- Top of page
- Material and Methods
- Supporting Information
Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40–60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from anti-CD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and, due to limited treatment options, the third greatest cause of cancer death. Despite numerous attempts at amelioration, curative treatments continue to exhibit a very high recurrence rate.
The liver possesses a specific immune system, which plays an intricate role in its functions and pathobiology, including the progressive evolution of chronic liver disease to fibrosis, cirrhosis and finally to HCC. Work in recent years has demonstrated that local and systemic immunity can be harnessed to fight hepatic neoplastic development, and a variety of evidence points toward immunotherapy as a promising strategy against HCC. The presence of lymphocytic infiltrates has been shown to correlate with improved overall survival of HCC patients after surgical resection. T lymphocytes specific for some tumor antigens have been detected in HCC patients. In some patients, conventional treatment has been shown to enhance immune responses, suggesting that immunotherapy, as an adjuvant to, or following primary treatments,[5, 6] could be beneficial for the patient, especially as a solution to the high recurrence risk.
Antigen specific immunotherapy could be a valid option for the treatment of HCC and a number of strategies are in development at a clinical level.[7, 8] Complementary to these approaches requiring a certain level of personalization (not all HCC express all tumor antigens), there is a place for nonspecific immunotherapy strategies: to block the suppressive mechanisms that arise during tumor development and impede the anti-tumor immune response, or to directly enhance this natural anti-tumor immune response. It is noteworthy that, to date, some of the most promising clinical results for HCC immunotherapy have been obtained by adoptive transfer of autologous T lymphocytes nonspecifically stimulated after surgical tumor resection.
CD137 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is expressed by many cell types including activated T cells, NK and NKT cells, dendritic cells, neutrophils or monocytes. The binding of its ligand CD137L to CD137 leads to the prolonged survival and sustained activation of CD8 T cells. Agonistic anti-CD137 monoclonal antibody fixation to CD137 enhances T-cell proliferation and cytokine production by T-helper lymphocytes, and protects CD8 T cells from activation-induced cell death.[12, 13] Agonistic anti-CD137 antibody induces regression of established tumors in various animal models,[14-16] and prevents tumor recurrence in a model of melanoma. In low tumor burden conditions this nonspecific immunotherapy strategy could be well-suited to the treatment of HCC in order to stimulate in situ the natural anti-tumor immune response. To test this hypothesis, we investigated the anti-tumor effect of an agonistic anti-CD137 antibody treatment on murine models of HCC for which the tumor develops within the liver of immunocompetent animals. We show the therapeutic efficacy of this approach. The mechanistic analyses demonstrate that the treatment mobilizes T cells, NK cells and allows the early and sustained recruitment of macrophages in the tumor nodules and that the immune response elicited by the treatment keeps potentially harmful regulatory cells at bay.
- Top of page
- Material and Methods
- Supporting Information
In the present work we demonstrate the therapeutic efficacy of agonistic anti-CD137 mAb in two models of in situ development of HCC, with a 65% reduction of the tumor burden in the orthotopic model. On the more stringent model of chemically induced HCC, a 42% reduction of the tumor burden was obtained after only three injections of 100 µg of the anti-CD137 mAb during 8 months of liver carcinogenesis.
In the orthotopic HCC model, the treatment induces a strong memory response, which protects the mice against a second presentation of tumor cells 3 months after the first, and after the anti-CD137 mAb injections. Moreover, adoptive transfer of splenocytes prepared from mice treated and cured using anti-CD137 mAb inhibits the tumor growth in recipient naive mice. These are very significant results, which reinforce the validity of this approach to treating tumor recurrence in the liver.
This effect is primarily mediated by NK and T lymphocytes. These two cell types are the most common effectors involved in the anti-tumor effect of the agonistic anti-CD137 mAb for various malignancies. In our model, the therapeutic effect of the anti-CD137 mAb treatment is limited to delayed tumor growth in CD4+ T cell-depleted mice, consistent with a role of these cells in enhancing the expansion and cytolytic function of tumor reactive CD8+ T cells and in the maintenance of a long-term memory response. Functionally, CD8+ and CD4+ T cells, and NK cells from the liver of treated mice have an increased capacity to secrete IFNγ. This could be responsible for the decreased percentage and number of NK cells in the liver and in the spleen. Indeed, it has recently been shown that CD137-induced IFNγ production downregulates NK cell development in bone marrow, leading to a decreased number of these cells in the periphery.
Anti-CD137 mAb treatment consistently led to 40–60% of the mice completely cured after two injections, and as early as 1 day after the second mAb injection, a specific proliferation of liver CD8+ and CD4+ T cells can be detected in only half the mice, though it is not yet possible to identify responder from non responder mice. On day 14, a time when responders already show a measurably reduced tumor burden, the responders have more liver-infiltrating CD8+ and CD4+ T cells than at day 9. At the same point the anti-tumor immune response seems to have “aborted” in the nonresponders.
