Search

Supplementary Table S4 shows respiratory rate of study participants at each visit, including immediately pre- and 30 minutes post-vaccination

Supplemental Methods

Supplemental Table 4. 2X2 table for CA125 and Anti-DISGTNTSRA.

Supplemental Table 5. 2X2 table for CA125 and Anti-CA125.

Supplemental Figure 2. OvCa patient survival based on CA125, anti-CA125, and anti-DISGTNTSRA status at time of diagnosis.

Patients with human papillomavirus type 16 (HPV16) infection and low-grade cervical dysplasia [low-grade squamous intraepithelial lesion (LSIL)/CIN1] or atypical squamous cells [atypical squamous cells of undetermined significance (ASC-US)/atypical squamous cells- cannot exclude high-grade squamous intraepithelial lesion (ASC-H)] require active surveillance for disease progression. A safe and effective immunotherapy to clear HPV16 is an unmet medical need. The safety run-in cohort of a randomized double-blind, placebo-controlled phase II trial of PVX2 [vaccination twice with HPV16-targeting pNGVL4a-Sig/E7(detox)/HSP70 plasmid and once with the HPV16 L2E7E6 fusion protein “TA-CIN”] as immunotherapy for patients with HPV16+ ASC-US, ASC-H, or LSIL/CIN1 (NCT03911076) was recently completed. The primary objective of this cohort was to determine the safety and tolerability of PVX2 vaccination. Subjects were confirmed to have HPV16 infection and LSIL/CIN1, ASC-US, or ASC-H. Adverse events were evaluated using Common Terminology Criteria for Adverse Events v5.0. HPV typing by HPV16 18/45 Aptima Assay was done at baseline, month 6, and month 12, with simultaneous cytology analysis. Cervical biopsies and endocervical curettage were performed at baseline and month 6. In the safety run-in cohort 12 eligible patients were enrolled. Each received three monthly immunizations. One was lost to follow-up after week 12. There were no serious adverse events. A total of five adverse events were noted by 4 patients; 4 were considered not vaccine-related, and one ‘unlikely related’ by the investigator. At month 6, 45% (5/11) of participants converted to HPV16-negative and 2 others developed CIN2+ and received a loop electrosurgical excision procedure. At month 12, 64% (7/11) were HPV16-negative, including those HPV16-negative at month 6. In conclusion, PVX2 immunotherapy was well tolerated and associated with viral regression, supporting further testing.Prevention Relevance:This safety run-in study cohort suggests that PVX2 immunotherapy is well tolerated in the target population and is sufficiently safe to warrant further clinical testing in a randomized study. The combined vaccines may facilitate higher-than-expected rate of human papillomavirus type 16 viral clearance 6 and 12 months after treatment, although this requires validation.

OvCa and normal plasma reactivity to DISGTNTSRA by ELISA.

High-grade epithelial ovarian cancer kills more women than any other gynecologic cancer and is rarely diagnosed at an early stage. We sought to identify tumor-associated antigens (TAA) as candidate diagnostic and/or immunotherapeutic targets by taking advantage of tumor autoantibody responses in individuals with ovarian cancer. Plasma-derived IgG from a pool of five patients with advanced ovarian cancer was subjected to iterative biopanning using a library of bacteriophage MS2 virus-like particles (MS2-VLPs) displaying diverse short random peptides. After two rounds of biopanning, we analyzed the selectant population of MS2-VLPs by Ion Torrent deep sequencing. One of the top 25 most abundant peptides identified (DISGTNTSRA) had sequence similarity to cancer antigen 125 (CA125/MUC16), a well-known ovarian cancer–associated antigen. Mice immunized with MS2-DISGTNTSRA generated antibodies that cross-reacted with purified soluble CA125 from ovarian cancer cells but not membrane-bound CA125, indicating that the DISGTNTSRA peptide was a CA125/MUC16 peptide mimic of soluble CA125. Preoperative ovarian cancer patient plasma (n = 100) was assessed for anti-DISGTNTSRA, anti-CA125, and CA125. Patients with normal CA125 (35 IU/mL). A statistically significant survival advantage was observed for patients who had either normal CA125 and/or higher concentrations of antibodies to CA125 at the time of diagnosis. These data show the feasibility of using deep sequence–coupled biopanning to identify TAA autoantibody responses from cancer patient plasma and suggest a possible antibody-mediated mechanism for low CA125 plasma concentrations in some ovarian cancer patients. Cancer Immunol Res; 4(2); 157–64. ©2015 AACR.

