Institutional Review Board for Baylor College of Medicine and Affiliated Hospitals Protocol Number: H-54674 Status: Approved Initial Submit Date: 11/8/2023 Approval Period: 2/5/2024 - 12/26/2024 Section Aa: Title & PI A1. Main Title A PHASE 2 STUDY OF HPV L1 V ACCINE IN COMBINA TION WITH IMIQUIMOD AND METFORMIN IN CER VICAL, VAGINAL, AND VUL VAR CANCERS A2. Principal Investigator Name: JAN SUNDE Phone: 7137986848 Id: 206551 Fax: 832 8259356 Department: OB-GYN: ADMINISTRA TIVE Email: sunde@bcm.edu Center: Mail Stn: 125084 A3. Administrative Contact Name: KENESHIA LANE Phone: Id: 232877 Fax: Email: 232877@bcm.edu Mail Stn: A3a. Financial Conflict of Interest Does any member of study personnel (Investigator (including investigator's spouse and/or dependent children)) that are involved in the design, conduct, or reporting of the research have a Significant Financial Interest (SFI) that would reasonably appear to be af fected by the research for which funding is sought and/or associated with an entity/business that would reasonably appear to be af fected by the research? No Section Ab: General Information A4. Co-Investigators Name: MICHELLE LUDWIG Phone: 7135663757 Id: 183182 Fax: 713 5663758 Department: RADIA TION ONCOLOGY Email: msludwig@bcm.edu Center: Mail Stn: BCM71 1 Name: SHELL Y SHARMA Phone: 9013555672 Id: 188978 Fax: Department: RADIA TION ONCOLOGY Email: shellys@bcm.edu Center: Mail Stn: BCM360 Name: SIMONE HERNANDEZ RUANO Phone: 8328244190 Id: 191164 Fax: 832 8257946 Department: OB-GYN: ADMINISTRA TIVE Email: shruano@bcm.edu Center: Mail Stn: BCM610 8/23/24, 10:39 AM Human Protocol Report https://brain.bcm.edu/esp1/reports/Human/Protocol.asp?protocol=486486 1/16 Name: KATHLEEN PENNINGT ON Phone: 8328244190 Id: 191410 Fax: 832 8257946 Department: OB-GYN: ADMINISTRA TIVE Email: kapennin@bcm.edu Center: Mail Stn: BCM610 Name: TRACIL YN HALL Phone: 7137982452 Id: 195219 Fax: Department: OB-GYN: ADMINISTRA TIVE Email: trhall@bcm.edu Center: Mail Stn: 125084 Name: BERKLEY ANDERSON Phone: Id: 198728 Fax: Department: OB-GYN: ADMINISTRA TIVE Email: bakingma@bcm.edu Center: Mail Stn: Name: ANTHONY COST ALES Phone: 7137983495 Id: 208504 Fax: Department: OB-GYN: ADMINISTRA TIVE Email: costales@bcm.edu Center: Mail Stn: 125084 Name: CLAIRE HOPPENOT Phone: 7137985165 Id: 231402 Fax: Department: OB-GYN: ADMINISTRA TIVE Email: 231402@bcm.edu Center: Mail Stn: 125084 Name: JON FUSON Phone: Id: 246863 Fax: Department: OB-GYN: ADMINISTRA TIVE Email: u246863@bcm.edu Center: Mail Stn: A5. Funding Source: Baylor College of Medicine (Internal Funding Only) A6a. Institution(s) where work will be performed: Baylor St. Luke's Medical Center (BSLMC) Harris Health System- Smith Clinic A6b. Research conducted outside of the United States: Country: Facility/Institution: Contact/Investigator: Phone Number: If documentation of assurances has not been sent to the Of fice of Research, please explain: A7. Research Category: Cancer - Adult A8. Therapeutic Intent Does this trial have therapeutic intent? Yes A9. ClinicalTrials.gov Registration Does this protocol/trial require registration on ClinicalT rials.gov due to it: meeting the definition of an Applicable Clinical Trial, being required under the terms and conditions of an award, or being proposed to be published in ICMJE journals? Yes Who will be responsible for registering and maintaining the registration of this Applicable Clinical Trial? The BCM PI will register the trial because either: - the trial is BCM PI-initiated,8/23/24, 10:39 AM Human Protocol Report https://brain.bcm.edu/esp1/reports/Human/Protocol.asp?protocol=486486 2/16 - BCM is the lead site of this multicenter trial, or , - the industry sponsor has instructed the BCM PI to register the trial, or , - registration of this trail is required as a term and condition of the reward by the funding agency . ClinicalT rials.gov Identifier: NCT Section B: Exempt Request B. Exempt From IRB Review Not Applicable Section C: Background Information Each year in the United States, about 13,000 new cases of cervical cancer are diagnosed and about 4,000 women die of this cancer . Hispanic women have the highest rates of developing cervical cancer , and Black women have the highest rates of dying from cervical cancer (Cervical Cancer Statistics CDC, 2023). HPV is thought to be responsible for more than 90% of anal and cervical cancers (Cancers Associated with Human Papillomavirus (HPV), 2023). Despite our best ef forts, persistent/recurrent disease continues to occur in 30-40% of locally advanced cervical patients in clinical trials and in our patient population at Ben Taub (SGO2023) (SGO202302). Radiation has been the mainstay for treatment of locally advanced cervical and vulvar cancer for over a century , with addition of chemotherapy as a radiation sensitizer following an NCI Clinical Announcement in 1999. Recently , immunotherapy has been added in patients positive for PD-L1 in advanced stage cancers (1,2). Vaginal and vulvar cancers, which are also often HPV related, are very rare. Together , they account for about 7% of all gynecologic cancers diagnosed in the U.S. (Basic Information About V aginal and V ulvar Cancers | CDC, 2023). There is little clinical trial data studying vulvar cancer and vaginal cancer since they are rare, but they are typically treated with chemoradiation, and pembrolizumab if PD-L1 positive, in the same manner as squamous cell cervical cancer . Given that all of these disease processes can be HPV related, inclusion of these patient populations is planned if an eligible patient is identified. W e are just beginning to understand some of the roles of the immune system in fighting cancer , even though intratumoral injection was first reported by Dr . Coley in 1898 and termed “Coley toxin” (3). This was the first immunotherapy reported and consisted of extracts from bacteria responsible for erysipelas (Streptococcus pneumoniae and Serratia marcescens) which were injected into various skin cancers with significant success but this technique fell out of favor to radiation therapy due to toxicity and inconsistent responses (4). This trial is designed to stimulate tumor immunity through the use of sequential, targeted vaccinations of the FDA- approved, HPV 9-valent recombinat vaccine injected during and after radiation of the tumor , in conjunction with immunity modulating imiquimod and metformin. Advanced HPV related cancers are believed to replicate under the influence of “early genes”, but the late gene, HPV -L1 protein, has been reported to have low levels of expression in advanced cervical cancers and HPV 9-valent vaccine may produce an ef fective anti-tumor response in cervical cancer (5). Imiquimod, an FDA-approved Toll-like receptor 7 (TLR7) agonist, has been successfully used as a vaccine adjuvant improving anti-tumor immune responses (6), and vaccines used in conjunction with radiation therapy improve the recognition and uptake of both DNA viruses and radiation induced tumor- associated antigens (neoantigens) (7). This TLR7-agonist facilitates antigen processing of tumor-specific antigens in regional lymph nodes (8). The evaluation of combinations of immunotherapy with other treatment modalities to improve ef ficacy has been an active area of research in the last several years. Immunotherapy is now combined with chemotherapy in initial therapy for patients with metastatic or recurrent cervical cancer , and has been found to be synergistic with radiotherapy in a mouse model and in a recent clinical trial for locally advanced disease (9). Imiquimod, alone and with HPV vaccination, has demonstrated ef ficacy in eradicating pre-invasive HPV lesions of the vulva and the cervix (10, 1 1, 12). Investigators using preclinical models have found that intratumoral vaccination combined with radiation af fects the tumor microenvironment to improve the response to treatment (13) and also improves the abscopal response (14). Additional reported unanticipated benefits of immune treatment include the finding that intramuscular HPV vaccination alone decreases recurrence of both treated pre-invasive HPV related disease and vulvar carcinoma (15,16). Extensive work is ongoing in basic science investigating intratumoral immune response, with a few examples as noted: Imiquimod has been shown to reduce lesion size in conjunction with HPV vaccination in mice (6) and intratumoral vaccination with a HPV vaccine in a TLR vector led to a better response with lesion eradication in up to 50% of mice compared to IM vaccination (17). A recent renewed interest in intratumoral immune treatments in humans has led to multiple early phase human cancer trials, with multiple dif ferent agents, including: cervical intratumoral hydrogen peroxide based radiosensitizer treatment (18), cervical intratumoral oncolytic virus (14), melanoma intratumoral oncolytic virus (19), melanoma imiquimod intratumoral oncolytic vaccine (20), colon intratumoral influenza vaccine (21), and liver intratumoral8/23/24, 10:39 AM Human Protocol Report https://brain.bcm.edu/esp1/reports/Human/Protocol.asp?protocol=486486 3/16 oncolytic virus (22) as examples. Other examples of intratumoral immunotherapy for non-malignant conditions include: intralesional HPV vaccine combined with Candida antigen, which has been used for