Lymph Node–Targeted Immunotherapy Mediates Potent Immunity Resulting in Regression of Isolated or Metastatic Human Papillomavirus–Transformed Tumors

Results

Intra–lymph node immunization with E7 peptide and pI:C elicits protective immunity against HPV-transformed tumor challenge

Direct lymph node injection of the HPV 16 E7 49-57 (RAHYNIVTF) epitope peptide in combination with pI:C dsRNA adjuvant resulted in robust antigen-specific T-cell responses (in the range of 15% of the total circulating CD8⁺ Tcells) in mice as measured by tetramer analysis (Fig. 1A and B). In contrast, E7 peptide or pI:C alone were not effective at inducing specific immunity (Fig. 1B). To assess the prophylactic nature of this vaccination approach, immunized mice were inoculated s.c. (day 40) with 10⁵ HPV 16–transformed E7-expressing C3.43 cells and no evidence of tumor growth was detected up to 120 days. In addition, 90% of the mice remained protected following a second tumor challenge on day 120, with tumor progression significantly delayed in those few animals that did develop tumors (Fig. 1C).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Intra–lymph node immunization with E7 peptide and pI:C elicits robust immunity protective against tumor challenge. Flow cytometry dot plots (A) comparing the tetramer response from an immunized (E7 + pI:C) and naïve control mouse representative of data shown in B. The results are expressed as the frequency of E7 tetramer⁺ CD8⁺ T cells relative to the total CD8⁺ T-cell population measured in peripheral blood 10 d after the completion of the immunization protocol. Intralymphatic vaccination with E7 49-57 HPV antigen and pI:C resulted in substantial E7-specific CD8⁺ T-cell responses, whereas pI:C or E7 49-57 peptide alone had no significant impact on immune response when compared with tumor control or naïve mice (B). Columns, mean E7 tetramer response for each group (n = 10); bars, SEM. HPV 16 E7 49-57 antigen–specific immune response correlated with tumor protection (C). Immunization of mice with E7 49-57 peptide and pI:C (n = 20) resulted in complete protection from s.c. challenge with 10⁵ HPV-transformed C3.43 tumor cells compared with tumor control mice (n = 20) and 90% protection following a tumor rechallenge compared with second group of tumor control mice (n = 3).

Therapeutic intra–lymph node immunization with E7 peptide and pI:C mediates immunologic regression of solid tumor

Next, we evaluated this approach against established C3.43 tumors to assess the efficacy of intra–lymph node immunotherapy. When tumors were clinically evident and palpable, mice were immunized with E7 peptide and pI:C in lymph nodes, or by s.c. injection with an equivalent amount of vaccine on day 7, 10, 21, and 24. Tetramer results (day 31) indicated that only mice immunized via lymph node injection generated statistically significant (P < 0.0001) E7 49-57–specific immune responses, with an average of 14.5% compared with s.c. dosed and unimmunized control mice (Fig. 2A), with average responses of 1.0% and 0.7%, respectively. As shown in Fig. 2B, tumors in mice immunized by lymph node injection began to regress on day 15, coinciding with elevated immunity, and resulting in 84% of animals in complete tumor remission by day 40. This response was significantly superior to that of animals dosed s.c. (P < 0.003) whose tumor progression was delayed compared with tumor controls. Nevertheless, this apparently modest response in mice dosed s.c. resulted in 32% of animals in complete remission (Fig. 2B). Untreated tumor-bearing mice displayed background levels of E7 tetramer staining (Fig. 2A), potentially reflecting exposure to endogenous tumor antigen, although their tumors progressed exponentially without regression, as expected (Fig. 2B). Complementing these findings, intranodal immunization ultimately translated to significantly enhanced long-term survival compared with s.c. immunized (P = 0.0004) or tumor control (P = 0.0001) mice (Fig. 2C). In an independent study, intranodal vaccination with E7 peptide or pI:C adjuvant alone offered little, if any, therapeutic benefit when compared with the combination of E7 peptide + pI:C (Supplementary Fig. S1A and B). Therefore, a key prerequisite to achieve a substantial amplification of immunity (Figs. 1B and 2A), tumor regression (Supplementary Fig. S1A; Fig. 2B), and survival (Supplementary Fig. S1B; Fig. 2C) was the direct intra–lymph node administration of E7 peptide and pI:C adjuvant, which could not be reproduced by E7 peptide alone, pI:C alone, or s.c. administration of the vaccine.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Intra–lymph node immunization with E7 peptide and pI:C mediates immunologic regression of solid tumor. In a therapeutic model of HPV 16, the antitumor efficacy of intranodal versus s.c. dosing was compared. C57BL/6 mice were challenged s.c. with 10⁵ C3.43 HPV tumor cells on day 0 and then immunized with a mixture of E7 49-57 peptide and pI:C in each bilateral inguinal lymph node (n = 19) or an equivalent amount of vaccine s.c. (n = 19) on day 7, 10, 21, and 24. The immune response was measured by E7 49-57 tetramer staining on day 31 from peripheral blood (A) and tumor size for each group was calculated and compared with untreated tumor challenged control (n = 19) mice (B). Lymph node–immunized mice generated statistically significant E7 49-57–specific immune responses with an average of 14.5% tetramer-positive CD8+ T cells (A) compared with s.c. dosed mice (P < 0.0001). In addition, tumors in mice immunized in the lymph node began to regress on day 15 resulting in 84% of animals in remission at day 40 (B). This response was significantly superior to animals dosed s.c. (P < 0.003) whose tumor progression was only delayed compared with tumor controls but resulted in 32% of animals in disease remission. Untreated tumor control mice displayed background levels of E7 tetramer staining (A) and their tumors progressed exponentially without regression as expected (B). Log-rank statistical tests confirmed that survival in the E7 + pI:C group was significantly prolonged when compared with animals immunized s.c. (P = 0.0004) or when compared with tumor controls (P = 0.0001; C).

We then showed that immune regression of established tumors depends on both the magnitude of immune response and the tumor size upon initiation of vaccination. The results summarized in Table 1 show that immunization early in the course of disease resulted in a much higher percentage of complete remissions (69%). In addition, a post hoc analysis of measured immune response versus tumor outcome in earlier stage disease showed a clear correlation between the frequency of specific T cells induced following day 7 immunization and tumor regression (Supplementary Fig. S2A and B).

When immunization was initiated later in the course of disease when tumors were considerably larger, the apparent impact of immunotherapy on tumor progression was minimal, despite the induction of substantial immune responses (Table 1).

CD4⁺/CD25⁺/FoxP3^HI Tregs correlate with impaired function of TILs within advanced tumors and are reduced following CTX treatment

These results led us to hypothesize that either the tumor-specific T cells generated by this approach may lack sufficient homing signals to migrate into established tumors of larger size, or alternatively, that immune checkpoints within the tumor microenvironment may be limiting the effector function of the T cells despite effective local recruitment. To address this question, mice were immunized on day 20, 24, 34, and 38 following inoculation with C3.43 tumors; TILs were isolated from the tumors of E7 peptide + pI:C or PBS + pI:C–immunized mice (as control); and their phenotype and functional status were assessed by flow cytometry. In Fig. 3A (top right dot plot), TILs costained with E7 tetramer and anti-CD8 identified a large population of E7-specific T cells, clearly demonstrating their ability to infiltrate tumors following E7 peptide + pI:C immunization. However, E7-specific TILs were not detected in tumors isolated from PBS + pI:C–immunized mice, showing the scarcity of endogenous T cells reactive against this epitope in the absence of immunization (Fig. 3A, top left dot plot). Furthermore, the functional status of these HPV-16 antigen–specific TILs was evaluated ex vivo by E7 49-57 peptide stimulation, resulting in intracellular IFN-γ production measured by flow cytometry. In Fig. 3A (bottom), a significant proportion of E7-specific TILs (∼36%) could produce IFN-γ cytokine upon ex vivo peptide stimulation, demonstrating a latent functional competency of the TILs. These data confirmed that E7-specific T cells resulting from intralymphatic immunization did migrate to established tumors and were functionally competent when tested ex vivo, but did not formally show their in situ functionality upon antigen engagement. To measure the TILs' function in situ, we used an in vivo CFSE cytotoxicity assay. To that aim, mice were inoculated with C3.43 tumors (day 0), immunized with E7 peptide + pI:C or PBS + pI:C control vaccine (day 20, 24, 34, and 38), and then challenged i.v. with an equal ratio of CFSE^high (HPV 16 E7 49-57 labeled) and CFSE^low (MART-1/Melan A 26-35 labeled) syngeneic splenocytes. Mononuclear cells from tumor and spleen were isolated 18 h later and target-specific lysis was analyzed by flow cytometry. Interestingly, E7 + pI:C–immunized mice with progressing tumors effectively cleared the HPV 16 E7 49-57–pulsed target cells in spleen, but not effectively within tumors (Fig. 3B). These results led us to hypothesize that immune checkpoints, such as the one represented by CD4⁺/CD25⁺/FoxP3^HI Tregs, likely to be present in high frequency in established tumors, may be responsible for this T-cell–suppressive effect (16). To assess this, we analyzed the CD4⁺/CD25⁺/FoxP3^HI Treg frequency by flow cytometry in spleens of immunized mice whose tumors were progressing compared with naïve mice or mice whose tumors had regressed (Fig. 3C). Mice with progressing tumors had ∼3-fold more Tregs in spleen than naïve or cured animals. In addition, a high frequency of tumor-infiltrating CD4⁺ T cells and CD4⁺/CD25⁺/FoxP3^HI Tregs could be detected in mice with progressing disease, suggesting a potential explanation for the reduced efficacy of active immunotherapy in a more advanced disease setting (Fig. 3D, left bar). Previous studies have reported that CTX treatment can augment tumor vaccine immunity by reducing the frequency and abrogating the activity of Tregs in vivo (17–19). When immunized mice with progressive tumors were treated with a single i.p. injection of 100 mg/kg CTX, the frequency of Tregs in spleen (Fig. 3C) and the number of CD4⁺ T cells as well as CD4⁺/CD25⁺/FoxP3^HI Tregs in tumor (Fig. 3D, right bar) were significantly decreased. When directly tested in the in vivo CFSE cytotoxicity model, CTX treatment resulted in enhanced killing of E7 49-57 peptide–labeled syngeneic target cells in tumors of immunized mice (P = 0.03) and had no adverse effect on specific target lysis in spleen (Fig. 3B). These data provide a rationale for combining chemotherapy with immunotherapy for the treatment of late-stage tumors that may be only marginally affected by vaccination alone (Table 1).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

CD4⁺/CD25⁺/FoxP3^HI Tregs impair in situ function of TILs in advanced tumors and are reduced following CTX treatment. Mice were inoculated with 10⁵ HPV-16–transformed C3.43 tumor cells on day 0 and immunized with E7 49-57 HPV peptide and pI:C in bilateral inguinal lymph nodes on day 20, 24, 34, and 38 (n = 3). Immunization control mice received PBS + pI:C in bilateral inguinal lymph nodes on day 20, 24, 34, and 38 (n = 3). A, flow cytometry dot plots comparing the frequency (top) and the functional ability to produce IFN-γ (bottom) of E7 49-57 antigen–specific TILs from representative E7 + pI:C–immunized or tumor control (PBS + pI:C) mice. B, impaired in situ function of TILs measured by in vivo cytotoxicity assay. E7 + pI:C–immunized mice with progressing tumors cleared >90% of HPV 16 E7 49-57–labeled target cells in spleen, whereas TILs from the same mice had little cytotoxic effect on target cells within established tumors. CTX treatment (100 mg/kg, i.p.) in a second group of E7 + pI:C–immunized mice (n = 3) with progressive disease resulted in enhanced killing of specific target cells in tumors (P = 0.03) and had no adverse effect on target cell lysis in spleen. C, immunized mice bearing HPV-16–transformed tumors (n = 3) displayed ∼3-fold higher numbers of CD4⁺/CD25⁺/FoxP3⁺ Tregs in spleen compared with naïve mice (n = 3) or immunized mice whose tumors completely regressed (n = 3). The level of CD4⁺/CD25⁺/FoxP3⁺ Tregs could be reduced in the spleen (n = 3; C) and the levels of CD4⁺ and CD4⁺/CD25⁺/FoxP3⁺ cells could be reduced in tumor (D) of mice with progressive disease by a single i.p. injection of 100 mg/kg CTX (n = 3 mice/group).

Adjunctive therapy with CTX enables immunotherapy in an advanced disease setting

To test this hypothesis and determine if combination therapy with CTX would enable active immunotherapy in a more advanced disease setting, mice were inoculated s.c. with 10⁵ C3.43 cells, and treated with either CTX (30 mg/kg) alone on day 14 and 18, immunized with E7 + pI:C alone on day 20, 24, 34, and 38, or treated with CTX and then immunotherapy. The immune response, measured by E7 49-57 tetramer staining on day 45 from peripheral blood, showed that the immunized-only group (E7 + pI:C) displayed HPV-specific immune responses in the range of 20%, with no observed inhibition of immune response in animals treated with CTX + immunotherapy (Fig. 4A). Furthermore, CTX + immunotherapy induced significant tumor regression (P < 0.001) compared with immunotherapy alone, dose-matched chemotherapy alone, or untreated tumor controls (Fig. 4B). In an attempt to maximize the potency of the immunization regimen assisted by CTX and to generate a more robust response in a relatively advanced tumor setting, a second treatment cycle was initiated and Kaplan-Meier estimates of the survival function were obtained for each of the four treatment conditions (Fig. 4C). Based on log-rank analysis, the survival in the CTX + immunotherapy group was significantly longer than the survival in the control group (P < 0.0001), the CTX only group (P = 0.0188), and the immunotherapy (E7 + pI:C) only group (P = 0.0033). The median survival time in the CTX + immunotherapy group was also longer (80 days) compared with the CTX only group (68 days) and the immunotherapy only group (54 days) in a setting associated with rapid tumor progression and mortality (median survival of control tumor-bearing mice was 52 days). Furthermore, it was not surprising that CTX alone resulted in some delayed tumor progression compared with untreated tumor control mice (P = 0.01; Fig. 4B), although, as discussed above, this effect did not translate to a significant survival benefit (Fig. 4C). These findings show that the sequential administration of CTX and intralymphatic HPV vaccination significantly improved the disease outcome in later stage disease.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Adjunctive therapy with CTX enables immunotherapy in a more advanced disease setting. Mice were inoculated with 10⁵ HPV-16–transformed C3.43 tumor cells on day 0; treated with CTX on day 14 and 18 (n = 20); immunized with E7 49-57 HPV peptide and pI:C in bilateral inguinal lymph nodes on day 20, 24, 34, and 38 (n = 20); or treated with CTX then E7 + pI:C immunotherapy (n = 20). Immune response (A) and tumor progression (B) was compared with untreated tumor control mice (n = 20). The immune response following immunotherapy was measured by E7 49-57 tetramer staining on day 45 from peripheral blood. The immunized only group (E7 + pI:C) displayed HPV-specific immune responses in the range of 20% with no observed inhibition of immune response in animals treated with CTX + E7 + pI:C that generated a similar tetramer response. The naïve control (n = 5) and CTX control groups generated background levels of tetramer staining (A). CTX + E7 + pI:C induced significant tumor regression (P < 0.001) compared with E7 + pI:C immunotherapy alone, CTX alone, and untreated tumor controls (B). Evaluation of adjunctive therapy on animal survival (C). A second therapeutic cycle was administered with animals receiving CTX on day 46 and 50 (n = 20), lymph node immunization with E7 + pI:C on day 52, 56, 65, and 69 (n = 20), or treated with CTX + E7 + pI:C (n = 20). Gray arrows, CTX treatment; black arrows, immunization days. Log-rank statistical tests confirmed that survival in the CTX + E7 + pI:C group was significantly longer than survival in the control group (P < 0.0001, n = 20), the CTX only group (P = 0.0188), and the E7 + pI:C immunotherapy only group (P = 0.0033).

Effective control of rapidly progressing metastatic pulmonary tumor by intra–lymph node immunization with E7 peptide and pI:C

To test the potency of lymph node–targeted active immunotherapy in a setting of rapidly progressing metastatic disease, we injected mice i.v. with 5 × 10⁵ C3.43 cells and evaluated the disease outcome following E7 + pI:C vaccination. We compared three vaccine time courses beginning on days 1, 8, or 15 posttumor challenge to assess the therapeutic benefit of our immunotherapy method. When mice were immunized by lymph node injection with E7 + pI:C beginning on day 1 (Fig. 5A), day 8 (Fig. 5B), or day 15 (Fig. 5C), a significant beneficial impact on disease progression could be measured in all cases. More specifically, 89%, 78%, and 57% respectively, of the immunized mice were still alive at day 175 posttumor challenge. The survival outcome of E7 + pI:C–immunized mice was significantly superior to that of unimmunized tumor control and pI:C adjuvant–injected mice for the day 1 (Fig. 5A) and 8 (Fig. 5B) treatment regimens, with P values equal to 0.0007 and 0.001, respectively. For mice that received immunotherapy with E7 + pI:C on day 15 following tumor injection (Fig. 5C), the survival outcome was significantly better than that of tumor control mice (P = 0.032) and more favorable, although not statistically different, from the pI:C adjuvant–injected group that showed a subtle beneficial impact on disease progression. This result may be due to the immune-modulating effect of pI:C via activation of TLR3 downstream pathways on immune cells or “somatic” cells. In addition, tissue histology (Supplementary Fig. S3) showed no residual tumors in animals effectively treated with E7 + pI:C, whereas the control mice and the pI:C-injected mice developed multiple lung tumors during the same time interval, explaining their deteriorated clinical status. In addition, systematic histologic analysis of pulmonary tissue at day 8 after tumor challenge of naïve mice (the start of effective therapeutic immunization) clearly showed the presence of multiple tumors of limited size within the lung parenchyma (Supplementary Fig. S3, top). This provided a more quantitative perspective on the disease burden manageable by this immunization strategy.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

Control of pulmonary metastases by intra–lymph node immunization with E7 peptide and pI:C. Mice were injected i.v. with 5 × 10⁵ C3.43 tumor cells and then immunized with one of the following intranodal E7 + pI:C vaccine time courses: day 1, 4, 15, and 20 (A); day 8, 12, 22, and 26 (B); or day 15, 20, 29, and 33 (C). Survival curves for each group (A-C) are shown, and the outcome for E7 + pI:C–immunized mice (n = 9) was compared with pI:C only (n = 9) and untreated tumor control mice (n = 9). Log-rank statistical tests confirmed that survival in the E7 + pI:C group for each vaccine time course (A-C) was significantly longer than survival in the tumor control group (P = 0.0007, 0.001, and 0.032, respectively) and the pI:C only group when immunization began on day 1 (P = 0.006; A) and day 8 (P = 0.004; B) but not day 15 (P = 0.06; C).

In summary, active immunotherapy was highly effective at inducing clearance of pulmonary metastatic tumors and preventing overt disease and mortality in this rapidly progressing tumor setting.

Discussion

Cancers associated with “nonself”-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, coexposure 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 nonreplicating 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 (8–10, 13) and points to potential improvements for other protein or peptide antigen–based immunization platforms (20, 21). In addition, use of intra–lymph node injection with synthetic, nonreplicating 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 (22–32) 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 used 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 (Fig. 1). In addition, immunotherapy, initiated when tumors were palpable yet limited in size (7 d after tumor challenge), translated into a high rate of tumor regression and survival with antitumor efficacy dependent on the coadministration of both the antigen and the adjuvant (Supplementary Fig. S1; Fig. 2). 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 (Supplementary Fig. S2; Table 1). 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 (Table 1).

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: (a) CD4⁺/CD25⁺/Fox-P3 Tregs were present within tumors; (b) there was a correlation between the number of local Tregs and progressing or regressing tumor status; and (c) upon cotreatment with CTX, an alkylating agent known to interfere with Tregs (17–19), their number in spleen and tumor was significantly diminished (Fig. 3). 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 intratumoral 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 (Fig. 4). 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, 34). Our data also suggests that the immune microenvironments in tumor and secondary lymphoid organs are quite different, specifically with regard to the Tregs' impact on CD8⁺ T-cell function in tumor versus spleen, and we suggest the following possible explanations: (a) Treg cells within the tumor site are more active in suppressing the antitumor effector cells; (b) 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 interleukin-10, transforming growth factor β, and others; and/or (c) infiltrating antitumor effector T cells are more susceptible to the effect of Treg cells. Although 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 i.v. 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 2 weeks after tumor challenge resulted not only in robust induction of immunity, but also prevented disease progression to full-blown clinical manifestation compared with untreated controls (Fig. 5). 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 (Supplementary Fig. S3).

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 antitumor 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: (a) selection of relevant antigens to which patients can rapidly mount substantial immune responses; (b) use of strongly immunogenic vaccination approaches, such as targeted lymph node vaccination, which result in high-magnitude, functional antitumor T-cell responses; (c) selection of appropriate disease settings for preclinical and clinical testing that maximize the therapeutic potential; and (d) use of adjunctive therapy to overcome immune-suppressive mechanisms associated with larger tumors.

Disclosure of Potential Conflicts of Interest

M. Kast is on the advisory board of MannKind Corp.