Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma

Abstract

BackgroundNeoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation.MethodsThree neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient’s immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient’s TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing.ResultsSelected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation.ConclusionsWe performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.

Adoptive Cellular Therapy for Multiple Myeloma Using CAR- and TCR-Transgenic T Cells: Response and Resistance

“…Meanwhile, very high binding strength leads to deteriorated signaling, such as poor mitogen-activated protein kinase (MAPK) phosphorylation [151]. Isolating neoantigen-reactive MHC-class I-restricted TCR from CD8+ T cells from a malignant melanoma patient, we also detected a higher overall frequency of TCR of lower functional avidity in different patient tissues over the course of several years [152,153]. All these findings suggest a "goldilocks" window of optimal T cell activation for in vivo application [154,155].…”

“…No further precise criteria for the "best" TCR for ACT are established to date-if such a best receptor even exists, rather than different qualities necessary at different disease stages, antigen densities or under different TME conditions. The heterogeneity in functional avidity of different TCRs in the anti-tumor T cell pool from melanoma patients as mentioned above suggests, that a deeper understanding of favorable TCR qualities is necessary across different entities [150,152]. Understanding, which TCR clonotypes have the greatest impact on immediate tumor killing, but also long-term tumor control and memory formation, will be essential to choose the optimal combination of TCR for ACT improving patient survival.…”

“…The extremely patient-specific nature of most epitopes recognized on top of the TCR's HLArestriction claim for single-patient protocols promising high specificity and minimized toxicity. Despite the preclinical success and progressive identification of various neoantigenspecific TCR with anti-tumor effector functions [150,152,153,207], no results from clinical trials are available, so far. Currently, one single-arm first-in-human phase Ia/Ib trial is open and recruiting patients for investigating the efficacy of a single dose of neoantigen-specific TCRs with and without additional anti-PD1 treatment in locally advanced or metastasized solid tumors (NCT03970382).…”

Promise and challenges of clinical non-invasive T-cell tracking in the era of cancer immunotherapy

“…Disadvantage of the direct labelling approach is the need of highly trained personal with experience in good manufacturing practice (GMP), as well as cell handling at the treatment side. In addition, with respect to track endogenously stimulated T-cells, tumor-specific T-cells circulating in the peripheral blood may have low frequencies and migration of blood-derived labelled cells into the tumor is so far ill-defined [ 82 ].…”

Neoantigen-Reactive T Cells: The Driving Force behind Successful Melanoma Immunotherapy

“…The authors further hypothesized that neoantigen-specific TILs from non-responders expressed low avidity or affinity TCRs, leading to unfavorable transcriptional programs. However, the expression levels of tumor antigens and tumor microenvironments may also strongly influence the T cell differentiation, leading to different observations [ 154 , 160 ]. As a result, the further investigation of this hypothesis is required.…”