Exploring the predictive potential of programmed death ligand 1 expression in healthy organs and lymph nodes as measured by 18F-BMS986-192 PET: pooled analysis of data from four solid tumor types.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) can elicit anticancer immune responses, but predictive biomarkers are needed. We measured programmed death ligand 1 (PD-L1) expression in organs and lymph nodes using 18F-BMS-986192 positron emission tomography (PET)-imaging and looked for correlations with response and immune-related adverse events. METHODS: Four 18F-BMS-986192 PET studies in patients with melanoma, lung, pancreatic and oral cancer, receiving ICI treatment, were combined. Imaging data (organ standardized uptake value (SUV)mean, lymph node SUVmax) and clinical data (response to treatment and incidence of immune-related adverse events) were extracted. RESULTS: Baseline PD-L1 uptake in the spleen was on average higher in non-responding patients than in responders (spleen SUVmean 16.1±4.4 vs 12.5±3.4, p=0.02). This effect was strongest in lung cancer, and not observed in oral cancer. In the oral cancer cohort, benign tumor-draining lymph nodes (TDLNs) had higher PD-L1 uptake (SUVmax 3.3 IQR 2.5-3.9) compared with non-TDLNs (SUVmax 1.8, IQR 1.4-2.8 p=0.04). Furthermore, in the same cohort non-responders showed an increase in PD-L1 uptake in benign TDLNs on-treatment with ICIs (+15%), while for responders the PD-L1 uptake decreased (-11%). PD-L1 uptake did not predict immune-related adverse events, though elevated thyroid uptake on-treatment correlated with pre-existing thyroid disease or toxicity. CONCLUSION: PD-L1 PET uptake in the spleen is a potential negative predictor of response to ICIs. On-treatment with ICIs, PD-L1 uptake in benign TDLNs increases in non-responders, while it decreases in responders, potentially indicating a mechanism for resistance to ICIs in patients with oral cancer.

Comparative analysis of immune infiltrates in head and neck cancers across anatomical sites.

BACKGROUND: The response rate to immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) receptor is 13%-18% for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Detailed understanding of the tumor immune microenvironment (TIME) is crucial in order to explain and improve this response rate. HNSCCs arise at various anatomical locations including the oral cavity, hypopharynx, larynx and oropharynx. Studies directly comparing immune infiltration between anatomical sites are scarce. Since the distinct locations could drive deviating microenvironments, we questioned whether the immune composition varies across these HNSCC sites. METHODS: Here, we characterized the TIME of 76 fresh tumor specimens using flow cytometry and performed single-cell RNA-sequencing on nine head and neck tumor samples. RESULTS: We found major differences in the composition of the TIME between patients. When comparing anatomical sites: tumors originating from the oral cavity had higher T cell infiltrates than tumors from other anatomical sites. The percentage of tumor-infiltrating T-lymphocytes positive for the immune checkpoint PD-1 varied considerably between patients, with the highest fraction of PD-1+ T cells found in larynx squamous cell carcinomas (SCCs). While we had hypothesized that the anatomical sites of tumor origin would drive sample clustering, our data showed that the type of TIME was more dominant and was particularly driven by the fraction of T cells positive for PD-1. Moreover, a high proportion of PD-1+ CD8+ T cells associated with an improved overall survival. Using single-cell RNA-sequencing, we observed that PD-1 expression was highest in the CD8-ENTPD1 tissue resident memory T cell/exhausted T cell and CD4-CXCL13 type 1 T helper cell clusters. CONCLUSIONS: We found that oral cavity SCCs had the highest frequencies of T cells. We also observed considerable interpatient heterogeneity for PD-1 on T cells, with noticeably higher frequencies of PD-1+ CD4+ T helper cells in larynx SCCs. Within the entire cohort, a higher fraction of CD8+ T cells positive for PD-1 was linked to improved overall survival. Whether the fraction of PD-1+ T cells within the TIME enables immune checkpoint inhibitor response prediction for patients with head and neck cancer remains to be determined.

An Antibody-Drug Conjugate Directed to Tissue Factor Shows Preclinical Antitumor Activity in Head and Neck Cancer as a Single Agent and in Combination with Chemoradiotherapy.

