Combined immunotherapy with anti-PDL-1/PD-1 and anti-CD4 antibodies cures syngeneic disseminated neuroblastoma

Abstract

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immuneenhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4 + CD25 + Treg cells and other CD4 + CD25 − regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4 + T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.Immune checkpoints are fundamental for the physiological maintenance of tolerance and protection of tissues from the damage that an unregulated immune response may cause. It is now clear that immune checkpoint dysregulation represents an important mechanism through which tumors escape immune system recognition and progress 1 . Studies in preclinical tumor models and cancer patients have proven that the targeting of immune checkpoints restores a silenced anti-tumor immune response, which then becomes effective in eliminating tumor cells. Indeed, the monoclonal antibodies (mAbs) blocking immune checkpoints such as Cytotoxic T-Lymphocyte Antigen (CTLA)-4 and Programed Death-Ligand1 (PD-L1)/Programed Death-1 (PD-1) showed extraordinary anti-tumor effects in clinical trials leading to their approval as anti-cancer drugs in different metastatic cancers 2 . In particular, anti-PD-1 mAbs have shown unprecedented clinical activity in patients with metastatic melanoma 3-8 , non-small-cell lung carcinoma (NSCLC) 9,10 and renal cell carcinoma 11 , with a significant fraction of patients showing prolonged clinical benefit. The combination of anti-PD-1 and anti-CTLA-4 leads to a further increase in clinical responses in metastatic melanoma 12 . Synergistic therapeutic activity may relate to the different site of action of CTLA4 and PD-L1/PD-1-blocking agents, i.e. the secondary lymphoid organs and the peripheral tissues, respectively. In addition, CTLA-4 blockade induces a proliferative gene expression signature

Sensitizing the Tumor Microenvironment to Immune Checkpoint Therapy

“…Experiments in murine cancer models have consistently demonstrated the importance of T cells for the efficacy of ICB (27)(28)(29). In humans, secondary resistance to ICB has also been associated with T cell deficits, including mutations in cancer cells associated with decreased sensitivity to T cell-mediated killing, or reduced antigen presentation to T cells (30,31).…”

“…CD8 + T cells have been shown to be the anti-tumor effector cells, and sensitivity to ICB was enhanced in tumors enriched for CD8 + T cells reactive to clonal neoantigens (35). The differential role of CD8 + vs. CD4 + T cells is less clear, with conflicting outcomes in different tumor models (28,29). The interpretation of these results is difficult because CD4 depletion in mice not only depletes T effector cells, but also regulatory T cells (T-regs) (36).…”

Monitoring Immune Responses in Neuroblastoma Patients during Therapy

“…Nevertheless, the potency of CPis to induce a more potent anti-tumor immune response, and therefore potential induction of anti-tumor immunological memory, makes checkpoint inhibition a very interesting candidate as an adjuvant therapy in curative treatment settings. The utilization of CPis as a monotherapy in NBL has been investigated in multiple pre-clinical studies [42,[111][112][113]. These studies show no effect of CPi treatment on systemic NBL progression in vivo.…”

“…Absence of TILs and PD-L1 tumor expression provides a first rationale to CPi therapy resistance. Secondly, one of the immunomodulatory mechanisms of NBL is the upregulation of PD-L1 in response to IFN-γ [111,113]. This highlights a potential resistance mechanism against CPi therapy, as persistent IFN-γ production by activated T cells may lead to a continuous cascade causing even higher upregulation of the immune checkpoints resulting in T cell senescence.…”

Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA

“…For that, VLPs containing HIV-1 Gag and Env were injected intramuscularly in a prime-boost regimen into naïve BALB/c mice. Two days after each immunization, mice were treated with either PBS, an isotype control, or monoclonal antibodies directed against PD-1 or its ligands PD-L1 and PD-L2 according to published protocols ( Figure 1A) [23,24]. After the boosting immunization, however, we observed no significant differences regarding the levels of IgG1 ( Figure 1B) and IgG2a ( Figure 1C) between all experimental groups immunized with VLPs.…”