The studies presented here using the ALK5/AlK4 inhibitor, SM16, in three different syngeneic tumor models (AB12 mesothelioma, LKR and TC-1 lung carcinomas), in flank and orthotopic models, and with two different immune-activating therapies (Ad.E7 and Ad.INF-β) support the utility of combining inhibition of TGF-β signaling with immune-activating therapies. These results show that TGF-β receptor blockade via SM16 can augment a variety of adenovirus-based, immune-activating therapies in a spectrum of tumors. Similar results using a soluble TGF-β receptor () suggest that our findings are applicable to other TGF-β blocking strategies.
Analysis of the mechanism of action in each model highlight the similarities and differences between the response of these tumor models to these therapies. A common finding was an increase in intratumoral CD8+
T cells, that was augmented significantly in the combination treatments (). This effect is consistent with the previously described mechanisms of action for SM16, Ad.INF-β and Ad.E7 (18
). However, the increase in tumor-associated NK (NK1+
) and macrophages (CD11b+
) cells with SM16 alone, in both the AB12 and LKR models, identifies a novel response to TGF-β inhibition. Interestingly, tumor-associated CD11b+
cells were not increased by SM16 in the TC-1 model, suggesting inherently different immunosuppressive mechanisms may be in play in these different models. However, a further enhancement of CD11b+
populations by the combination treatments above the single agent treatments was a common feature in all three tumor models. These data support the utility of the combination treatments in models where different immunosuppressive mechanisms may be operative.
Another novel finding was the ability of TGF-β receptor blockade alone to cause significant alterations in the tumor microenvironment favoring T cell activation and the Th1 phenotype, as well as T cell and leukocyte infiltration. SM16 treatment induced the expression of the message levels for cytokines which are chemoattractive for T cells and NK cells (29
). An increase in tumor endothelial ICAM-1 expression and the enhanced expression of message levels for TNFα, INF-γ and IL-1β, cytokines which stimulate adhesion molecule expression, are also consistent with a process that would increase T cell and leukocyte infiltration (30
). It should be noted that our RT-PCR results did not show perfect correlation with intra-tumoral cytokine measurements, however, this could be due to difficulties in sample proteolysis and inefficient extraction from tumor specimens.
The SM16-induced increase in the percentage of activated CD8+
T cells (CD25+
), the increased Th1 cytokines, and the decreased arginase message levels also suggest that TGF-β blockade promotes a tumor environment promoting the activation of tumor-associated T cells. This is consistent with previous reports showing TGF-β induces macrophage arginase levels (31
) and is inhibitory to T cell activity via inhibition of INF-γ and perforins (12
). In the TC-1 model, Ad.E7 treatment alone increased tetramer-positive T cells in both the spleen and tumors, while SM16 had no effect on this T cell population in either location on its own. However, the combination of SM16 and Ad.E7 induced a further increase in tumor-associated, tetramer-positive cell above that induced by Ad.E7, with no significant change in splenic tetramer-positive cells. These results suggest that TGF-β receptor blockade does not increase the overall number of tetramer-positive T cells, but does increase either the trafficking or persistence in the tumor of this anti-tumor T cell population. These findings suggest that TGF–β is a key, proximal immunosuppressive modulator of the tumor microenvironment that can inhibit T cell and leukocyte trafficking, function, or persistence in tumors.
We have emphasized the role of TGF-β on immune cells, however, interpretation of our studies needs to take into account the complexity of the role of TGF-β in tumor biology (1
) since multiple cells within a tumor make, activate, and respond to TGF-β. For example, using genetic models of mammary carcinogenesis in mice that result in selective loss of TGF-β signaling in tumor cells, investigators have observed marked changes in the secretion of specific chemokines by tumor cells that appear to then alter the tumor-associated myeloid cell populations and the tumor microenvironment (32
). As another example, loss of TGF-β receptor expression in lung cancer cells has been associated with increased invasiveness and increased production of the chemokine CCL5 (34
). Our experiments use a “global” inhibitor that would presumably block TGF-β signaling in stromal cells, leukocytes, and in the tumor cells themselves (cell autonomous effects). It is currently unclear, how important the blockade of each cell type might be. Additional effects of TGF-β on tumor biology could also be involved in anti-tumor effects, such as a recent report suggesting that TGF-β could subvert the immune system in directly promoting tumor growth through interleukin-17 (35
One issue that should be considered with any type of inhibitor compound is that of specificity. As recently published (25
), the activity of SM16 (similar to other such inhibitors like SD-093 or SD208 (16
)) is primarily directed against ALK5 (Ki: 10 nM) and ALK4 (Ki: 1.5 nM), although there is some moderate off-target activity to Raf (IC50
1 uM) and p38/SAPKa (IC50
, 0.8 uM). To try to address the question of off-target effects, we performed a study with the Ad.E7 vaccine in combination with a completely different class of TGF-β blocking agent, a soluble Type I receptor (18
), and showed very similar effects as we saw with SM16 ().
Previous studies have shown increased immunogenicity and anti-tumor responses when TGF-β inhibition (mediated by antisense oligonucleotides or dominant negative receptors) is targeted to tumor cells and immune cell types, which are then used as vaccines or in adoptive transfer (36
). Some of these approaches are moving into clinical trials (41
). There are only a limited number of reports where systemic inhibitors of TGF-β have been combined with immunotherapy in the intact animal, however. Some success in a rat model of glioma has been achieved by combining intracranial injection of anti-sense TGF-β2 oligonucleotides with an irradiated tumor cell line (plus interferon-γ) (45
). Induction of anti-TGF-β antibodies by injection of plasmid DNA encoding a xenopus TGF-β5 gene increased the therapeutic efficacy of a tyrosinase-related protein-2 plasmid DNA vaccine (46
). Perhaps most relevant to this study, is a report by Kobie et al. (47
) showing that administration of an antibody against TGF-β enhanced the ability of an intratumorally injected DC vaccine to inhibit the growth of established mouse breast cancer cells.
These aforementioned studies, as well as others, have revealed the immunosuppressive effects of TGF-β in blocking systemic generation or function of anti-tumor T cells (16
) and mediating T regulatory cell activity (48
). However, the experiments presented here identify additional mechanisms directed at immune and inflammatory cell infiltration, function, or persistence that may play an important role when combining TGF-β inhibition with immune-promoting therapies. These local intratumoral effects of TGF-β blockade may be extremely important, since many cancer patients progress despite exhibiting relatively high percentages of circulating anti-tumor T-cells (i.e., in melanoma) or showing the presence of T cells surrounding tumor tissue (49
), supporting the idea that T-cells must be able to move into the tumor, survive there, and effectively exert their direct and indirect (macrophage and NK cell activation) anti-tumor activities to be effective (50
). Consistent with this idea, we have recently shown that after adoptive T-cell transfer, the number of cytotoxic T cells with tumors is enhanced after blockade of TGF-β receptor function (51
Individual immune-activating therapies, such as tumor and dendritic cell vaccines, adoptive immune cell transfer, and adenovirus-based therapies (including Ad.INF-β), are now being tested as single agents in clinical trials (52
). The work presented here, and that of others in the field, suggest that a greater potential for efficacy may be achieved by combining these therapies with treatments targeting key modulators of immunosuppression, such as TGF-β, in order to promote an “immune-friendly” tumor microenvironment.