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10.1.3 exploratory endpoints

The exploratory endpoints of the Phase II portion of the study are:
To determine if the use of HPV vaccine alone results in potentially 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 HPV vaccine alone
(Arm 2A) in combination with standard of care chemoradiation in HPV-SNSCC participants.
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10.2 sample size determination

The phase I portion of the trial will be conducted using 3+3 dose escalation design evaluating the safety of PRGN-2009 at two dose levels as monotherapy and at the RP2D of PRGN-2009 in combination with a fixed dose of M7824. Using this design up to 22 participants may be enrolled on the phase I portion of the study. In arm 2A, phase II, 20 participants with p16+ oropharyngeal cancer will be enrolled based on the following reasoning. Assume that a success for any individual participant will be defined as that participant experiencing a two-fold increase in CD3+ tumor infiltrating T cells post treatment compared with pre-treatment. With 20 participants, there would be 80.4% power to detect a difference between 50% and 80% of participants experiencing a success in arm 2A, with an exact binomial test using a one-sided 0.05 significance level. As an illustration, with 20 participants, if 15 participants were able to have a two-fold increase, the lower two-sided 90% confidence bound on 15/20 is 54.4% and the upper two-sided 90% confidence bound is 89.6%. Thus, 15/20 participants would demonstrate the intended magnitude of effect. If 14/20 had this outcome, the lower 90% two-sided bound is 49.2% and the upper two-sided 90% bound is 86.0%, essentially demonstrating nearly the same result. Expecting that up to 22 evaluable participants will enroll on the phase I portion of the study, that 20 evaluable p16+ oropharyngeal cancer participants will enroll on phase II 2A, and that up to 2 evaluable HPV-SNSCC participants may enroll on phase II 2A, a total of up to 44 evaluable participants will be planned to enroll on this protocol. To allow for a few inevaluable participants the accrual ceiling for this protocol will be set at 50 participants.
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10.3 populations for analyses

All participants who receive any investigational treatment will be evaluable for safety and toxicity evaluations. For phase II, only HPV-OPC participants will be evaluable for primary and secondary objective endpoints. HPV-SNSCC participants will be evaluable only for exploratory objectives. For phase II, participants who receive at least two doses of therapy and have an adequate pretreatment and posttreatment biopsy will be evaluable for immune infiltration assessments. For phase II, participants who receive at least two doses of immunotherapy and go on to receive standard definitive therapy will be evaluable for relapse free survival and overall survival assessments. For phase I and II participants, only those participants who have measurable disease present at baseline and have had their disease re-evaluated will be considered evaluable for response. (Note: Participants who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.)
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10.4 statistical analyses 10.4.1 general

The phase I portion of the study will be conducted as a safety assessment using a 3+3 dose escalation design. In addition, Arm 1B will be expanded to a total of 10 evaluable participants to gauge the preliminary efficacy of the combination of PRGN-2009 and M7824 in participants with advanced disease. The phase II portion of the study will be conducted to evaluate for a potentially significant increase in CD3+ tumor infiltrating lymphocytes with immunotherapy induction.
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10.4.2 analysis of the primary

Regarding the phase I portion of the study, participants will be evaluated with respect to the grades and types of toxicities obtained. The results will be presented descriptively and tabled if appropriate. Regarding the phase II portion of the study the rate of doubling of baseline CD3+ tumor infiltrating lymphocytes will be provided as well as the 95% confidence interval. 10.4.3 Analysis of the Secondary Endpoints For the phase I portion of the study data will be obtained on ORR, duration of response, PFS, OS, and ratio of participants that are hospitalized because of adverse events attributed to disease progression. 10.4.4 Duration of 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 PD is objectively documented and is evaluated using the Kaplan-Meier method. 10.4.4.1 Progression-Free Survival and Relapse-Free survival PFS and RFS will be evaluated using Kaplan-Meier methods. PFS will be defined as the time from the date of first treatment to the date of disease progression or death (any cause) whichever occurs first. RFS will be defined as the time from completion of standard chemoradiation to the date of disease recurrence or death (any cause) whichever occurs first. Subjects who do not have disease progression, disease recurrence or have not died at the end of follow up will be censored at the last known date the subject was progression or relapse free. 10.4.4.2 Objective Response Objective response is a complete or partial radiographic response as defined by RECIST 1.1 (Section **6.3.5**).
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10.4.4.3 overall survival

OS will be evaluated using Kaplan-Meier methods. OS will be defined as the time from the date of first treatment to the date of death (any cause). Subjects who are alive at the end of follow up will be censored at the last known date alive.
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10.4.5 safety analyses

Safety endpoints will be analyzed as summary statistics during treatment and/or as change scores from baseline assessments. AEs will be coded as defined in the Medical Dictionary for Regulatory Activities (MedDRA). All AEs will be recorded and tabulated following each treatment. AEs will be recorded by severity, frequency, and relationship to the study intervention and will be presented by System Organ Class (SOC) designations and preferred term groupings. Information on each AE will include start date, stop date, severity, relationship, expectedness, outcome, and duration. Adverse events leading to premature discontinuation from the study intervention and serious treatment-emergent AEs will be presented either in a Table or a Listing. In addition, overall safety will be assessed by descriptive analyses using tabulated frequencies of AEs by grade using CTCAE Version 5 within dose cohorts and for the overall study population in terms of treatment-emergent AEs, SAEs, and clinically significant changes in safety laboratory tests, physical examinations, ECGs, and vital signs. 10.4.6 Baseline Descriptive Statistics Baseline Characteristics will be described. 10.4.7 Planned Interim Analyses Interim assessment of safety will be made based on DLT assessments according to the 3+3 dose escalation design in the phase I portion of the study and the first six participants enrolled on the phase II portion of the study. 10.4.8 Tabulation of Individual Participant Data Individual responses in a cohort may be depicted within a larger group using a waterfall plot or spider diagram. 10.4.9 Exploratory Analyses (Immune Responses) Where feasible, exploratory immune analyses will be conducted to evaluate anti-tumor immune responses. Immune response will be assessed among all subjects treated in each cohort. The magnitude of immune responses will be described. A subject will be considered evaluable for immune response if they receive at least one dose of treatment. The percentage of subjects with a positive immune response will be evaluated by cohort. For flow cytometry analyses on PBMC
samples, Student T tests or Kruskal-Wallis and Wilcoxon rank sum tests as appropriate will be performed on percentages of TNF- and/or IFN- expressing cells among the different cohorts to determine any significant differences in cell populations. For antigen specific T cell responses, a positive immune response is defined by CMI reactivity in *ex vivo* stimulation using a flow cytometric readout (cytokine production or CD107 expression). Antigen-specific peptide challenge assays require a readout of >250 reactive T-cells/million cells above the background (53).
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11 collaborative agreements 11.1 cooperative

A CRADA (02666) is in place with EMD Serono for the supply of M7824. A CRADA (03209) is in place with Precigen for the supply of PRGN-2009.
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12 human subjects protections 12.1

Subjects from all racial/ethnic groups are eligible for this study if they meet the eligibility criteria. Efforts will be made to extend accrual to a representative population. Due to impaired cellular immunity which may affect the efficacy of treatment, participants with poorly controlled HIV as well as patents with detectable viral loads of hepatitis B and C will be excluded. Patients who do not accept blood transfusions will be excluded. As there is a risk of severe bleeding with this study drug, participants must be willing to receive blood transfusions if medically necessary for their own safety. Participants must be able to receive blood transfusions in order to minimize the risks of receiving M7824. In addition, these patients could compromise the scientific validity of the study. For example, death from blood loss could make it difficult to assess other aspects of the investigational immunotherapy's safety—a primary scientific goal in this early-phase immunotherapy trial, which are carried out in small numbers of participants.
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12.2 participation of children

Individuals under the age of 18 will not be eligible to participate in this study because of unknown toxicities in pediatric patients.
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12.3 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (Section **12.4.2**), all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation to assess ongoing capacity of the subjects and to identify an LAR as needed. Please see Section **12.5.1** for consent procedure.
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12.4 risk/benefit assessment 12.4.1 known

Some of the procedures performed on this study are not known to be associated with risk. These include urine tests and EKGs. Below are a list of procedures and study interventions that are associated with risk.
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12.4.1.1 risks associated with study

Potential adverse reactions attributable to the administration of the study drug utilized in this trial are discussed in Section 14. All care will be taken to minimize side effects, but they can be unpredictable in nature and severity. Participants will be examined and evaluated prior to enrollment. All evaluations to monitor the treatment of participants will be recorded in the medical record. If participants suffer any physical injury as a result of the participation in this study, immediate medical treatment is available at the Clinical Center, National Institutes of Health, Bethesda, Maryland. Participants may be harmed from being in this study by toxicity due to the drug or combination of drugs given during this study. M7824 is similar to immune check point inhibitors. There are preliminary data to suggest that not all patients benefit from immune check point inhibitors nor M7824. Additionally, there are preliminary data to suggest that an unexpectedly rapid progression of disease occurs in some patients receiving immunotherapy such as immune checkpoint inhibitors.
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12.4.1.2 risk of biopsies

All care will be taken to minimize risks that may be incurred by tumor sampling. However, there are procedure-related risks (such as bleeding, infection and visceral injury) that will be explained fully during informed consent. 12.4.1.3 Risks of Exposure to Ionizing Radiation
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12.4.1.3.1 phase i

This research study involves the possibility of 8 CT CAP scans, 2 brain CT scan, and 2 CT guided biopsies collected for research purposes for Phase I participants. The amount of radiation exposure is equal to approximately 10.6 rem. This level of exposure results in an increased risk of cancer.
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12.4.1.3.2 phase ii

This research study involves the possibility of 3 CT neck and chest scans for research purposes for Phase II participants with oropharyngeal cancer and 3 CT neck, chest, and skull scans for research purposes for Phase II participants with SNSCC. The amount of radiation exposure is equal to approximately 0.99 rem and 1.29 rem for Phase II participants with oropharyngeal cancer and SNSCC, respectively. This level of exposure could result in an increased risk of cancer.
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12.4.1.4 ct scan risk

CT scans create low levels of radiation, which has a small potential to cause cancer and other defects. However, the risk associated with scan is small. 12.4.1.5 Risks Due to Contrast Agents for CT Contrast agents can cause allergic reactions and kidney damage. Allergic reactions can include mild itching associated with hives but can also result in a serious life-threatening emergency from difficulty breathing. If this occurs, it is treatable.
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12.4.1.6 risk of mri

People are at risk for injury from the MRI magnet if they have some kinds of metal in their body. People with fear of confined spaces may become anxious during an MRI. Those with back problems may have back pain or discomfort from lying in the scanner. The noise from the scanner is loud enough to damage hearing, especially in people who already have hearing loss. There are no known long-term risks of MRI scans. 12.4.1.7 Risk of Gadolinium Enhanced MRI The gadolinium infusion may cause mild symptoms such as coldness in the arm during the injection, a metallic taste, headache, and nausea. There are risks of an IV catheter include bleeding, infection, or inflammation of the skin and vein with pain and swelling. Procedure-related risks from MRI and gadolinium enhanced MRI will be explained fully during informed consent. 12.4.1.8 Risks of Clinical Endoscopy and Nasopharyngo-laryngoscopy In this procedure, a physician gently places a small, thin, flexible endoscope with a camera through participant's nose to view their upper airway anatomy above the vocal cords. This procedure takes only a few minutes, is painless and does not require any sedation. It is a normal part of an ENT
exam. Risks of clinic endoscopy include epistaxis or vasovagal syncope, each of which occur in <1% of all participants. If participant's tumors are not accessible in the clinic or a biopsy in the clinic is deemed unsafe by the study doctor, biopsies may be performed in the operating room under general anesthesia. Side effects of general anesthesia may include temporary confusion, difficulty passing urine, bruising or soreness from the IV drip, nausea and vomiting, shivering and feeling cold, and sore throat. Risks of operative biopsies include post-op bleeding (<1% of all participants), dental damage from laryngoscopy instrumentation (<1% of all participants) and temporary tongue paresthesia/dysgeusia (<10% of all participants) that resolves within a few days. 12.4.1.9 Risk from Saliva Collection Saliva collection is not known to be associated with risk. There may be some discomfort from the oral rinse and gargle with saline.
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12.4.1.10 research blood collection risks

Risks of blood draws include pain and bruising in the area where the needle is placed, lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood cell count (anemia) can develop. Side effects of this investigational treatment may cause delay in scheduled curative therapy. A prior study has shown that people with oropharyngeal cancer who received treatment more than 10 weeks after their initial diagnosis did significantly worse than those who received treatment sooner. Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document and this protocol document. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents. The potential benefit to a participant that goes onto study is a reduction in the bulk of their tumor which may or may not have favorable impact on symptoms and/or survival. Locally advanced, metastatic or refractory/recurrent HPV associated malignancies (cervical, anal, oropharyngeal cancers, etc.) need improved therapy options. Preclinical studies suggest that the use of a combination of multiple immunotherapy agents may improve anti-tumor efficacy. Therefore, PRGN-2009 alone or in combination with M7824 may improve clinical outcome of participants with HPV associated malignancies. A number of clinically appropriate strategies to minimize risk to participants have been built into the protocol through the means of inclusion/exclusion criteria, monitoring strategies, and management guidelines. Overall, the potential benefits of PRGN-2009 alone or in combination with M7824 for this group of participants outweigh the risks associated with the proposed entry into this protocol with PRGN2009 alone or in combination with M7824.  
The informed consent document will be provided as a physical or electronic document to the participant or consent designee(s) for review prior to consenting. A designated study investigator will carefully explain the procedures and tests involved in this study, and the associated risks, discomforts and benefits. In order to minimize potential coercion, as much time as is needed to review the document will be given, including an opportunity to discuss it with friends, family members and/or other advisors, and to ask questions of any designated study investigator. A signed informed consent document will be obtained prior to entry onto the study. The initial consent process as well as re-consent, when required, may take place in person or remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with local policy, including HRPP Policy 303) per discretion of the designated study investigator and with the agreement of the participant/consent designee(s). Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant/consent designee, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant/consent designee will be informed of the private nature of the discussion and will be encouraged to relocate to a more private setting if needed. Consent will be documented with required signatures on the physical document (which includes the printout of an electronic document sent to participant) or as described below, with a manual (non-electronic) signature on the electronic document. When required, witness signature will be obtained similarly as described for the investigator and participant as described below.  
Manual (non-electronic) signature on electronic document When a manual signature on an electronic document is used for the documentation of consent at the NIH Clinical Center, this study will use the following to obtain the required signatures:  
Adobe platform (which is not 21 CFR Part 11 compliant); or iMedConsent platform (which is 21 CFR Part 11 compliant)
During the consent process, participants and investigators will view individual copies of the approved consent document on screens at their respective locations (if remote consent); the same screen may be used when in the same location but is not required. Both the investigator and the subject will sign the document using a finger, stylus, or mouse. Note: Refer to the CCR SOP PM-2, Obtaining and Documenting the Informed Consent Process for additional information (e.g., verification of participant identity when obtaining consent remotely) found at: https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=73203825.  
As there is an optional biopsy for research in this protocol, the participant/consent designee will be asked to sign a separate consent at the time of the procedure. If the participant/consent designee refuses the optional biopsy at that time, the refusal will be documented in the medical record and in the research record. 12.5.1 Consent Process for Adults Who Lack Capacity to Consent to Research Participation For participants addressed in Section **12.3**, an LAR will be identified consistent with Policy 403 and informed consent obtained from the LAR, as described in Section **12.5**.
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13 regulatory and operational considerations

