Demonstration of the Antitumor Activity of the iNKT Agonist ABX196, a Novel Enhancer of Cancer Immunotherapy, in Melanoma and Hepatocarcinoma Mouse Models.

Immune checkpoint blockers (ICB) provide a promising approach to antitumor immunotherapy through blockade of immunosuppressive pathways. The synthetic glycolipid, ABX196, is a potent stimulator of invariant natural killer T cells (iNKT), a small subset of regulatory lymphocytes, which are powerful enhancers of immunity when activated. ABX196 was investigated alone and in combination with chemotherapy and ICBs in a melanoma B16F10 tumor cell-bearing and an orthotopic Hepa 1-6 hepatocarcinoma (HCC) cell-bearing C57BL/6 mice model. In the melanoma model, immune response evaluation included immunofluorescence staining and detection by flow cytometry to identify anti-CD45, anti-CD8, anti-CD4, anti-CD3, anti-CD19, anti-FoxP3, CD1d tetramer, and anti-programmed cell death protein 1 (PD-1) markers. Analysis by MRI, liver weight, and IHC staining to detect CD4, CD8, F4/80, PD-1, programmed death-ligand 1, Ki67, and FoxP3 markers were used to measure antitumor response in the HCC model. Combination treatment with ABX196 and anti-PD-1 resulted in significant synergistic antitumor effects, reflected by the increase of CD8+ cells in the tumor and an increased ratio of CD8+ effector cells to FoxP3+ regulatory T cells (Treg) in mice with melanomas. ABX196 monotherapy and combination therapy resulted in antitumor effects in the HCC model. No significant differences in survival were demonstrated between monotherapy and combination therapy due to high response levels with either treatment. A synergistic combination effect was apparent when IFNγ was measured in peripheral blood, indicating sustained activation of iNKT cells. In both models, the antitumor effects were associated with a generation of a more advantageous T-effector to Treg cell ratio within the tumor, which could lead to in the proliferation and accumulation of cells that would otherwise be anergized. Using melanoma and HCC tumor models in mice, this study demonstrates the potential of ABX196, alone and in combination with anti-PD-1 antibody, as a novel strategy to overcome the immunosuppressive microenvironment and to produce antitumor activity.

Scherrer D ，Barrett N ，Teyton L ，Pearce T ，Nitcheu J ，Pouletty P ，Santo J ，Ehrlich HJ ... -  《-》

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas.

Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell-mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non-small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

Zhou G ，Sprengers D ，Boor PPC ，Doukas M ，Schutz H ，Mancham S ，Pedroza-Gonzalez A ，Polak WG ，de Jonge J ，Gaspersz M ，Dong H ，Thielemans K ，Pan Q ，IJzermans JNM ，Bruno MJ ，Kwekkeboom J ... -  《-》

Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3.

Stimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to immune-checkpoint blockade (ICB) can cast significant challenges to the therapeutic options for patients with hard-to-treat tumors. There is an unmet clinical need to establish new therapeutic approaches for mitigating ICB unresponsiveness in patients. In this study, we investigated the efficacy and role of low-dose antineoplastic agent SN-38 or metformin in sensitizing unresponsive tumors to respond to ICB therapy. We assessed the significant pathological relationships between PD-L1 and FOXO3 expression and between PD-L1 and c-Myc or STAT3 expression in patients with various tumors. We determined the efficacy of low-dose SN-38 or metformin in sensitizing unresponsive tumors to respond to anti-PD-1 therapy in a syngeneic tumor system. We deciphered novel therapeutic mechanisms underlying the SN-38 and anti-PD-1 therapy-mediated engagement of natural killer (NK) or CD8+ T cells to infiltrate tumors and boost antitumor immunity. We showed that PD-L1 protein level was inversely associated with FOXO3 protein level in patients with ovarian, breast, and hepatocellular tumors. Low-dose SN-38 or metformin abrogated PD-L1 protein expression, promoted FOXO3 protein level, and significantly increased the animal survival rate in syngeneic mouse tumor models. SN-38 or metformin sensitized unresponsive tumors responding to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the tumor microenvironment (TME) and secret interferon-γ and granzyme B to kill tumors. SN-38 suppressed the levels of c-Myc and STAT3 proteins, which controlled PD-L1 expression. FOXO3 was essential for SN38-mediated PD-L1 suppression. The expression of PD-L1 was compellingly linked to that of c-Myc or STAT3 in patients with the indicated tumors. We show that SN-38 or metformin can boost antitumor immunity in the TME by inhibiting c-Myc and STAT3 through FOXO3 activation. These results may provide novel insight into ameliorating patient response to overarching immunotherapy for tumors.

