Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form. 8 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 1 INTRODUCTION 1.1 STUDY O BJECTIVES 1.1.1 Primary Objectives Phase I in participants with recurrent/metastatic HPV positive cancer: Primary objective: To determine the safety and recommended phase II dose (RP2D) of PRGN-2009 (HPV vaccine) alone or in combination with M7824 administered at RP2D of 1200 mg q2w. Phase II in participants with newly diagnosed stage I (T1,2 N1)/II/III p16-positive oropharyngeal cancer (OPSCC) cancer: Primary objective: To determine if HPV vaccine alone (Arm 2A) is able to result in a  2-fold increase in CD3+ tumor infiltrating T cells post treatment compared with pre- treatment in p16-positive oropharyngeal cancer. 1.1.2 Secondary Objectives Phase I in participants with recurrent/metastatic HPV positive cancer: To assess overall response rate (ORR) according to RECIST 1.1. To assess progression-free survival time (PFS). To assess overall survival (OS). To assess duration of response. To assess ratio of participants that are hospitalized because of adverse events attributed to disease progression. Phase II in participants with newly diagnosed stage I (T1,2 N1)/II/III p16-positive oropharyngeal cancer (OPSCC) cancer: To determine if the use of PRGN-2009 alone results in significantly prolonged survival as compared to the expected 80% three-year historical survival for this population. To determine the 3-year overall and relapse-free survival rate for PRGN-2009 alone (Arm 2A) as neoadjuvant/ induction therapy before definitive standard of care therapy for this population. To assess the safety of the recommended phase II dose (RP2D) of PRGN-2009 (HPV vaccine) alone in this participant population. 1.1.3 Exploratory Objectives Phase I and Phase II To conduct exploratory immunologic studies to understand and improve the administered treatment, including: oTo examine the pharmacokinetics of bintrafusp alfa while on treatment. oTo determine the ORR per RECIST v1.1 of combination of PRGN-2009 and M7824 in participants with advanced HPV associated malignancies 9 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 Precigen, Inc. may analyze samples to be used for additional exploratory analyses, to further investigate: oHPV 16/18 -specific immune responses, activation status of the immune system, anti-tumor specific immune responses induced by the exposure to PRGN-2009. Phase II only To determine the rate of objective response following neoadjuvant/induction immunotherapy alone per RECIST 1.1. To assess changes in salivary HPV DNA from pre-treatment and compared to during neoadjuvant/induction therapy with PRGN-2009 (HPV vaccine) alone in participants with HPV-OPC. To determine the feasibility and safety of neoadjuvant treatment therapy with PRGN-2009 (HPV vaccine) alone in participants with HPV-SNSCC. To assess the differences in CD3+ tumor infiltrating lymphocytes following neoadjuvant therapy with PRGN-2009 (HPV vaccine) alone in participants with HPV-SNSCC. To determine the objective response rate following neoadjuvant therapy with PRGN-2009 (HPV vaccine) alone in participants with HPV-SNSCC. 1.2 BACKGROUND AND RATIONALE 1.2.1 HPV Associated Malignancies In the United States, there are more than 30,000 cases of HPV associated cancer annually (1) (Table 1). Metastatic HPV associated malignancies (cervical, anal, oropharyngeal cancers etc.) are often incurable and poorly palliated by standard therapies. Responses to chemotherapy are variable but generally short-lived with median PFS around 3 to 7 months (2-5). In a Gynecologic Oncology Group randomized trial comparing four cisplatin-based doublets as first line therapy for metastatic cervical cancer the response rates were 22-29% and median PFS was 4 to 6 months with median OS 10 to 13 months (6). The addition of bevacizumab to combination chemotherapy has been reported to increase OS by 3.7 months, but virtually all patients die of their disease within 2 years (7). Randomized trials of second line therapy are lacking but response rates for single agents are generally reported to be less than 20% (8). Early evidence suggests that immune checkpoint therapy also has a low response rate in this disease with a phase 1b trial (KEYNOTE 028) showing a 12.5% response rate (3/24 patients) and a phase II study showing a response