treatment of recalcitrant warts (23), and injection of compounds such as MMR vaccine and PPD leading to clearance of common warts in 80 and 60% of patients respectively (23). Based on published reports, the route of administration planned is not anticipated to increase the risk associated with use of the HPV vaccine (intratumoral) or the imiquimod (intravaginal) (24, 25) A large study of MV A E2 vaccinia injected directly into gynecologic lesions in 1 176 females reported grade 1 toxicities only , including headaches, flu symptoms, pyrexia, chills, abdominal ache, joint pain, similar to side ef fects noted with many vaccines (24). A meta-analysis of 463 patients treated with intra-vaginal imiquimod for cervical intraepithelial neoplasia (CIN) reported side ef fects like those reported by women treated with vulvar imiquimod, such as headache/migraine (57%) followed by fatigue (56%) and myalgia (55%), while the most common local side ef fect was vulvar pain/pruritus in 48% (25). Metformin is included in the planned regimen due to its immune ef fects. Diabetic patients treated with metformin have reduced incidence and better survival from cancer of many organs, including colorectal, liver , pancreatic, rectal, breast, prostate cancer and upper tract urothelial carcinoma (26). Metformin has been found to function in multiple ways to af fect cancer growth, including the activation of adenosine monophosphate-activated kinase (AMPK), and is also thought to effect the tumor microenvironment (26; 27), and may interact with immunotherapy (28). Subcutaneous (SC) administration of vaccines has been reported to stimulate more of a cell mediated immune response (29) compared to intramuscular delivery , so the vaccine will be administered both intratumoral and SC. The timing of treatment with radiation and immunotherapy appears to be of critical importance, with radiation producing an autologous “in-situ vaccine” specific to each cancer (30). In a recent case study report, use of a combination of several of the treatments reported above: intratumoral HPV vaccine in combination with topical TLR7 cream, imiquimod, and oral metformin, as well as zinc, and lactobacillus acidophilus, led to regression of recurrent, chemoradiation refractory , HPV -HR positive squamous cell cervical and vulvar cancers in 2 patients, resulting in complete resolution of these incurable tumors and clearance of HPV infection (31). Of note, one of the patients, a 89-year-old African American female demonstrated an abscopal response with resolution of extensive lung metastases. Two additional patients with chemoradiation refractory vulvar cancer have subsequently been of fered an of f- label treatment course with a similar protocol using HPV L1 vaccine, imiquimod and metformin prior to initiation of palliative treatment for their incurable disease. One patient, who began with a 20 cm initial tumor and had a 10 cm persistent necrotic mass following chemoradiation, has had a complete response based on PET/CT imaging, and is now 12 months out from treatment initiation. A second similar vulvar cancer patient who did not present for follow-up at 3 months after chemoradiation has had progression of disease, with the responsiveness of her tumor to the novel treatment thought to be adversely af fected by the delay in presentation. An additional locally advanced cervical cancer patient has received of f- label treatment before and following completion of chemoradiation. Though only 5 patients have been treated with this novel protocol which uses FDA-approved medications of f-label, these results are promising for treatment of persistent disease that is typically considered dif ficult to cure. As immunotherapy becomes incorporated into the standard of care, there remains room for improvement in these dif ficult-to-treat HPV related gynecologic cancers. This window-of-opportunity study will evaluate the immune changes within the tumor microenvironment and validate why the combination immunotherapy treatment elicited such a robust immune response which led to complete resolution of the cancer , in comparison to the standard of care, chemoradiation. If the combination immunotherapy used together with chemoradiation elicits a more robust antitumor response, there is the potential that the addition of these FDA approved medications to chemoradiation could be a new standard of treatment for squamous cell carcinoma of the cervix and vulva. References 1. Chung, H.C., et al., Ef ficacy and Safety of Pembrolizumab in Previously Treated Advanced Cervical Cancer: Results From the Phase II KEYNOTE-158 Study . J Clin Oncol, 2019. 37(17): p. 1470-1478.