Estelle Dransart

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.

Induction of resident memory T cells enhances the efficacy of cancer vaccine

Tissue-resident memory T cells (Trm) represent a new subset of long-lived memory T cells that remain in tissue and do not recirculate. Although they are considered as early immune effectors in infectious diseases, their role in cancer immunosurveillance remains unknown. In a preclinical model of head and neck cancer, we show that intranasal vaccination with a mucosal vector, the B subunit of Shiga toxin, induces local Trm and inhibits tumour growth. As Trm do not recirculate, we demonstrate their crucial role in the efficacy of cancer vaccine with parabiosis experiments. Blockade of TFGβ decreases the induction of Trm after mucosal vaccine immunization, resulting in the lower efficacy of cancer vaccine. In order to extrapolate this role of Trm in humans, we show that the number of Trm correlates with a better overall survival in lung cancer in multivariate analysis. The induction of Trm may represent a new surrogate biomarker for the efficacy of cancer vaccine. This study also argues for the development of vaccine strategies designed to elicit them.

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)–based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8+ T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8+ T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials. Mol Cancer Ther; 14(6); 1336–45. ©2015 AACR.

In Vivo Tumor Targeting by the B-Subunit of Shiga Toxin

Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB) binds specifically to the glycosphingolipid Gb3, overex-pressed in membranes of certain tumor cells, and enters these cells through the retrograde pathway. Therefore, STxB binding to Gb3 receptors may be useful for cell-specific vectorization or imaging purposes. Here we labeled STxB with a fluorophore to evaluate its potential as an in vivo cell-specific targeting reagent in two different models of human colorectal carcinoma. Fluorescent STxB was administered systemically to xenografted nude mice, and its biodistribution was studied by optical imaging. The use of fluorescent STxB allowed the combination of the macroscopic observations with analyses at the cellular level using confocal microscopy. After administration, the fluorescent STxB was slowly eliminated by renal excretion. However, it accumulated in the tumor area. Furthermore, STxB was demonstrated to enter the Gb3-expressing tumoral cells, as well as the epithelial cells of the neovascularization and the monocytes and macrophages surrounding the xenografts.

Tumor Delivery of Ultrasound Contrast Agents Using Shiga Toxin B Subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.

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.

Vaccine-induced tumor regression requires a dynamic cooperation between T cells and myeloid cells at the tumor site

Most cancer immunotherapies under present investigation are based on the belief that cytotoxic T cells are the most important anti-tumoral immune cells, whereas intra-tumoral macrophages would rather play a pro-tumoral role. We have challenged this antagonistic point of view and searched for collaborative contributions by tumor-infiltrating T cells and macrophages, reminiscent of those observed in anti-infectious responses. We demonstrate that, in a model of therapeutic vaccination, cooperation between myeloid cells and T cells is indeed required for tumor rejection. Vaccination elicited an early rise of CD11b+ myeloid cells that preceded and conditioned the intra-tumoral accumulation of CD8+ T cells. Conversely, CD8+ T cells and IFNγ production activated myeloid cells were required for tumor regression. A 4-fold reduction of CD8+ T cell infiltrate in CXCR3KO mice did not prevent tumor regression, whereas a reduction of tumor-infiltrating myeloid cells significantly interfered with vaccine efficiency. We show that macrophages from regressing tumors can kill tumor cells in two ways: phagocytosis and TNFα release. Altogether, our data suggest new strategies to improve the efficiency of cancer immunotherapies, by promoting intra-tumoral cooperation between macrophages and T cells.

A new delivery system for auristatin in STxB-drug conjugate therapy

A key challenge in anticancer therapy is to gain control over the biodistribution of cytotoxic drugs. The most promising strategy consists in conjugating drugs to tumor-targeting carriers, thereby combining high cytotoxic activity and specific delivery. To target Gb3-positive cancer cells, we exploit the non-toxic B-subunit of Shiga toxin (STxB). Here, we have conjugated STxB to highly potent auristatin derivatives (MMA). A former linker was optimized to ensure proper drug-release upon reaching reducing environments in target cells, followed by a self-immolation step. Two conjugates were successfully obtained, and inxa0vitro assays demonstrated the potential of this targeting system for the selective elimination of Gb3-positive tumors.

Intracellular trafficking of Shiga‐toxin‐B‐subunit‐functionalized spherulites

Background information. Spherulites are multi‐lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B‐subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non‐toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures.