*** In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as disease progression. Note: Participants with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be reported as “symptomatic deterioration.” Every effort should be made to document the objective progression even after discontinuation of treatment. 6.3.5.4 Duration of Response Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. 6.3.5.5 Progression-Free Survival PFS is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first. 6.3.6 Immune Response Evaluation 6.3.6.1 Tumor Biopsies: Biopsies obtained will be stained for multiple immune cell markers (CD4, CD8, CD3, PD-1, PD-L1) as well as tumor cell markers (pancytokeratin) using an automated multiplex immunofluorescence (MxIF) staining protocol (Opal™ method). This method explained below uses tissue sparingly and ensures simultaneous staining of different markers in the same tissue plain. An average of 5 consecutive biopsy recuts (4-5-micron thick formalin fixed paraffin embedded tissue sections (FFPE) mounted on 25 x 75mm, charged glass slides) or unstained slides (US) per each biopsy time point is needed for this analysis. Hematoxylin and Eosin (H&E) staining (done on last or deepest recut level) will be examined by a research pathologist (GMB TIME Laboratory), for presence of tumor and tissue integrity. 6.3.6.2 Staining Protocol: Formalin-fixed paraffin-embedded (FFPE) tumor sections (US) will be immuno-stained using Opal Multiplex Automation IHC Detection Kits on the BOND RX from Leica Biosystems' BOND RX. First, sections will be subjected to deparaffinization and antigen retrieval (HIER). Subsequent Opal staining of each antigen occurs as follows: slides will be 63 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 blocked with blocking buffer (Akoya/Leica) for 10 min then incubated with primary antibodies at optimized concentrations (GMB TIME Laboratory) followed by Opal HRP polymer (or secondary antibody) and one of the Opal fluorophores. Individual antibody complexes will be stripped after each round of antigen detection. After the final stripping step, DAPI (4',6- diamidino-2- phenylindole) counterstain will be applied and slides will be removed from the BOND RX for cover slipping. The Opal method is based on PerkinElmer’s TSA® (Tyramide Signal Amplification) Plus reagents and offers many advantages compared to conventional indirect immunofluorescence labeling, including brighter signals for faster scanning, balanced signals for higher level multiplexing, signals that do not photobleach, and freedom from species-based interference. The Opal method is explained in more details in the attachment (Opal_Assay_Development_Guide_Aug2017). 6.3.6.3 Digital Imaging Protocol: Once all antigens of interest are stained, a multiplex scanning protocol will be created on Vectra Polaris scanner (Akoya/Perkinelmer), to allow for whole slide imaging at 10-20X. This is followed by higher resolution scan of regions of interest (ROIs) picked (5 on average) or whole slide analysis (if feasible) by research pathologist (GMB TIME Laboratory). The scans generated at high resolution are called multispectral images (MSIs) and are available in .im3 format. These can only be analyzed using inform software (available in GMB TIME Laboratory). The software allows for spectral unmixing based on a spectral library (built based on single reference stains) and artificial intelligence based unmixing algorithm. Individual ROIs will be segmented based on nuclear counterstain DAPI, and cells will be counted based on expression of various markers (CD3, CD4, CD8) in a binary fashion allowing for objective measurements of density, absolute counts, and percentages of each cell type. Averages of representative ROIs or whole areas of interest (if feasible) for each time point will be generated for statistical analysis. 6.4 TOXICITY CRITERIA The following adverse event management guidelines are intended to ensure the safety of each participant while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm ). 7 NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN 7.1 DEFINITIONS Please refer to definitions provided in Policy 801: Reporting Research Events found at: https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835#Policies&Guidance- 800Series-ComplianceandResearchEventReportingRequirements. 7.2 OHSRP OFFICE OF COMPLIANCE AND TRAINING / IRB REPORTING 7.2.1 Expedited Reporting Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found at: 64 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835#Policies&Guidance- 800Series-ComplianceandResearchEventReportingRequirements. Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies. 7.2.2 IRB Requirements for PI Reporting at Continuing Review Please refer to the reporting requirements in Policy 801: Reporting Research Events found: at: https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835#Policies&Guidance- 800Series-ComplianceandResearchEventReportingRequirements. 7.3 NCI CLINICAL DIRECTOR R EPORTING Problems expeditiously reviewed by the OHSRP in the NIH eIRB system will also be reported to the NCI Clinical Director/designee; therefore, a separate submission for these reports is not necessary. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to NCICCRQA@mail.nih.gov within one business day of learning of the death. 