Supplemental Table 1. Top 25 ranked peptides from Round 2 of selection with OvCa patient IgG.

Supplemental Table 2. DISGTNTSRA peptide alignment with CA125/MUC16 (accession # NP_078966). Amino acid position is indicated by numerical position and alignment indicates the sequence similarity of the peptide (top) to CA125/MUC16 (bottom).

Weight gain in survivors compared to cancer-free women, by BRCA1/2 carrier status.

Supplementary Figure 7:Detection of CD103 and s1p1 expression by HPV16 E7-specific CD8+ T cells after intracervicovaginal vaccination Supplementary Figure 8: Comparison of HPV16 E7-specific CD8+ T cells induced by pNGVL4a-Sig/E7(detox)/HSP70 intracervicovaginal vaccination followed by either intracervicovaginal or subcutaneous TA-HPV boost

XLSX file - 125K, Drugs with 40-100% inhibition.

Purpose: Radiotherapy kills cancer cells by causing DNA damage, and stimulates a systemic antitumor immune response by releasing tumor antigen and endogenous adjuvant within the tumor microenvironment. However, radiotherapy also induces the recruitment of immunosuppressive myeloid cells, which can interfere with the antitumor immune responses elicited by apoptotic tumor cells. We hypothesized that local delivery of vaccine following radiotherapy will lead to the priming of antigen-specific CTL immune responses and render immunosuppressive myeloid cells susceptible to killing by the activated CTLs.Experimental Design: Using several antigenic systems, we tested whether intratumoral injection of antigenic peptide/protein in irradiated tumors would be able to prime CTLs as well as load myeloid cells with antigen, rendering them susceptible to antigen-specific CTL killing.Results: We show that by combining radiotherapy and targeted antigenic peptide delivery to the tumor, the adjuvant effect generated by radiotherapy itself was sufficient to elicit the priming and expansion of antigen-specific CTLs, through the type I IFN-dependent pathway, leading to synergistic therapeutic antitumor effects compared with either treatment alone. In addition, using two different types of transgenic mice, we demonstrated that CTL-mediated killing of stromal cells in tumors by our approach is important for tumor control. Finally, we confirmed the efficacy of this approach in our preclinical model using two clinically tested therapeutic human papilloma virus (HPV) vaccines.Conclusions: These data serve as an important foundation for the future clinical translation of radiotherapy combined with a clinically tested therapeutic HPV vaccine for the control of HPV-associated cancers. Clin Cancer Res; 20(3); 644–57. ©2013 AACR.

Comparison of anti-tumor effect generated by either pNGVL4a-Sig/E7(detox)/HSP70 intracervicovaginal vaccination followed by intracervicovaginal TA-HPV boost or intracervicovaginal TA-HPV vaccination

XLSX file - 165K, Response of Panc502 to 125 anti-neoplastic drugs.

