Search

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti-CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (T

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.

Background The sentinel lymph node (SLN) is the first node to receive lymphatic drainage from the primary tumor and the site where naive T cells are first primed. As such it is of great importance in initiating an effective anti-tumor immune response and an attractive target for immunomodulatory agents. Pre-clinical studies have reported that i.t. administration of anti-CTLA-4 is as effective in inducing tumor eradication as systemic delivery, without the risk of treatment related side effects. However, it remains unclear whether this is due primarily to modulation of the tumor microenvironment or of tumor-draining lymph nodes (TDLN). Here, we have evaluated the safety, tolerability and immunomodulatory effects in the SLN and peripheral blood mononuclear cells (PBMC) of anti-CTLA-4/tremelimumab, delivered locally at the tumor excision site in patients with early-stage melanoma. This unique setting (post tumor excision but prior to SLN biopsy) allowed us to clinically assess the role of TDLN in the biological efficacy of CTLA-4 blockade. Methods In this phase I dose-escalation trial, patients with clinical stage I-II melanoma received one intradermal injection of tremelimumab at four dose levels (2, 5, 10 [n=3 each] or 20 mg [n=4]) around the primary excision site of the tumor, seven days prior to re-excision and SLN biopsy. Flow cytometry was performed to study viable cells from melanoma SLN and PBMC (prior to tremelimumab administration [day 0], and at 7 days, 3 weeks and 3 months after tremelimumab injection). Systemic melanoma antigen (MART-1/NY-ESO-1)-specific T cells responses were assessed by IFN-γ ELISPOT assay. Results Intradermal delivery of tremelimumab was safe and well tolerated. In terms of biological efficacy it selectively induced profound and durable decreases in Treg frequencies in both SLN and PBMC, decreased systemic MDSC rates, activated migratory dendritic cell subsets in the SLN, and induced T cell activation (by HLA-DR and ICOS up-regulation), both in SLN and PBMC. Moreover, systemic anti-melanoma T cell responses were induced (n=5) or boosted (n=2), in association with T cell activation and central-memory T cell differentiation. Of note, tumor recurrences so far were only observed in two patients who did not develop a systemic anti-tumor T cell response. Conclusions These findings indicate that i.d. administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, they demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and warrant the development of TDLN-targeted delivery methods for anti-CTLA-4. Acknowledgements This study received funding from the Harry J. Lloyd Charitable Trust; tremelimumab was provided by Pfizer Inc. Trial Registration NCT04274816 Ethics Approval The study was approved by the Medical Ethics Committee of the VU University Medical Center and Spaarne Gasthuis.

BackgroundWe 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.MethodsImmune 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.ResultsAlthough 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.ConclusionEx 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.

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. Electronic supplementary material The online version of this article (10.1186/s40425-019-0605-1) contains supplementary material, which is available to authorized users.

Background: Primary treatment for patients with intermittent claudication is exercise therapy. Diabetes mellitus (DM) is a frequently occurring comorbidity in patients with intermittent claudication, and in these patients, exercise tolerance is decreased. However, there is little literature about the increase in walking distance after supervised exercise therapy (SET) in patients with both intermittent claudication and DM. The objective of this study was to determine the effectiveness of SET for intermittent claudication in patients with DM. Methods: Consecutive patients with intermittent claudication who started SET were included. Exclusion criteria were Rutherford stage 4 to 6 and the inability to perform the standardized treadmill test. SET was administered according to the guidelines of the Royal Dutch Society for Physiotherapy. At baseline and at 1, 3, and 6 months of follow-up, a standardized treadmill exercise test was-performed. The primary outcome measurement was the absolute claudication distance (ACD). Results: We included 775 patients, of whom 230 had DM (29.7%). At 6 months of follow-up, data of 440 patients were available. Both ACD at baseline and at 6 months of follow-up were significantly lower in patients with DM (P

