Cancers associated with ‘non-self’ tumor antigens linked to the pathogenesis of the disease, such as HPV antigen-expressing tumors, remain particularly appealing therapeutic opportunities due to the increased likelihood of eliciting effective immunity. Nevertheless, despite promising evidence obtained preclinically, some encouraging data in the clinic and efforts to design next generation approaches (2
), there are still no approved therapeutic cancer vaccines and, in the particular case of HPV tumors, there are no investigational agents in late-stage clinical development.
Herein, we provided evidence supporting the use of a novel immunization platform leading to a robust induction of T cells against a relevant TAA (HPV 16 E7), with the resulting T cell repertoire effectively dominated by functional peptide-specific CD8+
T cells, recognizing a murine epitope (E7 49-57). More specifically, co-exposure of T cells within the lymph node microenvironment to antigen and a synthetic TLR3 ligand (pI:C) as a prototype adjuvant (15
) yielded a high frequency of specific CD8+
MHC class-I restricted T cells (in the range of 1/10 CD8+
T cells), otherwise achievable in mice only by infection with select viruses or by genetic manipulation. This builds on the previously reported findings that intra-lymph node administration of non-replicating immunizing vectors such as peptides and plasmids, which typically have limited pharmacokinetic and pharmacodynamic effects if delivered using conventional parenteral means, greatly improves on the magnitude of immune response (8
) and points to potential improvements for other protein or peptide antigen-based immunization platforms (20
). In addition, use of intra-lymph node injection with synthetic, non-replicating vectors offers a straightforward means to achieve a substantial expansion of TAA-specific T cells, rather than using more complex immunization platforms such as liposomes, viral and bacterial vectors, or cell-based approaches (22
) that may be associated with significant translational challenges and safety or manufacturing issues. Furthermore, even when a substantial magnitude of immunity is generated against a TAA, it is not clear whether there are intrinsic limitations to active immunotherapy associated with specific clinical settings. Thus, the question arises: Could improved cancer immunotherapy regimens be effective alone or would they require combination with traditional treatment approaches (e.g., chemotherapy) to be efficacious in a range of disease settings?
To address this question, we utilized an HPV tumor model that served the purpose of exploring key aspects of the translatability of the lymph node immunization technology described above, although certain inherent limitations of the model should be noted, such as the relevance of the select mouse HPV epitope to man. In this model, lymph node immunization with E7 + pI:C prevented tumor formation in a prophylactic setting and was associated with persistent immune memory (). In addition, immunotherapy, initiated when tumors were palpable yet limited in size (7 days after tumor challenge), translated into a high rate of tumor regression and survival with anti-tumor efficacy dependent on the co-administration of both the antigen and the adjuvant ( and Supplemental Fig. 1
). By retrospectively stratifying treated mice into two groups - those that displayed an objective tumor response and those whose tumors continued to progress - a significant difference in epitope-specific CD8+
T cell response emerged, illustrating the critical importance of generating a substantial tumor antigen-specific immune response as a prerequisite for tumor regression ( and Supplemental Fig. 2
). A systematic analysis of the efficacy of therapeutic vaccination at later time points following tumor challenge (days 14, 20, and 28) showed that, despite achieving immune responses of similar magnitude, the impact on tumor progression was quite limited when tumors at the initiation of vaccination were larger ().
To address this, we evaluated potential mechanisms responsible for treatment resistance in bulky disease. Using a CFSE in vivo
cytotoxicity assay, we directly showed that tumor-specific T cells in the spleen cleared antigen-expressing target cells rapidly but were unable to do so within the tumor of the same animal, despite their local recruitment in substantial numbers and their ex vivo
functionality. These data highlight the importance of the tumor microenvironment in regulating the activity of tumor-specific T cells and was confirmed by the following observations: i) CD4+
/Fox-P3 Tregs were present within tumors; ii) there was a correlation between the number of local Tregs and progressing or regressing tumor status; and iii) upon co-treatment with CTX, an alkylating agent known to interfere with Tregs (17
), their number in spleen and tumor was significantly diminished (). Integrating tumor vaccines with standard chemotherapeutic drugs is a highly attractive approach due to the wide use of cytotoxic chemotherapy in the treatment of most malignancies, and thus we combined CTX with immunotherapy in an attempt to treat late-stage cancer. Chemoimmunotherapy with CTX was accompanied by enhancement of intra-tumoral activity of specific T cells, significant tumor growth suppression, and increased survival of treated mice in late-stage cancer following a second therapeutic immunization cycle (). This strategy was supported by a recent study showing that continuous immunization against a select tumor antigen resulted in sustained and elevated immunity (10
). Although we have not formally generated evidence ruling out other immune escape mechanisms within tumors, the enabling effect of CTX relative to vaccination strongly suggested that this combination approach rendered E7-expressing tumor cells susceptible to immune-mediated clearance by changing the tumor milieu, resulting in a lower frequency of Tregs. In synergy with this interpretation, there is an accumulating body of evidence in support of the effect of CTX on Tregs and its significant immune modulating activity in relation to cancer vaccines (33
). Our data also suggests that the immune microenvironments in tumor and secondary lymphoid organs are quite different, specifically in regard to the Tregs' impact on CD8+
T cell function in tumor versus spleen, and we suggest the following possible explanations: i) Treg cells within the tumor site are more active in suppressing the anti-tumor effector cells; ii) Tregs within the tumor microenvironment act in concert with other immune suppressive mechanisms that are not active in spleen, as in the excess production of suppressive cytokines such as IL-10, TGFβ and others; and/or iii) infiltrating anti-tumor effector T cells are more susceptible to the effect of Treg cells. While these possibilities are not mutually exclusive and alternate mechanisms may also be at work, the results shown here do clearly support the use of CTX to augment immunotherapy in late-stage disease, with potential translational value.
Finally, we tested the applicability of this immunization platform in a setting of rapidly progressing pulmonary metastatic disease. Mice infused intravenously with HPV-transformed C3.43 cells rapidly developed multiple lung tumors (evident by microscopy as early as 8 days after challenge), progressed expeditiously, and became moribund within several weeks. Initiating therapeutic lymph-node vaccination within two weeks after tumor challenge resulted not only in robust induction of immunity, but also prevented disease progression to full-blown clinical manifestation as compared to untreated controls (). This was mirrored by the lack of residual tumors assessed macroscopically and microscopically, supporting an immune-mediated clearance of tumor cells and progressing tumor lesions within the lung parenchyma (Supplemental Fig. 3
In conclusion, innovative immunotherapy approaches hold great promise for the treatment of cancer by harnessing a patient's immune system to eradicate their malignant neoplasms. Our findings describe a straightforward method to achieve substantial anti-tumor immunity and point to disease scenarios where immunotherapy may have the greatest potential, whether as a stand-alone therapy or combined with traditional treatments. The results of our study emphasize four key complementary aspects important to the development and translation of effective active cancer immunotherapies: i) selection of relevant antigens to which patients can rapidly mount substantial immune responses; ii) use of strongly immunogenic vaccination approaches, such as targeted lymph node vaccination, that result in high-magnitude, functional anti-tumor T cell responses; iii) selection of appropriate disease settings for preclinical and clinical testing that maximize the therapeutic potential; and iv) use of adjunctive therapy to overcome immune-suppressive mechanisms associated with larger tumors.