rate of 13.3% (13/98 patients) to pembrolizumab in patients with recurrent or metastatic cervical cancer (9). Table 1: Estimated annual incidence of HPV associated cancers in the US Site Incidence of HPV associated cancers Cases attributed to HPV (%) Oropharyngeal 15,738 11,000 (70.1%) Cervix 11,771 10,700 (90.6%) Vulvar 3554 2400 (68.8%) 10 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 Site Incidence of HPV associated cancers Cases attributed to HPV (%) Vaginal 802 600 (75%) Penis 1168 700 (63.3%) Rectal (squamous cell)750 700 (91.1%) Anus 5,010 4,600 (91.1%) Total 38,793 30,700 (79.1%) For metastatic oropharyngeal cancer, the best estimates of the chemotherapy responsiveness are inferred from looking at the oropharyngeal site in subset analyses from clinical trials for head and neck cancers. In a pivotal clinical trial that established platinum, 5-fluorouracil (5-FU), plus cetuximab as first line therapy in head and neck cancer, patients with oropharyngeal tumors experienced PFS of 4 to 6 months and OS of 8 to 11 months (3). Immune checkpoint therapy has become the standard second line therapy for metastatic oropharyngeal cancer but response rates are still low with this therapy. As an example, the phase 1b trial (KEYNOTE-012) of pembrolizumab which led to its FDA approval for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) as second line therapy had a 17.7% response rate (34/192 patients). Response rates were slightly better in HPV positive HNSCC (21.9%) (10), but still occurred in only a minority of the patients. More recently pembrolizumab had been FDA approved as first line therapy for metastatic PD-L1 expressing HNSCC but the response rate for pembrolizumab alone in this setting is still only 16.9%. In regard to metastatic anal cancer, only a handful of randomized trials have been performed in the last 30 years. In the metastatic setting, most of the evidence is limited to small phase II trials, retrospective series, and case reports. A recent retrospective series looking at 77 patients, 44 (55%) of whom received 5-FU in combination with cisplatin, 24 (31%) of whom received carboplatin + paclitaxel, and 11 (14%) of whom received another regimen showed a median PFS of 7 months and median OS of 22 months (5). As with HNSCC, recent early phase trials evaluating immune checkpoint therapy in this disease have shown that responses here too are limited to around 20% of patients treated. A recent phase II trial of nivolumab for metastatic squamous cell anal cancer showed a 21% response rate (7/33 patients) and a recent phase IB trial (KEYNOTE-028) of pembrolizumab showed a 20% response rate (5/25 patients) (11). In a recently published phase II trial, Massarelli et al. evaluated the combination of a therapeutic HPV vaccine and Nivolumab (12). In contrast to previous single therapy immune checkpoint inhibitor trials, they found an overall response rate of 33% showing the potential benefit of Immuno-Oncology (I/O) combination therapy for these diseases. Human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) now represents the most common HPV-associated malignancy in the United States and the incidence is rising (13, 14). Over 80% of OPSCC is HPV-positive, and nearly all HPV-positive OPSCC are caused by HPV 16 or 18. Definitive standard-of-care treatment for patients with stage II/III HPV- positive oropharyngeal head and neck squamous cell carcinoma most commonly includes multi- 11 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 modality treatment consisting of concurrent chemotherapy and radiation (CRT) (15). Surgery is rarely utilized due to the morbidity associated with resection of oropharyngeal tissues. Despite maximum treatment, 20% of patients with stage II and 35% of patients with stage III disease will die within 4 years of diagnosis ( Figure 1)(16). In patients cured of their disease, treatment often results in dysfunctional swallowing, taste, and speech as well as in extensive tissue fibrosis and pain (17). Patients with HPV-positive OPSCC are diagnosed at a younger age, making the long- term morbidity associated with concurrent CRT particularly concerning. Induction treatment strategies that have the goals of both decreasing disease recurrence and increasing survival permit the future study of definitive treatment de-intensification (18). Attempts to de-intensify treatment