Head and neck squamous cell carcinoma (HNSCC) is a solid tumor type that arises in the squamous epithelial cells lining the mucosal surfaces of the upper aerodigestive tract. Long-term survival of patients with advanced disease stage remains disappointing with current treatment options. We show that tissue factor is abundantly expressed on patient-derived HNSCC cell lines, xenograft tumor material, and tumor biopsies from patients with HNSCC. Tisotumab vedotin (TV) is an antibody-drug conjugate (ADC) directed to tissue factor, a protein expressed in many solid tumors. HNSCC cells and xenograft tumors were efficiently eliminated in vitro and in vivo with TV-monotherapy compared with treatment with a control antibody conjugated to monomethyl auristatin E (MMAE). Antitumor activity of TV was also tested in vivo in combination with chemoradiotherapy, standard of care for patients with advanced stage HNSCC tumors outside the oral cavity. Preclinical studies showed that by adding TV to chemoradiotherapy, survival was markedly improved, and TV, not radiotherapy or chemotherapy, was the main driver of antitumor activity. Interestingly, TV-induced cell death in xenograft tumors showed an influx of macrophages indicative of a potential immune-mediated mode-of-action. In conclusion, on the basis of these preclinical data, TV may be a novel treatment modality for patients suffering from head and neck cancer and is hypothesized to improve efficacy of chemoradiotherapy. SIGNIFICANCE: This work shows preclinical in vitro and in vivo antitumor activity of the antibody-drug conjugate Tisotumab vedotin in head and neck cancer models, and enhanced activity in combination with chemoradiotherapy, supporting further clinical development for this cancer type.

A Bispecific γδ T-cell Engager Targeting EGFR Activates a Potent Vγ9Vδ2 T cell-Mediated Immune Response against EGFR-Expressing Tumors.

Vγ9Vδ2 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing Vγ9Vδ2 T cells to EGFR-expressing tumors. An EGFR-Vδ2 bispecific T-cell engager (bsTCE) was generated, and its capacity to activate Vγ9Vδ2 T cells and trigger antitumor activity was tested in multiple in vitro, in vivo, and ex vivo models. Studies to explore safety were conducted using cross-reactive surrogate engagers in nonhuman primates (NHP). We found that Vγ9Vδ2 T cells from peripheral blood and tumor specimens of patients with EGFR+ cancers had a distinct immune checkpoint expression profile characterized by low levels of PD-1, LAG-3, and TIM-3. Vγ9Vδ2 T cells could be activated by EGFR-Vδ2 bsTCEs to mediate lysis of various EGFR+ patient-derived tumor samples, and substantial tumor growth inhibition and improved survival were observed in in vivo xenograft mouse models using peripheral blood mononuclear cells (PBMC) as effector cells. EGFR-Vδ2 bsTCEs exerted preferential activity toward EGFR+ tumor cells and induced downstream activation of CD4+ and CD8+ T cells and natural killer (NK) cells without concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs. Administration of fully cross-reactive and half-life extended surrogate engagers to NHPs did not trigger signals in the safety parameters that were assessed. Considering the effector and immune-activating properties of Vγ9Vδ2 T cells, the preclinical efficacy data and acceptable safety profile reported here provide a solid basis for testing EGFR-Vδ2 bsTCEs in patients with EGFR+ malignancies.

Tumor-educated Tregs drive organ-specific metastasis in breast cancer by impairing NK cells in the lymph node niche.

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (Tregs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive Tregs during primary tumor growth. Tumor-educated Tregs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as Treg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that Tregs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis. In line, an increased Treg/NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent.

Comparison of the tumor microenvironments of squamous cell carcinoma at different anatomical locations within the upper aerodigestive tract in relation to response to ICI therapy.

Immunotherapy with immune checkpoint inhibitors (ICI) has improved treatment outcomes in many cancer types and has focused attention on cancer immunity and the role of the tumor microenvironment (TME). Studies into efficacy of immunotherapy and TME are generally restricted to tumors in one anatomical location, while the histological type may have substantial influence on the contexture of the TME, perhaps more so than anatomical location, and subsequently to the response to immunotherapy. This review aims to focus on the TME in ICI-treated tumors of the same histological type, namely carcinogen-induced squamous cell carcinoma developing within the aerodigestive tract, at three locations, i.e. head and neck (HNSCC), esophagus (ESCC) and lung (LUSC).

NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.

Palmitoylated antigens for the induction of anti-tumor CD8+ T cells and enhanced tumor recognition.