This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable cause. Written notification, documenting the reason for study suspension or termination, will be provided by the suspending or terminating party to study participants, investigator, funding agency, the Investigational New Drug (IND) sponsor and regulatory authorities. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be informed of changes to study visit schedule. Circumstances that may warrant termination or suspension include, but are not limited to:  
Determination of unexpected, significant, or unacceptable risk to participants Demonstration of efficacy that would warrant stopping Insufficient compliance to protocol requirements Data that are not sufficiently complete and/or evaluable Determination that the primary endpoint has been met Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB and as applicable Food and Drug Administration (FDA). 13.2 QUALITY ASSURANCE AND QUALITY C**ONTROL** The clinical site will perform internal quality management of study conduct, data and biological specimen collection, documentation and completion. An individualized quality management plan will be developed to describe a site's quality management. Quality control (QC) procedures will be implemented beginning with the data entry system and data QC checks that will be run on the database will be generated. Any missing data or data anomalies will be communicated to the site for clarification/resolution. Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is conducted and data are generated, and biological specimens are collected, documented (recorded), and reported in compliance with the protocol, International Conference on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). The investigational site will provide direct access to all trial related sites, source data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities.
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13.3 conflict of interest policy

The independence of this study from any actual or perceived influence, such as by the pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to their participation in the design and conduct of this trial. The study leadership in conjunction with the NCI has established policies and procedures for all study group members to disclose all conflicts of interest and will establish a mechanism for the management of all reported dualities of interest.
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13.4 confidentiality and privacy

Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participants. Therefore, the study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. All research activities will be conducted in as private a setting as possible. The study monitor, other authorized representatives of the sponsor, representatives of the Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records
(office, clinic, or hospital) and pharmacy records for the participants in this study. The clinical study site will permit access to such records. The study participant's contact information will be securely stored at the clinical site for internal use during the study. At the end of the study, all records will continue to be kept in a secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. Study participant research data, which is for purposes of statistical analysis and scientific reporting, will be stored at the NCI CCR. This will not include the participant's contact or identifying information. Rather, individual participants and their research data will be identified by a unique study identification number. The study data entry and study management systems used by the clinical sites and by NCI CCR research staff will be secured and password protected. At the end of the study, all study databases will be archived at the NIH. To further protect the privacy of study participants, a Certificate of Confidentiality has been issued by the National Institutes of Health (NIH). This certificate protects identifiable research information from forced disclosure. It allows the investigator and others who have access to research records to refuse to disclose identifying information on research participation in any civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from being compelled to disclose information that would identify research participants, Certificates of Confidentiality help achieve the research objectives and promote participation in studies by helping assure confidentiality and privacy to participants.
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14 pharmaceutical information 14.1 prgn-2009

PRGN-2009 will be provided by Precigen through a CRADA with NCI to Pharmacy. All study interventions will be given at NIH Clinical Center and documented in the electronic medical record. The investigator or designee (e.g., pharmacist) will maintain an ongoing inventory of the investigational product supply according to standard site procedures. The investigational product will be dispensed at the direction of an investigator for administration to a study participant enrolled on the clinical trial. Disposal of expired or unused product will be returned to the manufacturer or disposed of according to standard site procedures based on agreement between the manufacturer and the site.
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14.1.2 toxicity

PRGN-2009 has not been tested in humans. PRGN-2009 antigen was successfully evaluated in three different types of *in vitro* analyses designed to identify potential oncogenicity of novel DNA in mammalian cells. The HPV PRGN-2009 antigen did not induce an oncogenic profile in the host cells in any assay, whereas cells transformed with wild type HPV oncogene versions unambiguously displayed morphological and phenotypic changes consistent with oncogenesis, both qualitatively and quantitatively. This data provides confidence that the HPV PRGN-2009 molecular vaccine candidate is safe for use in mammalian cells and will not induce aberrant growth consistent with carcinogenesis.  
PRGN-2009 is a non-replicating, non-integrating adenoviral vector.  Since the parental adenovirus is classified as biosafety level 2 (BSL2), PRGN-2009 should be handled as BSL2 material for disposal of all materials that were used in the dose preparation.  Avoid generation of aerosols.  
Spills of PRGN-2009 should be decontaminated using a solution of 10% bleach. This is a first in human study with PRGN-2009. Possible side effects may include injection side reaction, fever, flu like symptoms (e.g., fatigue, headache, muscle aches), poor appetite, increase in blood sugar, and skin rash.
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table 8 summary of treatment-emergent

| PRCN-2009, 1 x 10¹¹ PU   |  PRCN-2009, 5 x 1011 PU   | PRCN-2009, 5 x 10¹¹ PU   |            |            |           |            |
|--------------------------|---------------------------|--------------------------|------------|------------|-----------|------------|
| + M7824, 1200mg (n=6)    |                           |                          |            |            |           |            |
| (n=3)                    | (n=3)                     |                          |            |            |           |            |
| # of events              | # of subjects             |                          |            |            |           |            |
| # of                     | # of                      | # of                     | # of       |            |           |            |
| subjects                 | erents                    | subjects                 | erents     |            |           |            |
| All adverse              | 72                        | 3/3                      | 20         | 3/3 (100%) | 64        | 6/6 (100%) |
| events                   | (100%)                    |                          |            |            |           |            |
| 3/3                      |                           |                          |            |            |           |            |
| Grade 1                  | 44                        | 11                       | 3/3 (100%) | 41         | 5/6 (83%) |            |
| (100%)                   |                           |                          |            |            |           |            |
| 3/3                      |                           |                          |            |            |           |            |
| Grade 2                  | 3/3 (100%)                | 5/6 (83%)                |            |            |           |            |
| 19                       | 9                         | 18                       |            |            |           |            |
| (100%)                   |                           |                          |            |            |           |            |
| 2/3 (67%)                | 26 (33%)                  |                          |            |            |           |            |
| Grade 3                  | 9                         | 0                        | 0/3 (0%)   | 3          |           |            |
| 0/3 (0%)                 | 0/3 (0%)                  | 1/6 (17%)                |            |            |           |            |
| Grade 4                  | 0                         | 0                        | i          |            |           |            |
| Grade 5                  | 0                         | 0/3 (0%)                 | 0          | 0/3 (0%)   | i         | 1/6 (17%)  |
| PRCN-2009                | 2/3 (67%)                 | 5/6 (83%)                |            |            |           |            |
| 30                       | 3/3                       | 4                        | 23         |            |           |            |
| (100%)                   |                           |                          |            |            |           |            |
| treatment                |                           |                          |            |            |           |            |
| related AE               |                           |                          |            |            |           |            |
| PRCN-2009                | 0                         | 0/3 (0%)                 | 0          | 0/3 (0%)   | 0         | 0/6 (0%)   |
| treatment                |                           |                          |            |            |           |            |
| related AE               |                           |                          |            |            |           |            |
| ≥grade 3                 |                           |                          |            |            |           |            |
| All serious AE           | 6                         | 2/3 (67%)                | i          | 1/3 (33%)  | 4         | 26 (33%)   |
| 0/3 (0%)                 | 0/6 (0%)                  |                          |            |            |           |            |
| Treatment                | 0                         | 0/3 (0%)                 | 0          | 0          |           |            |
| related Serious          |                           |                          |            |            |           |            |
| AE                       |                           |                          |            |            |           |            |
| DLT                      | 0                         | 0/3 (0%)                 | 0          | 0/3 (0%)   | 0         | 0/6 (0%)   |  
Table 9: Adverse Events Related to PRGN-2009 Treatment by System-Organ Class in Phase 1/2 Study in Patients with HPV+ Malignancies (NCT04432597) (Reference: Table 8 in PRGN-2009 IB Version 2)  
| PRCN-2009,                                           |                    |            |    |    |
|------------------------------------------------------|--------------------|------------|----|----|
| 5 x 10¹¹ PU +                                        |                    |            |    |    |
| Severity                                             | PRCN-2009.         | PRCN-2009. |    |    |
| (CTCAE                                               | M7824, 1200mg      |            |    |    |
| l x 10¹¹ PU (n=3)                                    | 5 x 10 11 PU (n=3) |            |    |    |
| Number of Events                                     | Grade)             | (n=5)      |    |    |
| General disorders and administration site conditions |                    |            |    |    |
| 6                                                    | 2                  |            |    |    |
| l                                                    |                    |            |    |    |
| Fatigue                                              | 2                  | l          |    |    |
| Fever                                                | !                  | l          |    |    |
| l                                                    | 3                  | 10         |    |    |
| Flu like symptoms                                    | 2                  | 18         | 2  | 4  |
| Injection site                                       | i                  |            |    |    |
| reaction                                             | l                  |            |    |    |
| Metabolism and Nutrition Disorders                   |                    |            |    |    |
| Anorexia                                             | l                  |            |    |    |
| Hyperglycemia                                        |                    |            |    |    |
| Nervous system disorders                             |                    |            |    |    |
| Headache                                             |                    |            |    |    |
| Dysgeusia                                            |                    |            |    |    |
| Skin and subcutaneous tissue disorders               |                    |            |    |    |
| Rash maculo-                                         | i                  |            |    |    |
| papular                                              |                    |            |    |    |  
14.1.3  Formulation and Preparation Precigen will provide the following materials:
o o The concentration of the PRGN-2009 in supplied vials is 5 x 10E11 PU/ml; o PRGN-2009 vials (manufactured by ABL) - 1.0 ml/vial (extractable volume; frozen);
o Final formulation buffer (FFB) Diluent vials (manufactured by ABL) - 4.0 ml/vial (frozen); and o Sterile empty glass vials (manufactured by ALK) for preparing dilutions (if required).
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14.1.3.1 stability and storage

PRGN-2009 (manufactured by ABL) is provided as a sterile, frozen, injectable liquid that has been aseptically filled into crystal zenith (CZ) vials fitted with a butyl rubber stopper and an aluminum flip-cap seal. PRGN-2009 must be stored in an ultracold freezer in the temperature range of -60 to -90°C. Each vial contains 1.0 ml (extractable volume) of PRGN-2009 at a target concentration of 5 x 10¹¹ PU/ml. Therefore, each vial contains approximately 5 x 10¹¹ PU.
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14.1.3.2 diluent

