Natural killer cell-directed therapies: moving from unexpected results to successful strategies.

Natural killer (NK) cells influence innate and adaptive immune host defenses. Existing data indicate that manipulating the balance between inhibitory and activating NK receptor signals, the sensitivity of target cells to NK cell-mediated apoptosis, and NK cell cross-talk with dendritic cells might hold therapeutic promise. Efforts to modulate NK cell trafficking into inflamed tissues and/or lymph nodes, and to counteract NK cell suppressors, might also prove fruitful in the clinic. However, deeper investigation into the benefits of combination therapy, greater understanding of the functional distinctions between NK cell subsets, and design of new tools to monitor NK cell activity are needed to strengthen our ability to harness the power of NK cells for therapeutic aims.

Infiltrating and peripheral immune cell analysis in advanced gastric cancer according to the Lauren classification and its prognostic significance.

BACKGROUND: The correlation between immune cells and the Lauren classification subtypes and their prognostic impact in advanced gastric cancer (AGC) are unknown. METHODS: Circulating natural killer (NK) cells, CD4+ and CD8+ T cells, regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were quantified in peripheral blood mononuclear cells (PBMCs) from 67 patients with untreated AGC enrolled in the PRODIGE 17-ACCORD 20 trial. CD56+ cells (NK), CD8+, and FoxP3+ (Treg) tumor-infiltrating lymphocytes (TILs) were assessed in tumor samples. RESULTS: Circulating NK and Treg proportions were significantly lower in patients with diffuse/mixed-type AGC (n = 27) than those with intestinal type (n = 40; median 6.3% vs 11.5%; p = 0.02 and median 3.3% vs 5.2%; p = 0.03, respectively). Proportions of circulating MDSC, CD4+ and CD8+ T cells were not associated with one pathological type. Among tumor-infiltrating cells, CD8+ T cells, but not NK or FoxP3+ cells, were significantly lower in diffuse/mixed-type AGC (median 21 vs 59 cells/field; p = 0.009). Patients with high circulating NK cell counts (> 17%) had a better overall survival than those with < 17% (HR 0.40; 95% CI [0.15-1.06]; p = 0.04). Patients with high CD8+ TIL counts (> 31 cells/field) had significantly longer overall survival (HR 0.44; 95% CI [0.21-0.92]; p = 0.02). The prognostic value of CD8+ TILs was maintained after adjustment for confounding factors, including the Lauren classification (HR = 0.42; 95% CI [0.18-0.96]; p = 0.039). CONCLUSION: Diffuse/mixed-type AGC has lower rates of CD8+ TILs and circulating NK cells and Tregs than the intestinal type. This "cold tumor" phenotype may be associated with a worse outcome.

IL-15 Trans-Signaling with the Superagonist RLI Promotes Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of PD-1 Antagonists.

Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.

[Immune system and tumors]. / Système immunitaire et tumeurs.

Despite having been much debated, it is now well established that the immune system plays an essential role in the fight against cancer. In this article, we will highlight the implication of the immune system in the control of tumor growth and describe the major components of the immune system involved in the antitumoral immune response. The immune system, while exerting pressure on tumor cells, also will play a pro-tumoral role by sculpting the immunogenicity of tumors cells as they develop. Finally, we will illustrate the numerous mechanisms of immune suppression that take place within the tumoral microenvironment which allow tumor cells to escape control from the immune system. The increasingly precise knowledge of the brakes to an effective antitumor immune response allows the development of immunotherapy strategies more and more innovating and promising of hope.

NK cells from pleural effusions are potent antitumor effector cells.

Natural killer (NK) cells express a set of activating and inhibitory receptors which, after interaction with their ligands, determine whether or not the target cell will be lysed. Many studies have clearly demonstrated that NK cells have the capacity to lyse stressed cells (such as tumor or virally-infected cells). However, NK cells that infiltrate tumors usually exhibit phenotypic and functional defects. In this issue of the European Journal of Immunology, Vacca et al. [Eur. J. Immunol. 2013. 43: 550-561] show that NK cells in pleural effusions of primary and metastatic tumors of various origins are not anergic, possibly because the downregulation of activating receptors and the upregulation of inhibitory receptors does not occur, as previously reported for tumor NK cells. Another major finding of this study is the capacity of these pleural NK cells to respond to IL-2 stimulation, as the authors demonstrate that pleural NK cells stimulated by IL-2 in long-term culture acquire the capacity to lyse autologous tumor cells isolated from pleural effusions. These results support the treatment of primary or metastatic pleural tumors with IL-2 or other innovative strategies currently being developed to stimulate NK cells in cancer patients as discussed in this Commentary.

First-Line LV5FU2 with or without Aflibercept in Patients with Non-Resectable Metastatic Colorectal Cancer: A Randomized Phase II Trial (PRODIGE 25-FFCD-FOLFA).

