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Abstract
Background Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease largely stemming from human papilloma virus infection, tobacco and alcohol intake, and inherited mutations resulting in a relatively high tumor mutational burden.1 HNSCC is considered amenable to immunotherapy and a subset of patients have a comparatively dense immune infiltrate.2Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) is one promising therapeutic strategy that harnesses a polyclonal population of T cells with enriched specificity and reactivity toward tumor-derived antigens.
Methods DNA and RNA were extracted from HNSCC followed by whole exome and RNA sequencing. Long peptides (25mers) were custom synthesized spanning neoantigens predicted to bind major histocompatibility complex (MHC) proteins. TIL expanded from HNSCC were co-cultured with autologous antigen presenting cells (APCs) loaded with neoantigen peptides. TIL were analyzed for increased surface expression of OX40 and 4-1BB by flow cytometry, and secretion of granzyme B, interferon gamma (IFNg), and tumor necrosis factor alpha (TNFa). Clonotype analysis was completed by T cell receptor (TCR) sequencing. Autologous patient-derived xenograft (PDX)-bearing mice received ACT with TIL and tumor area was measured until endpoint analysis of T cell infiltration by flow cytometry. Immunopeptidomics analysis by mass spectrometry was performed on MHC-bound peptides on APCs.
Results Across eight HNSCC samples, an average of 139 somatic mutations were discovered (range 56–232) and a mean of 101 25mers were predicted to bind to each patient’s respective MHC (range 43–160). Systematic analysis of the first patient sample revealed a candidate neoantigen ‘hit’ resulting in activation of 45% of CD4+ TIL by OX40+/4-1BB+ induction, while CD8+ TIL remained unchanged. Neoantigen-reactive TIL secreted granzyme B, IFNg, and TNFa, in a dose-dependent manner, which was abrogated in the presence of HLA-DQ blockade. TCR sequencing revealed an oligoclonal T cell population. Pilot non-clinical ACT experiments in mice demonstrated TIL engraftment and tumor infiltration. Initial APC immunopeptidomics analysis found nine unique epitopes bound to MHC Class I and 17 peptides presented by MHC Class II following neoantigen pulse. Additional sample analysis is ongoing.
Conclusions Neoantigen-reactive TIL can be selected and expanded from solid tumors.3 Here, we show that expanded HNSCC TIL contain neoantigen-reactive effector T cells. Ongoing studies aim to expand this investigation to better understand the landscape of T cell reactivity and the dynamic antigenic and cellular determinants involved in the anti-tumor immune response. Ultimately, we plan to determine the potential therapeutic benefit of an enriched antigen-reactive TIL-ACT product for HNSCC patients.
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Ethics Approval MCC18510 was approved by Chesapeake IRB approval number Pro00015586 and all participants gave informed consent before taking part.
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