Final formulation buffer (FFB; manufactured by ABL) will be used as the diluent for PRGN-2009 and will be supplied by Precigen. Diluent is provided as a sterile, frozen, injectable liquid that has been aseptically filled into crystal zenith (CZ) vials fitted with a butyl rubber stopper and an aluminum flip-cap seal. Each vial contains 4.0 ml (extractable volume) of PRGN-2009. NOTE: no other diluents other than FFB should be used with PRGN-2009. Two different doses of PRGN-2009 will be evaluated during the phase I dose escalation phase of the trial:
Initial dose: 1 x 1011 PU - A single vial of PRGN-2009 and a single vial of Diluent will be used for dilution and administration of the initial dose of 1 x 1011 PU. See instructions below for preparation of the initial dose.  
Maximum dose: 5 x 1011 PU - A single vial will be used for administration of the maximum dose of 5 x 1011 PU. No pooling of vials will be required. A Diluent vial will not be needed for the 5 x 1011 PU dose.
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14.1.4 pharmacy preparation

Follow institutional procedures for handling of a BSL-2 organism. Since the PRGN-2009, is provided with a butyl rubber stopper with aluminum flip-tear seal, these materials do not required handling within a biological safety cabinet. Care should be taken to avoid creation of aerosols during syringe withdrawals through the butyl rubber stopper. If institutional policies dictate, the steps involving a syringe transfer should be conducted within a biological safety cabinet. Spills of PRGN-2009 should be decontaminated using a solution of 10% bleach. All materials should be disposed of according to institutional procedures for a BSL-2 organism.
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14.1.5 vaccine preparation

Thawing - For each subject, a single vial of PRGN-2009 should be removed from the ultracold freezer and rapidly thawed in a 37C water bath until the contents are just thawed (avoid prolonged exposure of the thawed vial to the 37C water bath). Once thawed, do not refreeze. After thawing, vials should be wiped down with 70% IPA. Thawed vials of PRGN-2009 can be stored for up to 2 hours at ambient temperature prior to administration. Do not store thawed vials on wet ice or in contact with a cold pack or inside a refrigerator. Dilution of PRGN-2009 and storage of the diluted product at ambient temperature is addressed in the pharmacy manual, which should be referenced if dilution is required. Visually inspect the vialed products before use. If the appearance does not match the appearance listed below, contact the manufacturer (Precigen) and quarantine the vial(s) until further guidance is provided. Do not use the product if they appear to be damaged in any way.  
o PRGN-2009 should appear to be a clear to slightly opalescent, colorless liquid and should be free of visible particulates.  
o FFB should appear to be a clear, colorless liquid and should be free of visible particulates.  
Two different doses of PRGN-2009 will be evaluated during the dose escalation phase of the trial:
Preparation of the 1 x 1011 PU dose - A single vial of PRGN-2009 will be thawed and diluted 1:5 using the Diluent provided by Precigen, prior to administration to a subject as follows. After thawing, 0.4 ml of PRGN-2009 should be aseptically withdrawn from the vial and transferred into an empty sterile glass vial (provided by Precigen) using an appropriately sized sterile syringe. It is permissible to push air into the vial before withdrawing the specified volumes, as long as aerosols are not created. Next, 1.6 ml of thawed Diluent will be aseptically withdrawn from the Diluent vial using an appropriately sized sterile syringe and this volume will be transferred into the glass vial into which the 0.4 ml of PRGN-2009 has been transferred in the previous step. The contents should be gently swirled to thoroughly mix (do not vigorously shake). A volume of 1.0 ml of the diluted PRGN-2009 will then be aseptically withdrawn using an appropriately sized sterile syringe and administered for the initial dose of 1 x 1011 PU. The remaining contents of the PRGN-2009 vial, the remaining contents of the Diluent vial, the glass dilution vial, and used syringes should be discarded as biohazardous waste. The syringe containing PRGN2009 can be stored at ambient temperature after preparation. The total time for exposure of PRGN-2009 to ambient temperature after thawing the vial is 2 hours, including dilution, syringe filling and administration. Do not store prepared PRGN-2009 syringe on wet ice or in a refrigerator. Also, do not share vials of PRGN-2009 or Diluent between subjects.  
Preparation of the 5 x 1011 PU dose - The top dose of 5 x 1011 PU will be obtained by aseptically withdrawing 1.0 ml of PRGN-2009 from a single thawed vial using an appropriately sized sterile syringe. One ml of the undiluted PRGN-2009 will be administered for the top dose of 5 x 1011 PU. The empty PRGN-2009 vial should be discarded as biohazardous waste. The total time for exposure of PRGN-2009 to ambient temperature after thawing the vial is 2 hours, including syringe filling and administration. Do not store on wet ice or in a refrigerator.
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14.1.6 stability and storage