Chung YM ，Khan PP ，Wang H ，Tsai WB ，Qiao Y ，Yu B ，Larrick JW ，Hu MC ... -  《Journal for ImmunoTherapy of Cancer》

B Cells Are Required to Generate Optimal Anti-Melanoma Immunity in Response to Checkpoint Blockade.

Immunotherapies such as checkpoint blockade therapies are known to enhance anti-melanoma CD8+ T cell immunity, but only a fraction of patients treated with these therapies achieve durable immune response and disease control. It may be that CD8+ T cells need help from other immune cells to generate effective and long-lasting anti-tumor immunity or that CD8+ T cells alone are insufficient for complete tumor regression and cure. Melanoma contains significant numbers of B cells; however, the role of B cells in anti-melanoma immunity is controversial. In this study, B16 melanoma mouse models were used to determine the role of B cells in anti-melanoma immunity. C57BL/6 mice, B cell knockout (KO) C57BL/6 mice, anti-CD19, and anti-CXCL13 antibody-treated C57BL/6 mice were used to determine treatment efficacy and generation of tumor-specific CD8+ T cells in response to PD-L1 blockade alone or combination with TLR-7/8 activation. Whole transcriptome analysis was performed on the tumors from B cell depleted and WT mice, untreated or treated with anti-PD-L1. Both CD40-positive and CD40-negative B cells were isolated from tumors of TLR-7/8 agonist-treated wild-type mice and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1 and TLR-7/8 agonist. Therapeutic efficacy was determined in the presence of activated or inactivated B cells. Microarray analysis was performed on TLR-7/8-treated tumors to look for the B cell signatures. We found B cells were required to enhance the therapeutic efficacy of monotherapy with anti-PD-L1 antibody and combination therapy with anti-PD-L1 antibody plus TLR-7/8 agonist. However, B cells were not essential for anti-CTLA-4 antibody activity. Interestingly, CD40-positive but not CD40-negative B cells contributed to anti-melanoma immunity. In addition, melanoma patients' TCGA data showed that the presence of B cell chemokine CXCL13 and B cells together with CD8+ T cells in tumors were strongly associated with improved overall survival. Our transcriptome data suggest that the absence of B cells enhances immune checkpoints expression in the tumors microenvironment. These results revealed the importance of B cells in the generation of effective anti-melanoma immunity in response to PD-1-PD-L1 blockade immunotherapy. Our findings may facilitate the design of more effective anti-melanoma immunotherapy.

Singh S ，Roszik J ，Saini N ，Singh VK ，Bavisi K ，Wang Z ，Vien LT ，Yang Z ，Kundu S ，Davis RE ，Bover L ，Diab A ，Neelapu SS ，Overwijk WW ，Rai K ，Singh M ... -  《-》

Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 T cells in a poorly immunogenic tumor model.

Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti-4-1BB/anti-PD-1 combination with that of the anti-PD-1/anti-LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti-PD-1 and anti-4-1BB concomitantly, while combining anti-PD-1 with anti-LAG-3 led to a modest degree of tumor suppression. The activity of the anti-4-1BB/anti-PD-1 combination was dependent on IFNγ and CD8(+) T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8(+) T cells by anti-4-1BB/anti-PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8(+)/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8(+) CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti-4-1BB/anti-PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients.

Chen S ，Lee LF ，Fisher TS ，Jessen B ，Elliott M ，Evering W ，Logronio K ，Tu GH ，Tsaparikos K ，Li X ，Wang H ，Ying C ，Xiong M ，VanArsdale T ，Lin JC ... -  《-》