PI decision to end the study. Participant requests to be withdrawn from study. Participant lost to follow-up. Investigator discretion. Death. Completion of 5-year follow up period. (Only for Phase II) 3.6.3 Lost to Follow-up A participant will be considered lost to follow-up if he or she fails to return for 4 scheduled visits and is unable to be contacted by the study site staff. The following actions must be taken if a participant fails to return to the clinic for a required study visit: The site will attempt to contact the participant and reschedule the missed visit within the next 2 weeks and counsel the participant on the importance of maintaining the assigned visit schedule and ascertain if the participant wishes to and/or should continue in the study. Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, an IRB approved certified letter to the participant’s last known mailing address or local equivalent methods). These contact attempts should be documented in the participant’s medical record or study file. Should the participant continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up. 4 CONCOMITANT MEDICATIONS/MEASURES 4.1 PERMITTED MEDICATIONS Any medications (other than those excluded by the clinical trial protocol) that are considered necessary to protect subject welfare or alleviate symptoms and will not interfere with the trial medication may be given at the Investigator’s discretion. Palliative radiotherapy delivered in a normal organ-sparing technique may be administered during the trial. The assessment of PD will not be based on the necessity for palliative radiotherapy. 4.2 PROHIBITED MEDICATIONS The following treatments should not be administered during the trial: Other immunotherapies or immunosuppressive drugs (for example, chemotherapy or systemic corticosteroids except for prophylaxis or treatment of allergic reactions, endocrine replacement therapy at low dose prednisone [≤ 10 mg daily] or equivalent, for the treatment of irAEs, or for short courses (≤ 14 days) as appropriate medical therapy for 47 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 unrelated medical conditions (e.g., asthma). Steroids with no or minimal systemic effect (topical, inhalation) are allowed. Prophylactic use of corticosteroids for infusion related reactions. Corticosteroid administration prior to CT scans in participants with intravenous contrast allergy is allowed. Any live vaccine therapies for the prevention of infectious disease. Administration of inactivated vaccines is allowed (for example, inactivated influenza vaccines or locally approved COVID vaccines). Systemic anticancer treatment (except standard of care therapy for phase II participants). 48 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 5 CORRELATIVE STUDIES FOR RESEARCH 5.1 BIOSPECIMEN COLLECTION Test/assayVolume (approx.) per TimepointType of tube aCollection pointLocation of specimen analysis Standard and 123 immune cell subsets by FACSLTIB [Storage at Clinical Services Program (CSP)] Functional Analysis of immune cell subsets by FACSLTIB (Storage at CSP) Antigen Specific Immune Response by cytokine staining assayLTIB (Storage at CSP) T cell clonality by immuno-seq platformLTIB and NCI Frederick Genomic Core Facility (Storage at CSP) RNA expression level of 770 genes60-80 mL blood for PBMCsSodium heparin (green top) tubes LTIB and NCI Frederick Genomic Core Facility Soluble Factors (to include sCD27 and sCD40 ligand) by ELISA 8 mL blood for serumSST LTIB (Storage at CSP) M7824 Pharmacokinetics4 mL blood for serumSST EMD Serono (Storage at BPC) ADA by ELISA 4 mL blood for serumSST EMD Serono (Storage at BPC) AVA by ELISA 1 mL blood for serumSST Precigen (Storage at BPC) Immune Markers by IHCTumor samplesN/A GMB TIME LAB (Storage at LP, CSP) Single-cell Proteomic analysisTumor samplesN/ASee Study Calendar Precigen (Storage at LP, CSP) 49 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 Test/assayVolume (approx.) per TimepointType of tube aCollection pointLocation of specimen analysis Immune transcriptomic analysis Tumor samplesN/A Precigen (Storage at LP, CSP) RNA expression level of 770 genesTumor samplesN/A LTIB and NCI Frederick Genomic Core Facility (Storage at LP, CSP) HPV status by PCR of DNATumor samplesN/A Dr. Norberg’s Team (Storage at LP, CSP) Circulating free tumor DNA (cftDNA) by PCR systemPlasma EDTA (lavender top)LTIB and NCI Frederick Genomic Core Facility (Storage at CSP) T cell clonality by immunoseq platformTumor samplesN/A LTIB and NCI Frederick Genomic Core Facility (Storage at LP, CSP) Salivary HPV DNA 15-20 mL 