of HPV-positive OPSCC by replacing cisplatin chemotherapy with the EGFR monoclonal antibody (mAb) cetuximab have failed (15). Stage I (AJCC 8th edition) includes patients with T0-2 and N0-1 disease. Treatment within Stage I is not uniform. Recommended treatment for T1-2 N0 disease is definitive radiotherapy or surgical resection with or without ipsilateral/bilateral neck lymph node dissection and risk-stratified adjuvant treatment. Recommended treatment for T1-2 N1 with 1 lymph node (ipsilateral) not larger than 3 cm is definitive chemoradiotherapy or radiotherapy or surgical resection with or without ipsilateral/bilateral neck lymph node dissection and risk-stratified adjuvant treatment. Recommended treatment for T1-2 N1 with 1 lymph node (ipsilateral) larger than 3 cm or 2 or more ipsilateral lymph nodes none larger than 6 cm follows the paradigm of Stage II disease, with definitive chemoradiotherapy or resection plus adjuvant treatment. Consequently, while the prognosis for Stage I patients is better than Stage II and III, there is a need for de-intensification of treatment in Stage I T1-2 N1 disease. In this clinical trial, we are studying immunotherapy consisting of PRGN-2009, an investigational HPV vaccine with therapeutic intent with or without M7824, a dual PD-L1 and TGF beta inhibitor, first as a phase I study to assess safety and tolerability, then as a phase II induction “window of opportunity” study with treatment before and after definitive therapy for patients with newly diagnosed stage I (T1,2 N1)/II/III HPV-positive OPSCC. 1.2.2 Sinonasal Squamous Cell Cancer Sinonasal Squamous Cell Cancer (SNSCC) comprises 3-5% of all head and neck squamous cell cancers. Approximately 50% arise in the nasal cavity and the rest in the paranasal sinuses, and are usually not accompanied by nodal metastases at diagnosis (~10-20%). Emerging evidence supports a potential role for HPV in a subset of SNSCC, with studies reporting ~30% of SNSCC having HPV present, more often so in the nonkeratinizing SCC subtype, with HPV-16 the most common genotype, and less commonly genotypes HPV-18, 31 and 33. Contrary to HPV-OPC it has been reported that is more common in patients who are smoking or were smokers. Importantly, similarly to HPV-OPC, HPV positivity appears to be associated with improved prognosis, with a 5-year OS for HPV-SNSCC ranging 68.1-80.0% vs. 31-51.5% in HPV negative SNSCC. Treatment for newly diagnosed tumors consists of surgical resection with postoperative radiotherapy, but in the case of advanced stage tumors treatment is not well defined, and induction approaches are promising. For incurable tumors treatment follows the paradigm of HNSCC. In this trial we are planning to test an induction strategy in a small number of newly diagnosed HPV- SNSCC patients, following the design utilized for the HPV-OPC patient cohort. 12 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 Figure 1: Stage-specific overall survival based upon AJCC 8th Edition Classification 1.2.3 T cell dysfunction in HPV-positive OPSCC HPV-positive OPSCCs are infiltrated by exhausted and functionally suppressed HPV-specific T cells (19-21). This demonstrates that although an adaptive immune response against one or more HPV antigens develops, these T cells are ineffective at controlling the growth of antigen- expressing tumors. This also suggests that reversing exhaustion in or the suppression of existing HPV-specific T cells may induce HPV antigen-specific anti-tumor immunity. When ligated by programmed death-ligand 1 (PD-L1), signaling through programmed death-receptor 1 (PD-1) on T cells is a significant mechanism of T cell exhaustion (22). 1.2.4 M7824 M7824 is a novel bifunctional fusion protein composed of a fully human IgG1 mAb against PD- L1 fused to the soluble extracellular domain of human transforming growth factor-beta (TGF)- receptor II which functions as a TGF “trap”. Functionally, M7824 both blocks PD-L1 and neutralizes TGF within the tumor microenvironment (TME). Expression of PD-1/PD-L1 pathway components is present in approximately two-thirds of HPV-positive OPSCCs (23, 24). Immune checkpoint blockade of the PD-1/PD-L1 pathway with pembrolizumab is currently FDA-approved for the first-line treatment of recurrent/metastatic HPV-positive or -negative head and neck SCC in patients with a combined positive score (CPS) of >1 (24, 25) (NCT02358031). TGF is a major driver of local suppression of both T cell and NK cell function and is overexpressed in HPV- associated malignancies (26, 27). Through dual PD-L1 blockade and neutralization of TGF, M7824 induced superior tumor growth inhibition in multiple pre-clinical, syngeneic models of cancer (28, 29). 