Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor-Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display

Abstract

Tumor-infiltrating lymphocytes (TIL) are considered enriched for T cells recognizing shared tumor antigens or mutation-derived neoepitopes. We performed exome sequencing and HLA-A * 02:01 epitope prediction from tumor cell lines from two HLA-A2-positive melanoma patients whose TIL displayed strong tumor reactivity. The potential neoepitopes were screened for recognition using autologous TIL by immunological assays and presentation on tumor major histocompatibility complex class I (MHC-I) molecules by Poisson detection mass spectrometry (MS). TIL from the patients recognized 5/181 and 3/49 of the predicted neoepitopes, respectively. MS screening detected 3/181 neoepitopes on tumor MHC-I from the first patient but only one was also among those recognized by TIL. Consequently, TIL enriched for neoepitope specificity failed to recognize tumor cells, despite being activated by peptides. For the second patient, only after IFN-γ treatment of the tumor cells was one of 49 predicted neoepitopes detected by MS, and this coincided with recognition by TIL sorted for the same specificity. Importantly, specific T cells could be expanded from patient and donor peripheral blood mononuclear cells (PBMC) for all neoepitopes recognized by TIL and/or detected on tumor MHC-I. In summary, stimulating the appropriate inflammatory environment within tumors may promote neoepitope MHC presentation while expanding T cells in blood may circumvent lack of specific TIL. The discordance in detection between physical and functional methods revealed here can be rationalized and used to improve neoantigen-targeted T cell immunotherapy.

Sensitive and quantitative detection of MHC-I displayed neoepitopes using a semi-automated workflow and TOMAHAQ mass spectrometry

“…At least nine of these novel neoepitopes have been tested and found to be immunogenic by our group, using peptide vaccination (14) and/or RNA lipoplex vaccination (data not shown). This represents a dramatic increase in our ability to detect neoepitopes, as within our untargeted MHC-I assay only two endogenous neoepitopes were observed (not counting those from the over-expressed neoantigen), a result that is typical for detection of neoepitopes in untargeted analyses performed in our lab and others (7, 33, 40). Our synthetic mixture experiments also allow us to assign approximate limits of detection for each neoepitope, providing information on the relative detectability of each, and therefore whether the absence of a given neoepitope may be due to lack of abundance or difficulty of detection.…”

“…CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.16.423097 doi: bioRxiv preprint 29 neoepitopes in untargeted analyses performed in our lab and others (7,33,40). Our synthetic mixture experiments also allow us to assign approximate limits of detection for each neoepitope, providing information on the relative detectability of each, and therefore whether the absence of a given neoepitope may be due to lack of abundance or difficulty of detection.…”

Chemical complementarity between immune receptors and cancer mutants, independent of antigen presentation protein binding, is associated with increased survival rates

“…Having said that, there would also remain the question of whether TCR-neoantigen complementarity and good HLA binding together represent the most clear survival distinctions. This is a particularly apt question given recent work calling into question the relevance of very high neoantigen-HLA binding alone as a successful predictor of T-cell activation in the cancer setting [31] .…”

Metabolic optimization of adoptive T cell transfer cancer immunotherapy: A historical overview

“…Specific anti-near-self T cell clones should be expandable extracorporeally in quantities that, when reintroduced, would suffice to target autologous cancer cells-a process that has become known as adoptive T cell transfer (ACT). [8][9][10][11] Indeed, it is the cytotoxic CD8 + T cell population, rather than the predominantly regulatory CD4 + T cell population, that is most responsive to near-self antigens. 12 The technology has been improved by the employment of checkpoint inhibitors for 'immune checkpoint blockade' 13 and depletion of patients' existing in vivo T cell populations (lympho-depletive regimes) 14 prior to infusion of their own T cells, 15 which may have been engineered to match tumour antigens.…”

“…16,17 While tumour infiltrating lymphocytes have long been studied in this context, 8 peripheral blood mononuclear cells (PBMC) are proving a better T cell source since they are at a developmental stage less prone to metabolic 'exhaustion', an antigen dose-dependent tolerogenic process that is likely in tumour microenvironments. 10,11,18 As with peripheral immune system T cells generally, cultured PBMC can be activated to transform and divide by lectins. This polyclonal activation process shares many features with the monoclonal activation of an individual T cell by the specific antigen (pMHC) that can be bound by its T cell receptor (TCR).…”