Induction of tumor-specific cytotoxic CD8+ T cells (CTLs) via immunization relies on the presentation of tumor-associated peptides in major histocompatibility complex (MHC) class I molecules by dendritic cells (DCs). To achieve presentation of exogenous peptides into MHC class I, cytosolic processing and cross-presentation are required. Vaccination strategies aiming to induce tumor-specific CD8+ T cells via this exogenous route therefore pose a challenge. In this study, we describe improved CD8+ T cell induction and in vivo tumor suppression of mono-palmitic acid-modified (C16:0) antigenic peptides, which can be attributed to their unique processing route, efficient receptor-independent integration within lipid bilayers, and continuous intracellular accumulation and presentation through MHC class I. We propose that this membrane-integrating feature of palmitoylated peptides can be exploited as a tool for quick and efficient antigen enrichment and MHC class I loading. Importantly, both DCs and non-professional antigen-presenting cells (APCs), similar to tumor cells, facilitate anti-tumor immunity by efficient CTL priming via DCs and effective recognition of tumors through enhanced presentation of antigens.

Immunotherapy Goes Local: The Central Role of Lymph Nodes in Driving Tumor Infiltration and Efficacy.

Immune checkpoint blockade (ICB) has changed the therapeutic landscape of oncology but its impact is limited by primary or secondary resistance. ICB resistance has been related to a lack of T cells infiltrating into the tumor. Strategies to overcome this hurdle have so far focused on the tumor microenvironment, but have mostly overlooked the role of tumor-draining lymph nodes (TDLN). Whereas for CTLA-4 blockade TDLN have long since been implicated due to its perceived mechanism-of-action involving T cell priming, only recently has evidence been emerging showing TDLN to be vital for the efficacy of PD-1 blockade as well. TDLN are targeted by developing tumors to create an immune suppressed pre-metastatic niche which can lead to priming of dysfunctional antitumor T cells. In this review, we will discuss the evidence that therapeutic targeting of TDLN may ensure sufficient antitumor T cell activation and subsequent tumor infiltration to facilitate effective ICB. Indeed, waves of tumor-specific, proliferating stem cell-like, or progenitor exhausted T cells, either newly primed or reinvigorated in TDLN, are vital for PD-1 blockade efficacy. Both tumor-derived migratory dendritic cell (DC) subsets and DC subsets residing in TDLN, and an interplay between them, have been implicated in the induction of these T cells, their imprinting for homing and subsequent tumor control. We propose that therapeutic approaches, involving local delivery of immune modulatory agents for optimal access to TDLN, aimed at overcoming hampered DC activation, will enable ICB by promoting T cell recruitment to the tumor, both in early and in advanced stages of cancer.

Oncolytic Adenovirus ORCA-010 Activates Proinflammatory Myeloid Cells and Facilitates T Cell Recruitment and Activation by PD-1 Blockade in Melanoma.

Immune checkpoint inhibitors have advanced the treatment of melanoma. Nevertheless, a majority of patients are resistant, or develop resistance, to immune checkpoint blockade, which may be related to prevailing immune suppression by myeloid regulatory cells in the tumor microenvironment (TME). ORCA-010 is a novel oncolytic adenovirus that selectively replicates in, and lyses, cancer cells. We previously showed that ORCA-010 can activate melanoma-exposed conventional dendritic cells (cDCs). To study the effect of ORCA-010 on melanoma-conditioned macrophage development, we used an in vitro co-culture model of human monocytes with melanoma cell lines. We observed a selective survival and polarization of monocytes into M2-like macrophages (CD14+CD80-CD163+) in co-cultures with cell lines that expressed macrophage colony-stimulating factor. Oncolysis of these melanoma cell lines, effected by ORCA-010, activated the resulting macrophages and converted them to a more proinflammatory state, evidenced by higher levels of PD-L1, CD80, and CD86 and an enhanced capacity to prime allogenic T cells and induce a type-1 T cell response. To assess the effect of ORCA-010 on myeloid subset distribution and activation in vivo, ORCA-010 was intratumorally injected and tested for T cell activation and recruitment in the human adenovirus nonpermissive B16-OVA mouse melanoma model. While systemic PD-1 blockade in this model in itself did not modulate myeloid or T cell subset distribution and activation, when it was preceded by i.t. injection of ORCA-010, this induced an increased rate and activation state of CD8α+ cDC1, both in the TME and in the spleen. Observed increased rates of activated CD8+ T cells, expressing CD69 and PD-1, were related to both increased CD8α+ cDC1 rates and M1/M2 shifts in tumor and spleen. In conclusion, the myeloid modulatory properties of ORCA-010 in melanoma, resulting in recruitment and activation of T cells, could enhance the antitumor efficacy of PD-1 blockade.

