Alain Gey

PD-1–Expressing Tumor-Infiltrating T Cells Are a Favorable Prognostic Biomarker in HPV-Associated Head and Neck Cancer

Head and neck cancers positive for human papillomavirus (HPV) have a more favorable clinical outcome than HPV-negative cancers, but it is unknown why this is the case. We hypothesized that prognosis was affected by intrinsic features of HPV-infected tumor cells or differences in host immune response. In this study, we focused on a comparison of regulatory Foxp3(+) T cells and programmed death-1 (PD-1)(+) T cells in the microenvironment of tumors that were positive or negative for HPV, in two groups that were matched for various clinical and biologic parameters. HPV-positive head and neck cancers were more heavily infiltrated by regulatory T cells and PD-1(+) T cells and the levels of PD-1(+) cells were positively correlated with a favorable clinical outcome. In explaining this paradoxical result, we showed that these PD-1(+) T cells expressed activation markers and were functional after blockade of the PD-1-PD-L1 axis in vitro. Approximately 50% of PD-1(+) tumor-infiltrating T cells lacked Tim-3 expression and may indeed represent activated T cells. In mice, administration of a cancer vaccine increased PD-1 on T cells with concomitant tumor regression. In this setting, PD-1 blockade synergized with vaccine in eliciting antitumor efficacy. Our findings prompt a need to revisit the significance of PD-1-infiltrating T cells in cancer, where we suggest that PD-1 detection may reflect a previous immune response against tumors that might be reactivated by PD-1/PD-L1 blockade.

A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self antigens.

Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.

Better understanding tumor–host interaction in head and neck cancer to improve the design and development of immunotherapeutic strategies

Head and neck cancers are heavily infiltrated by immune cells, the significance of which is complex. The natural immune response against head and neck tumors, including anti‐human papillomavirus (HPV) T cells, and humoral responses has been clearly documented. However, during the course of tumor progression, co‐option of the immune system by tumor cells for their own advantage and increased resistance of tumor cells to immune attack also occur. Inflammation and immune subversion to support angiogenesis are key factors promoting tumor growth. Only a better understanding of this tumor–host interaction will permit a rational design of new immunotherapeutic approaches combining immunostimulation with drugs endowed with the ability to counteract immunoevasion mechanisms.

In vivo induction of functional FcγRI (CD64) on neutrophils and modulation of blood cytokine mRNA levels in cancer patients treated with G-CSF (rMetHuG-CSF)

Neutrophils from 13 children who received G‐CSF for the collection of peripheral blood progenitors while they were in haematological steady state were studied at various times after G‐CSF injection for FcγR expression (FcγRI or CD 64, FcγRII or CD32, and FcγRIII or CD16) and for their ability to exert antibody‐dependent cell cytotoxicity (ADCC) through FcγRI. Changes in IFNγ, IL8, IL10, MCP1 and TNFα mRNA levels in peripheral blood cells were also studied 4 h and 24 h after the first G‐CSF injection. FcγRI expression increased strongly after 24 h and then remained at the same level throughout treatment. In contrast, FcγRIII expression sharply decreased at day 1 and diminished even further thereafter. No change in FcγRII was observed. ADCC exerted by neutrophils through FcγRI started to increase after 24 h with the peak level at day 5. Cytokine mRNA analyses indicated a reproducible and strong increase of IL8 mRNA (11/13 children) after 24 h, whereas the changes in the mRNA levels of the other cytokines tested were more heterogenous (IFNγ: three; IL10: six; MCP1: five; TNFα: four, of the 13 children). Therefore this study opens the way to an optimized therapeutic schedule for the combined use of G‐CSF and monoclonal antibodies in adjuvant immuno‐intervention.

Mechanisms of action and rationale for the use of checkpoint inhibitors in cancer

The large family of costimulatory molecules plays a crucial role in regulation of the immune response. These molecules modulate TCR signalling via phosphorylation cascades. Some of the coinhibitory members of this family, such as PD-1 and CTLA-4, already constitute approved targets in cancer therapy and, since 2011, have opened a new area of antitumour immunotherapy. Many antibodies targeting other inhibitory receptors (Tim-3, VISTA, Lag-3 and so on) or activating costimulatory molecules (OX40, GITR and so on) are under evaluation. These antibodies have multiple mechanisms of action. At the cellular level, these antibodies restore the activation signalling pathway and reprogram T cell metabolism. Tumour cells become resistant to apoptosis when an intracellular PD-L1 signalling is blocked. CD8+ T cells are considered to be the main effectors of the blockade of inhibitory receptors. Certain CD8+ T cell subsets, such as non-hyperexhausted (CD28+, T-bethigh, PD-1int), follicular-like (CXCR-5+) or resident memory CD8+ T cells, are more prone to be reactivated by anti-PD-1/PD-L1 monoclonal antibody (mAb). In the future, the challenge will be to rationally combine drugs able to make the tumour microenvironment more permissive to immunotherapy in order to potentiate its clinical activity.