0.9% NaCl rinse50 mL conical tubeLTIB and NCI Frederick Genomic Core Facility (Storage at CSP) a Please note that tubes and media may be substituted based on availability with the permission of the PI or laboratory investigator. Research blood samples will be sent either to the Clinical Services Program – Leidos Biomedical Research, Inc. (CSP) (Section 5.2.1) or the Blood Processing Core (BPC) (Section 5.2.2) for barcoding, initial processing and storage. Tissue will be sent to the Laboratory of Pathology as described in Section 5.2.3. Additional tissue for research may also be stored in CSP (Section 5.2.1). From these facilities, coded, linked samples will be sent to the designated labs for analysis upon request. 5.1.1 Immune Phenotyping Exploratory immunologic studies will be conducted to evaluate the study drug’s effect on the immune response before and after treatment, to gain insight into potential biomarkers, and help improve the administered therapy. Blood will be collected as per the Study Calendar. The following immune assays may be performed at the Laboratory of Tumor Immunology and Biology (LTIB) at the NCI’s Center for Cancer Research (CCR) in select participants where adequate samples are available: 1. PBMCs may be analyzed for changes in standard immune cell types (CD4 and CD8 T cells, natural killer [NK] cells, regulatory T cells [Tregs], myeloid-derived suppressor cells 50 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 [MDSCs], and dendritic cells) as well as 123 immune cell subsets, using multi-color flow cytometry. 2. PBMCs from selected subjects may be analyzed for function of specific immune cell subsets, including CD4 and CD8 T cells, NK cells, Tregs, and MDSCs using flow-based assays. 3. PBMCs may be analyzed for tumor antigen-specific immune responses using an intracellular cytokine staining assay. PBMCs will be stimulated in vitro with overlapping 15-mer peptide pools encoding the tumor-associated antigens HPV 16 E6 and E7 and HPV18 E6 and E7; control peptide pools will involve the use of human leukocyte antigen peptide as a negative control and CEFT peptide mix as a positive control. CEFT is a mixture of peptides of CMV, Epstein-Barr virus, influenza, and tetanus toxin. Post- stimulation analyses of CD4 and CD8 T cells will involve the production of IFN-γ, IL-2, TNF, and the degranulation marker CD107a. If sufficient PBMCs are available, assays may also be performed for the development of T cells to other tumor-associated antigens. 5.1.2 Soluble Factors Samples for soluble factor analysis will be collected as per the Study Calendar. Sera may be analyzed pre- and post-therapy for the following soluble factors: sCD27, sCD40 ligand using commercial ELISA kits. Sera may be analyzed for changes in cytokines (IFN-γ, IL-10, IL-12, IL-2, IL-4, etc.), chemokines, antibodies, tumor-associated antigens, and/or other markers using ELISA or multiplexed assays (e.g., Mesoscale, Luminex, cytokine bead array). 5.1.3 AVA Anti-vector antibodies (AVA) are one mechanism of neutralization and inefficacy of viral vectors. Longitudinal detection and titer measurement of AVA to the vaccine adenoviral vector will assist in characterizing cases of loss of efficacy. 5.1.4 M7824 Pharmacokinetics PK measurements of M7824 will be taken in Arm 1B only to collect data which will provide insight into population PKs of M7824 in participants receiving these novel combinations. The schedule is in the Study Calendar. Serum M7824 PK measurements will be done at EMD Serono. 5.1.5 ADA Anti-Drug Antibody (ADA) development is an accepted mechanism of loss of efficacy of administered human monoclonal antibodies. Measuring titers will ensure that lack of efficacy of M7824 is not due to ADA development. The schedule is in the Study Calendar – samples will be collected in Arm 1B only. The investigation will be done by EMD Serono using ELISA. 51 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 5.1.6 Tumor Tissue Analyses for Immune Markers Analyses may be performed in pre-treatment vs. post-treatment (at Week 4-5 for phase II participants) tumor tissue. Where available, archival tumor samples may be requested for pre- treatment analysis (preferably tissue samples from the last 6 months). For participants with lesions amenable to biopsy, two biopsies may be performed at baseline and at Week 4-5 (phase II participants) or Week 9 (phase I participants). Tumor samples will be sent to the Laboratory of Pathology for disease evaluation; remaining samples will be used for research. Tissue samples for research may also be stored in CSP as described in Section 5.2.1. Where available, study of immune infiltration as well as PD-L1 status within the tumor microenvironment pre vs. post treatment by IHC and/or multiplex immunofluorescence may be performed by the GMB TIME Laboratory. 