A Bispecific Antibody Antagonizes Prosurvival CD40 Signaling and Promotes Vγ9Vδ2 T cell-Mediated Antitumor Responses in Human B-cell Malignancies.

Novel T cell-based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by Vγ9Vδ2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific Vγ9Vδ2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD40L-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of Vγ9Vδ2 T cells in the presence of CD40+ tumor cells induced potent Vγ9Vδ2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific γδ T-cell engager demonstrated lysis of leukemic cells by autologous Vγ9Vδ2 T cells present in patient-derived samples. Taken together, our CD40 bispecific γδ T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent Vγ9Vδ2 T cell-dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

At the Crossroads of Molecular Biology and Immunology: Molecular Pathways for Immunological Targeting of Head and Neck Squamous Cell Carcinoma.

Background: Recent advances in immunotherapy for head and neck squamous cell carcinoma (HNSCC) have led to implementation of anti-programmed death receptor 1 (PD-1) immunotherapy to standard of care for recurrent/metastatic HNSCC. However, the majority of tumors do not respond to these therapies, indicating that these tumors are not immunogenic or other immunosuppressive mechanisms might be at play. Aim: Given their role in carcinogenesis as well as in immune modulation, we discuss the relation between the STAT3, PI3K/AKT/mTOR and Wnt signaling pathways to identify potential targets to empower the immune response against HNSCC. Results: We focused on three pathways. First, STAT3 is often overactivated in HNSCC and induces the secretion of immunosuppressive cytokines, thereby promoting recruitment of immune suppressive regulatory T cells and myeloid-derived suppressor cells to the tumor microenvironment (TME) while hampering the development of dendritic cells. Second, PI3K/AKT/mTOR mutational activation results in increased tumor proliferation but could also be important in HNSCC immune evasion due to the downregulation of components in the antigen-processing machinery. Third, canonical Wnt signaling is overactivated in >20% of HNSCC and could be an interesting pleotropic target since it is related to increased tumor cell proliferation and the development of an immunosuppressive HNSCC TME. Conclusion: The molecular pathology of HNSCC is complex and heterogeneous, varying between sites and disease etiology (i.e., HPV). The in HNSCC widely affected signaling pathways STAT3, PI3K/AKT/mTOR and Wnt are implicated in some of the very mechanisms underlying immune evasion of HNSCC, thereby representing promising targets to possibly facilitate immunotherapy response.

Breast cancer-induced immune suppression in the sentinel lymph node is effectively countered by CpG-B in conjunction with inhibition of the JAK2/STAT3 pathway.

BACKGROUND: We previously showed selectively hampered activation of lymph node-resident (LNR) dendritic cell (DC) subsets in the breast cancer (BrC) sentinel lymph node (SLN) to precede a state of profound T cell anergy. Reactivating these DC subsets by intratumoral delivery of the Toll-like receptor-9 (TLR9) agonist CpG-B could potentially offer a promising immune therapeutic strategy to combat this immune suppression and prevent disease spread. Unfortunately, CpG-B can limit its own immune stimulatory activity through direct TLR9-mediated activation of signal transducer and activator of transcription 3 (STAT3), pinpointed as a key regulator of immune suppression in the tumor microenvironment. Here, we have investigated whether in vitro exposure to CpG-B, with or without simultaneous inhibition of STAT3 signaling, could overcome immune suppression in BrC SLN. METHODS: Immune modulatory effects of CpG-B (CPG7909) with or without the JAK2/STAT3 inhibitor (STAT3i) AG490 were assessed in ex vivo cultured BrC SLN-derived single-cell suspensions (N=29). Multiparameter flow cytometric analyses were conducted for DC and T cell subset characterization and assessment of (intracellular) cytokine profiles. T cell reactivity against the BrC-associated antigen Mammaglobin-A was determined by means of interferon-γ ELISPOT assay. RESULTS: Although CpG-B alone induced activation of all DC subsets, combined inhibition of the JAK2/STAT3 pathway resulted in superior DC maturation (ie, increased CD83 expression), with most profound activation and maturation of LNR DC subsets. Furthermore, combined CpG-B and JAK2/STAT3 inhibition promoted Th1 skewing by counterbalancing the CpG-induced Th2/regulatory T cell response and significantly enhanced Mammaglobin-A specific T cell reactivity. CONCLUSION: Ex vivo immune modulation of the SLN by CpG-B and simultaneous JAK2/STAT3 inhibition can effectively overcome BrC-induced immune suppression by preferential activation of LNR DC, ultimately restoring type 1-mediated antitumor immunity, thereby securing a BrC-specific T cell response. These findings provide a clear rationale for clinical exploration of SLN-immune potentiation through local CpG/STAT3i administration in patients with BrC.

