and pulsed with the candidate GTSF1-peptide prior to co-incubation with PBMC. These co-cultures received a previously optimized dosing schedule of cytokines which enables enrichment of antigen-specific T cells present at initial frequencies &lt; 1:1,000,000. Antigen-specific T cells were subsequently sorted from co-cultures, expanded, and evaluated for cytotoxic activity against a panel of lymphoid malignancy cell lines with confirmed protein expression of GTSF1 and Class I MHC. We have confirmed the activity of GTSF1-targeting CTLs in flow cytometry-based co-culture cytotoxicity assays against at least 3 such cell lines spanning both T- and B-cell malignancies. In contrast, GTSF1-targeting CTLs did not demonstrate cytotoxic activity against a negative control lymphoma cell line with the corresponding HLA-encoded MHC but lacking GTSF1 expression.

We demonstrated a pathway for developing novel cellular therapies for a rare lymphoma subtype through the identification and targeting of a tumor-associated antigen which is over-expressed across multiple hematologic malignancies. The successful isolation of GTSF1 antigen-specific CTLs from multiple donors provides proof-of-concept for the feasibility of GTSF1-targeted ETC therapy for patients with the corresponding HLA restriction and preserved class I MHC presentation. We are also characterizing the T cell receptor (TCR) sequences and specificities of these GTSF1-targeting CTL products towards the development of adoptive TCR T cell therapy. Finally, we are pursuing additional CTCL-associated antigens (restricted by other HLA subtypes) that were identified by our immunopeptidome analysis to increase coverage of patient HLA diversity.