Supplementary Figures S1-S4 from Ubiquitin Proteasome System Stress Underlies Synergistic Killing of Ovarian Cancer Cells by Bortezomib and a Novel HDAC6 Inhibitor

Supplementary Figure S2 from Delivery of Telomerase Reverse Transcriptase Small Interfering RNA in Complex with Positively Charged Single-Walled Carbon Nanotubes Suppresses Tumor Growth

Purpose: To determine whether -CONH-(CH2)6-NH3+Cl− functionalized single-walled carbon nanotubes (SWNT) carrying complexed small interfering RNA (siRNA) can enter into tumor cells, wherein they release the siRNA to silence the targeted gene.Experimental Design: -CONH-(CH2)6-NH3+Cl− was used to mediate the conjugation of telomerase reverse transcriptase (TERT) siRNA to SWNTs. The ability of TERT siRNA delivered via SWNT complexes to silence the expression of TERT was assessed by their effects on the proliferation and growth of tumor cells both in vitro and in mouse models.Results: The functionalized SWNTs -CONH-(CH2)6-NH3+Cl− could facilitate the coupling of siRNAs that specifically target murine TERT expression to form the mTERT siRNA:SWNT+ complex. These functionalized SWNTs rapidly entered three cultured murine tumor cell lines, suppressed mTERT expression, and produced growth arrest. Injection of mTERT siRNA:SWNT+ complexes into s.c. Lewis lung tumors reduced tumor growth. Furthermore, human TERT siRNA:SWNT+ complexes also suppressed the growth of human HeLa cells both in vitro and when injected into tumors in nude mice.Conclusions: -CONH-(CH2)6-NH3+Cl− functionalized SWNTs carry complexed siRNA into tumor cells, wherein they release the siRNA from the nanotube sidewalls to silence the targeted gene. The -CONH-(CH2)6-NH3+Cl− functionalized SWNTs may represent a new class of molecular transporters applicable for siRNA therapeutics.

PDF file - 81K, Characterization of tumor infiltrating T cells following treatment with systemic DNA vaccination and locally applied imiquimod.

Purpose: Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)–infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high-grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T-cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164).Experimental Design: Here, we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than intramuscular (IM) delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16+ cervical cancer (TC-1 luc).Results: We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8+ T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8+ T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8+ T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8+ T-cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract.Conclusions: Our results support future clinical translation using cervicovaginal TA-HPV vaccination. Clin Cancer Res; 22(3); 657–69. ©2015 AACR.See related commentary by Nizard et al., p. 530

Local and systemic immune responses produced by different vaccination routes

PDF file - 112KB

PDF file - 116K, In vivo response of xenograftted tumors.

PDF file - 129K

PDF file - 504KB, RA-9 inhibits tumor growth in vivo.

Purpose: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells.Experimental Design: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts.Results: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)–approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs.Conclusion: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. Clin Cancer Res; 19(5); 1139–46. ©2012 AACR.

Purpose: Elevated metabolic activity of ovarian cancer cells causes increased ubiquitin-proteasome-system (UPS) stress, resulting in their greater sensitivity to the toxic effects of proteasomal inhibition. The proteasomes and a potentially compensatory histone deacetylase 6 (HDAC6)-dependent lysosomal pathway mediate eukaryotic protein turnover. We hypothesized that up-regulation of the HDAC6-dependent lysosomal pathway occurs in response to UPS stress and proteasomal inhibition, and thus, ovarian cancer cell death can be triggered most effectively by coinhibition of both the proteasome- and HDAC6-dependent protein degradation pathways.Experimental Design: To address this hypothesis, we examined HDAC6 expression patterns in normal and cancerous ovarian tissues and used a novel HDAC6-specific inhibitor, NK84, to address HDAC6 function in ovarian cancer.Results: Abnormally high levels of HDAC6 are expressed by ovarian cancer cells in situ and in culture relative to benign epithelium and immortalized ovarian surface epithelium, respectively. Specific HDAC6 inhibition acts in synergy with the proteasome inhibitor Bortezomib (PS-341) to cause selective apoptotic cell death of ovarian cancer cells at doses that do not cause significant toxicity when used individually. Levels of UPS stress regulate the sensitivity of ovarian cancer cells to proteasome/HDAC6 inhibition. Pharmacologic inhibition of HDAC6 also reduces ovarian cancer cell spreading and migration consistent with its known function in regulating microtubule polymerization via deacetylation of α-tubulin.Conclusion: Our results suggest the elevation of both the proteasomal and alternate HDAC6-dependent proteolytic pathways in ovarian cancer and the potential of combined inhibition of proteasome and HDAC6 as a therapy for ovarian cancer.