We observe an early variation in liver macrophages after anti-CD137 mAb treatment. Macrophages can, depending on their polarization, either favor or limit the host immune response. The M1 phenotype is induced by IFNγ and is associated with anti-tumor activity and iNOS expression. The M2 phenotype is associated with tissue remodeling, anti-inflammatory cytokine secretion and arginase-1 expression. Presence of tumor infiltrating macrophages in HCC after resection has been correlated with both good and bad prognosis.[26, 27] In absence of treatment, we observed that macrophages are present in the periphery of the tumor nodules but not inside the nodules. The anti-CD137 mAb treatment allows the macrophages to penetrate within tumors early in their development and the sustained infiltration of tumor nodules with macrophages is correlated with a favorable outcome for the animals. Macrophage behavior is largely coordinated by cytokine/chemokine signaling and consistent with this, CCL2 (MCP-1), a chemokine known to have a major role in the recruitment and activation of macrophages, as well as IL6 proinflammatory cytokine, are found in increased amounts in the liver of responder mice at an early time point. This concords with results obtained in human HCC, where the expression of IL-6 and of CCL2 have been associated with a good prognosis. Finally, we demonstrate that the tumor infiltrating macrophages express iNOS, a marker of M1 proinflammatory/anti tumoral polarization.
Regulatory immune cells might also be involved in the early arrest of anti-tumor immune response in the non responder mice. Increased populations of MDSC and Tregs have been found in patients with HCC.[29, 30] We observed that the liver populations of MDSC and Treg cells increase along with tumor development. This significant infiltration of regulatory cells does not happen in animals responding to the anti-CD137 mAb treatment, suggesting that in these animals the tumor environment is skewed toward a proinflammatory, efficacious anti-tumor immune response, maybe as a consequence of the action of the infiltrating M1 macrophages.
To summarize these results, we propose a model in which HCC development is accompanied both by a natural anti-tumor immune response (T lymphocytes and NK cells) and by suppressive strategies to overcome this immune response (MDSC and Treg cells). In some mice, the anti-CD137 mAb treatment tips the balance towards an efficacious anti-tumor response, and the early events identified for this to happen are a significant infiltrate of CD8+ T cells, a massive IFNγ secretion potential by various populations of NPC, and a sustained recruitment within the nodules and an activation of pro-inflammatory macrophages, which in turn increase the inflammatory environment surrounding the tumor nodules. In other mice, however, the immune response induced by the treatment is subdued, leading to an increased infiltration of regulatory immune cells, MDSC, Treg cells, which in turn inhibit the anti-tumor immune response and lead to further tumor growth. DEN-induced liver carcinogenesis is accompanied with local and systemic inflammatory reactions, with an accumulation of macrophages and lymphocytes and its clinical relevance is well established.[31, 32] Thus the mechanisms underlying the therapeutic effect might be comparable to that shown in the orthotopic model.
Anti-CD137 mAb has been used in combination with various other strategies in the treatment of other tumors.[33-35] Recently, the combination of anti-CD137 mAb with other immunostimulatory antibodies (anti-PD-L1 and anti-OX40) has been shown to lead to an extension of the survival in a transgenic model of HCC, however, none of the immunostimulatory Abs alone generated a therapeutic effect. The difference in efficacy of the treatment with the anti-CD137 mAb between this study and ours could be due to the difference in the HCC models, which are of different “aggressiveness.” Indeed, the DEN treatment will lead to the initiation of some hepatocytes, and in a comparable manner, only a limited number of tumor Hepa1.6 cells are injected in the liver in the orthotopic model, whereas in the transgenic model, all the hepatocytes express the c-myc oncogene.
Increased frequency of MDSC has been demonstrated in patients with HCC, as well as in various murin models of the disease.[30, 36] We show here that the depletion of these cells with an anti-Gr1 Ab produces a modest therapeutic effect, however, the combination of the anti-CD137 mAb and anti-Gr1 Ab, leads to 80% of recovery. Although there is little neutrophil infiltration in the present model, we cannot exclude a role of these cells since they are also depleted by the anti-Gr1 Ab. Further more specific deletion experiments will be necessary to conclude on this matter.
Finally, there have been some reports of liver toxicity associated with the anti-CD137 mAb treatment. A phase II clinical trial (NCT00612664) of the BMS-663513, the humanized anti-CD137 mAb, as a second line treatment of melanoma patients, was terminated due to unusual high incidence of hepatitis. Liver toxicity was also observed in some mouse models[39, 40] and not in others, nor in ours. These differences could be due to the antibody doses, and/or to the number and frequency of injections, which vary from one study to another, and require further in-depth analysis. A new clinical phase 1 study is currently evaluating the safety, and tolerability of low doses of BMS-663513 (NCT01471210).
In conclusion, this study demonstrates that stimulation of CD137 is a promising strategy for HCC immunotherapy. Our results provide justification for further investigation into the use of agonistic anti-CD137 mAb associated with first line conventional HCC treatments, and they also suggest that concomitant inhibition of regulatory immune cells may further increase anti-tumor immunity. Such a combination should be considered as a potential therapeutic option for treating HCC patients.