Here we demonstrated that the blockade of TGF-β by a monoclonal antibody (1D11) and a CTL-inducing peptide vaccine synergistically induced anti-tumor immunity in a subcutaneous TC1 tumor model, supporting our hypothesis that removing negative regulators of the immune system enhances tumor vaccine efficacy. These results are consistent with a study with a recombinant Listeria vaccine (33
) and anti-TGF-β antibody (2G7), a recent publication with a recombinant adenoviral vaccine and SM16, a small type I TGF-β receptor kinase inhibitor that blocks SMAD phosphorylation (34
) and a peptide vaccine with 1D11 in glioma model (Ueda et al., manuscript submitted along with this manuscript).
TGF-β has been shown to influence multiple aspects of the tumor microenvironment, including suppression of local immunity. In addition, TGF-β may also play a critical role in negative feedback mechanisms in immune responses to avoid self-destruction caused by over-zealous immune responses. This role is revealed in mice genetically ablated of TGF-β or TGF-β receptor genes (15
). For example, TGF-β1 KO mice show a severe wasting syndrome and multiple organ failure as a result of autoimmune inflammation (15
). Similarly, mice expressing a dominant negative type II TGF-β receptor only on T cells develop a severe autoimmune disease characterized by inflammation in multiple organs and development of autoantibodies (16
). In contrast, long-term treatment of mice with anti-TGF-β 1D11 showed no immune dysregulation (21
). These differences in terms of phenotype of mice genetically ablated of TGF-β or TGF-β receptor gene and of mice chronically treated with 1D11, which binds only activated TGF-β suggest limited access of 1D11 to active TGF-β in vivo.
Dominant negative type II TGF-β receptor transgenic mice also show strong natural tumor immune responses and reject highly invasive tumors, suggesting that despite the fact that TGF-β suppresses almost all lymphoid populations and myeloid cells such as macrophages and DCs (35
), one critical target of TGF-β to support tumor growth is T cells (36
). Therefore, we hypothesized that even if anti-TGF-β alone is not enough to facilitate natural tumor immunosurveillance, blockade of TGF-β at the time of T cell induction by a vaccine should enhance vaccine efficacy. The results that anti-TGF-β significantly enhanced vaccine efficacy as well as anti-tumor CD8+
cell activity measured by in vivo CTL assay in the TC1 tumor model demonstrated that this hypothesis was correct at least in this tumor model.
In studies to design a T cell-inducing vaccine, the efficacy should be determined by both quantity (magnitude) and quality of the responses induced. The quality can be represented in multiple ways, such as cytokine profile and functional avidity. The functional avidity of T cells has been shown to play a critical role in tumor immunity as well as in infectious disease settings (30
T cells with higher functional avidity which can recognize a lower number of antigen-MHC complexes on the target cell surface (37
) and kill targets faster (30
) have been shown to be more effective for tumor rejection (30
). Therefore, we asked whether enhancement of vaccine efficacy by anti-TGF-β is due to induction of increased numbers of high functional avidity CD8+
T cells. The results showed that combination treatment induced an increased number of tumor antigen-specific CD8+
T cells with high functional avidity as measured by intracellular IFN-γ staining and in vivo CTL assay. However, the proportion of high functional avidity cells did not seem to change, so the increased absolute number of high functional avidity CD8+
T cells was due to the greater number of antigen-specific CD8+
T cells induced, including both high and low avidity cells. This observation suggests that there is still room for improvement of the vaccines efficacy by means of inducing a better quality of response. Adding reagents that can skew the response toward higher functional avidity may be worth considering. Recently it was suggested that IL-15 incorporated into a vaccine could induce higher functional avidity CD8+
T cells (41
). Thus, combination of anti-TGF-β and IL-15 together with the vaccine might be a possible way to further improve vaccine efficacy.