Fluropyrimidine monotherapy is an option for some patients with inoperable metastatic colorectal cancer. Unlike bevacizumab, the addition of aflibercept, an antibody acting as an anti-angiogenic agent, has never been evaluated in this context. The aim of the study was to determine whether aflibercept could increase the efficacy of fluoropyrimidine monotherapy without increasing toxicity. This multicenter phase II non-comparative trial evaluated the addition of aflibercept to infusional 5-fluorouracil/folinic acid (LV5FU2 regimen) as first-line treatment in patients unfit to receive doublet cytotoxic chemotherapy. The primary endpoint was 6-month progression-free survival (PFS). The clinical hypotheses expected a PFS rate at 6 months of over 40% (60% expected). A total of 117 patients, with a median age of 81 years, were included: 59 in arm A (LV5FU2-aflibercept) and 58 in arm B (LV5FU2 alone). Six-month PFS was 54.7% in both arms (90% CI 42.5-66.5 in both). Median overall survival was 21.8 months (arm A) and 25.1 months (arm B). Overall toxicity was more common in arm A: grade ≥ 3 toxicity in 82% versus 58.2%. Given the 6-month PFS, the study can be considered positive. However, the toxicity of aflibercept in this population was high, and continuation of the trial into phase III is not envisaged.

A novel dendritic cell subset involved in tumor immunosurveillance.

The interferon (IFN)-gamma-induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-gamma is not the conventional NK cell but a subset of B220(+)Ly6C(-) dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220(+)NK1.1(+) dendritic cells secrete high levels of IFN-gamma and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2(-/-)Il2rg(-/-) mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.

Modulation of immunity by antiangiogenic molecules in cancer.

In the last decades a new class of therapeutic drugs have been developed that block tumor angiogenesis. These antiangiogenic molecules, which target VEGF or VEGFR, PDGFR, and c-kit, can act not only on endothelial cells but also on immune cells. Some antiangiogenic molecules inhibit the development of immunosuppressive mechanisms developed by the tumors to escape the immune system (such as regulatory T cells, myeloid-derived suppressor cells, and immunosuppressive cytokines). These immunomodulatory effects must be characterized in detail to enable a better prescription of these treatments. In this paper we will focus on the impact of anti-angiogenic drugs on immunosuppression and their potential combination with immunotherapeutic strategies. Interestingly, immune parameters or their modulation during treatment could serve as potential biomarkers of response or resistance to anti-angiogenic therapies.

Synergistic effect of combining sunitinib with a peptide-based vaccine in cancer treatment after microenvironment remodeling.

Although it has proven difficult to demonstrate the clinical efficacy of therapeutic vaccination as a monotherapy in advanced cancers, its combination with an immunomodulatory treatment to reduce intra-tumor immunosuppression and improve vaccine efficacy is a very promising strategy. In this context, we are studying the combination of a vaccine composed of peptides of the tumor antigen survivin (SVX vaccine) with the anti-angiogenic agent sunitinib in a colorectal carcinoma model. To this end, we have been focusing on administration scheduling and have highlighted a therapeutic synergy between SVX vaccine and sunitinib when the vaccine was administered at the end of anti-angiogenic treatment. In this setting, a prolonged control of tumor growth associated with an important percentage of complete tumor regression was observed. Studying the remodeling induced by each therapy on the immunological and angiogenic tumor microenvironment over time we observed, during sunitinib treatment, a transient increase in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and a decrease in NK cells in the tumor microenvironment. In contrast, after sunitinib treatment was stopped, a decrease in PMN-MDSC populations has been observed in the tumor, associated with an increase in NK cells, pericyte coverage of tumor vessels and CD8+ T cell population and functionality. In conclusion, sunitinib treatment results in the promotion of an immune-favorable tumor microenvironment that can guide the optimal sequence of vaccine and anti-angiogenic combination to reinforce their synergy.

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner.

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell-mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)-beta, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-beta-/- T reg cells into nude mice suppressed NK cell-mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell-mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.

Dendritic cells and innate defense against tumor cells.

Tumor growth results from a delicate balance between intrinsic dysregulation of oncogenes, tumor suppressor and stability genes counteracted by extrinsic defenses composed of immune cells shaping tumor immunogenicity. Although immune subversion might be the ultimate outcome of this process, a complex network of cellular interactions take place eventually leading to tumor specific cognate immune responses. The links between innate and cognate antitumor immunity eliciting protective T cell responses are instigated by cytokines, chemokines and damage associated molecular patterns. The intricate differentiation pathway whereby dendritic cells could undergo an efficient maturation program in the tumor microenvironment appears crucial. We will discuss the role of innate effectors and cancer therapies in the process of defense against tumor cells.

Direct and Indirect Modulation of T Cells by VEGF-A Counteracted by Anti-Angiogenic Treatment.