1.2.5 Clinical Experience with M7824 in HPV associated malignancies The first in human dose escalation study of M7824 in patients with advanced solid tumors was conducted in the CCR (29). This work demonstrated on-target PD-L1 saturation and reduction of 13 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 circulating TGF, early evidence of clinical efficacy including in a number of patients with HPV associated malignancies, and an acceptable safety profile. Continuation of this work in patients Figure 2: Waterfall plot of confirmed best overall responses in patients with immune checkpoint inhibitor-naïve, recurrent/metastatic HPV-positive OPSCC (orange). with metastatic HPV-associated cancers which was presented at AACR 2019 revealed a 35% response rate in 43 patients including a 43% response rate in 14 patients with p16+ OPSCC (Figure 2). These response rates are notably higher than the 12-24% response rate seen with blockade of PD-1 or PD-L1 alone for HPV-associated malignancies (e.g., cervical, anal, P16+ oropharyngeal) in the literature (25, 30-35). In addition, the median survival of the 43 enrolled patients with HPV-associated malignancies was 16.2 months and the median survival of the 36 enrolled patients with HPV positive malignancies was not reached after 16.8 months of follow-up. It should be noted that both of these median survivals are substantially longer than the median survival reported for similar populations receiving PD-1 or PD-L1 inhibitors alone (7.5-11.5 months) in the literature (25, 32, 33, 35-37) . Adverse events for M7824 were similar to those experienced with pembrolizumab or nivolumab, with the addition of the development of low-grade mucosal bleeding and keratoacanthomas (21%) known to be associated with systemic TGF blockade. Treatment with M7824 induced the expansion of peripheral blood HPV 16 antigen-specific T cells to a greater degree in patients with a best overall response of CR, PR or SD compared to patients that had a best overall response of PD (Figure 3). These data suggested that M7824 could reverse suppression of HPV antigen- specific immunity in patients harboring HPV-positive malignancies refractory to standard treatments. 14 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 Figure 3: PBMC from patients treated with M7824 were assayed for HPV 16 antigen-specific cytokine production or CD107a positivity 1.2.6 HPV 16/18 gorilla adenovirus vaccine In addition to reversing exhaustion and local immunosuppression of existing antigen-specific T cells, a complimentary approach to induce anti-cancer immunity is to induce de novo T cell responses with therapeutic vaccines ( 38). The use of a therapeutic vaccine may be ideal for virally- induced cancers as viral oncogenes required for malignant transformation are known (E6 and E7 for high-risk HPV). Human adenoviral vaccine platforms are effective at delivering nucleic acid payload to dendritic cells and can induce antigen-specific T cell immunity following as few as two treatments but can be limited by the presence of pre-existing immunity to the vaccine platform itself. Gorilla adenovirus vaccine platforms retain the advantages of human adenoviruses including the ability to delete genetic regions to ensure replication incompetence and allow for insertion of transgenes of interest, but are not recognized by human sera from healthy donors (39). Whole genes or long synthetic peptides of interest can be inserted into adenoviral constructs to allow the processing of naturally processed and presented T cell antigens by antigen presenting cells. This allows for treatment of all patients with HPV-positive malignancy without HLA restriction. In collaboration with Precigen, Inc., a gorilla adenovirus vaccine that encodes HPV T cell antigen epitopes from HPV 16 and 18 (hereto referred to as PRGN-2009) has been developed. PRGN-2009 was developed from the base gorilla adenoviral vector GC46. The adenovirus from which the GC46 adenovector is built was originally isolated from a healthy African