PRGN-2009 did not induce an oncogenic profile in any assay. 16 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 Figure 4: Peripheral PBMC from WT B6 mice bearing TC-1 tumors were assessed for HPV 16 E6 antigen-specific responses following exposure to overlapping peptide. HPV4 = PRGN-2009. Figure 5: (A) NSG B2M-/- mice bearing SiHa tumors were immune humanized with HLA matched PBMC and mice were treated with PRGN-2009. (B) Following the second vaccine administration, SiHa tumors were assessed for CD8+ T cell infiltration by flow cytometry. PBS vs Ad-HPV: P<0.0001 Empty vector vs HPV: P = 0.09 Pre-clinically, PRGN-2009 induced the formation of peripheral blood HPV 16 E6 antigen-specific responses in wild-type C57BL/6 mice bearing HPV 16 E6/E7 positive lung SCCs (TC-1 tumors). No HPV 16 E6 antigen-specific T cell responses were measurable in the absence of PRGN-2009 (Figure 4). Additionally, mice bearing HPV 16-positive SiHa cervical cancers were immune humanized with HLA-matched human PBMC and treated with PRGN-2009 (Figure 5). Treatment 17 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 resulted in tumor growth inhibition and increased tumor infiltration of CD8+ T cells. These data demonstrated that PRGN-2009 is capable of inducing HPV antigen-specific immune responses and anti-tumor immunity sufficient to inhibit tumor growth. 1.2.7 Rationale for combination of PRGN-2009 and M7824 The rationale for combination therapy includes induction of antigen-specific T cell responses with vaccine that can then be fully activated with the addition of immune checkpoint blockade. In a phase II trial, ISA101, a synthetic long-HPV 16 E6/E7 peptide vaccine, was combined with nivolumab in patients with incurable HPV 16-positive cancer (12). This study demonstrated an objective response rate of 33% and an acceptable safety profile. Two phase I studies have demonstrated the safety and feasibility of combining therapeutic vaccines targeting different antigens with PD-1 mAb treatment, along with correlative immunologic responses, in patients with melanoma (42, 43). In addition, there is an ongoing clinical trial at CCR evaluating an adenoviral vaccine targeting CEA (ETBX-011) in combination with Avelumab and standard of care chemotherapy (FOLFOX) for patients with metastatic colorectal cancer (NCT03050814). To date 23 patients have been enrolled on this study of which 15 patients have received the combination of all study agents including the adenoviral vaccine and Avelumab. One patient had grade 2 arthritis which was managed with a steroid taper. Otherwise no notable immune related toxicities have been observed in the 15 patients receiving vaccine and Avelumab. The specific combination of PRGN-2009 and M7824 was tested in wild-type C57BL/6 mice bearing TC-1 tumors (Figure 6). The addition of M7824 further enhanced tumor growth inhibition observed with PRGN-2009 alone. Cumulatively, these results indicate that the addition of M7824 to potentially therapeutic vaccination is both safe and feasible in the clinical setting as well as induces tumor growth inhibition in pre-clinical models of HPV-positive malignancy. Figure 6: C57BL/6 mice bearing TC-1 tumors were treated with PRGN-2009 alone or in combination with M7824 and assessed for primary tumor growth. PBS vs Ad-HPV: P<0.0001 PBS vs Ad-HPV + M7824: P<0.0001 1.2.8 Phase I study for participants with advanced HPV associated malignancies To first assess the safety and feasibility of treatment with PRGN-2009 alone or in combination with M7824, we plan to implement a sequential phase I study of dose-escalating PRGN-2009 (two dose levels) alone or in combination with a fixed-dose of M7824. Participants with 18 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 recurrent/metastatic HPV-positive malignancy will receive two dose levels (1x1011 and 5x1011 VP) of PRGN-2009, followed by an evaluation of the RP2D dose of PRGN-2009 in combination with 1200 mg (RP2D) of M7824.until progression or unacceptable toxicity. This design will allow the determination of safety and tolerability of PRGN-2009 alone and in combination with M7824 and allow each participant the opportunity to benefit from combination treatment. The phase I portion of this study will allow immune checkpoint naïve or refractory participants. 1.2.9 Phase II window of opportunity study for participants with newly diagnosed stage I (T1,2 N1)/II/III p16-positive OPSCC Once the safety and tolerability of PRGN-2009 alone or in combination with M7824 is established in the phase I portion of the study, we plan to implement a two arm induction, Phase II window- of-opportunity study of PRGN-2009 (Arm 2A) or PRGN-2009 plus M7824 (Arm 2B) in participants with newly diagnosed, previously untreated stage I (T1,2 N1)/II/III p16-positive OPSCC who plan to receive definitive therapy. Induction or neoadjuvant immunotherapy for newly diagnosed, OPSCC is an area of active study in an attempt to decrease locoregional failure and the development of distant metastasis after completion of definitive treatment. Participants will receive PRGN-2009 or PRGN-2009 plus M7824 before therapy. Upon completion of the neoadjuvant or induction immunotherapy treatment participants will be referred back to their referring providers for standard of care definitive therapy. Participants diagnosed with a new stage II/III p16-positive OPSCC with planned definitive therapy typically wait 4-6 weeks for initiation of treatment due to radiation mapping and simulation and scheduling for chemotherapy (44). During this “window of opportunity” participants receive no treatment for their cancer.