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An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression

Abstract

We have investigated if immunotherapy against human papilloma virus (HPV) using a viral gene delivery platform to immunize against HPV 16 genes E6 and E7 (Ad5 [E1-, E2b-]-E6/E7) combined with programmed death-ligand 1 (PD-1) blockade could increase therapeutic effect as compared to the vaccine alone. Ad5 [E1-, E2b-]-E6/E7 as a single agent induced HPV-E6/E7 cell-mediated immunity. Immunotherapy using Ad5 [E1-, E2b-]-E6/E7 resulted in clearance of small tumors and an overall survival benefit in mice with larger established tumors. When immunotherapy was combined with immune checkpoint blockade, an increased level of anti-tumor activity against large tumors was observed. Analysis of the tumor microenvironment in Ad5 [E1-, E2b-]-E6/E7 treated mice revealed elevated CD8+ tumor infiltrating lymphocytes (TILs); however, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1+ TILs. When Ad5 [E1-, E2b-]-E6/E7 immunotherapy was combined with anti-PD-1 antibody, we observed CD8+ TILs at the same level but a reduction in tumor PD-L1 expression on tumor cells and reduced PD-1+ TILs providing a mechanism by which combination therapy favors a tumor clearance state and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials.

Introduction

High-risk human papillomavirus (HPV) such as HPV type-16 is associated with the etiology of cervical and more than 90% of HPV-related head and neck squamous cell carcinomas.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Preventive vaccines such as Human Papillomavirus Bivalent [Types 16 and 18] Vaccine, Recombinant and Human Papillomavirus Quadrivalent [Types 6, 11, 16 and 18] Vaccine, Recombinant are a primary defense against HPV-associated cancers by preventing infection with the virus but reports indicate that they are not effective for active immunotherapy of established disease.12, 13, 14, 15 Immunotherapeutic treatment of HPV-induced malignances has been investigated by us and others. The HPV early 6 (E6) and early 7 (E7) genes are expressed at high levels in HPV-induced cancers and are involved in the immortalization of primary human epidermal cells.3, 8, 9, 10, 11 Thus, these are ideal targets for tumor-specific immunotherapy because unlike many other tumor-associated antigens these viral antigens are “non-self” and thus do not have the potential to induce autoimmunity.3, 8, 9, 10, 11

We have developed and previously reported on an immunotherapeutic vaccine to treat or prevent HPV-associated cancers.16 The immunotherapeutic was comprised of a gene delivery vehicle (Ad5 [E1-, E2b-]) carrying modified genes for HPV type-16 E6 and E7.16 The HPV-E6 and E7 genes were modified to render them non-oncogenic while retaining the antigenicity necessary to produce an immune response against HPV-induced tumors.16 We have also reported that these mutations lack the capacity to degrade p53, pRb, and PTPN13 and we have incorporated the modified non-oncogenic HPV-E6/E7 genes into our vaccine (Ad5 [E1-, E2b-]-E6/E7). The Ad5 [E1-, E2b-]-E6/E7 retains the ability to induce an HPV-specific cell-mediated immune (CMI) response and synergizes with standard clinical therapy, enhancing immune-mediated clearance of an HPV-E6/E7 expressing tumor in vivo. In a murine model of HPV-E6/E7 expressing tumors, we investigated the Ad5 [E1-, E2b-]-E6/E7 product for immunogenicity and anti-tumor activity.16 We observed that multiple homologous immunizations with Ad5 [E1-, E2b-]-E6/E7 induced significant CMI responses directed against HPV-E6/E7 antigens as assessed by IFN-γ and IL-2 secreting lymphocytes in an ELISpot assay. Importantly, when combined with chemotherapy/radiation treatment in HPV-E6/E7 expressing tumor bearing mice, immunotherapy treatment with Ad5 [E1-, E2b-]-E6/E7 resulted in significant improvement in overall survival as compared to control mice that received chemotherapy/radiation alone.16

Programmed death-ligand 1 (PD-L1) and programmed cell death protein-1 (PD-1) interact as immune checkpoints and this interaction is a major tolerance mechanism which results in the blunting of anti-tumor immune responses and subsequent tumor progression.17, 18, 19, 20 PD-1 is present on activated T cells and PD-L1, the primary ligand of PD-1, is often expressed on tumor cells and antigen-presenting cells (APC) as well as other cells, including B cells.21 Significant expression of PD-L1 has been demonstrated on various human tumors including HPV-associated head and neck cancers.22, 23, 24, 25 PD-L1 interacts with PD-1 on T cells inhibiting T cell activation and cytotoxic T lymphocyte (CTL) mediated lysis.19, 20 Previous studies have shown an improved therapeutic benefit by combining immunotherapeutic vaccination with blocking of immune checkpoints.26, 27, 28 Herein, we investigate if Ad5 [E1-, E2b-]-E6/E7 immunizations combined with PD-1 blockade can increase an anti-tumor effect. Also, we further characterize the CMI response induced by the Ad5 [E1-, E2b-]-E6/E7 vaccine and determine the kinetics of an anti-tumor response to evaluate the therapeutic potential of treating small versus large established tumors. To investigate a possible mechanism of action, we evaluate the relationship between the levels of effector T cells and suppressor T cells within the parenchyma of the tumor and characterize lymphocyte populations and expression of co-inhibitory molecules that may play a role in the observed anti-tumor responses.

Methods

Mice

Specific pathogen-free (SPF), female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) aged 8–10 weeks were housed in animal facilities at the Infectious Disease Research Institute (IDRI) (Seattle, WA, USA). All procedures were conducted according to Institutional Animal Care and Usage Committee (IACUC) approved protocols.

Viral construction

Ad5 [E1-, E2b-]-E6/E7 was constructed and produced as previously described.16, 29 Briefly, the transgenes were sub-cloned into the Ad5 [E1-, E2b-] vector using a homologous recombination-based approach and the replication deficient virus was propagated in the E.C7 packaging cell line, CsCl2 purified, and titered as previously described.29 Viral infectious titer was determined as plaque forming units (PFU) on an E.C7 cell monolayer. The viral particle (VP) concentration was determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 and 280 nm (ViraQuest, North Liberty, IA). As a vector control, we employed Ad5 [E1-, E2b-]-null, which is the Ad5 platform backbone with no transgene insert.

Immunization and splenocyte preparation

Female C57BL/6 mice (n=5/group) were injected subcutaneously (SQ) with varying doses of Ad5 [E1-, E2b-]-E6/E7 or Ad5 [E1-, E2b-]-null. Doses were administered in 25 μl injection buffer (20 mM HEPES with 3% sucrose) and mice were immunized three times at 14-day intervals. Fourteen days after the final injection, spleens and sera were collected. Serum from mice was frozen at −20 °C until evaluation. Suspensions of splenocytes were generated by disrupting the spleen capsule and gently pressing the contents through a 70 μm nylon cell strainer (BD Falcon, San Jose, CA). Red blood cells were lysed by the addition of red cell lysis buffer (Sigma-Aldrich, St Louis, MO) and after lysis, the splenocytes were washed twice in R10 (RPMI 1640 supplemented with L-glutamine (2 mM), HEPES (20 mM) (Corning, Corning, NY), penicillin (100 U/ml) and streptomycin (100 μg/mL) (Hyclone, GE Healthcare Life Sciences, Logan, UT), and 10% fetal bovine serum (Hyclone). Splenocytes were assayed for cytokine production by ELISpot and flow cytometry as described below.

Enzyme-linked immunosorbent spot (ELISpot) assay

HPV-E6 and E7 specific interferon-γ (IFN-γ) secreting T cells were determined by ELISpot assays using freshly isolated mouse splenocytes prepared as described above. The ELISpot assay was performed according to the manufacturer’s specifications (Affymetrix Bioscience, San Diego, CA). Pools of overlapping peptides spanning the entire coding sequences of HPV-E6 and E7 were synthesized as 15-mers with 11-amino acid overlaps (JPT GmbH, Berlin, Germany) and lyophilized peptide pools were dissolved in DMSO. Splenocytes (2 × 105 cells) were stimulated with 2 μg/ml/peptide of overlapping 15-mer peptides in pools derived from E6 or E7. Cells were stimulated with Concanavalin A (Con A) at a concentration of 0.06 μg/per well as a positive control. Overlapping 15-mer complete peptide pools derived from SIV-Nef (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were used as irrelevant peptide controls. The numbers of Spot Forming Cells (SFC) were determined using an Immunospot ELISpot plate reader (Cellular Technology, Shaker Heights, OH) and results reported as the number of SFC per 106 splenocytes.

Intracellular cytokine stimulation

Splenocytes were prepared as described for the ELISpot assay above. Stimulation assays were performed using 106 live splenocytes per well in 96-well U-bottom plates. Splenocytes in R10 media were stimulated by the addition of HPV-E6, HPV E7, or SIV-Nef peptide pools at 2 μg/ml/peptide for 6 h at 37 °C in 5% CO2, with protein transport inhibitor (GolgiStop, BD) added two hours after initiation of incubation. Stimulated splenocytes were then stained for lymphocyte surface markers CD8α and CD4, fixed with paraformaldehyde, permeabilized, and stained for intracellular accumulation of IFN-γ and TNF-α. Fluorescent-conjugated antibodies against mouse CD8α (clone 53-6.7), CD4 (clone RM4-5), IFN-γ (clone XMG1.2), and TNF-α (clone MP6-XT22) were purchased from BD and staining was performed in the presence of anti-CD16/CD32 (clone 2.4G2). Flow cytometry was performed using an Accuri C6 Flow Cytometer (BD) and analyzed using BD Accuri C6 Software.

Tumor immunotherapy

For in vivo tumor immunotherapy studies, female C57BL/6 mice, 8–10 weeks old, were implanted with 2 × 105 TC-1 HPV-E6/E7 expressing tumor cells (kindly provided by Dr Andre Lieber, University of Washington) SQ in the left flank. Mice were treated three times at 7-day intervals with SQ injections of 1010 VP Ad5 [E1-, E2b-]-E6/E7. Control mice were injected with 1010 VP Ad5 [E1-, E2b-]-null under the same protocol. In combinational studies, mice were given 100 μg of rat anti-PD-1 (clone RMP1-14) or an isotype rat control antibody (clone 2A3) IP at the same time as immunization. Rat anti-PD-1 antibody and rat IgG2a isotype control antibodies were purchased from BioXcell (Lebanon, NH). Tumor size was measured by two opposing dimensions (a, b) and volume was calculated according to the formula V=(a2 × b)/2 where a was the shorter dimension.30 Animals were euthanized when tumors reached 1500 mm3 or when tumors became ulcerated.

