untitled h1
untitled h2

Version Date: *05.12.2023* Abbreviated Title: Combo Immunotx in HPV Cancers NIH Protocol \#: 20C0045 Version Date: May 12, 2023 NCT Number: NCT04287868 RSC Number: 2744 Title: Phase I/II Trial of Combination Immunotherapy in Subjects with Advanced HPV
Associated Malignancies NCI Principal Investigator: Charalampos Floudas, MD, DMSc, MS
Center for Immuno-Oncology (CIO) Center for Cancer Research (CCR) National Cancer Institute (NCI) 10 Center Drive Building 10, Room 7N240A Bethesda, MD 20892 Phone: 240-858-3032 Email: charalampos.floudas@nih.gov  
| Drug Name:    | PDS0101           | MSB0011359C       | NHS-IL12          |
|---------------|-------------------|-------------------|-------------------|
| (M7824)       | (M9241)           |                   |                   |
| IND Number:   | 019267            | 019267            | 019267            |
| Sponsor:      | Center for Cancer | Center for Cancer | Center for Cancer |
| Research, NCI | Research, NCI     | Research, NCI     |                   |
| Manufacturer: | PDS Biotechnology | EMD Serono, Inc.  | EMD Serono, Inc.  |
| Supplier:     | PDS Biotechnology | EMD Serono, Inc.  | EMD Serono, Inc.  |  
Commercial Agents: None 1
untitled h1
precis

Background:
- Metastatic or refractory/recurrent HPV associated malignancies (cervical, anal, oropharyngeal cancers etc.) are poorly palliated by standard therapies. There is an unmet need for active treatments for these tumors.  
- In a phase I trial of M7824 (NCT02517398) 15 out of 43 (34.9%) participants with HPV
associated malignancies had radiographic tumor responses according to RECIST 1.1 or iRECIST.  
- While the response rate observed with M7824 appears to be higher than single agent PD-1 inhibitors alone (15-20%), the majority of patients with these diseases still do not seem to benefit from immunotherapy.  
- Preclinical studies suggest that the use of a combination of multiple immunotherapy agents may have improved anti-tumor efficacy.  
o Specifically, preclinical studies have shown that the combination of three immunotherapy agents (1) a therapeutic vaccine against HPV positive cancers (PDS0101), (2) a bifunctional fusion protein targeting PD-L1 and TGF beta (M7824), and (3) a tumor targeted immunocytokine (NHS-IL12) produces greater anti-tumor activity than any single or dual combination of these agents.
untitled h1
objectives

- To evaluate the objective response rate (ORR) according to Response Evaluation Criteria (RECIST 1.1) of the combination of (1) a therapeutic vaccine against HPV positive cancers (PDS0101), (2) a tumor targeted immunocytokine (NHS-IL12) and (3) a bifunctional fusion protein targeting PD-L1 and TGF beta (M7824) in subjects with checkpoint naïve advanced HPV associated malignancies.  
Eligibility:
- Age > 18 years old.  
- Subjects with cytologically or histologically confirmed locally advanced or metastatic HPV associated malignancies:
o Cervical cancers; o P16+ Oropharyngeal cancers; o Anal cancers; o Vulvar, vaginal, penile, and squamous cell rectal cancers; o Other locally advanced or metastatic solid tumors (e.g., lung, esophagus) that are known HPV+.  
- Prior first line systemic therapy is required unless the participant declines standard treatment after appropriate counseling has been provided.  
- Subjects must have measurable disease.
untitled h1
design

- This is a phase I/II trial of combination immunotherapy.  
- The trial will be conducted using a Simon optimal two-stage design.  
- Participants will receive HPV vaccine + NHS-IL12 + M7824.  
- The first six participants will be evaluable for dose limiting toxicities (DLTs) and accrual will only continue to 8 participants who have not been treated with checkpoint inhibitors if less than 2 out of the first 6 participants experience a DLT.  
- If three or more out of eight participants who have not been treated with checkpoint inhibitors have objective responses accrual will be expanded to enroll 20 evaluable participants.  
- Patients with cervical cancer with prior pelvic radiation and boost brachytherapy will be enrolled in a separate cohort to evaluate safety and preliminary evidence of efficacy.  
3  
| TABLE OF CONTENTS                                                                          |                                                                                                                                                              |
|--------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|
| hPRÉCIS                                                                                    |                                                                                                                                                              |
| TABLE OF CONTENTS                                                                          |                                                                                                                                                              |
| STATEMENT OF COMPLIANCE                                                                    |                                                                                                                                                              |
| 1.                                                                                         | INTRODUCTION .                                                                                                                                               |
| 1.1                                                                                        | Study Objectives .                                                                                                                                           |
| 1.2                                                                                        | Background and Rationale ................................................................................................................................. 8 |
| 2                                                                                          | ELIGIBILITY ASSESSMENT AND ENROLLMENT.                                                                                                                       |
| Eligibility Criteria .                                                                     |                                                                                                                                                              |
| 2.1                                                                                        |                                                                                                                                                              |
| 2.2                                                                                        | Screening Evaluation                                                                                                                                         |
| 2.3                                                                                        | Participant Registration and Status Update Procedures .......................................................... 34                                          |
| 2.4                                                                                        | Baseline Evaluation                                                                                                                                          |
| 3                                                                                          | STUDY IMPLEMENTATION .                                                                                                                                       |
| 3.1                                                                                        | Study Design .                                                                                                                                               |
| Drug Administration                                                                        |                                                                                                                                                              |
| 3.2                                                                                        | Study Intervention Compliance                                                                                                                                |
| 3.3                                                                                        |                                                                                                                                                              |
| 3.4                                                                                        | Dose Modifications .                                                                                                                                         |
| 3.5                                                                                        | Study Calendar .                                                                                                                                             |
| Cost and Compensation                                                                      |                                                                                                                                                              |
| 3.6                                                                                        |                                                                                                                                                              |
| 3.7                                                                                        | Criteria for Removal from Protocol Therapy and Off Study Criteria .................................. 53                                                      |
| 4                                                                                          | CONCOMITANT MEDICATIONS/MEASURES .                                                                                                                           |
| The Following Treatments Should Not Be Administered During the Trial................... 54 |                                                                                                                                                              |
| 4.1                                                                                        |                                                                                                                                                              |
| s                                                                                          | CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES ........ 55                                                                                         |
| 5.1                                                                                        | Biospecimen Collection .                                                                                                                                     |
| 5.2                                                                                        | Samples for Genetic/Genomic Analysis .                                                                                                                       |
| Sample Storage, Tracking and Disposition .                                                 |                                                                                                                                                              |
| 5.3                                                                                        |                                                                                                                                                              |
| 6                                                                                          | DATA COLLECTION AND EVALUATION                                                                                                                               |
| 6.1                                                                                        | DATA COLLECTION                                                                                                                                              |
| 6.2                                                                                        | DATA SHARING Plans .                                                                                                                                         |
| 6.3                                                                                        | Response Criteria                                                                                                                                            |
| 6.4                                                                                        | Toxicity Criteria                                                                                                                                            |
| 7                                                                                          | NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN.. 68                                                                                            |  
4  
Version Date: *05.12.2023*  
| Version Date: 05.12.2023 7.1 Definitions 68 7.2 OHSRP Office of Compliance and Training / IRB Reporting 68 7.3 NIH Required Data and Safety Monitoring Plan 69 8 SPONSOR PROTOCOL/SAFETY REPORTING 70 8.1 Definitions 70 8.2 Assessment of Safety Events 71 8.3 Reporting of Serious Adverse Events 72 8.4 Waiver of expedited reporting to CCR 72 8.5 Safety Reporting Criteria to the Pharmaceutical Collaborators 72 8.6 Reporting Pregnancy 72 8.7 Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND 73 8.8 Sponsor Protocol Deviation Reporting 73 9 CLINICAL MONITORING PLAN 74 10 STATISTICAL CONSIDERATIONS 74 10.1 Statistical Hypothesis 74 10.2 Sample Size Determination 75 10.3 Populations for Analyses 76 10.4 Statistical Analyses 76 11 COLLABORATIVE AGREEMENTS 78 11.1 Cooperative Research and Development Agreement (CRADA) 78 12 HUMAN SUBJECTS PROTECTIONS 78 12.1 Rationale For Subject Selection 78 12.2 Participation of Children 78 12.3 Participation of Subjects Unable to Give Consent 78 12.4 Risks/Benefit Assessment 79 12.5 Consent Process and Documentation 80 13 REGULATORY AND OPERATIONAL CONSIDERATIONS 82 13.1 Study Discontinuation and Closure 82 13.2 Quality Assurance and Quality Control 82 13.3 Conflict of Interest Policy 82 13.4 Confidentiality and Privacy 83 14 PHARMACEUTICAL INFORMATION 83   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
5 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023  
| PDS0101 (IND # 019267) .   |                                                                                                                                                          |
|----------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------|
| 14.1                       |                                                                                                                                                          |
| 14.2                       | M7824 (MSB0011359C) (IND # 019267) ............................................................................................ 85                       |
| 14.3                       | NS-IL12 (IND # 019267) .............................................................................................................................. 89 |
| 15 REFERENCES              |                                                                                                                                                          |
| 16 APPENDICIES .           |                                                                                                                                                          |
| 16.1                       | Appendix A Performance Status Criteria ................................................................................... 95                            |  
6
untitled h1
statement of compliance

The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical Practice (ICH GCP) and the following:
- United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects Protection and ICH GCP Training. The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form.
untitled h1
1. introduction 1.1 study objectives