PRGN-2009 and Diluent are provided in cardboard cartons containing 10 vials of frozen liquid. Each 10-pack carton is provided within a sealed, labeled Mylar pouch. The sealed pouch is shipped inside a cardboard box that contains dry ice so that the contents will remain frozen during shipment. This Mylar pouch protects the vials from direct exposure to dry ice vapors. PRGN-2009 and Diluent will be shipped to the clinical site from SriSai Biopharmaceutical Solutions, Frederick, MD.  
After receipt, the package should be opened and the contents inspected to confirm that a quantity of dry ice remains and that the Mylar pouch is intact. (Report to Precigen any issues observed when receiving and opening a package). The Mylarenclosed carton of 10 vials should be immediately transferred to an ultracold freezer and stored in the range of -60 to -90C. This freezer should be appropriately monitored for temperature excursions. Any excursions that occur outside the recommended storage temperature range for PRGN-2009 should be documented and reported to the Sponsor.  
The carton can remain within the Mylar pouch until a vial of PRGN-2009 is required for administration to a subject. After the Mylar pouch is opened, the carton should be quickly returned to the ultracold freezer to avoid exposure of PRGN-2009 to ambient temperature. After the pouch is opened it can be disposed of and the cardboard carton can be stored directly within the ultracold freezer. Do not expose unprotected vials (without Mylar pouch) directly to dry ice vapors after opening and removing the pouch.
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14.2 m7824 (ind #19628 )

M7824 is manufactured and supplied for the trial by EMD Serono Research and Development Institute to Pharmacy. All study interventions will be given at NIH Clinical Center and documented in the electronic medical record. The investigator or designee (e.g., pharmacist) will maintain an ongoing inventory of the investigational product supply according to standard site procedures. The investigational product will be dispensed at the direction of an investigator for administration to a study participant enrolled on the clinical trial. Disposal of expired or unused product will be returned to the manufacturer or disposed of according to standard site procedures based on agreement between the manufacturer and the site.
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14.2.2 toxicity

The immunoglobulin portion of M7824 molecule is identical to avelumab (Bavencio). Respective warnings and precautions for grade 2 or higher immune-mediated pneumonitis, immune-mediated colitis, immune-mediated endocrinopathies, immune-mediated hepatitis) and infusion reactions are included in the prescribing for Bavencio (bavencio.com). Participants will be pre-medicated to prophylax against infusions reactions. The following additionally significant immune-mediated adverse reactions have occurred in less than 1% of 1738 participants treated with BAVENCIO: myocarditis with fatal cases, myositis, psoriasis, arthritis, exfoliative dermatitis, erythema multiforme, pemphigoid, hypopituitarism, uveitis, Guillain-Barré syndrome, and systemic inflammatory response. The above irAEs are all considered an anticipated risk of treatment with M7824 and thus will not be considered DLTs. In a phase 1, open-label 3+3 dose-escalation study of M7824 in 16 participants, 3 participants experienced grade 3 drug-related adverse events including skin infection secondary to grade 2 bullous pemphigoid, lipase increased, and colitis with associated anemia. There were no grade 4 - 5 treatment related adverse events. Please see table below for details.  
| 3 mg/kg (n =  3)   | 10 mg/kg (n = 3)   | 20 mg/kg (n = 7)   | Total (n = 16)   |         |        |        |       |
|--------------------|--------------------|--------------------|------------------|---------|--------|--------|-------|
| Any                | Grade              | Any                | Grade            | Any     | Grade  | Any    | Grade |
| Grade              | 3                  | Grade              | 3                | Grade   | 3      | Grade  | 3     |
| Participants with  | 2                  | 1                  | (33.3)           | 0 (0.0) | 4      |        |       |
| 1                  | 2                  | 7                  | 3                |         |        |        |       |
| any event**        | (66.7)             | (33.3)             | (57.1)           | (28.6)  | (43.8) | (18.8) |       |  
| Version Date: 04.25.2023                                                                | 3 mg/kg (n =  3)   | 10 mg/kg (n = 3)   | 20 mg/kg (n = 7)   | Total (n = 16)   |       |       |       |
|-----------------------------------------------------------------------------------------|--------------------|--------------------|--------------------|------------------|-------|-------|-------|
| Any                                                                                     | Grade              | Any                | Grade              | Any              | Grade | Any   | Grade |
| Grade                                                                                   | 3                  | Grade              | 3                  | Grade            | 3     | Grade | 3     |
| Anemia                                                                                  | 1                  | 1                  |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            | 1 (6.3)            |                    |                  |       |       |       |
| (14.3)                                                                                  |                    |                    |                    |                  |       |       |       |
| Bullous                                                                                 | 1                  |                    |                    |                  |       |       |       |
| (33.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| pemphigoid  Colitis                                                                     | 1                  | 1                  |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            | 1 (6.3)            |                    |                  |       |       |       |
| (14.3)                                                                                  |                    |                    |                    |                  |       |       |       |
| Dermatitis                                                                              | 1                  |                    |                    |                  |       |       |       |
| (33.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| acneiform Dyspnea                                                                       | 1                  |                    |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| exertional*** Hyperthyroidism                                                           | 1                  |                    |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| Hypophosphatemia                                                                        | 1                  |                    |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| Hypothyroidism                                                                          | 1                  | 1                  | 2                  |                  |       |       |       |
| (33.3)                                                                                  | (14.3)             | (12.5)             |                    |                  |       |       |       |
| Infusion-related                                                                        | 1                  |                    |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| reaction Keratoacanthoma                                                                | 1                  |                    |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| Lipase increase                                                                         | 1                  | 1                  |                    |                  |       |       |       |
| (14.3)                                                                                  | 1 (6.3)            | 1 (6.3)            |                    |                  |       |       |       |
| (14.3)                                                                                  |                    |                    |                    |                  |       |       |       |
| Nausea                                                                                  | 1                  |                    |                    |                  |       |       |       |
| (33.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| Pruritus                                                                                | 1                  |                    |                    |                  |       |       |       |
| (33.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| Rash maculopapular                                                                                         | 1                  | 1                  | 2                  |                  |       |       |       |
| (33.3)                                                                                  | (33.3)             | (12.5)             |                    |                  |       |       |       |
| Skin infection                                                                          | 1                  | 1                  |                    |                  |       |       |       |
| (33.3)                                                                                  | 1 (6.3)            | 1 (6.3)            |                    |                  |       |       |       |
| (33.3)                                                                                  |                    |                    |                    |                  |       |       |       |
| Vomiting                                                                                | 1                  |                    |                    |                  |       |       |       |
| (33.3)                                                                                  | 1 (6.3)            |                    |                    |                  |       |       |       |
| **There were no treatment-related AEs in the 3 participants treated with 1 mg/kg M7824. |                    |                    |                    |                  |       |       |       |  
***The differential for this dyspnea was pneumonitis vs. lymphangitic spread of disease (disease progression). As of August 2017, > 500 participants have been treated with M7824 across multiple solid tumor expansion cohorts. The safety profile is consistent with other monotherapy checkpoint inhibitors, with the exception of keratoacanthomas and cutaneous squamous cell carcinomas which have occurred in approximately 3-5% of participants and are well managed with surgical excision. These lesions have not been a criterion for treatment discontinuation, but thus far have all spontaneously regressed following treatment discontinuation. In addition, after discussion among NCI investigators on multiple protocols using M7824 bleeding events ranging from low grade gingival bleeding and epistaxis to more serious hemoptysis, GI bleeding and hematuria have been observed. Some of these events can be attributed to bleeding events related to cancer directly. However, there remains the possibility that M7824 may increase the overall risk of bleeding in ways that may not be directly related to direct tumor bleeding. It is hypothesized that this possible increased bleeding risk may be due to TGF beta inhibition which has an effect on angiogenesis. Accordingly, participants will be notified of the same possible risk in the informed consent document for this study (e.g., gum bleeding, nose bleeds, coughing up blood, blood in their urine, or blood in the stool).  
Potential risks include anemia, alterations in wound, healing or repair of tissue damage, and embryofetal toxicity. In addition, at least 2 instances of nodular regenerative hyperplasia have been observed with the use of this agent. 14.2.3 Formulation and Preparation M7824 is provided as a sterile liquid formulation and packaged at a 10 mg/mL concentration in USP/ Ph Eur type I 50R vials that are filled with drug product solution to allow an extractable volume of 60 mL (600 mg/60 mL). The vials are closed with rubber stoppers in serum format complying with USP and Ph Eur with an aluminum crimp seal closure. Each single-use vial contains 600mg of M7824, formulated as 10mg/mL of active, 6% (w/v) Trehalose, 40 mM NaCl, 5 mM Methionine, 0.05% (w/v) Tween 20, 10 mM LHistidine at pH 5.5. The liquid formulation is diluted directly with 0.9% sodium chloride solution for injection. The estimated volumes of delivery are anticipated to be no more than 250mL. The verified concentration range in the infusion solution is 0.16 mg/mL to 9.6 mg/mL.
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14.2.4 stability and storage

