PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non–small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity.

Adequate spontaneous activation of tumor-specific T lymphocytes in tumor-bearing hosts is rare, despite the expression of tumor antigens that are potentially highly immunogenic. For example, failure of the immune system to raise competent responses against established tumors expressing the human adenovirus E1A-antigen allows this tumor to grow in immunocompetent mice. We show that systemic in vivo administration of agonistic anti-CD40 antibodies into tumor-bearing mice results in tumor eradication mediated by CD8 ؉ T cells. Treatment resulted in a strong expansion and systemic accumulation of E1A-specific CTL and depended on CD40 expression on host cells, as the tumor was CD40 ؊ , and therapy failed in CD40-deficient mice. Local intratumoral administration of anti-CD40 mAb is equally effective in licensing strong, systemic CTL immunity, resulting in the clearance of distant tumor nodules. Our data indicate that the immune response after cancerhost interactions can be directed toward competence, leading to the cure of established tumors merely by delivery of a CD40-dependent ''license to kill'' signal.M ost solid tumors express MHC class I molecules but lack costimulatory molecules essential for appropriate CTL activation (1, 2). Therefore, presentation of tumor-derived antigens by professional antigen-presenting cells (APCs) is most likely required for optimal tumor-specific T cell induction (3-6). Such activation of naïve T cells is called cross-priming and was first demonstrated by Bevan (7). As naïve T cells are thought to recirculate within the lymphoid system, cross-presentation provides the immune system with a means to detect and respond to antigens that are expressed only in the periphery.An important factor determining the outcome of immune responses is the level of antigen expressed in the periphery (8). In the case of relatively low levels of antigen, antigen is not presented at sufficient levels to activate naïve T cells. This situation is associated with ignorance of the antigen by the immune system. In the case of higher antigen-expression levels, antigen will be (cross-)presented in sufficient quantities to be detected by naïve T cells. In this case, antigen-recognition can either lead to tolerance or immunity (9, 10). The outcome of antigen recognition by naïve T cells, i.e., tolerance or immunity, is thought to be the consequence of the activation state of professional APCs that (cross-)present the antigen. This activation state is strongly influenced by inflammatory stimuli as well as the action of CD4 ϩ T helper (Th) cells.Studies on the requirement of CD4 ϩ Th cells in cross-priming of cytotoxic T lymphocytes (CTL) showed that both Th cells and CTLs must recognize antigens presented on the same APC (11,12). The interaction between Th cell and APC is sufficient to convert the APC to a state that allows priming of antigen-specific CTL (13, 14), which explains the observation that infusion of antigen-specific Th cells can rescue autoreactive CTL from deletion, resulting in CTL-mediated autoimmun...

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Purpose: Blockade of CTLA-4 by antibodies has potentiated antitumor T-cell responses in both preclinical models and clinical trials. However, treatment with CTLA-4 blocking antibodies is associated with autoimmune and inflammatory side effects. In this study, we propose a novel administration method for CTLA-4 blocking antibodies as monotherapy.Experimental Design: We use different preclinical mouse models of cancer to investigate the local administration of CTLA-4 blocking antibody and its effect on cancer progression and the antitumor T-cell response.Results: By injecting the antibodies in a subcutaneous slow-release delivery formulation in the tumor area, we show that an eight-fold lower dose of antibody is as effective in inducing tumor eradication as systemic delivery. A lower dose and slow release of the antibody results in thousand-fold decreased levels of antibody in the serum, reducing adverse events and the risk of autoimmunity. The main target and effector cells of the CTLA-4 blockade treatment in the studied tumor models are tumor-specific endogenous CD8 þ T cells that are capable of eradicating also distant tumors, whereas CD4 þ T cells do not play a prominent role in the antibody-mediated tumor eradication. Conclusions: Injecting CTLA-4 blocking antibody in a slow-release formulation close to the tumor is an effective way of activating the antitumor T-cell response. This administration method is associated with very low serum levels of antibody, which decreases the risk of treatment-induced side effects. These results call for exploration of a similar delivery principle in clinical settings.

Purpose: Immunotherapy against tumors with anti-CD40 agonistic antibodies has been extensively studied in preclinical animal models and recently also in clinical trials. Although promising results have been obtained, antibody (Ab)-related toxicity has been a limiting factor. We reasoned that strict local activation of tumor-specific CD8 T cells through stimulation of CD40 on the dendritic cells (DC) in the tumor area while excluding systemic stimulation might be sufficient for effective tumor eradication and can limit systemic toxicity.Experimental Design: Preclinical in vivo models for immunogenic tumors were used to investigate the potential of delivering a nontoxic dose of agonistic anti-CD40 Ab to the tumor region, including draining lymph node, in a slow-release formulation (montanide).Results: The delivery of anti-CD40 monoclonal Ab, formulated in slow-release Montanide ISA-51, reprograms CTLs by inducing local but not systemic DC activation, resulting in effective tumor-specific CTL responses that eradicate local and distant tumors. Adverse side effects, assayed by organ histology and liver enzymes in the blood, were much lower after local anti-CD40 Ab delivery than systemic administration. The local delivery of anti-CD40 Ab activates only CTLs against antigens presented in the tumor-draining area, because unrelated distant tumors expressing different tumor antigens were not eradicated.Conclusions: These results establish a novel therapeutic principle that local delivery and slow release of agonistic anti-CD40 Ab to the tumor-draining area effectively activates local tumor-specific CD8 T cells to become systemic effectors without causing systemic toxicity or nonspecific CTL activation. These findings have important implications for the use of anti-CD40 therapies in patients.

The fate of naive CD8+ T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c+ APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8+ CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.

Immunotherapy with PD-1/PD-L1-blocking antibodies is clinically effective for several tumor types, but the mechanism is not fully understood. PD-L1 expression on tumor biopsies is generally regarded as an inclusion criterion for this cancer therapy. Here, we describe the PD-L1-blocking therapeutic responses of preclinical tumors in which PD-L1 expression was removed from cancer cells, but not from immune infiltrate. Lack of PD-L1 expression on malignant cells delayed tumor outgrowth in a CD8+ T cell-mediated fashion, showing the importance of this molecule in immune suppression. PD-L1 expression was evident on myeloid-infiltrating cells in the microenvironment of these tumors and targeting stromal PD-L1 with blocking antibody therapy had additional antitumor effect, demonstrating that PD-L1 on both malignant cells and immune cells is involved in the mechanism of immunotherapeutic antibodies. Importantly, comparable results were obtained with PD-1-blocking therapy. These findings have implications for inclusion of cancer patients in PD-1/PD-L1 blockade immunotherapies.

Targeting of antigens to dendritic cells (DCs) to induce strong cellular immune response can be established by loading in a nano‐sized carrier and keeping the antigen associated with the particles until they are internalized by DCs. In the present study, a model antigen (ovalbumin, OVA) is immobilized in cationic dextran nanogels via disulfide bonds. These bonds are stable in the extracellular environment but are reduced in the cytosol of DCs due to the presence of glutathione. Reversible immobilization of OVA in the nanogels is demonstrated by the fact that hardly any release of the protein occurred at pH 7 in the absence of glutathione, whereas rapid release of OVA occurs once the nanogels are incubated in buffer with glutathione. Furthermore, these OVA conjugated nanogels show intracellular release of the antigen in DCs and boost the MHC class I antigen presentation, demonstrating the feasibility of this concept for the aimed intracellular antigen delivery.