Abstract 1513: A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy
Authors: Hall MacLean , Innamarato Pat , Mackay Amy , Branthoover Holly , Alfaro Alex , Rich Carolyn , Richards Allison , Hensel Jonathan , Beatty Matthew , Mullinax John , Sarnaik Amod A. , Pilon-Thomas Shari 
Published: July 2021

Abstract
Immunotherapy for cancer has long been focused on the generation of CD8+ cytotoxic T lymphocyte responses, independent of their dynamic CD4+ T cell counterpart. One promising approach, adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), has yielded response rates ranging from 28-55%. Although lasting and complete responses have been achieved, there is substantial opportunity for improvement. Investigation into the role of CD4+ TIL in this setting remains critically underexplored. CD4+ T cells recognize tumor antigen presented on MHC Class II either directly on tumor cells or indirectly through antigen presenting cells (APCs) and are able to elicit potent anti-tumor responses under the appropriate conditions. Here, we present a case study of a metastatic melanoma patient who received adoptive transfer of a predominantly (88%) CD4+ TIL product. This patient demonstrated a complete response (CR) to therapy despite a lack of detection of IFNg in the infusion product in vitro when these TIL were co-cultured with autologous tumor prior to ACT. Tumor recognition was also absent when CD8+ TIL were isolated and stimulated directly with HLA-matched tumor lines, indicating a lack of recognition of shared melanoma antigens presented on MHC Class I. Longitudinal analysis of the peripheral blood of this patient confirmed that the infused CD4+ TIL persisted after therapy for at least six weeks. Whole exome sequencing (WES) performed on the TIL surgical specimen discovered 88 non-synonymous single nucleotide variants (SNVs) as candidate neoantigens. Predicted binding of the resulting mutant peptides to autologous HLA molecules generated a predominantly MHC Class II restricted profile, with 81.8% of variants capable of MHC Class II presentation and greater than half exclusive to MHC Class II only. CD4+ TIL were screened for tumor antigen recognition by upregulation of OX40 and 41BB after stimulation with autologous APCs loaded with mutant peptides. Nearly half (49.2%) of CD4+ TIL responded to tumor-derived peptides. These CD4+ TIL were then sorted into tumor-reactive and non-reactive subsets for further clonal analysis of phenotype and transcriptional profile (scRNASeq) of these T cells in order to characterize the nature of the CD4+ TIL response to tumor antigen. Overall, thorough interrogation of this patient's case study demonstrated evidence of CD4+ TIL involvement in a complete clinical response after ACT. Ongoing studies will define the precise role of tumor-reactive CD4+ T cells in the anti-tumor immune response and provide the framework for future investigation into their function and therapeutic efficacy.
Citation Format: MacLean Hall, Pat Innamarato, Amy Mackay, Holly Branthoover, Alex Alfaro, Carolyn Rich, Allison Richards, Jonathan Hensel, Matthew Beatty, John Mullinax, Amod A. Sarnaik, Shari Pilon-Thomas. A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1513.