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 reg ) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted ( N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods.

Background: We previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8 + T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration., Methods: Re-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles., Results: Significant increases in CD83 + , CD14 + , CD68 + , and CD123 + APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123 + BDCA2 + plasmacytoid dendritic cells (DCs) and BDCA3/CD141 + CLEC9A + type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14 + APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14 + monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration., Conclusion: The CpG-B-induced concerted recruitment of cDC1 and CD14 + APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Whereas TLR9 agonists are recognized as powerful stimulators of antitumor immunity, GM-CSF has had mixed reviews. In previously reported randomized trials we assessed the effects of local immune modulation in early-stage melanoma with CpG-B alone or with GM-CSF. Here we discuss the added value of GM-CSF and show sex-related differences., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Melanoma exerts immune-suppressive effects to facilitate tumor progression and metastatic spread. We studied these effects on dendritic cell (DC) and T-cell subsets in 36 melanoma sentinel lymph node (SLN) from 28 stage I-III melanoma patients and determined their clinical significance. Four conventional DC subsets, plasmacytoid DCs, and CD4 + , CD8 + , and regulatory T cells (Tregs), were analyzed by flow cytometry. We correlated these data to clinical parameters and determined their effect on local and distant melanoma recurrence, with a median follow-up of 75 months. In stage I and II melanoma, increased Breslow thickness (i.e., invasion depth of the primary melanoma) was associated with progressive suppression of skin-derived migratory CD1a + DC subsets. In contrast, LN-resident DC subsets and T cells were only affected once metastasis to the SLN had occurred. In stage III patients, increased CD4:CD8 ratios in concert with the accumulation of Tregs resulted in decreased CD8:Treg ratios. On follow-up, lower frequencies of migratory DC subsets proved related to local melanoma recurrence, whereas reduced maturation of LN-resident DC subsets was associated with distant recurrence and melanoma-specific survival. In conclusion, melanoma-mediated suppression of migratory DC subsets in the SLN precedes local spread, whereas suppression of LN-resident DC subsets follows regional spread and precedes further melanoma dissemination to distant sites. This study offers a rationale to target migratory as well as LN-resident DC subsets for early immunotherapeutic interventions to prevent melanoma recurrence and spread. Cancer Immunol Res; 5(11); 969-77. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Although risk of recurrence after surgical removal of clinical stage I-II melanoma is considerable, there is no adjuvant therapy with proven efficacy. Here, we provide clinical evidence that a local conditioning regimen, aimed at immunologic arming of the tumor-draining lymph nodes, may provide durable protection against disease recurrence (median follow-up, 88.8 months). Experimental Design: In two randomized phase II trials, patients, diagnosed with stage I-II melanoma after excision of the primary tumor, received local injections at the primary tumor excision site within 7 days preceding re-excision and sentinel lymph node (SLN) biopsy of either a saline placebo ( n = 22) or low-dose CpG type B (CpG-B) with ( n = 9) or without ( n = 21) low-dose GM-CSF. Results: CpG-B treatment was shown to be safe, to boost locoregional and systemic immunity, to be associated with lower rates of tumor-involved SLN (10% vs. 36% in controls, P = 0.04), and, at a median follow-up of 88.8 months, to profoundly improve recurrence-free survival ( P = 0.008), even for patients with histologically confirmed (i.e., pathologic) stage I-II disease ( P = 0.02). Conclusions: Potentially offering durable protection, local low-dose CpG-B administration in early-stage melanoma provides an adjuvant treatment option for a large group of patients currently going untreated despite being at considerable risk for disease recurrence. Once validated in a larger randomized phase III trial, this nontoxic immunopotentiating regimen may prove clinically transformative. Clin Cancer Res; 23(19); 5679-86. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. In an effort to determine the optimal way to strengthen immune defenses, 28 clinical stage I-II melanoma patients were randomized in a 3-arm Phase II study to receive, prior to excision and sampling of the SLN, i.d. injections of saline or low-dose CpG-B (CpG), alone or combined with GM-CSF (GM), around the melanoma excision site. We previously described the combined administration of these DC-targeting agents to result in activation and recruitment of potentially cross-presenting BDCA3(+) DCs to the SLN. In this report we describe the effects on effector and regulatory T and NK cell subsets. Local low-dose CpG administration resulted in lower CD4/CD8 ratios, Th1 skewing, increased frequencies of melanoma-specific CD8(+) T cells and possible recruitment of effector NK cells, irrespective of GM co-administration. These immune-potentiating effects were counterbalanced by increased IL-10 production by T cells and significantly higher levels of FoxP3 and CTLA4 in regulatory T cells (Tregs) with correspondingly higher suppressive activity in the SLN. Notably, CpG ± GM-administered patients showed significantly lower numbers of SLN metastases (saline: 4/9, CpG + GM: 1/9, CpG: 0/10, p = 0.04). These findings indicate that i.d. delivery of low-dose CpG ± GM potentially arms the SLN of early-stage melanoma patients against metastatic spread, but that antitumor efficacy may be further boosted by counteracting the collateral activation of Tregs.