Analysis of tumor infiltrating cells (TILs) by flow cytometry

Four groups of 8–10 week old female C57BL/6 mice (n=5/group) were implanted with 2 × 105 TC-1 tumor cells SQ in the left flank at day 0. Two of these groups were immunized SQ with 1010 VP Ad5 [E1-, E2b-]-null vector control and the other two groups SQ with 1010 VP Ad5 [E1-, E2b-]-E6/E7 vaccine. These immunizations were administered twice at 7-day intervals starting on day 12. In addition to immunizations, mice in one Ad5 [E1-, E2b-]-E6/E7 group and one Ad5 [E1-, E2b-]-null group were administered 100 μg rat anti-PD-1 (clone RMP1-14) SQ at days 12 and 16 and 100 μg hamster anti-PD-1 (clone J43) at days 19 and 23 to increase the effective dose of anti-PD-1. To control for treatment with these checkpoint inhibitors, mice in the remaining Ad5 [E1-, E2b-]-E6/E7 and Ad5 [E1-, E2b-]-null groups were administered the relevant rat and hamster control IgG antibodies on the same days. Hamster anti-PD-1 antibody and isotype control were purchased from BioXcell. At day 27, tumors were measured, excised, and weighed. Tumors were minced and digested with a mixture of collagenase IV (1 mg/ml), hyaluronidase (100 μg/ml), and DNase IV (200 U/ml) in Hank’s Balanced Salt Solution (HBSS) at room temperature for 30 min and rotating at 80 rpm. Enzymes were purchased from Sigma-Aldrich. After digestion, the tumor suspension was placed through a 70 μm nylon cell strainer and centrifuged. Red cells were removed by the addition of red cell lysis buffer (Sigma-Aldrich) and after lysis, the tumor suspensions were washed twice in phosphate buffered saline (PBS) containing 1% (w/v) bovine serum albumin and resuspended in fluorescent activated cell sorting (FACS) buffer (PBS pH 7.2, 1% fetal bovine serum, and 2 mM EDTA) for staining. Fluorescent-conjugated antibodies against CD45 (30-F11), CD4 (RM4-5), and PD-L1 (MIH5) were purchased from BD. Fluorescent-conjugated antibodies against CD8β (H35-17.2), CD25 (PC61.5), FoxP3 (FJK-16 s), PD-1 (RMP1-30), LAG-3 (C9B7W), and CTLA-4 (UC10-4B9) were all purchased from eBioscience. Surface staining was performed for 30 min at 4 °C in 100 μl FACS buffer containing anti-CD16/CD32 (clone 2.4G2). Stained cells were washed in FACS buffer, fixed with paraformaldehyde, and (if needed) permeabilized in permeabilization buffer (eBioscience) before staining with fluorescent-conjugated anti-FoxP3 or anti-CTLA-4 for 60 min at 4 °C in 100 μl permeabilization buffer containing anti-CD16/CD32 (clone 2.4G2). Cells were washed with permeabilization buffer, washed back into FACS buffer, and a fixed volume of each sample was analyzed by flow cytometry using a BD Accuri C6 flow cytometer. Tumor cells were defined as CD45 events in a scatter gate that includes small and large cells. CD4+ TILs were defined as CD45+CD4+ events in a lymphocyte scatter gate. CD8+ TILs were defined as CD45+CD8β+ events in a lymphocyte scatter gate. Regulatory T cells (Tregs) were defined as CD45+CD4+CD25+FoxP3+ events in a lymphocyte scatter gate. Effector CD4+ T cells were defined as CD45+CD4+CD25FoxP3 events in a lymphocyte scatter gate. Isotype-matched control antibodies were used to determine positive expression of FoxP3, PD-L1, PD-1, LAG-3, and CTLA-4. Flow cytometry was performed using an Accuri C6 Flow Cytometer (BD) and analyzed in BD Accuri C6 Software.

Results

HPV-E6/E7 specific cell-mediated immune responses induced by Ad5 [E1-, E2b-]-E6/E7

A study was performed to determine the effect of increasing doses of Ad5 [E1-, E2b-]-E6/E7 immunizations on the induction of CMI responses in mice. Groups of C57BL/6 mice (n=5/group) were immunized SQ three times at 14-day intervals with 108, 109, or 1010 VP Ad5 [E1-, E2b-]-E6/E7. Control mice received 108 VP, 109 VP, or 1010 VP Ad5 [E1-, E2b-]-null (empty vector controls). Two weeks after the last immunization, splenocyte CMI responses were assessed by ELISpot analysis for IFN-γ secreting cells. A dose effect was observed and the highest CMI response level was obtained by immunizations with 1010 VP Ad5 [E1-, E2b-]-E6/E7 (Figure 1). No responses were detected in control mice injected with Ad5 [E1-, E2b-]-null (data not shown).

Figure 1
figure1

CMI dose response of Ad5 [E1-, E2b-]-E6/E7. C57BL/6 mice (n=5/group) were immunized three times at 14-day intervals with doses of 108, 109 or 1010 VP Ad5 [E1-, E2b-]-E6/E7. Fourteen days after the final immunization, splenocytes were assessed by ELISpot analysis. The greatest induction of CMI was achieved with the 1010 VP dose. For positive controls, splenocytes were exposed to concanavalin A in all ELISpot assays as described (data not shown). The error bars depict the standard error of the mean (s.e.m.).

Intracellular accumulation of IFN-γ and TNF-α in both CD8α+ and CD4+ splenocytes populations was also determined in mice immunized with 1010 VP Ad5 [E1-, E2b-]-E6/E7. Intracellular cytokine staining (ICS) after stimulation with overlapping peptide pools revealed E6 and E7 antigen-specific IFN-γ accumulation in CD8α+ lymphocytes isolated from all mice immunized with Ad5 [E1-, E2b-]-E6/E7 (Figure 2a). Peptide-stimulated splenocytes were also stained for the intracellular accumulation of TNF-α, and we were able to detect significant populations of multifunctional (IFN-γ+TNF-α+) CD8α+ splenocytes specific for both E6 and E7 (Figure 2b).