7.4 INSTITUTIONAL BIOSAFETY C OMMITTEE (IBC) REPORTING CRITERIA 7.4.1 Serious Adverse Event Reports to IBC The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or life-threatening experience associated with the use of PRGN-2009 as soon as possible but in no event later than 7 calendar days of initial receipt of the information. Serious adverse events that are unexpected and associated with the use of the PRGN-2009, but are not fatal or life-threatening, must be reported to the NIH IBC as soon as possible, but not later than 15 calendar days after the investigator’s initial receipt of the information. Adverse events may be reported by using the FDA Form 3500a. 7.4.2 Annual Reports to IBC Within 60 days after the one-year anniversary of the date on which the IBC approved the initial protocol, and after each subsequent anniversary until the trial is completed, the Principal Investigator (or delegate) shall submit the information described below. Alternatively, the IRB continuing review report can be sent to the IBC in lieu of a separate report. Please include the IBC protocol number on the report. 7.4.3 Clinical Trial Information A brief summary of the status of the trial in progress or completed during the previous year. The summary is required to include the following information: the title and purpose of the trial; clinical site; the Principal Investigator; clinical protocol identifiers; 65 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 participant population (such as disease indication and general age group, e.g., adult or pediatric); the total number of participants planned for inclusion in the trial; the number entered into the trial to date whose participation in the trial was completed; and the number who dropped out of the trial with a brief description of the reasons; the status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, or fully completed; and if the trial has been completed, a brief description of any study results. 7.4.4 Progress Report and Data Analysis Information obtained during the previous year's clinical and non-clinical investigations, including: a narrative or tabular summary showing the most frequent and most serious adverse experiences by body system; a summary of all serious adverse events submitted during the past year; a summary of serious adverse events that were expected or considered to have causes not associated with the use of the gene transfer product such as disease progression or concurrent medications; if any deaths have occurred, the number of participants who died during participation in the investigation and causes of death; and a brief description of any information obtained that is pertinent to an understanding of the gene transfer product’s actions, including, for example, information about dose-response, information from controlled trials, and information about bioavailability. 7.5 NIH REQUIRED DATA AND SAFETY MONITORING PLAN 7.5.1 Principal Investigator/Research Team The clinical research team will meet on a regular basis (approximately weekly) when participants are being actively treated on the trial to discuss each participant. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior participants. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. Events meeting requirements for expedited reporting as described in Section 7.2.1 will be submitted within the appropriate timelines. The principal investigator will review adverse event and response data on each participant to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff. 66 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 8 SPONSOR PROTOCOL / SAFETY REPORTING 8.1 DEFINITIONS 8.1.1 Adverse Event Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product (ICH E6 (R2)). 8.1.2 Serious Adverse Event (SAE) An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following: Death; A life-threatening adverse event (see Section 8.1.3); Inpatient hospitalization or prolongation of existing hospitalization; oA hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event. oA hospitalization/admission that is solely driven by non-medical reasons (e.g., hospitalization for participant convenience) is not considered a serious adverse event. oEmergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria. Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions; A congenital anomaly/birth defect; Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. 8.1.3 Life-threatening An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death (21CFR312.32). 8.1.4 Severity The severity of each Adverse Event will be assessed utilizing the CTCAE version 5.0. 67 Abbreviated Title: Combo Immunotx in HPV OPSCC Version Date: 04.25.2023 8.1.5 Relationship to Study Product All AEs will have their relationship to study product assessed using the terms: related or not related. Related – There is a reasonable possibility that the study product caused the adverse event. Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event. Not Related – There is not a reasonable possibility that the administration of the study product caused the event. 8.1.6 Adverse Events of Special Interest (AESI) Adverse events of special interest (AESIs) are serious or nonserious AEs that are of clinical interest and should be closely followed. AESIs include following: Infusion-related reactions including immediate hypersensitivity. Immune-related adverse events. TGFβ inhibition mediated skin reactions. Anemia.