Karolina Malecek

T-cell receptor affinity and avidity defines antitumor response and autoimmunity in T-cell immunotherapy

T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a unique self-antigen system comprising seven human melanoma gp100(209–217)-specific TCRs spanning physiological affinities (1–100 μM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 µM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.

2D TCR–pMHC–CD8 kinetics determines T-cell responses in a self-antigen-specific TCR system

Two‐dimensional (2D) kinetic analysis directly measures molecular interactions at cell–cell junctions, thereby incorporating inherent cellular effects. By comparison, three‐dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T‐cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T‐cell receptors (TCRs) interacting with a melanoma self‐antigen peptide (gp100209–217) bound to peptide‐major histocompatibility complex in the absence and presence of co‐receptor CD8. We found that while 3D parameters are inadequate to predict T‐cell function, 2D parameters (that do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T‐cell function. Thus, our data support the general notion that 2D parameters of TCR–peptide‐major histocompatibility complex–CD8 interactions determine T‐cell responsiveness and suggest a potential 2D‐based strategy to screen TCRs for tumor immunotherapy.

Engineering improved T cell receptors using an alanine-scan guided T cell display selection system.

T cell receptors (TCRs) on T cells recognize peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells and this interaction determines the T cell immune response. Due to negative selection, naturally occurring TCRs bind self (tumor) peptides with low affinity and have a much higher affinity for foreign antigens. This complicates isolation of naturally occurring, high affinity TCRs that mediate more effective tumor rejection for therapeutic purposes. An attractive approach to resolve this issue is to engineer high affinity TCRs in vitro using phage, yeast or mammalian TCR display systems. A caveat of these systems is that they rely on a large library by random mutagenesis due to the lack of knowledge regarding the specific interactions between the TCR and pMHC. We have focused on the mammalian retroviral display system because it uniquely allows for direct comparison of TCR-pMHC-binding properties with T-cell activation outcomes. Through an alanine-scanning approach, we are able to quickly map the key amino acid residues directly involved in TCR-pMHC interactions thereby significantly reducing the library size. Using this method, we demonstrate that for a self-antigen-specific human TCR (R6C12) the key residues for pMHC binding are located in the CDR3β region. This information was used as a basis for designing an efficacious TCR CDR3α library that allowed for selection of TCRs with higher avidity than the wild-type as evaluated through binding and activation experiments. This is a direct approach to target specific TCR residues in TCR library design to efficiently engineer high avidity TCRs that may potentially be used to enhance adoptive immunotherapy treatments.

Specific Increase in Potency via Structure-Based Design of a TCR

Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive “self” proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in ∼30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag–MHC (peptide–MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.

Retroviral transduction of T-cell receptors in mouse T-cells.

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity). It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6. Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

Impact of aging on host immune response and survival in melanoma: an analysis of 3 patient cohorts.

BackgroundAge has been reported as an independent prognostic factor for melanoma-specific survival (MSS). We tested the hypothesis that age impacts the host anti-tumor immune response, accounting for age-specific survival outcomes in three unique melanoma patient cohorts.MethodsWe queried the U.S. population-based Surveillance, Epidemiology, and End Results Program (SEER), the prospective tertiary care hospital-based Interdisciplinary Melanoma Cooperative Group (IMCG) biorepository, and the Cancer Genome Atlas (TCGA) biospecimen database to test the association of patient age at time of melanoma diagnosis with clinicopathologic features and survival outcomes. Age groups were defined as ≤45 (young), 46–65 (intermediate), and >65 (older). Each age group in the IMCG and TCGA cohorts was stratified by tumor infiltrating lymphocyte (TIL) measurements and tested for association with MSS. Differential expression of 594 immunoregulatory genes was assessed in a subset of primary melanomas in the IMCG and TCGA cohorts using an integrative pathway analysis.ResultsWe analyzed 304, 476 (SEER), 1241 (IMCG), and 292 (TCGA) patients. Increasing age at melanoma diagnosis in both the SEER and IMCG cohorts demonstrated a positive correlation with tumor thickness, ulceration, stage, and mortality, however age in the TCGA cohort did not correlate with mortality. Older age was associated with shorter MSS in all three cohorts. When the young age group in both the IMCG and TCGA cohorts was stratified by TIL status, there were no differences in MSS. However, older IMCG patients with brisk TILs and intermediate aged TCGA patients with high lymphocyte scores (3–6) had improved MSS. Gene expression analysis revealed top pathways (T cell trafficking, communication, and differentiation) and top upstream regulators (CD3, CD28, IFNG, and STAT3) that significantly changed with age in 84 IMCG and 43 TCGA primary melanomas.ConclusionsOlder age at time of melanoma diagnosis is associated with shorter MSS, however age’s association with clinicopathologic features is dependent upon specific characteristics of the study population. TIL as a read-out of the host immune response may have greater prognostic impact in patients older than age 45. Recognition of age-related factors negatively impacting host immune responses may provide new insights into therapeutic strategies for the elderly.

The expression quantitative trait loci in immune pathways and their effect on cutaneous melanoma prognosis

Purpose: The identification of personalized germline markers with biologic relevance for the prediction of cutaneous melanoma prognosis is highly demanded but to date, it has been largely unsuccessful. As melanoma progression is controlled by host immunity, here we present a novel approach interrogating immunoregulatory pathways using the genome-wide maps of expression quantitative trait loci (eQTL) to reveal biologically relevant germline variants modulating cutaneous melanoma outcomes. Experimental Design: Using whole genome eQTL data from a healthy population, we identified 385 variants significantly impacting the expression of 268 immune-relevant genes. The 40 most significant eQTLs were tested in a prospective cohort of 1,221 patients with cutaneous melanoma for their association with overall (OS) and recurrence-free survival using Cox regression models. Results: We identified highly significant associations with better melanoma OS for rs6673928, impacting IL19 expression (HR, 0.56; 95% CI, 0.41–0.77; P = 0.0002) and rs6695772, controlling the expression of BATF3 (HR, 1.64; 95% CI, 1.19–2.24; P = 0.0019). Both associations map in the previously suspected melanoma prognostic locus at 1q32. Furthermore, we show that their combined effect on melanoma OS is substantially enhanced reaching the level of clinical applicability (HR, 1.92; 95% CI, 1.43–2.60; P = 2.38e-5). Conclusions: Our unique approach of interrogating lymphocyte-specific eQTLs reveals novel and biologically relevant immunomodulatory eQTL predictors of cutaneous melanoma prognosis that are independent of current histopathologic markers. The significantly enhanced combined effect of identified eQTLs suggests the personalized utilization of both SNPs in a clinical setting, strongly indicating the promise of the proposed design for the discovery of prognostic or risk germline markers in other cancers. Clin Cancer Res; 22(13); 3268–80. ©2016 AACR.