- To evaluate the objective response rate (ORR) according to Response Evaluation Criteria
(RECIST 1.1) of the combination of (1) a therapeutic vaccine against HPV positive cancers (PDS0101), (2) a tumor targeted immunocytokine NHS-IL12 (M9241) and (3) a bifunctional fusion protein targeting PD-L1 and TGF beta (M7824) in subjects with checkpoint naïve advanced HPV associated malignancies.  
1.1.2 Secondary Objectives 1.1.3 Exploratory Objectives
- To conduct exploratory immunologic studies to understand and improve the administered treatment, including:
o peripheral immune subset analysis before and on treatment; 7
- To evaluate the safety of the combination of (1) a therapeutic vaccine against HPV
positive cancers (PDS0101), (2) a tumor targeted immunocytokine NHS-IL12 (M9241)
and (3) a bifunctional fusion protein targeting PD-L1 and TGF beta (M7824) in subjects with advanced HPV associated malignancies.  
- To assess progression-free survival time (PFS) according to RECIST 1.1. - To assess overall survival (OS).  
- To assess duration of response and ratio of participants that are hospitalized because of adverse events attributed to disease progression.  
- To evaluate the safety of the combination at a reduced dose level in patients with cervical cancer with prior pelvic radiation and boost brachytherapy where a potential higher risk of grade 3 hematuria may exist with the full starting doses of both M7824 and NHS-IL12.  
o soluble factors circulation (e.g., sCD27 and sCD40 ligand) before and on treatment; o tumor tissue immune infiltration before and after treatment.  
- To assess responses to therapy using iRECIST.  
- To assess responses to therapy by HPV serotype or PD-L1 expression.  
- To assess correlation between circulating tumor DNA and response to therapy
- To assess response to therapy in checkpoint refractory disease
- To assess response to therapy by HLA type
- To assess response using reduced doses of NHS-IL12 or M7824
untitled h1
1.2 background and rationale 1.2.1