Interestingly while anti-TGF-β has been shown to reduce tumor growth in multiple tumor models (1
), anti-TGF-β,. 1D11 alone did not show any impact on growth of the TC1 tumor in vivo. This observation is consistent with a previous study in TC1 tumor model using different anti-TGF-β antibody, 2G7, specific to TGF-β1 (33
) and a recent study with 1D11 in a mouse glioma model (Ueda et al., a manuscript submitted along with this manuscript). At least three potential explanations can be given for the differences in anti-TGF-β effect. One is that in the TC1 model, tumors do not use TGF-β as a major mechanism to evade the immune system although we detected 2257±125.4 pg/ml of TGF-β1 produced in a 3 day culture supernatant. This idea may be supported by the results suggesting that at least three immunosuppressive immunological components, Treg cells, IL-17 producing T cells and NKT cells, in which TGF-β plays a role, are not involved in the immune suppression occurring in this tumor model. This idea may also be supported by our unpublished observation that extending the period of anti-TGF-β treatment after vaccination did not improve the clinical outcome compared with treating for only two weeks after the vaccination, suggesting that anti-TGF-β is required only when TGF-β is induced as a negative feedback mechanism to terminate the activation of T cells induced by the vaccine. The second possibility is that the anti-TGF-β antibody that binds only active TGF-β does not have enough access to active TGF-β playing a critical role in tumor growth, so that the activities of TGF-β are not well suppressed in the TC1 model. In fact, while a previous study with anti-TGF-β1, 2G7, and another study with 1D11 (Ueda et al., a manuscript submitted along with this manuscript) agreed with the result of present study, a recent study with orally-administered SM16, a small type I TGF-β receptor kinase inhibitor which may have better distribution in vivo, in the TC1 tumor model showed that a single agent treatment with SM16 significantly slowed tumor growth. The third possibility is that despite the fact that TC1 cells are carrying foreign viral antigens, the tumor cells are not sufficiently immunogenic to induce any T cell immune responses that can be revealed by removing the negative suppressor. Regardless of the reason for not seeing the effect of anti-TGF-β in a single agent treatment, the results presented in the present study strongly suggest that combination of anti-TGF-β with a vaccine is an attractive approach to treat cancers.
As mentioned above, recently Kim et al conducted a study to examine the effect of a small molecule TGF-β receptor kinase inhibitor on adenoviral vaccines (34
). In that study, an adenoviral vector expressing HPV16 E7 was used as a vaccine in the TC1 tumor model. Although the overall conclusion from both studies was that a vaccine and TGF-β antagonist synergize to induce anti-tumor immunity, there are several interesting differences between two studies. First, although there was a significant effect of SM16 on tumor growth with a single agent treatment, 1D11 alone did not show any effect on tumor growth. As noted, this may be because of their size and tissue accessibility or their target specificity for receptor vs for activated TGF-β. This difference also made the synergy more striking in the case of 1D11, because simple additivity would have no impact. Second, the distribution of vaccine-induced tumor-specific CD8+
T cells was different. Kim at al, observed a concentration of T cells just in tumors. In the present study, although we did not assess the number of intratumoral T cells, a similar increase of tumor antigen-specific T cells was observed in both spleens and tumor draining lymph nodes. Third, in the present study, the level of E749-57
T cells and tumor antigen-specific lytic activity measured by in vivo CTL assay was hardly detected in spleens and tumor draining lymph nodes of untreated tumor-bearing mice, while more than 1% of CD8+
T cells were E749-57
in the previous study. These differences may be caused by the differences in the nature of TGF-β/TGF-β receptor antagonists and the vaccines used in each study and may provide important insights when these reagents are translated into the clinic.
In the present study, we could not identify a single immunosuppressive mechanism abrogated by anti-TGF-β sufficient to account for the synergistic enhancement of vaccine efficacy, even though we examined several possible mechanisms, such as Treg cells and IL-17-producing cells that can be induced by TGF-β Further, NKT cells alone were not sufficient to account for the source of TGF-β. Although it is possible that other immunological mechanisms involving TGF-β play a critical role in the TC1 tumor model to suppress vaccine efficacy, it may also be possible that it is a combination of multiple mechanisms. Adhesion molecules on endothelial cells are critical for tumor-specific T cells homing to the right place (44
). Inhibition of TGF-β signaling can change the expression levels of adhesion molecules (34
). Therefore, one effect of anti-TGF-β may be on the tumor microenvironment recruiting T cells induced by the vaccine. This may also depend on the nature of the vaccine combined with anti-TGF-β or on the type of tumors studied, as suppression of Treg cells seemed be a mechanism of action in other studies (33
) (Ueda et al., a manuscript submitted along with this manuscript). This issue should be further explored in future studies.
Currently, the human equivalent of 1D11, called GC1008, is in a phase I clinical trial in human cancer patients (22
). Similar to the studies with 1D11 in mice, GC1008 showed minimal adverse effects in the patients. The study presented here, in conjuction with the data presented by Ueda et al. (companion manuscript), provide the preclinical basis and rationale for the use of pan-neutralizing anti-TGF-β antibodies in combination with tumor vaccines for the treatment of cancer patients.