Soluble interleukin-2 receptor and metalloproteinase-9 expression in head and neck cancer: prognostic value and analysis of their relationships

In a series of 84 head and neck patients, a statistically significant correlation was observed between high serum soluble interleukin (IL)‐2 receptor alpha (sIL‐2Rα) (P = 0·034) and metalloproteinase‐9 (MMP‐9) concentrations (P = 0·036) at diagnosis and a shorter survival of these patients. As MMP‐9 has been shown to mediate cleavage of IL‐2Rα (CD25) by preactivated T cells, we looked for a relationship between MMP‐9 expression and soluble IL‐2Rα serum concentrations in these cancer patients. We did not find any correlation between intratumoral expression of MMP‐9 or serum MMP‐9 concentrations and serum sIL‐2Rα levels. These results led us to reassess the role of MMP‐9 in the release of sIL‐2Rα. Treatment of Kit225 leukaemic cells with recombinant MMP‐9 slightly decreased membrane CD25 expression and was associated with an increased concentration of sIL‐2Rα in the supernatants. However, using a selective inhibitor of MMP‐9 we did not succeed in specifically inhibiting the release of sIL‐2Rα by the Kit225 cell line or by phytohaemagglutinin (PHA)‐activated peripheral blood mononuclear cells. In addition, in a preclinical mouse model, basal serum sIL‐2Rα concentrations and sIL‐2Rα production by activated cells were not altered in MMP‐9‐deficient mice compared to wild‐type mice. Interestingly, a broad spectrum metalloproteinase inhibitor inhibited the release of sIL‐2Rα by PHA‐activated peripheral blood mononuclear cells, suggesting that in contrast with current views concerning the major role of MMP‐9 in the cleavage of membrane IL‐2Rα, other proteases are involved in the shedding of sIL‐2Rα. MMP‐9 and sIL‐2Rα appear therefore as independent prognostic markers in head and neck cancers.

Granulocytic myeloid-derived suppressor cells inversely correlate with plasma arginine and overall survival in critically ill patients

Critically ill patients display a state of immunosuppression that has been attributed in part to decreased plasma arginine concentrations. However, we and other authors have failed to demonstrate a clinical benefit of L‐arginine supplementation. We hypothesize that, in these critically ill patients, these low plasma arginine levels may be secondary to the presence of granulocytic myeloid‐derived suppressor cells (gMDSC), which express arginase known to convert arginine into nitric oxide (NO) and citrulline. Indeed, in a series of 28 non‐surgical critically ill patients, we showed a dramatic increase in gMDSC compared to healthy subjects (P = 0·0002). A significant inverse correlation was observed between arginine levels and gMDSC (P = 0·01). As expected, gMDSC expressed arginase preferentially in these patients. Patients with high gMDSC levels on admission to the medical intensive care unit (MICU) presented an increased risk of death at day 7 after admission (P = 0·02). In contrast, neither plasma arginine levels, monocytic MDSC levels nor neutrophil levels were associated with overall survival at day 7. No relationship was found between body mass index (BMI) or simplified acute physiology score (SAPS) score, sequential organ failure assessment (SOFA) score or gMDSC levels, eliminating a possible bias concerning the direct prognostic role of these cells. As gMDSC exert their immunosuppressive activity via multiple mechanisms [production of prostaglandin E2 (PGE2), interleukin (IL)‐10, arginase, etc.], it may be more relevant to target these cells, rather than simply supplementing with L‐arginine to improve immunosuppression and its clinical consequences observed in critically ill patients.