5.1.7 Single-cell proteomic analysis Where available, tumor tissue single-cell proteomic analysis of immune and signaling pathways may be performed with the Isoplexis Single Cell Functional proteomic platform by Precigen. 5.1.8 Immune transcriptomic analysis Where available, tumor tissue immune transcriptomic analysis may be performed with the Nanostring platform by Precigen. 5.1.9 Additional Biomarker Studies De-identified, coded blood (PBMC, plasma, and/or serum) or tissue samples may be sent to Precigen Inc., the manufacturer of PRGN-2009, to further investigate HPV 16/18 -specific immune responses, activation status of the immune system (e.g., leukocyte subsets, exhaustion, cytokines profile), antigen cascade, anti-tumor specific immune responses induced by the exposure to PRGN-2009, and potential predictive/prognostic biomarker candidates related to the drug and/or cancer (e.g., specificity of tumor infiltrating cells), and results of these analyses will be shared with the NCI. These analyses are exploratory and dependent on the quality and availability of sufficient materials. The panel of biomarkers might be adjusted based on results from ongoing research; therefore, any remaining samples can be stored at Precigen and can be used for future exploratory research on the drug and/or disease-related aspects. Only remaining samples will be used to this purpose, no additional samples will be collected. 5.1.10 Assessment of HPV status In participants with available tumor tissue (either archival or by optional biopsy), HPV testing will be performed using the Roche Cobas or Becton Dickinson HPV PCR based DNA assay, if no prior HPV testing of the tumor has been performed. This will be done through collaboration with Dr. Norberg’s Team. 5.1.11 Circulating tumor DNA If sufficient plasma is available, select participant samples may be analyzed for circulating tumor DNA (LTIB and NCI Frederick Genomic Core Facility). Plasma DNA will be isolated with an automated purification system. The circulating tumor/HPV DNA will be quantified with a digital droplet PCR system from Bio-Rad to obtain precise quantification. This is just quantification by PCR. There is no DNA sequencing. 52 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 5.1.12 RNA and T-cell Receptor Clonality Analysis of Blood and Tumor Tissue Where possible, RNA expression and T-cell receptor clonality analysis will be done on the peripheral blood as well as archived tumor tissue or optional biopsies to help further evaluate changes in immune response and RNA expression levels with treatment as well as to determine tumor and infiltrating lymphocyte characteristics which may be predictive of response to treatment. In addition, these analyses will also be used to gauge resistance mechanisms and additional targets for future therapy. Coded, linked samples may be analyzed for RNA expression levels using the Nanostring platform and T-cell receptor clonality using the ImmunoSeq platform (LTIB and NCI Frederick Genomic Core Facility). NCI Fredrick Genomic Core Facility: Leidos Biomedical Research, Inc: ATRF, Rm C3016 8560 Progress Drive Frederick, MD 21701 Ph. 301-846-7677 5.1.13 Salivary HPV DNA Participants in Phase 2 Cohort 2 with p16-positive oropharyngeal cancer will have collection of saliva by mouth rinse and gargle with 15-20 mL 0.9% NaCl for 30 seconds and spitting into the collection tube (timepoints per Study Calendar). Where possible, saliva samples will be analyzed for HPV DNA detection and quantification (LTIB and NCI Frederick Genomic Core Facility). Salivary HPV DNA will be quantified with digital droplet PCR; no sequencing is involved. 5.2 SAMPLE STORAGE, TRACKING AND DISPOSITION Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. Samples will not be sent outside the National Institutes for Health (NIH) without appropriate approvals and/or agreements, if required. 5.2.1 Sample Management and Storage at Clinical Services Program – Leidos Biomedical Research, Inc. (CSP) Clinical Services Program - Leidos Biomedical Research, Inc. Attn: Theresa Burks 1050 Boyles Street Bldg. 496/Room 121 Frederick, MD 21702 On days samples are drawn, Jen Bangh at CSP (part of NCI Frederick Central Repositories) should be notified (phone: [301] 846-5893; fax: [301] 846-6222). She will arrange same-day courier delivery of the specimens.