M7824 must be stored at 2℃ to 8℃ until use. Product stored at room temperature for extended periods of time might be subject to degradation. M7824 must not be frozen. Rough shaking of the reconstituted solution must be avoided. The chemical and physical in-use stability for the infusion solution of M7824 in 0.9% sodium chloride for injection has been demonstrated for a total of 72 hours at room temperature; however, from a microbiological point of view, the diluted solution should be used immediately and is not intended to be stored unless dilution has taken place in controlled and validated aseptic conditions. No other drugs should be added to the infusion containers containing M7824. See Manual of Preparation of approved ancillary supplies. 14.2.5 Administration Procedures See Section **3.2.2**. 14.2.6 Incompatibilities Not available.
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15 references

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| 16   | APPENDIX A: PERFORMANCE STATUS CRITERIA ECOG Performance Status Scale Karnofsky Performance Scale Grade Descriptions Percent Description 100 Normal, no complaints, no  evidence of disease. Normal activity. Fully active, able  0 to carry on all pre-disease  Able to carry on normal activity;  performance without restriction. 90 minor signs or symptoms of  disease. Normal activity with effort;  80 some signs or symptoms of  disease. 1 Symptoms, but ambulatory.  Restricted in physically strenuous  activity, but ambulatory and able  to carry out work of a light or  sedentary nature (e.g., light  housework, office work). 70 Cares for self, unable to carry on  normal activity or to do active  work. Requires occasional assistance,  In bed <50% of the time.  60 but is able to care for most of  Ambulatory and capable of all  his/her needs. 2 self-care, but unable to carry out  any work activities. Up and about  more than 50% of waking hours. 50 Requires considerable assistance  and frequent medical care. 40 Disabled, requires special care  and assistance. In bed >50% of the time. Capable  of only limited self-care, confined  to bed or chair more than 50% of  Severely disabled,  waking hours. 30 hospitalization indicated. Death  not imminent. 3 20 Very sick, hospitalization  indicated. Death not imminent. 4 100% bedridden. Completely  disabled. Cannot carry on any  self-care. Totally confined to bed  or chair. 10 Moribund, fatal processes  progressing rapidly. 5 Dead. 0 Dead.   |
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