gorilla. The wild type adenovirus was fully sequenced and, the adenovector genome, was isolated and inserted into plasmid DNA. Therefore, the GC46 adenovector is completely separated from the virus isolation process. The GC46 adenovirus vector is closely related to and clusters phylogenetically with the human species C adenovirus based on hexon, DNA polymerase and E4 ORF6 protein sequence comparisons (40) . The seroprevalence of GC46 is less than 6% in the USA and the seropositive titers were low (below 100 IC 90). In comparison, the seroprevalence of Ad5 is 57% with most of the seropositive 15 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 individuals having high titers (above 200 IC 90; (41)). In addition, comparative studies from human sera samples from Sub-Saharan Africa also confirmed the rare and weak pre-existing neutralizing activity in the human population. High level pre-existing seroprevalence to Ad5 has been shown to limit the effectiveness of Ad5 based adenovectors in clinical studies. Therefore, the rare and weak pre-existing neutralizing activity to GC46 provides an advantage for GC46 based adenovector use in clinical applications. The cloned GC46 genome was engineered to have deletions of two essential regions of the adenovector, E1 and E4. Deletion of these regions removes several essential elements necessary for GC46 replication. Therefore, PRGN-2009 is not replication competent. PRGN-2009, as well as other GC46 adenovectors with E1 and E4 deletions have been evaluated for growth in human cell lines and were found to only replicate in Precigen’s complementing manufacturing cell lines, ORF6 and M2A. The multi-deleted, replication incompetent adenovector platform technology was first developed using Ad5 based adenovectors. These similarly deleted, constructed and manufactured Ad5 adenovectors were used for clinical development of multiple molecular vaccine and gene therapy molecules administered to over 3,000 clinical study participants. HPV-16 and HPV-18 are the genotypes of HPV most frequently associated with cervical, head and neck, and other cancers and, as such, infections with either genotype are considered “high risk” for the development of cancer. In HPV-16 and HPV-18, two primary oncoproteins, E6 and E7, are constitutively expressed by HPV-associated tumors and are critical for the induction and maintenance of cellular transformation in HPV-infected cells. Recent evidence suggests the E5 protein also affects viral transformation. The HPV antigen expressed in PRGN-2009 was designed for optimum immunogenicity. Guided by bioinformatics analysis and in silico protein engineering, 32 key immunogenic peptides were selected which included CTL specific peptides from E6 (HPV- 16/-18), E7 (HPV-16/-18), and E5 (HPV16) and 3 unique agonist peptides (40) for this multi- epitope antigen design. These 35 CTL peptide sequences were grafted to a human ankyrin repeat protein scaffold enabling protein linker sequences embedded between the peptides wherein this scaffold retains it tertiary structure displaying the HPV epitopes. Designed shuffling of the peptides prevents any reformation of E6 and E7 oncogenic protein potential and HPV protein viral function. PRGN-2009 Drug Substance (DS) is manufactured at Advanced Bioscience Laboratories (ABL) utilizing a standardized Adenoviral vector manufacturing process that was developed at Precigen and transferred to ABL. The production cell line is Precigen’s proprietary 293-ORF6 cell line, which complements for both adenoviral E1 and E4 ORF6 to enable efficient production of the replication incompetent GC46 adenoviral vector. The 293-ORF6 cells are cultured in serum free suspension in shaker flasks and infected with PRGN-2009 Master Virus Bank (MVB) at a multiplicity of infection (MOI) of 100 PU/cell. The PRGN-2009 culture harvest is downstream processed and purified using three rounds of cesium chloride (CsCl) density gradient ultracentrifugation to yield a highly purified PRGN-2009 DS material. Manufacture of the PRGN- 2009 DP commences with the thaw of frozen PRGN-2009 DS, which is then sterile-filtered and filled into Diakyo Crystal Zenith (CZ) vials to create the DP, which is stored in freezer boxes in a secure freezer at -60ºC to -90ºC. PRGN-2009 has been tested in three independent in vitro analyses to assess oncogenic potential in mammalian cells.