Melanoma-induced suppression of dendritic cells (DC) in the sentinel lymph node (SLN) interferes with the generation of protective antitumor immunity. In an effort to strengthen immune defense against metastatic spread, we performed a three-arm phase II study comprising 28 patients with stage I-II melanoma randomized to receive intradermal injections around the primary tumor excision site of saline or low-dose CpG-B, alone or combined with GM-CSF, before excision of the SLNs. After pathologic examination, 5 patients were diagnosed with stage III melanoma based on the presence of tumor cells in the SLNs. Combined CpG/GM-CSF administration resulted in enhanced maturation of all identifiable conventional (cDC) and plasmacytoid (pDC) DC subsets and selectively induced increased frequencies of SLN-resident BDCA3/CD141(+) cDC subsets that also expressed the C-type lectin receptor CLEC9A. Correlative in vivo analyses and in vitro studies provided evidence that these subsets were derived from BDCA3(+) cDC precursors in the blood that were recruited to the SLNs in a type I IFN-dependent manner and subsequently matured under the combined influence of CpG and GM-CSF. In line with their reported functional abilities, frequencies of in vivo CpG/GM-CSF-induced BDCA3/CD141(+) DCs correlated with increased ex vivo cross-presenting capacity of SLN suspensions. Combined local CpG/GM-CSF delivery thus supports protective antimelanoma immunity through concerted activation of pDC and cDC subsets and recruitment of BDCA3(+) cDC subsets with T cell-stimulatory and cross-priming abilities., (©2015 American Association for Cancer Research.)

We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8(+) T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8(+) T cells. Moreover, recall and melanoma antigen-specific CD8(+) T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo-primed specific CD8(+) T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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 (Treg) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted (N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti–CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods. Under the skin: Although systemic immune checkpoint blockade (ICB) displays therapeutic efficacy in cancers, it induces immune-related adverse events (irAEs). Avoiding irAEs is crucial for effective ICB. Van Pul et al. performed a phase 1 clinical trial testing the effects of an intradermal injection of anti–CTLA-4 at the site of primary tumor excision in patients with early-stage melanoma. Seven of 13 patients immunologically responded to the treatment, with little to no irAEs. Responders had more tumor antigen–specific T cells in the blood, increased migratory DC activation in the sentinel lymph node (SLN), and decreased Tregs in both the SLN and blood. Thus, intradermal anti–CTLA-4 after primary tumor excision in melanoma induced a favorable immune response and has promise as a treatment option for patients with early-stage melanoma. [ABSTRACT FROM AUTHOR]

Whereas TLR9 agonists are recognized as powerful stimulators of antitumor immunity, GM-CSF has had mixed reviews. In previously reported randomized trials we assessed the effects of local immune modulation in early-stage melanoma with CpG-B alone or with GM-CSF. Here we discuss the added value of GM-CSF and show sex-related differences. [ABSTRACT FROM AUTHOR]

Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. In an effort to determine the optimal way to strengthen immune defenses, 28 clinical stage I-II melanoma patients were randomized in a 3-arm Phase II study to receive, prior to excision and sampling of the SLN, i.d. injections of saline or low-dose CpG-B (CpG), alone or combined with GM-CSF (GM), around the melanoma excision site. We previously described the combined administration of these DC-targeting agents to result in activation and recruitment of potentially cross-presenting BDCA3 DCs to the SLN. In this report we describe the effects on effector and regulatory T and NK cell subsets. Local low-dose CpG administration resulted in lower CD4/CD8 ratios, Th1 skewing, increased frequencies of melanoma-specific CD8 T cells and possible recruitment of effector NK cells, irrespective of GM co-administration. These immune-potentiating effects were counterbalanced by increased IL-10 production by T cells and significantly higher levels of FoxP3 and CTLA4 in regulatory T cells (Tregs) with correspondingly higher suppressive activity in the SLN. Notably, CpG ± GM-administered patients showed significantly lower numbers of SLN metastases (saline: 4/9, CpG + GM: 1/9, CpG: 0/10, p = 0.04). These findings indicate that i.d. delivery of low-dose CpG ± GM potentially arms the SLN of early-stage melanoma patients against metastatic spread, but that antitumor efficacy may be further boosted by counteracting the collateral activation of Tregs. [ABSTRACT FROM AUTHOR]

To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a+ subsets were identified as most likely skin-derived CD11cint Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11chi dermal DCs with variable expression of langerin. Two other CD1a- LN-residing cDC subsets were characterized as CD14-BDCA3hiCD103- and CD14+BDCA3loCD103+, respectively. Whereas the CD1a+ skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFNγ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a- cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies. [ABSTRACT FROM AUTHOR]