The Immune Microenvironment in Head and Neck Squamous Cell Carcinoma: on Subsets and Subsites.

PURPOSE: To understand why some patients respond to immunotherapy but many do not, a clear picture of the tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) is key. Here we review the current understanding on the immune composition per HNSCC subsite, the importance of the tumor's etiology and the prognostic power of specific immune cells. RECENT FINDINGS: Large cohort data are mostly based on deconvolution of transcriptional databases. Studies focusing on infiltrate localization often entail small cohorts, a mixture of HNSCC subsites, or focus on a single immune marker rather than the interaction between cells within the TME. Conclusions on the prognostic impact of specific immune cells in HNSCC are hampered by the use of heterogeneous or small cohorts. To move forward, the field should focus on deciphering the immune composition per HNSCC subsite, in powered cohorts and considering the molecular diversity in this disease.

Lipo-Based Vaccines as an Approach to Target Dendritic Cells for Induction of T- and iNKT Cell Responses.

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8+ T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8+ and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (LeY) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8+ T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing LeY also showed priming of MART-126-35L specific CD8+ T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8+ T- and iNKT cells. These liposomes present a new vaccination strategy against tumors.

Constitutively active GSK3ß as a means to bolster dendritic cell functionality in the face of tumour-mediated immune suppression.

In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3ß (GSK3ß) as a pivotal kinase in both DC development and suppression. GSK3ß inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3ß induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3ß activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.

Glycan-Modified Apoptotic Melanoma-Derived Extracellular Vesicles as Antigen Source for Anti-Tumor Vaccination

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

Selectively hampered activation of lymph node-resident dendritic cells precedes profound T cell suppression and metastatic spread in the breast cancer sentinel lymph node.

BACKGROUND: Immune regulated pathways influence both breast cancer (BrC) development and response to (neo)adjuvant chemotherapy. The sentinel lymph node (SLN), as the first metastatic site, is also the first site where BrC-induced suppression of immune effector subsets occurs. Since intricate knowledge of the phenotypic and functional status of these immune effector subsets is lacking, we set out to map the immune landscape of BrC SLN. METHODS: Viable LN cells from BrC SLN (n = 58) were used for detailed flowcytometry-assisted mapping of the immune landscape of BrC SLN in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n = 17). Findings were related to clinicopathological characteristics. RESULTS: Our data show that BrC-induced immune suppression in tumor-involved SLN, as evidenced by increased Treg and MDSC rates as well as by a generalized state of T cell anergy, coincides with hampered activation of LN-resident (LNR) dendritic cell (DC) subsets rather than of migratory DC subsets. Importantly, suppression of these LN-resident DC subsets preceded profoundly disabled T cell effector functions in tumor-involved SLN. Furthermore, we provide evidence that the suppressed state of LNR-cDC is not only related to nodal involvement but is also related to high-risk breast cancer subtypes that lack expression of hormone receptors and may be a negative predictor of disease-free survival. CONCLUSION: These data thus provide new insights in the mechanisms underlying loco-regional immune suppression induced by BrC and how these relate to clinical outcome. They identify the LNR-cDC subset as a pivotal regulatory node in cellular immune suppressive pathways and therefore as a promising therapeutic target to combat immune suppression and secure the induction of effective antitumor immunity, e.g. in combination with neo-adjuvant chemotherapy. .

Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer.

The immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous - single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+ T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85-22% of CD8+ cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+ T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy.