Identification of tumor antigens as potential target antigens for immunotherapy by serological expression cloning

The presence of tumor infiltrating T cells has been shown to be associated with a favorable prognosis in different tumor types. Several strategies have been developed to identify relevant tumor antigens which can be used for active immunotherapy strategies. The SEREX technique (serological analysis of cDNA expression libraries) identifies tumor antigens based on a spontaneous humoral immune response in cancer patients. This technique is not limited to tumor types that can be grown in cell culture or depends on established T cell clones recognizing the autologous tumor. Several steps of analysis are mandatory to evaluate SEREX-defined antigens before they become new target antigens for active immunotherapy: expression analysis; serological analysis with sera from tumor patients and normal individuals; identification of potential peptide epitopes for CD8 T cells and evaluation in T cell assays. This article summarizes our approach of antigen identification and evaluation giving the example of the recently cloned breast cancer antigen NY-BR-

Integrated Akt/PKB Signaling in Immunomodulation and Its Potential Role in Cancer Immunotherapy

T cell development and maturation involve a variety of defined and coordinated developmental stages under the control of a variety of signaling networks. They function as the major mediator in cell-based immunity that defends against pathogen infections and executes immune surveillance against tumor cells. Protein kinase B (PKB, also called Akt) is central to multiple signaling pathways and transduces extracellular signals to dictate cellular responses towards proliferation, migration, anti-apoptosis, and maintenance of metabolic homeostasis. Although the prosurvival function of PKB was thought to be responsible for most of the functions regulated by PKB, emerging evidence has started to dissect its role in immunomodulation. More importantly, hyperactivation of PKB in cancer stroma frequently occurs in patients treated clinically with targeted cancer therapies, where it acts as a key mediator involved in the trapping of host immune cells in the vicinity of tumors, which supports cancer cell invasion and the escape of cancer cells from host immune surveillance. Encouragingly, recent studies have shown that inhibition of PKB improves the recognition of cancer cells by the host immune system, indicating a potential clinical strategy to rekindle the suppressed host immune response through the specific targeting of PKB. In this review, we explore how PKB signaling contributes to T cell development and cellular immune responses and discuss the mechanistic roles that PKB plays in the creation of immunosuppressive conditions and the escaping of immune recognition in the microenvironment of cance

Changes of peripheral T cell subsets in melanoma patients with immune-related adverse events

IntroductionImmunotherapies have improved the prognosis of many cancer patients including patients with advanced melanoma. Immune checkpoint receptors including CTLA-4 and PD-1 have been established as main therapeutic targets for immunotherapy of melanoma. Although monotherapy is effective in melanoma patients, a dual therapy approach has been shown to be most effective. Dual checkpoint blockade, however, increases substantially the risk for immune-related adverse events (irAEs).MethodsIn this study, we characterized peripheral immune cell subsets in patients with anti-PD-1 monotherapy and with dual immune receptors blockade targeting PD-1 and CTLA-4.ResultsWe found differences in peripheral T cells between patients who developed severe immune-related side effects and patients with mild irAEs. We identified several mainly changes in CD8+ T cell subsets in patients with severe irAE under dual PD-1 and CTLA-4 blockade.DiscussionThis work suggests that peripheral immune cell dynamics could be associated with severe immune-related side effects in patients receiving immune checkpoint inhibitors. These changes could be used as future biomarkers in early diagnosis of irAEs

Pre-treatment comorbidities, C-reactive protein and eosinophil count, and immune-related adverse events as predictors of survival with checkpoint inhibition for multiple tumour entities

BACKGROUND The development of immune-related adverse events (irAEs) may be associated with clinical efficacy of checkpoint inhibitors (CPIs) in patients with cancer. We therefore investigated the effect of irAEs and pre-treatment parameters on outcome in a large, real-life patient cohort. METHODS We performed a single-centre, retrospective, observational study including patients who received CPIs from 2011 to 2018 and followed until 2021. The primary outcome was overall survival, and the secondary outcome was the development of irAEs. RESULTS In total, 229 patients with different tumour entities (41% non-small cell lung cancer [NSCLC], 29% melanoma) received a total of 282 CPI treatment courses (ipilimumab, nivolumab, pembrolizumab or atezolizumab). Thirty-four percent of patients developed irAEs (of these 17% had CTCAE Grade ≥3). Factors independently associated with mortality were pre-treatment CRP ≥10 mg/L (hazard ratio [HR] 2.064, p = 0.0003), comorbidity measured by Charlson comorbidity index (HR 1.149, p = 0.014) and irAEs (HR 0.644, p = 0.036) (age-adjusted, n = 216). Baseline eosinophil count ≤0.2 × 10$^{9}$ /L was a further independent predictor of mortality (age-, CRP-, CCI- and irAE-adjusted HR = 2.252, p = 0.002, n = 166). Anti-CTLA-4 use (p < 0.001), and pre-treatment CRP <10 mg/L were independently associated with irAE occurrence (p = 0.037). CONCLUSIONS We found an independent association between irAE occurrence and improved overall survival in a real-life cohort spanning multiple tumour entities and treatment regimens. Pre-treatment comorbidities, CRP and eosinophil count represent potential markers for predicting treatment response

PD-1+ natural killer cells in human non-small cell lung cancer can be activated by PD-1/PD-L1 blockade

Natural killer (NK) cells are critically involved in anti-tumor immunity by targeting tumor cells. In this study, we show that intratumoral NK cells from NSCLC patients expressed elevated levels of the immune checkpoint receptor PD-1 on their cell surface. In contrast to the expression of activating receptors, PD-1; +; NK cells co-expressed more inhibitory receptors compared to PD-1; -; NK cells. Intratumoral NK cells were less functional compared to peripheral NK cells, and this dysfunction correlated with PD-1 expression. Tumor cells expressing PD-L1 inhibited the functionality of PD-1; +; NK cells in ex vivo models and induced PD-1 clustering at the immunological synapse between NK cells and tumor cells. Notably, treatment with PD-1 blockade was able to reverse PD-L1-mediated inhibition of PD-1; +; NK cells. Our findings highlight the therapeutic potential of PD-1; +; NK cells in immune checkpoint blockade and could guide the development of NK cell-stimulating agents in combination with PD-1 blockade

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restriction

The microtubule-depolymerizing agent ansamitocin P3 programs dendritic cells toward enhanced anti-tumor immunity

In addition to direct tumor cell cytotoxicity, chemotherapy can mediate tumor reduction through immune modulation of the tumor microenvironment to promote anti-tumor immunity. Mature dendritic cells (DCs) play key roles in priming robust immune responses in tumor-bearing hosts. Here, we screened a panel of 21 anticancer agents with defined molecular targets for their ability to induce direct maturation of DCs. We identified ansamitocin P3, a microtubule-depolymerizing agent, as a potent inducer of phenotypic and functional maturation of DCs. Exposure of both murine spleen-derived and human monocyte-derived DCs to ansamitocin P3 triggered up-regulation of maturation markers and production of pro-inflammatory cytokines, resulting in an enhanced T cell stimulatory capacity. Local administration of ansamitocin P3 induced maturation of skin Langerhans cells in vivo and promoted antigen uptake and extensive homing of tumor-resident DCs to tumor-draining lymph nodes. When used as an adjuvant in a specific vaccination approach, ansamitocin P3 dramatically increased activation of antigen-specific T cells. Finally, we demonstrate that ansamitocin P3, due to its immunomodulatory properties, acts in synergy with antibody-mediated blockade of the T cell inhibitory receptors PD-1 and CTLA-4. The combination treatment was most effective and induced durable growth inhibition of established tumors. Mechanistically, we observed a reduced regulatory T cell frequency and improved T cell effector function at the tumor site. Taken together, our study unravels an immune-based anti-tumor mechanism exploited by microtubule-depolymerizing agents, including ansamitocin P3, and paves the way for future clinical trials combining this class of agents with immunotherapy

Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient's response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discusse

Degeneracy of Antigen Recognition as the Molecular Basis for the High Frequency of Naive A2/Melan-A Peptide Multimer+ CD8+ T Cells in Humans

In contrast with the low frequency of most single epitope reactive T cells in the preimmune repertoire, up to 1 of 1,000 naive CD8+ T cells from A2+ individuals specifically bind fluorescent A2/peptide multimers incorporating the A27L analogue of the immunodominant 26–35 peptide from the melanocyte differentiation and melanoma associated antigen Melan-A. This represents the only naive antigen-specific T cell repertoire accessible to direct analysis in humans up to date. To get insight into the molecular basis for the selection and maintenance of such an abundant repertoire, we analyzed the functional diversity of T cells composing this repertoire ex vivo at the clonal level. Surprisingly, we found a significant proportion of multimer+ clonotypes that failed to recognize both Melan-A analogue and parental peptides in a functional assay but efficiently recognized peptides from proteins of self- or pathogen origin selected for their potential functional cross-reactivity with Melan-A. Consistent with these data, multimers incorporating some of the most frequently recognized peptides specifically stained a proportion of naive CD8+ T cells similar to that observed with Melan-A multimers. Altogether these results indicate that the high frequency of Melan-A multimer+ T cells can be explained by the existence of largely cross-reactive subsets of naive CD8+ T cells displaying multiple specificities