Background: This study prospectively examines weight gain in breast cancer survivors compared with cancer-free women from a familial risk cohort.Methods: Absolute and percent weight change over 4 years was compared among 303 breast cancer survivors and 307 cancer-free women matched on age and menopausal status, from the same familial risk cohort. Linear and logistic regression was used to estimate the association between survivor status and weight gain.Results: Overall, breast cancer survivors gained significantly more weight [β = 3.06 pounds; 95% confidence intervals (CI), 0.94–5.17] than cancer-free women. Significant weight gain was observed in survivors diagnosed less than 5 years prior to baseline (β = 3.81 pounds; 95% CI, 1.22–6.29) and women with estrogen receptor (ER)-negative tumors (β = 7.26 pounds; 95% CI, 2.23–12.30). Furthermore, survivors treated with chemotherapy were 2.1 times more likely to gain at least 11 pounds during follow-up compared with cancer-free women (OR, 2.10; 95% CI, 1.21–3.63). Weight gain was even greater among survivors who took statins while undergoing chemotherapy treatment (Pinteraction = 0.01).Conclusion: This is the first study to demonstrate that weight gain is an important issue in breast cancer survivors with a familial risk. In the first five years posttreatment, breast cancer survivors gain weight at a faster rate than cancer-free women, particularly after chemotherapy and statin use but not after hormone therapy alone.Impact: Our findings provide support for the development of weight gain interventions for young breast cancer survivors with a familial risk. Cancer Epidemiol Biomarkers Prev; 24(8); 1262–9. ©2015 AACR.

PDF file - 941K, Characterization of E7-specific CD8+ T cell activation in tumor-bearing mice treated with radiation and intratumoral vaccination with E7 peptide.

Characterization of MAdCAM-1 expression in cervicovaginal tissue after vaccination

PDF file - 722KB, Dose- and time-dependent accumulation of poly-ubiquitinated proteins following RA-9 exposure.

PDF file - 809K, Evaluation of antitumor effect after radiation combined with intratumoral E7 peptide treatment in TLR9-/- and WT C57BL/6 mice.

PDF file - 87K, Breeding strategy for production and maintenance of the SCID hprt-null mouse.

Analysis of HPV16 E7-specific CD8+ T cells after intracervicovaginal vaccination with or without CD4+ T cell depletion

Comparison of HPV16 E7-specific CD8+ T cells induced by HPV16 E7 long peptide after either intramuscular or intracervicovaginal vaccination

PDF file - 105K, Pharmagram of Panc502 to all drugs in the JHDL.

PDF file - 1656K, Characterization of the distribution of CD11b+ myeloid cells and CD11b+ Gr-1+ MDSCs in tumor, spleen and DLN after radiation.

Supplemental Figure Legend

Purpose: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7.Experimental Design: Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance.Results: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8+ T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+ CD8+ T cells to the genital tract.Conclusions: Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. Clin Cancer Res; 20(21); 5456–67. ©2014 AACR.

PDF file - 1505K, Selection of pancreatic cancer cell line, Panc502.

PDF file - 70K, E7-specific T cells efficiently migrated from circulation to the genital tract following imiquimod application.

PDF file - 2157K, Characterization of different subsets of MDSCs in spleens, DLNs and tumors following the various treatments.

PDF file - 732KB, UPR stress levels in ovarian surface epithelial cells (OSEs).

PDF file - 1893K, FM108 ovarian cell line production.

PDF file - 272KB, Effect of RA-9 treatment on GCN2 expression levels.

PDF file - 122KB, Effect of RA-9 treatment on total body weight.