A therapeutic Her2/neu vaccine targeting dendritic cells preferentially inhibits the growth of low Her2/neu-expressing tumor in HLA-A2 transgenic mice

Purpose: E75, a peptide derived from the Her2/neu protein, is the most clinically advanced vaccine approach against breast cancer. In this study, we aimed to optimize the E75 vaccine using a delivery vector targeting dendritic cells, the B-subunit of Shiga toxin (STxB), and to assess the role of various parameters (Her2/neu expression, combination with trastuzumab) in the efficacy of this cancer vaccine in a relevant preclinical model. Experimental Design: We compared the differential ability of the free E75 peptide or the STxB-E75 vaccine to elicit CD8+ T cells, and the impact of the vaccine on murine HLA-A2 tumors expressing low or high levels of Her2/neu. Results: STxB-E75 synergized with granulocyte macrophage colony-stimulating factors and CpG and proved to be more efficient than the free E75 peptide in the induction of multifunctional and high-avidity E75-specific anti-CD8+ T cells resulting in a potent tumor protection in HLA-A2 transgenic mice. High expression of HER2/neu inhibited the expression of HLA-class I molecules, leading to a poor recognition of human or murine tumors by E75-specific cytotoxic CD8+ T cells. In line with these results, STxB-E75 preferentially inhibited the growth of HLA-A2 tumors expressing low levels of Her2/neu. Coadministration of anti-Her2/neu mAb potentiated this effect. Conclusions: STxB-E75 vaccine is a potent candidate to be tested in patients with low Her2/neu–expressing tumors. It could also be indicated in patients expressing high levels of Her2/neu and low intratumoral T-cell infiltration to boost the recruitment of T cells—a key parameter in the efficacy of anti-Her2/neu mAb therapy. Clin Cancer Res; 22(16); 4133–44. ©2016 AACR.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Abstract 2504: A therapeutic Her2-Neu cancer vaccine alone or in combination with anti-Her2 mAb inhibits tumor growth in HLA-A2 transgenic mice

Her2/neu is a validated target in cancer, as various antibodies (Abs) against this antigen have demonstrated their clinical efficacy in patients presenting either breast or gastric cancers. Various preclinical and clinical studies showed that the combination of anti-Her2 Abs and vaccines aiming to induce anti-Her2-T cells synergized in promoting the regression of Her2 expressing tumors. The Her2 369-377 peptide (E75) has been shown to be immunogenic in humans and an E75 peptide vaccine is currently being tested in a phase III clinical trial. We have developed a vector based on the non-toxic B subunit of Shiga toxin (STxB) which targets antigens directly to dendritic cells and favours cross-presentation of exogenous antigens to CD8 + T cells. We coupled the E75 peptide to STxB and showed that immunization of HLA-A2 transgenic mice with STxB-E75 elicits statistically significant higher levels of anti-Her2-neu CD8 + T cells (> 3%) detected by tetramer analysis, than the use of the non vectorized E75 peptide ( + T cells induced by STxB-E75 were functional, cytotoxic in vivo and of higher avidity, than those elicited by the E75 peptide. We then proceeded to set up a human (h) Her2/neu-HLA-A2 expressing tumor model (B16) in HLA-A2 transgenic mice for vaccination with STxB-E75. We demonstrated than STxB-E75 was more efficient to inhibit the growth of hHer2-HLA-A2 tumor in humanized HLA-A2 TG mice, than the E75 peptide in both a prophylactic and a therapeutic setting. As control, STxB-E75 had no influence in the growth of hHer2 negative HLA-A2-B16 tumor. We then observed that the anti-Her2 CD8 + T cells elicited in HLA-A2 transgenic mice recognized human breast tumor cell lines expressing either high or low levels of hHer2/neu, as well as the HLA-A2 B16 clones expressing low levels of hHer2. To assess the relevance of this capacity of STxB-E75 to detect low levels of Her2, we grafted the HLA-A2 B16 clone expressing low levels of hHer2/neu on HLA-A2 transgenic mice. The combination of STxB-E75 and anti-Her2 mAb (4D5) was more efficient to inhibit the tumor growth, than the use of the vaccine or antibody alone. The body of experiments presented in this work clearly demonstrated that the STxB-E75 vaccine is a potent candidate to be tested in the clinics for Her2 expressing tumor. In addition, a synergy was observed for the combination of anti-Her2/neu mAb and STxB-E75 vaccine for the regression of low expressing Her2/neu tumor. Citation Format: Thi Tran, Mariana De Oliveira Diniz, Estelle Dransart, Alain Gey, Sylvie Godefroy, Craig Sibley, Ludger Johannes, Eric Tartour. A therapeutic Her2-Neu cancer vaccine alone or in combination with anti-Her2 mAb inhibits tumor growth in HLA-A2 transgenic mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2504. doi:10.1158/1538-7445.AM2015-2504