Meeting abstracts Breast cancer (BrC)-derived factors inhibit proper development and activation of dendritic cells (DC), which in turn promote the expansion of regulatory T cells (Tregs), thus contributing to tumor progression and spread. In two separate BrC sentinel lymph node (SLN) cohorts, one

Meeting abstracts Since it is the site of initial immune activation and priming of antigen-specific T cells, as well as the first location to which tumor cells metastasize, our research focuses on understanding the immune status and tumor-induced suppression within breast cancer (BrCa)-draining

While systemic immune checkpoint blockade is unsurpassed in the induction of clinically effective T cell immunity in advanced-stage melanoma patients, the risk of autoimmune-related side effects precludes its application in earlier stages. In a Phase-1 dose escalation study we have studied the safety, feasibility and immunological effects of intradermal delivery of a single low dose of anti-CTLA-4/tremelimumab at the primary tumor excision site of patients with clinically Stage I-II melanoma. Dose escalation (4x3 cohorts of 2, 5, 10 and 20 mg) has been concluded with one additional patient enrolled in a 20 mg expansion cohort. Breslow thickness ranged from 0.9 to 6 mm (mean 1.98 mm). Examination of the sentinel lymph node (SLN) revealed (micro-)metastases in five of the patients. The tremelimub administration was found to be safe without apparent side effects except for one case of localized vitiligo at the highest (20 mg) dose level. Of note, potent immunological effects were observed in equal measure at all dose levels. In all patients a decrease in systemic rates of activated Tregs (aTregs, defined as CD4+CD25hiFoxP3hi T cells) was observed after 7 days of treatment (p Specific T cell reactivity against long peptides derived from the melanoma antigens NY-ESO-1 and MART-1 was tested after in vitro re-stimulation in an IFNγ Elispot assay. In 7/13 patients evidence was found of tremelimumab-induced increases in systemic T cell rates, which remained elevated up to 12 weeks post-treatment. Remarkably, 5/13 patients showed pre-existent NY-ESO-1 reactivity, which in all cases had decreased by day 7, and in 3/5 patients had increased over baseline levels by day 21. T cell reactivity to either NY-ESO-1 or MART-1 was observed in 4/4 tested SLN. Of note, both NY-ESO-1 and MART-1 reactive T cells were detected in the SLN of the patient experiencing an isolated incidence of vitiligo after receiving 20 mg tremelimumab. In conclusion, local administration of a single low dose of anti-CTLA-4/tremelimumab has proven safe and effective in terms of attenuating aTreg levels in peripheral blood and boosting systemic antitumor immunity. As such it may offer an attractive, and up to now sorely lacking, adjuvant treatment option for early-stage melanoma patients at risk for tumor recurrence. Citation Format: Anita G.M. Stam, Kim M. van Pul, Bas D. Koster, Dafni Chondronasiou, Sinéad M. Lougheed, M. Petrousjka van den Tol, Karin Jooss, Ronald J.C.L.M. Vuylsteke, Alfons J.M. van den Eertwegh, Tanja D. de Gruijl. Single low-dose administration of anti-CTLA-4/tremelimumab at the tumor excision site boosts systemic antitumor immunity in early-stage melanoma patients: Results from a Phase-1 study. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B138.

The sentinel lymph node (SLN) is an emerging focus for immunological research in breast cancer. Cryopreservation of SLN single-cell suspensions allows for simultaneous phenotypic multi-parameter analyses and minimizes operator dependent variability. This is of particular importance for immunomonitoring of large multicenter trials. However, little data are available regarding the influence of cryopreservation on phenotypic characteristics of lymph node dendritic cells and T cells. In this study we assessed the feasibility of cryopreservation of viable SLN cell samples for flowcytometric analysis, by comparing quantitative analyses of SLN cell samples after freeze-thawing with direct analysis of fresh SLN cell samples. SLN were collected from nine breast cancer patients. From each SLN cell sample, half was used for immediate analysis and half was analyzed after cryopreservation and thawing. Conventional dendritic cell (cDC) and T cell subsets were quantified and phenotypically characterized by flow cytometry. The observed frequencies of both CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subsets showed significant correlation between the fresh and frozen-thawed samples. Similar high correlations were found for CD83 and CD86 expression markers on the more frequent (>0.2%) CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subset, but not on the low-frequency (

Background Increasing evidence suggests that immune regulated pathways influence both breast cancer (BrC) development and response to (neo) adjuvant chemotherapeutic treatment. The sentinel lymph node (SLN) is the first site of BrC induced suppression of immune effector subsets, rendering them unable to mount an effective anti-tumor response. Since detailed knowledge of the functional status of these immune effector subsets is lacking, we compared the immune status of BrC SLN with healthy lymph nodes (HLN) using viable cell samples and correlated our findings to several clinico-pathological characteristics. Additionally we investigated if ex vivo conditioning with the Toll-like receptor-9 ligand CpG-B with or without simultaneous inhibition of JAK2/STAT3 signaling (a downstream signaling pathway implicated as the ‘master switch’ in tumor induced immune suppression) could overcome the observed immune suppression in BrC SLN. Methods Viable SLN cells were obtained from 40 clinically node negative BrC patients. Axillary HLN were obtained from 17 BRCA-1 or -2 patients undergoing a prophylactic mastectomy. Frequencies and activation state of dendritic cell (DC) subsets and regulatory T cells (Treg) were determined by extensive multi-color flowcytometric analyses. Additionally, SLN cells from 12 BrC patients were cultured in absence and presence of CpG-B (PF-3512676) and the combination of CpG-B with a JAK2-STAT3 inhibitor (AG-490). Results Of the 40 BrC SLN 11 contained metastasis. We found clear evidence of BrC-related immune suppression, as significantly higher Treg frequencies were observed in metastasis negative BrC SLN as compared to HLN. These frequencies further increased in metastasis positive BrC SLN. Activation state (by expression of the co-stimulatory molecules CD40, CD83 and CD86) of lymph node resident (LNR)-DC subsets (both CD11c+ myeloid DC and CD123+BDCA2+ plasmacytoid DC), but not of CD1a+ migratory subsets, was significantly lower in BrC SLN as compared to HLN. Additionally, in BrC SLN, activation state of these LNR-DC subsets was further reduced upon metastatic involvement and in non-luminal BrC subtypes (triple negative and HER-2+) as compared to luminal BrC subtypes (ER and/or PR positive). We previously showed in early-stage melanoma patients that local treatment with CpG-B led to the preferential activation of LNR-DC subsets. Similarly, ex vivo targeting of BrC SLN with CpG and CpG + JAK2/STAT3 inhibition resulted primarily in significantly enhanced activation of LNR-DC subsets, with superior effects of combined CpG and JAK2/STAT3 targeting evidenced by significantly higher CD83 expression. Conclusion BrC induced immune suppression in SLN seems to be primarily mediated by hampered activation of LNR-DC subsets, which was most effectively overcome (ex-vivo) with the immune activating compound CpG-B in combination with a small-molecule inhibitor of the immune suppressive JAK2/STAT3 signaling pathway (AG490). This combined immune targeting strategy might be of clinical benefit in enhancing effectiveness of conventional (neo)adjuvant chemotherapeutic treatment, especially in -more immune suppressed- non-luminal BrC subtypes with known poor prognosis. Citation Format: Kim M van Pul, Ronald JCLM Vuylsteke, Sinéad M Lougheed, Lisette EA te Velde, Petrousjka van den Tol, Emiel J Th Rutgers, Hein BAC Stockmann, Tanja D de Gruijl. Breast cancer related immune suppression in the sentinel lymph node can be effectively countered by combined CpG-B administration and JAK2/STAT3 inhibition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-01.