Vascular endothelial growth factor A is known to play a central role in tumor angiogenesis. Several studies showed that VEGF-A is also an immunosuppressive factor. In tumor-bearing hosts, VEGF-A can modulate immune cells (DC, MDSC, TAM) to induce the accumulation of regulatory T-cells while simultaneously inhibiting T-cell functions. Furthermore, VEGFR-2 expression on activated T-cells and FoxP3high regulatory T-cells also allow a direct effect of VEGF-A. Anti-angiogenic agents targeting VEGF-A/VEGFR contribute to limit tumor-induced immunosuppression. Based on interesting preclinical studies, many clinical trials have been conducted to investigate the efficacy of anti-VEGF-A/VEGFR treatments combined with immune checkpoint blockade leading to the approvement of these associations in different tumor locations. In this review, we focus on the impact of VEGF-A on immune cells especially regulatory and effector T-cells and different therapeutic strategies to restore an antitumor immunity.

Targeting HGF/c-Met Axis Decreases Circulating Regulatory T Cells Accumulation in Gastric Cancer Patients.

Elucidating mechanisms involved in tumor-induced immunosuppression is of great interest since it could help to improve cancer immunotherapy efficacy. Here we show that Hepatocyte Growth Factor (HGF), a pro-tumoral and proangiogenic factor, and its receptor c-Met are involved in regulatory T cells (Treg) accumulation in the peripheral blood of gastric cancer (GC) patients. We observed that c-Met is expressed on circulating monocytes from GC patients. The elevated expression on monocytes is associated with clinical parameters linked to an aggressive disease phenotype and correlates with a worse prognosis. Monocyte-derived dendritic cells from GC patients differentiated in the presence of HGF adopt a regulatory phenotype with a lower expression of co-stimulatory molecules, impaired maturation capacities, and an increased ability to produce interleukin-10 and to induce Treg differentiation in vitro. In the MEGA-ACCORD20-PRODIGE17 trial, GC patients received an anti-HGF antibody treatment (rilotumumab), which had been described to have an anti-angiogenic activity by decreasing proliferation of endothelial cells and tube formation. Rilotumumab decreased circulating Treg in GC patients. Thus, we identified that HGF indirectly triggers Treg accumulation via c-Met-expressing monocytes in the peripheral blood of GC patients. Our study provides arguments for potential alternative use of HGF/c-Met targeted therapies based on their immunomodulatory properties which could lead to the development of new therapeutic associations in cancer patients, for example with immune checkpoint inhibitors.

Decrease of Pro-Angiogenic Monocytes Predicts Clinical Response to Anti-Angiogenic Treatment in Patients with Metastatic Renal Cell Carcinoma.

The modulation of subpopulations of pro-angiogenic monocytes (VEGFR-1+CD14 and Tie2+CD14) was analyzed in an ancillary study from the prospective PazopanIb versus Sunitinib patient preferenCE Study (PISCES) (NCT01064310), where metastatic renal cell carcinoma (mRCC) patients were treated with two anti-angiogenic drugs, either sunitinib or pazopanib. Blood samples from 86 patients were collected prospectively at baseline (T1), and at 10 weeks (T2) and 20 weeks (T3) after starting anti-angiogenic therapy. Various subpopulations of myeloid cells (monocytes, VEGFR-1+CD14 and Tie2+CD14 cells) decreased during treatment. When patients were divided into two subgroups with a decrease (defined as a >20% reduction from baseline value) (group 1) or not (group 2) at T3 for VEGFR-1+CD14 cells, group 1 patients presented a median PFS and OS of 24 months and 37 months, respectively, compared with a median PFS of 9 months (p = 0.032) and a median OS of 16 months (p = 0.033) in group 2 patients. The reduction in Tie2+CD14 at T3 predicted a benefit in OS at 18 months after therapy (p = 0.04). In conclusion, in this prospective clinical trial, a significant decrease in subpopulations of pro-angiogenic monocytes was associated with clinical response to anti-angiogenic drugs in patients with mRCC.

[Current advances in immunotherapy in ovarian cancer]. / Les avancées actuelles de l'immunothérapie dans le cancer de l'ovaire.

Ovarian cancers express highly immunogenic tissue-specific antigens. The resulting immune infiltration is a major prognostic factor. There is therefore a strong biological rationale for the development of immunotherapy in ovarian cancer. However, based on Phase I and II clinical trials data, the efficacy of anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors (ICPIs) remains limited in monotherapy in heavily pre-treated patients. Currently, the identification of predictive biomarkers of response and resistance is one of the major areas of research. Identifying effective combination of anti-PD-1 or anti-PD-L1 with other anticancer agents is another clinical need. Several combinations were evaluated. The association of ICPIs with chemotherapy (anthracyclines or carboplatin+paclitaxel) is disappointing (JAVELIN studies). The association with PARP inhibitors, anti-angiogenic agents and CTLA-4 inhibitors seems promising. Other immune therapies such as cell therapies (adoptive transfer of intra-tumor lymphocytes, CAR T cells or vaccines from dendritic cells) could be the future of immunotherapy in ovarian cancer but only early phase studies clinical data is available at this time.

Therapeutic Cancer Vaccine and Combinations With Antiangiogenic Therapies and Immune Checkpoint Blockade.

Considering the high importance of immune surveillance and immune escape in the evolution of cancer, the development of immunotherapeutic strategies has become a major field of research in recent decades. The considerable therapeutic breakthrough observed when targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the induction and proper activation of an immune response against cancer. In this context, therapeutic vaccination, which can induce a specific immune response against tumor antigens, is an important approach to consider. However, this strategy has its advantages and limits. Considering its low clinical efficacy, approaches combining therapeutic cancer vaccine strategies with other immunotherapies or targeted therapies have been emphasized. This review will list different cancer vaccines, with an emphasis on their targets. We highlight the results and limits of vaccine strategies and then describe strategies that combine therapeutic vaccines and antiangiogenic therapies or immune checkpoint blockade. Antiangiogenic therapies and immune checkpoint blockade are of proven clinical efficacy for some indications, but are limited by toxicity and the development of resistance. Their combination with therapeutic vaccines could be a way to improve therapeutic outcome by specifically stimulating the immune system and considering a global approach to tumor microenvironment remodeling.

High Therapeutic Efficacy of a New Survivin LSP-Cancer Vaccine Containing CD4+ and CD8+ T-Cell Epitopes.

The efficacy of an antitumoral vaccine relies both on the choice of the antigen targeted and on its design. The tumor antigen survivin is an attractive target to develop therapeutic cancer vaccines because of its restricted over-expression and vital functions in most human tumors. Accordingly, several clinical trials targeting survivin in various cancer indications have been conducted. Most of them relied on short peptide-based vaccines and showed promising, but limited clinical results. In this study, we investigated the immunogenicity and therapeutic efficacy of a new long synthetic peptide (LSP)-based cancer vaccine targeting the tumor antigen survivin (SVX). This SVX vaccine is composed of three long synthetic peptides containing several CD4+ and CD8+ T-cell epitopes, which bind to various HLA class II and class I molecules. Studies in healthy individuals showed CD4+ and CD8+ T-cell immunogenicity of SVX peptides in human, irrespective of the individual's HLA types. Importantly, high frequencies of spontaneous T-cell precursors specific to SVX peptides were also detected in the blood of various cancer patients, demonstrating the absence of tolerance against these peptides. We then demonstrated SVX vaccine's high therapeutic efficacy against four different established murine tumor models, associated with its capacity to generate both specific cytotoxic CD8+ and multifunctional Th1 CD4+ T-cell responses. When tumors were eradicated, generated memory T-cell responses protected against rechallenge allowing long-term protection against relapses. Treatment with SVX vaccine was also found to reshape the tumor microenvironment by increasing the tumor infiltration of both CD4+ and CD8+ T cells but not Treg cells therefore tipping the balance toward a highly efficient immune response. These results highlight that this LSP-based SVX vaccine appears as a promising cancer vaccine and warrants its further clinical development.

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects.

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

BCR/ABL promotes dendritic cell-mediated natural killer cell activation.

BCR/ABL fusion gene, encoding a paradigmatic tyrosine kinase involved in chronic myelogenous leukemia (CML), can modulate the expression of genes involved in natural killer (NK) cell target recognition. Recent reports outline the role of allogeneic antileukemic NK effectors in the graft-versus-leukemia effect but the regulation of NK cell activation in the setting of graft-versus-leukemia effect remains unknown. Here we show that dendritic cells derived from monocytes of CML patients are selectively endowed with NK cell stimulatory capacity in vitro. We further show, using a gene transfer approach in mouse bone marrow progenitors, that ABL/ABL is necessary to promote dendritic cell-mediated NK cell activation. The dendritic cell/NK cell cross-talk in ABL/ABL-induced CML seems unique because JunB or IFN consensus sequence binding protein loss of functions, associated with other myeloproliferative disorders, do not promote dendritic cell-mediated NK cell activation. NK cell activation by leukemic dendritic cells involves NKG2D activating receptors and is blocked by imatinib mesylate. Indeed, ABL/ABL translocation enhances the expression levels of the NKG2D ligands on dendritic cells, which is counteracted by imatinib mesylate. Altogether, the clonal ABL/ABL dendritic cells display the unique and selective ability to activate NK cells and may participate in the NK cell control of CML. This study also highlights the deleterious role of imatinib mesylate at the dendritic cell level for NK cell activation.