Figure 2
figure2

Activated splenocyte populations after immunization of mice with Ad5 [E1-, E2b-]-E6/E7. C57BL/6 mice (n=5/group) were immunized three times at two week intervals with 1010 VP Ad5 [E1-, E2b-]-E6/E7 (black bars). Controls received 1010 VP Ad5 [E1-, E2b-]-null (white bar). Splenocytes were collected 14 days after the final immunization and were assessed by flow cytometry for (a) CD8α+IFN-γ+ and (b) CD8α+IFN-γ+TNF-α+ cells. For positive controls, splenocytes were exposed to PMA/ionomycin (data not shown). The error bars depict the s.e.m.

Treatment of HPV-E6/E7 expressing tumors

We investigated the anti-tumor effect of immunotherapy treatment in mice bearing HPV-E6/E7 TC-1 tumors. These tumor cells expressed PD-L1 as assessed by flow cytometry analysis. When labeled with PE-conjugated anti-PD-L1, the TC-1 cells had a median fluorescent intensity (MFI) of 537 whereas cells labeled with a PE-conjugated isotype control antibody had an MFI of 184, demonstrating the presence of the immune suppressive PD-L1 on the surface of the TC-1 cells (data not shown). Two groups of C57BL/6 mice (n=5/group) were inoculated with 2 × 105 TC-1 tumor cells SQ into the right subcostal area on day 0. On days 1, 8, and 14 mice were treated by SQ injections of 1010 VP Ad5 [E1-, E2b-]-null (vector control) or 1010 VP Ad5 [E1-, E2b-]-E6/E7. All mice were monitored for tumor size and tumor volumes were calculated. Mice immunized with Ad5 [E1-, E2b-]-E6/E7 had significantly smaller tumors than control mice beginning on day 12 (P<0.01) and remained significantly smaller for the remainder of the experiment (P<0.02), including 3 of 5 mice showing complete tumor regression (Figure 3a). Tumors in mice from the vector control treated group began reaching the threshold for euthanasia starting on day 26 and all mice in this group were euthanized by day 33, whereas mice in the Ad5 [E1-, E2b-]-E6/E7 treated group were all alive with complete tumor regression of small tumors (<150 mm3) at the end of experiment on day 36 (Figure 3b).

Figure 3
figure3

Immunotherapy of non-palpable HPV-E6/E7 expressing tumors with Ad5 [E1-, E2b-]-E6/E7. C57BL/6 mice (n=5/group) were implanted on day 0 with 2 × 105 TC-1 tumor cells and administered 1010 VP Ad5 [E1-, E2b-]-null (vector control) or 1010 VP Ad5 [E1-, E2b-]-E6/E7 on days 1, 8 and 15 as indicated by arrows. (a) Tumor size was determined and volumes calculated according to the formula V=(a2 × b)/2. Analysis of significance was performed between experimental and vector control groups using unpaired t-tests and significance is denoted by * (P<0.05) and ** (P<0.01). Error bars represent the standard error of the means. (b) Survival curves were plotted and compared using the Mantel-Cox test. Significance is denoted by ** (P<0.01).

To determine if immunotherapy with Ad5 [E1-, E2b-]-E6/E7 was effective against larger tumors, we implanted TC-1 tumor cells tumors as described above in two groups of C57BL/6 mice (n=4/group) and then delayed weekly treatment with Ad5 [E1-, E2b-]-E6/E7 for 6 days post tumor implantation, at a time when tumors were small but palpable. Mice beginning treatment on day 6 initially demonstrated tumor growth similar to the control group; however, beginning on day 16, tumor regression was observed (Figure 4a). The tumors in mice that began treatment on day 6 were significantly smaller (P<0.05) than the control group beginning on day 20 and 3 of 4 mice had complete regression by day 27. Ad5 [E1-, E2b-]-E6/E7 administration beginning on day 6 also conferred a significant survival benefit (P<0.01) (Figure 4b).

Figure 4
figure4

Immunotherapy of small palpable HPV-E6/E7 expressing tumors with Ad5 [E1-, E2b-]-E6/E7. C57BL/6 mice (n=4/group) were implanted on day 0 with 2 × 105 TC-1 tumor cells and administered 1010 VP Ad5 [E1-, E2b-]-null (vector control) or 1010 VP Ad5 [E1-, E2b-]-E6/E7 on days 6, 13 and 20 as indicated by arrows. (a) Tumor size was determined and volumes calculated according to the formula V=(a2 × b)/2. On day 23, mice were euthanized from the vector control group and no analyses of significance could be performed after this time point. This is denoted by a dashed line. Analysis of significance was performed between experimental and vector control groups using unpaired t-tests and significance is denoted by ** (P<0.01). Error bars represent the standard error of the means. (b) Survival curves were plotted and compared using the Mantel-Cox test. Significance is denoted by ** (P<0.01).

Finally, to determine if immunotherapy with Ad5 [E1-, E2b-]-E6/E7 was effective against large established tumors, we implanted TC-1 tumor cells tumors as described above in two groups of C57BL/6 mice (n=4/group) then delayed weekly treatment with Ad5 [E1-, E2b-]-E6/E7 until 13 days post tumor implantation, when tumors were ~100 mm3. In this treatment group, we also observed initial tumor growth similar to the control group but some mice in the control group reached euthanasia criteria on day 23, preventing analysis of significance at further time points (Figure 5a). However, tumor volumes in the Ad5 [E1-, E2b-]-E6/E7 treated group were below the euthanasia threshold through day 29, at which point tumors from all mice in the vector control group had exceeded 1500 mm3 and were euthanized (Figure 5b). These results indicate that in the TC-1 tumor model the Ad5 [E1-, E2b-]-E6/E7 immunotherapeutic was a potent inhibitor of tumor growth and lead to significant overall survival benefit, however complete clearance of tumors was only observed when treatment was initiated in smaller tumors. Furthermore, these results demonstrate that, despite the presence of immune suppressing PD-L1 on tumor cells, immunotherapeutic treatment with Ad5 [E1-, E2b-]-E6/E7 resulted in significant inhibition of tumor growth.

Figure 5
figure5

Immunotherapy of large established HPV-E6/E7 expressing tumors with Ad5 [E1-, E2b-]-E6/E7. C57BL/6 mice (n=4/group) were implanted on day 0 with 2 × 105 TC-1 tumor cells and administered 1010 VP Ad5 [E1-, E2b-]-null (vector control) or 1010 VP Ad5 [E1-, E2b-]-E6/E7 on days 13, 20 and 27 as indicated by arrows. (a) Tumor size was determined and volumes calculated according to the formula V=(a2 × b)/2. On day 23, mice were euthanized from the vector control group and no analyses of significance could be performed after this time point. This is denoted by a dashed line. Analysis of significance was performed between experimental and vector control groups using unpaired t-tests. Error bars represent the standard error of the means. (b) Survival curves were plotted and compared using the Mantel-Cox test. Significance is denoted by ** (P<0.01).

Combination immunotherapy with immune checkpoint inhibition

To determine if we could improve the therapeutic effect of Ad5 [E1-, E2b-]-E6/E7 in the setting of large tumors, we co-administered anti-PD-1 antibody. Four groups of mice (n=7/group) were implanted with 2 × 105 TC-1 tumor cells on day 0 and beginning on day 10 the mice received weekly administrations of SQ 1010 VP Ad5 [E1-, E2b-]-E6/E7 plus IP 100 μg anti-PD-1, 1010 VP Ad5 [E1-, E2b-]-null plus 100 μg anti-PD-1, 1010 VP Ad5 [E1-, E2b-]-E6/E7 plus 100 μg rat IgG2a isotype control, or 1010 VP Ad5 [E1-, E2b-]-null plus 100 μg rat IgG2a isotype control. Tumor size was monitored over time and mice were euthanized when tumor size exceeded 1500 mm3 or when tumor ulceration was present. Control mice that received Ad5 [E1-, E2b-]-null plus 100 μg rat IgG2a isotype control (Figure 6a) and mice treated with Ad5 [E1-, E2b-]-null plus 100 μg anti-PD-1 (Figure 6b) exhibited a similar tumor growth pattern (Figure 6b). No significant survival benefit was observed between these two groups. Mice that received Ad5 [E1-, E2b-]-E6/E7 plus rat IgG2a isotype control had a delayed tumor growth pattern as compared to the controls and 2 of the mice had tumor regressions to near baseline level at day 52 post tumor implantation (Figure 6c). Four of the 7 mice that received Ad5 [E1-, E2b-]-E6/E7 and anti-PD-1 had tumor regression starting at day 25, and two of these resulted in tumor clearance through the end of experiment at day 53 (Figure 6d).

Figure 6
figure6

Anti-tumor activity observed with Ad5 [E1-, E2b-]-E6/E7 and anti-PD-1 combination immunotherapy. C57BL/6 mice (n=7/group) were inoculated on day 0 with 2 × 105 TC-1 tumor cells and administered treatments on days 10, 17, and 24 with Ad5 [E1-, E2b-]-null (vector control) plus isotype control rat IgG, (b) Ad5 [E1-, E2b-]-null plus anti-PD-1, (c) Ad5 [E1-, E2b-]-E6/E7 plus isotype control rat IgG or (d) Ad5 [E1-, E2b-]-E6/E7 plus anti-PD-1. Ad5 [E1-, E2b-]-E6/E7 and Ad5 [E1-, E2b-]-null were administered at 1010 VP per dose and control rat IgG and rat anti-PD-1 antibody were administered at 100 μg per dose. Tumor size was determined and volumes calculated according to the formula V=(a2 × b)/2. Mice were terminated if tumor volumes exceeded 1500 mm3 or due to excessive ulceration. Tumor growth kinetics represents individual mice in each group.

Mice treated with Ad5 [E1-, E2b-]-E6/E7 plus rat IgG2a isotype control (Figure 7) also experienced a survival benefit with 28.6% of the animals surviving at termination of the study whereas 100% of the control mice (Ad5 [E1-, E2b-]-null plus rat IgG2a isotype control) and the Ad5 [E1-, E2b-]-null plus anti-PD-1 treated mice had to be terminated by day 28 and 32, respectively (Figure 7). Mice treated with both Ad5 [E1-, E2b-]-E6/E7 and anti-PD-1 antibody had the greatest treatment benefit (Figure 7), demonstrating delayed tumor growth and a significant improvement (P≤0.0006) in survival as compared to the controls.

Figure 7
figure7

Overall survival of mice treated with Ad5 [E1-, E2b-]-E6/E7 or vaccine plus anti-PD-1. C57BL/6 mice (n=7/group) were treated as those in Figure 6 and observed for survival. The experiment was terminated on day 52 following tumor implantation. Mice treated with Ad5 [E1-, E2b-]-E6/E7 and control antibody exhibited significantly (P<0.008) longer survival as compared to both groups of control mice (Ad5 [E1-, E2b-]-null and control antibody or Ad5 [E1-, E2b-]-null and anti-PD-1). Two of 7 (29%) Ad5 [E1-, E2b-]-E6/E7 and control antibody treated mice remained alive at day 52. Mice treated with Ad5 [E1-, E2b-]-E6/E7 plus anti PD-1 exhibited significantly (P<0.0006) longer survival as compared to both groups of controls. 4 of 7 (57%) Ad5 [E1-, E2b-]-E6/E7 plus anti-PD-1 treated mice remained alive at day 52.

We did observe that mouse anti-rat IgG antibody responses were induced by the second injection (endpoint antibody titer 1:200 by ELISA, data not shown) with rat anti-PD-1 antibody and these responses were dramatically increased by the third injection (endpoint antibody titer 1:4000 to 1:8000 by ELISA, data not shown). This anti-rat antibody response may explain why no anti-tumor activity was observed after injections with anti-PD-1 antibody alone. Also, it is likely that the first and possibly the second injections of anti-PD-1 antibody combined with Ad5 [E1-, E2b-]-E6/E7 immunotherapy were effective but the third injection with anti-PD-1 was effectively neutralized by the induced mouse anti-rat IgG response.

Tumor microenvironment following combination immunotherapy

To analyze cell populations that contributed to delayed tumor growth and survival in Ad5 [E1-, E2b-]-E6/E7 treated mice, we analyzed tumor infiltrating lymphocytes (TILs) by flow cytometry. Four groups of mice were implanted with 2 × 105 TC-1 cells and began treatment 10 days later with two weekly immunizations of Ad5 [E1-, E2b-]-E6/E7 plus PD-1 antibody. On day 27 whole tumors were collected and processed as described in the materials and methods. The number of infiltrating CD8+ T cells per mg of tumor was significantly increased in the Ad5 [E1-, E2b-]-E6/E7 treated groups as compared to the groups that received Ad5 [E1-, E2b-]-null (Figure 8c). Anti-PD-1 antibody treatment had little or no effect on the number of infiltrating CD8+ T cells (Figure 8c). There was no difference between any of the four groups, in terms of the number of infiltrating Tregs (CD4+CD25+Foxp3+) per mg of tumor (Figure 8b). However, the increase in CD8+ T cells led to a decrease in the Treg:CD8+ T cell ratio in the tumor microenvironment when the mice were treated with the Ad5 [E1-, E2b-]-E6/E7 vaccine or Ad5 [E1-, E2b-]-E6/E7 vaccine plus anti-PD-1 antibody treatment (Figure 8a).

Figure 8
figure8

Ad5 [E1-, E2b-]-E6/E7 promotes the recruitment of CD8+ TIL into TC-1 tumors. C57BL/6 mice (n=5/group) were implanted with 2 × 105 TC-1 tumor cells. Twelve days after implantation mice began treatment with Ad5 [E1-, E2b-]-null empty vector plus control IgG (white), Ad5 [E1-, E2b-]-null plus anti-PD-1 (gray), Ad5 [E1-, E2b-]-E6/E7 plus control IgG (hatch), or Ad5 [E1-, E2b-]-E6/E7 plus anti-PD-1 (black). Vaccine was administered SQ weekly and anti-PD-1 antibodies were administered IP every 3-4 days until all mice were euthanized and tumors were analyzed on day 27. Ad5 [E1-, E2b-]-E6/E7 treatment significantly decreases the ratio of Treg/CD8+ TIL (a). This reduction is not driven by a reduction in the number of Tregs (b) but through an increase in the number of CD8+ TILs (c). Analysis of significance was performed using unpaired t-tests and significance is denoted by ns (P>0.05), * (P<0.05), ** (P<0.01), *** (P<0.001), or **** (P<0.0001).

To further study the synergistic/additive effect of anti-PD-1 antibody to Ad5 [E1-, E2b-]-E6/E7 immunotherapy, we examined the expression of PD-1, LAG-3, and CTLA-4 on TILs. The expression of these co-inhibitory molecules on T cells within the tumor microenvironment has been shown to down regulate activation of antigen-specific T cells.31, 32 Immunizations with Ad5 [E1-, E2b-]-E6/E7 plus control antibody treatment significantly increased the fraction of PD-1+ and LAG-3+ CD8+ TILs (Figure 9a and b), whereas, expression of these co-inhibitory molecules on CD4+ TILs was unaffected by this treatment (Figure 9a and b). The percentage of CD4+ and CD8+ TILs expressing CTLA-4 was not significantly affected by vaccine treatment (data not shown). Combining anti-PD-1 antibody injections with Ad5 [E1-, E2b-]-E6/E7 vaccine treatment resulted in a significant reduction in the fraction of PD-1+ CD8+ and CD4+ TILs, as compared with those found in tumors from mice treated with Ad5 [E1-, E2b-]-E6/E7 plus control antibody (P=0.0083 for CD8+ TILs and P=0.0016 for CD4+ TILs) (Figure 9a). Furthermore the fraction of PD-1+ CD8+ TILs was decreased to the level of expression observed in the Ad5 [E1-, E2b-]-null treated control groups, and the fraction of PD-1+ CD4+ TILs was significantly reduced to below that observed in the control groups (P=0.0016, Figure 9a). In addition, the percentage of LAG-3+ CD8+ TILs was also observed to decrease when the Ad5 [E1-, E2b-]-E6/E7 immunization was combined with the anti-PD-1 checkpoint inhibitor (P=0.0363, Figure 9b). Since it has previously been shown that vaccine treatment can enhance PD-L1 expression on tumor cells ex vivo,26 we examined the expression of PD-L1 on tumor cells. There was an augmentation in the median fluorescence intensity of PD-L1 on tumor cells after vaccine treatment (Figure 9c). However, PD-L1 expression was reduced in mice treated with the combination of Ad5 [E1-, E2b-]-E6/E7 and anti-PD-1 antibody, although this level was still significantly expressed above that observed in Ad5 [E1-, E2b-]-null treated control mice.

Figure 9
figure9

Ad5 [E1-, E2b-]-E6/E7 plus anti-PD-1 combination therapy promotes a pro-inflammatory tumor microenvironment. C57BL/6 mice (n=5/group) were tumor implanted, treated, and tumors were analyzed as in Figure 8, above. The frequency of PD-1+ CD4+ and CD8+ TILs (a), LAG-3+ CD8+ TILs (b), and the expression level of PD-L1 on tumor cells (c) is increased in tumors from mice treated with Ad5 [E1-, E2b-]-E6/E7. Tumors from mice treated with a combination of Ad5 [E1-, E2b-]-E6/E7 and anti-PD-1 have a significantly lower frequency of PD-1+ CD4+ and CD8+ TILs (a), a significantly lower frequency of LAG-3+ CD8+ TILs (b), and the expression level of PD-L1 on the tumor cells is significantly reduced (c), bringing these levels more in line with tumors from control mice. Analysis of significance was performed using unpaired t-tests and significance is denoted by ns (P>0.05), * (P<0.05), ** (P<0.01), or *** (P<0.001).

Discussion

Recent advances in the understanding and defining of immune co-inhibitory molecules, also known as immune checkpoints, are actively being applied to cancer immunotherapy. A balance between activation and inhibitory signals regulates the interaction between T lymphocytes and tumor cells, wherein T cell responses are initiated through antigen recognition by T-cell receptors (TCRs).18, 21 Blocking of immune checkpoints, such as the cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and PD-1, has been tested in cancer immunotherapy clinical trials and the results are encouraging.33, 34, 35, 36

In this report, we first tested the immunogenicity of the Ad5 [E1-, E2b-]-E6/E7 vaccine as a single agent. We observed that HPV-E6/E7 directed CMI responses were induced in a dose-dependent manner employing ELISpot assays to determine IFN-γ secreting splenocyte levels (Figure 1). Using the highest dose of vaccine, flow cytometry studies revealed that CD8+ lymphocytes expressing IFN-γ and TNF-α were detected and revealed that multifunctional T cells were induced with the Ad5 [E1-, E2b-]-E6/E7 vaccine (Figure 2), thus confirming that Ad5 [E1-, E2b-]-E6/E7 is effective at raising antigen-specific CMI responses against HPV-E6/E7 in this murine model.

Immunotherapy studies were then performed to test the anti-tumor activity induced by Ad5 [E1-, E2b-]-E6/E7. Using the HPV-E6/E7 expressing TC-1 murine tumor model we evaluated the efficacy of Ad5 [E1-, E2b-]-E6/E7 vaccine administration at three times post tumor implantation. If the initiation of immunotherapy began when tumors were not yet palpable, we observed that 60% of mice had complete tumor regression with overall survival at 100% (Figure 3). Similarly, if treatment began when tumors were small but palpable, we showed that the initial tumor growth tracked with those in the control group and that 3 of 4 mice had complete tumor regression and 100% overall survival (Figure 4). However, if treatment began when tumors were large and well-established, we observed that tumor growth also tracked with controls, but that inhibition of tumor growth was less striking and because tumors in control mice reached our predetermined euthanasia criteria, significance could only be detected by survival at the end of the experiment (Figure 5). These results indicated that immunotherapy with Ad5 [E1-, E2b-]-E6/E7 as a single agent could significantly impact tumor progression and induced anti-tumor activity; however immune suppression within more established tumors limited its efficacy. It also suggested that Ad5 [E1-, E2b-]-E6/E7 as a single immunotherapy may be most effective in patients with early disease and/or low tumor burden.

To determine if immune augmentation using anti-PD-1 antibody would enhance the anti-tumor effect in large established tumors, Ad5 [E1-, E2b-]-E6/E7 was combined with injections of anti-PD-1 antibody (Figures 6 and 7). Treatment with anti-PD-1 antibody alone had no anti-tumor effect. Mice treated with Ad5 [E1-, E2b-]-E6/E7 single immunotherapy responded with 29% of mice displaying tumor regression. Furthermore, we observed a synergistic trend when anti-PD-1 antibody was co-administered with Ad5 [E1-, E2b-]-E6/E7 as 57% of mice displayed tumor regression. We speculate that if we were able to utilize a mouse anti-PD-1 antibody, a more effective combination of immunotherapy may have been achieved; however, mouse anti-PD-1 antibody was not available for our use. Never the less, these studies demonstrated that combining Ad5 [E1-, E2b-]-E6/E7 immunotherapy with anti-PD-1 antibody resulted in a more effective form of anti-cancer treatment in this model of HPV-E6/E7 expressing tumors.

It has been reported that the balance between effector cells and regulatory T cells within the tumor itself can be a prognostic marker for tumor regression.27, 37 We analyzed TILs within the tumors of mice treated with Ad5 [E1-, E2b-]-E6/E7 alone or in combination with PD-1 inhibition. We observed that infiltrating CD8+ lymphocytes were increased within the tumors of mice treated with vaccine alone or in combination with anti-PD-1 as compared to controls (Figure 8). Since treatment with anti-PD-1 alone did not have this effect, this indicated that the increase in CD8+ T cells was driven by immunization with Ad5 [E1-, E2b-]-E6/E7. Although there were no differences observed in the numbers of infiltrating Treg cells, the increase in infiltrating CD8+ T cells in vaccine alone or anti-PD-1 antibody combination treated mice did result in decreased Treg/CD8+ TIL ratios (Figure 8).

Despite the increased homing of CD8+ T cells into the tumors, we found that treatment with vaccine alone upregulated the expression of several inhibitory immune checkpoint molecules, including PD-1 and lymphocyte activation gene-3 (LAG-3) on CD8+ TILs. More importantly, we observed the upregulation of PD-L1 on non-hematopoietic tumor cells. The upregulation of PD-L1 in response to tumor antigen-specific T cell infiltration has been reported and this has been hypothesized to be an important mechanism, which tumors employ to evade clearance by TILs.26, 38 Furthermore, tumor cell PD-L1 expression has been reported to correlate with clinical responses to nivolumab, an anti-PD-1 therapeutic,39 demonstrating that the expression of these checkpoints in response to antigen-specific vaccine may provide an opportunity for effective anti-tumor treatment with checkpoint inhibitors. In support of this, we showed that the tumor microenvironment in mice treated with Ad5 [E1-, E2b-]-E6/E7 plus anti-PD-1 antibody was much less suppressive than with vaccine alone, namely, we observed a reduction in PD-1+ and LAG-3+ TIL frequency, and noted that PD-L1 expression on tumor cells was reduced (Figure 9). A reduction in the number of PD-1+ TILs was important because we blocked this target resulting in an enhanced anti-tumor response. Interestingly, the fraction of LAG-3-expressing CD8+ TILs was also decreased when immunotherapy was combined with anti-PD-1 antibody. LAG-3 has been identified as a closely related molecule to CD4 that binds to Class II molecules.40 It can be expressed on human and murine NK cells, T regulatory cells, and activated T cells41, 42 and functions to negatively regulate T cell activation and limits the expansion of T cells through cell cycle arrest.43, 44 LAG-3 was shown to be expressed on TILs isolated from various carcinomas and ‘exhausted’ T cells in chronic viral infections.45, 46 Another study in ovarian cancer demonstrated a role for both PD-1 and LAG-3 in the co-inhibition of antigen-specific tumor infiltrating T cells.31 It was also shown that a significant proportion of antigen-specific CD8+ cells were LAG-3+PD-1+ and that they produced less IFN-γ and TNF-α than cells not expressing the co-inhibitory molecules.31 The data in this report further supports the notion that PD-1 and LAG-3 synergize to inhibit anti-tumor responses and injections with anti-PD-1 antibody may reverse this effect. Finally, we observed that PD-L1 expression was decreased on tumor cells from mice treated with combination immunotherapy. These tumor cells did not express PD-1 with vaccine treatment alone (data not shown) therefore the effect must not be a direct result of the anti-PD-1 antibody therapy, but rather driven by the specific removal of PD-1+ TILs. This strongly suggests that PD-L1 upregulation on tumor cells is driven by the influx of tumor antigen-specific PD-1+ TILs into the tumor microenvironment.

In summary, our data demonstrate that Ad5 [E1-, E2b-]-E6/E7 can induce HPV-E6/E7 directed CMI responses in a dose-dependent manner, which results in upregulation of PD-L1 on tumor cells. Multiple homologous immunizations in tumor bearing mice with the highest dose of vaccine resulted in significant anti-tumor activity and increased survival, particularly in mice bearing small tumors. Importantly, a greater degree of anti-tumor activity was achieved when immunotherapy with Ad5 [E1-, E2b-]-E6/E7 was combined with anti-PD-1 in mice with large tumors. Overall, immunizations with the Ad5 [E1-, E2b-]-E6/E7 vaccine combined with anti-PD-1 antibody results in an increase in CD8+ and CD4+ effector populations that have a less exhaustive/anergic phenotype and therefore favor the balance to a more pro-inflammatory state in the tumor microenvironment. The observation that the combined treatment was associated with reductions in large tumor mass indicates that immunotherapy with Ad5 [E1-, E2b-]-E6/E7 combined with anti-PD-1 antibody might increase clinical effectiveness during the immunotherapy of patients with HPV-associated head and neck or cervical cancers. Furthermore, our data suggests that clinical trials with the Ad5 [E1-, E2b-]-E6/E7 vaccine should be combined with a checkpoint inhibitor and remains a high priority.

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Acknowledgements

The authors thank Dr Winston Witcomb for management and care of the animals. We also thank Carol Jones for management of grant activities. This study was funded by Small Business Innovative Research (SBIR) Grants 1R43DE021973-01 and 2R44DE021973-02 from the National Institute of Dental and Cranial Research (NIDCR).

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Correspondence to E S Gabitzsch.

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AER, YL, JPB, ESG and FRJ are employees of Etubics and have equity and/or stock options in the company. JHL is a member of the Etubics Scientific Advisory Board and has stock options in the company.

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Rice, A., Latchman, Y., Balint, J. et al. An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression. Cancer Gene Ther 22, 454–462 (2015). https://doi.org/10.1038/cgt.2015.40

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