Here we report on the safety and feasibility of the topical TLR7 agonist imiquimod when used to condition vaccination sites for intradermal injection with recombinant cancer/testis antigen NY-ESO-1 in patients with malignant melanoma. The NY-ESO-1 protein / imiquimod vaccine regimen was well tolerated – only mild and transient side effects were observed, and all patients completed the study.
The vaccine combination successfully elicited both humoral and cellular immune responses in a significant fraction of patients. NY-ESO-1-specific antibody responses were detected in 4 of 9 patients (44%). However, antibody titers were significantly lower than those described in a previous study using intramuscular injection of NY-ESO-1 protein with the saponin-based adjuvant ISCOMATRIX (43
), as well as in a more recent study by our group using Montanide ISA-51 adjuvant and TLR9 agonist CpG 7909 administered subcutaneously with recombinant NY-ESO-1 (42
). In both these studies, all seronegative patients receiving NY-ESO-1 developed antibodies, and average maximum titers were 10-fold or more higher than what we observed here, suggesting that a protective carrier formulation providing a controlled release or depot effect may be required for optimal antibody responses. In the current study, imiquimod / NY-ESO-1 did, however, induce antibodies in a higher percentage (44%) of patients than those in the control group of the ISCOMATRIX study who received NY-ESO-1 protein alone without any adjuvant (25% of patients).(43
) Epitope mapping of the antibody responses using individual peptides confirmed that the immunogenic regions of the protein were similar to those found in patients with spontaneous NY-ESO-1 immunity, indicating that the vaccine is capable of inducing B cell responses against epitopes naturally presented by NY-ESO-1 positive tumors. We also have previously shown that these vaccine-induced NY-ESO-1 antibodies facilitated cross-presentation (42
In this study, NY-ESO-1 / imiquimod vaccination induced IFNγ-secreting NY-ESO-1-specific CD4+ T cells in 6 of 8 (75%) evaluable patients. These responses were clearly evident by intracellular cytokine staining following a one week in vitro presensitization with NY-ESO-1 overlapping peptides, but were not detectable in “ex vivo” ELISPOT assays (i.e., without pre-sensitization). In contrast, CD8+ T cell responses were not detectable using either method.
In the above mentioned ISCOMATRIX study, both CD4+ and CD8+ T cell responses to NY-ESO-1 were detectable in a subset of patients vaccinated with the ISCOMATRIX adjuvant, although a more prolonged in vitro pre-sensitization was used and ex vivo assays were not attempted, making a comparison with our study difficult.(43
) However, in our recent CpG / Montanide / NY-ESO-1 protein vaccine study almost all patients developed CD4+ T cell responses and approximately half had detectable CD8+ T cell responses as measured by the same vitro pre-sensitization assay described here. In addition, ex vivo assays showed CD4+ T cell responses in 67% of subjects and CD8+ T cell responses in 39%. (42
) These results indicate that CpG plus Montanide is a more potent adjuvant formulation than imiquimod alone, and is capable of cross-priming CD8+ T cell responses. Of note, CpG was administered mixed with the antigen/Montanide emulsion, whereas imiquimod was applied topically before and after antigen injection.
A possible explanation for the absence of CD8+ T cell immunity observed in our study, as well as for the weaker CD4+ T cell and antibody responses, may be the timing used for the application of imiquimod. In recent experiments in mice it was observed that when TLR agonists were provided prior to antigen encounter, cross presentation by APCs was impaired and cross priming inhibited due to premature APC maturation.(44
) Thus it is possible that the use of TLR agonists may be most effective at the time of, or immediately following, the delivery of antigen. The use of TLR activation stimuli may also require additional adjuvants or emulsifiers for optimal effect. In recent studies in non-human primates, the use of imidazoquinolines or CpG was most effective when these compounds were either covalently coupled to antigen or mixed in a water-oil emulsion,(20
) and Montanide emulsification in our above-mentioned study might have contributed significantly to the vaccine's strong immunogenicity (42
). In addition, it may prove that other imidazoquinolines such as resiquimod, which activates both TLR7 and TLR8, are more potent stimulators of immunity than imiquimod. (45
) We are currently addressing these issues in a follow-up clinical study.
One question raised by this study is whether similar results could have been obtained by vaccinating with NY-ESO-1 protein alone. Although we did not include an antigen-alone control arm, previous studies have confirmed that NY-ESO-1 or MAGE-3 proteins given alone are poorly immunogenic (43
), and that addition of an immunological adjuvant is essential for the induction of persistent immunological memory (48
). The above-mentioned NY-ESO-1 protein / ISCOMATRIX study included an NY-ESO-1 protein-alone control group of 16 patients, most of whom had resected melanoma. NY-ESO-1 protein was given intramuscularly at 4 week intervals at the same dose as in our study (100 mcg). However, the frequency of induction of NY-ESO-1 specific antibodies was less than we observed for NY-ESO-1 / imiquimod (25% compared to 44%), and the induction of CD4+ T cells reactive to NY-ESO-1 overlapping peptides was 0% compared to 75%. As in our study, CD8+ T cells reactive to NY-ESO-1 were not induced in the absence of ISCOMATRIX. These data suggest that topical imiquimod can enhance the induction of antibody and CD4+ T cell responses, but not CD8+ T cell responses.
To address the potential the mechanism of action of imiquimod in inducing vaccine responses, we show that topical application of imiquimod is associated with dermal mononuclear cell infiltrates that are composed largely of T cells, antigen-presenting cells (monocytes, macrophages, myeloid DCs and plasmacytoid DCs) and NK cells. Activated DCs (CD83+ and DC-LAMP+ cells) were also evident. Only rare B cells were seen. Although we were unable to further characterize the observed CD123+ cells by double-staining, we expect the majority of them to be plasmacytoid DCs, as we morphologically excluded basophils (which also express CD123). Our findings are in accordance with previous observations of recruitment and activation of myeloid and plasmacytoid DCs in the skin of patients whose skin tumors were treated with imiquimod,(18
) as well as observations of accumulation of plasmacytoid DC-like cells in the skin of mice after imiquimod treatment,(17
) and of “monocyte-macrophage-dendrocyte” dermal infiltrates in healthy skin chronically exposed to imiquimod.(49
) These observations suggest that the recruitment and activation of APCs at the vaccination site by imiquimod may lead to improved uptake and presentation of the injected protein antigen, promoting immunization.
In contrast to other studies, we did not observe a net loss of epidermal Langerhans cells following imiquimod application.(16
) These studies reported a decrease in CD1a+ Langerhans cells in the epidermis, suggestive of migration to draining lymph nodes, after imiquimod treatment for up to 10 days in mice and following chronic exposure over months in humans. The observed difference with our results may be due to a dose effect, as studies using the imidazoquinoline resiquimod in healthy volunteers demonstrated a decrease of CD1a+ cells only in the highest dose group.(50
) Furthermore, recent analyses have failed to detect TLR7 in Langerhans cells freshly purified from human skin, at least at the mRNA level (51
In summary, this study demonstrates the feasibility and excellent safety profile of a topically applied TLR agonist as adjuvant for a protein vaccine in cancer patients. Although no conclusions about imiquimod's additive effect can be drawn in the absence of a control arm, we show that the imiquimod/NY-ESO-1 combination elicits both humoral and CD4+ T cell responses in a significant fraction of patients. In light of these findings, further studies evaluating imiquimod's relative effect on the immunogenicity of this combination, including experiments evaluating the dose and timing of its application, are warranted.