In the United States, there are more than 30,000 cases of HPV associated cancer annually [1]
(**Table 1**). Metastatic HPV associated malignancies (cervical, anal, oropharyngeal cancers etc.)
are often incurable and poorly palliated by standard therapies. Responses to chemotherapy are variable but generally short-lived with median PFS around 3 to 7 months [2-5]. In a Gynecologic Oncology Group randomized trial comparing four cisplatin-based doublets as first line therapy for cervical cancer the response rates were 22-29% and median PFS was 4 to 6 months with median OS 10 to 13 months [6]. The addition of bevacizumab to combination chemotherapy has been reported to increase OS by 3.7 months, but virtually all patients die of their disease within 2 years [7]. Randomized trials of second line therapy are lacking but response rates for single agents are generally reported to be less than 20% [8]. Early evidence suggests that immune checkpoint therapy also has a low response rate in this disease with a phase 1b trial (KEYSTONE 028) showing a 12.5% response rate (3/24 patients) and a phase II study showing a response rate of 13.3% (13/98 patients) to pembrolizumab in patients with recurrent or metastatic cervical cancer [9].  
| Site          | Incidence of HPV associated cancers   | Cases attributed to HPV (%)   |
|---------------|---------------------------------------|-------------------------------|
| Oropharyngeal | 15,738                                | 11,000 (70.1%)                |
| Cervix        | 11,771                                | 10,700 (90.6%)                |
| Vulvar        | 3554                                  | 2400 (68.8%)                  |
| Vaginal       | 802                                   | 600 (75%)                     |
| Penis         | 1168                                  | 700 (63.3%)                   |  
| Site             | Incidence of HPV associated cancers   | Cases attributed to HPV (%)   |
|------------------|---------------------------------------|-------------------------------|
| Rectal (squamous | 750                                   | 700 (91.1%)                   |
| cell) Anus       | 5,010                                 | 4,600 (91.1%)                 |
| Total            | 38,793                                | 30,700 (79.1%)                |  
For oropharyngeal cancer, the best estimates of the chemotherapy responsiveness are inferred from looking at the oropharyngeal site in subset analyses from clinical trials for head and neck cancers. In a pivotal clinical trial that established platinum, 5-fluorouracil (5-FU), plus cetuximab as first line therapy in head and neck cancer, patients with oropharyngeal tumors experienced PFS of 4 to 6 months and OS of 8 to 11 months [3]. Immune checkpoint therapy has become the standard second line therapy for metastatic oropharyngeal cancer but response rates are still low with this therapy. As an example, the phase 1b trial (KEYNOTE-012) of pembrolizumab which led to its FDA approval for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) as second line therapy had a 17.7% response rate (34/192 patients). Response rates were slightly better in HPV positive HNSCC (21.9%) [10], but still occurred in only a minority of the patients. In regard to metastatic anal cancer, only a handful of randomized trials have been performed in the last 30 years. In the metastatic setting, most of the evidence is limited to small phase II trials, retrospective series, and case reports. A recent retrospective series looking at 77 patients, 44 (55%) of whom received 5-FU in combination with cisplatin, 24 (31%) of whom received carboplatin + paclitaxel, and 11 (14%) of whom received another regimen showed a median PFS of 7 months and median OS of 22 months [5]. As with HNSCC, recent early phase trials evaluating immune checkpoint therapy in this disease have shown that responses here too are limited to around 20% of patients treated. A recent phase II trial of nivolumab for metastatic squamous cell anal cancer showed a 21% response rate (7/33 patients) and a recent phase IB trial (KEYNOTE-028) of pembrolizumab showed a 20% response rate (5/25 patients) [11]. In a recently published phase II trial, Massarelli et al. evaluated the combination of a therapeutic HPV vaccine and Nivolumab [12]. In contrast to previous single therapy immune checkpoint inhibitor trials, they found an overall response rate of 33% showing the potential benefit of Immuno-Oncology (I/O) combination therapy for these diseases.  
Table 2 Response in patients treated with ISA101 and Nivolumab (Massarelli JAMA Oncol  
| Table 2. Response Overall, in Oropharyngeal Cancer, and by Treatment History"    |                                                                              |                   |                      |                                              |                 |                    |
|----------------------------------------------------------------------------------|------------------------------------------------------------------------------|-------------------|----------------------|----------------------------------------------|-----------------|--------------------|
| No. (%)                                                                          | Patients With Oropharyngeal Cancer                                           | Platin- and       |                      |                                              |                 |                    |
| Response per RECIST,                                                             | All Patients                                                                 | Platin-Refractory | Cetuximab-Refractory | Cetuximab-Refractory    Received Second-Line |                 |                    |
| version 1.1                                                                      | (N = 24)                                                                     | All (n = 22)      | Disease (n = 17)     | Disease (n = 8)                              | Disease (n = 6) | Treatment (n = 12) |
| Overall response rate                                                            | 8 (33)                                                                       | 8 (36)            | 6 (35)               | 5 (63)                                       | 3 (50)          | 5 (42)             |
| Complete response                                                                | 2 (8)                                                                        | 2 (9)             | 2 (12)               | 1 (13)                                       | 0               | 2 (17)             |
| Partial response                                                                 | 6 (25)                                                                       | 6 (27)            | 4 (24)               | 4 (50)                                       | 3 (50)          | 3 (25)             |
| Stable disease                                                                   | 3 (13)                                                                       | 2 (9)             | 1 (6)                | 0                                            | 1 (17)          | 1 (8)              |
| Disease control rateb                                                            | 1 (46)                                                                       | 10 (45)           | 7 (41)               | 5 (63)                                       | 4 (67)          | 6 (50)             |
| Progression of disease                                                           | 13 (54)                                                                      | 12 (55)           | 10 (59)              | 3 (38)                                       | 2 (33)          | 6 (50)             |
| Abbreviation: RECIST, Response Evaluation Criteria in Solid Turnors.             | tumors exhibited complete response, partial response, and stable disease and |                   |                      |                                              |                 |                    |
| a Refractory indicates progression of disease within 6 months of treatment.      | indicates lack of progression per RECIST.                                    |                   |                      |                                              |                 |                    |
| 6 Disease control rate is calculated by adding the percentages of patients whose |                                                                              |                   |                      |                                              |                 |                    |  
Unfortunately, little data exists evaluating immunotherapy for rarer HPV associated malignancies including metastatic vulvar, vaginal, penile, squamous cell rectal or neuroendocrine cervical cancer.
untitled h1
1.2.2 rationale for combination of

Preclinical studies are now revealing that an effective immuno-oncology strategy for enhancing anti-tumor responses is the use of multiple immune-mediating agents, each targeting different components of the immune system. These include: (a) induction of an immune response via vaccination directed against tumor-associated antigens, (b) reduction of immunosuppressive entities in the TME by the use of anti-PD1/PDL1 MAb checkpoint inhibitors, and/or reduction of immunosuppressive cytokines such as TGF-β with the use of a bifunctional anti-PDL1/TGF-β2 "Trap" designated M7824, and (c) potentiation of the immune response in the TME by the use of the tumor targeting NHS-IL12 immunocytokine. The LTIB, in collaboration with the GMB, has been able to develop and/or co-develop these agents via a series of CRADAs. Each has been interrogated in a series of preclinical studies and in Phase I/II clinical studies in the CCR as well as by other investigators. Preclinical studies and early clinical studies are demonstrating the relative lack of additional toxicities with enhanced clinical benefit employing combinations of 2 of these agents. Preclinical studies are now demonstrating that the combined use of agents from each of the immune-mediating categories described above elicits the most immune-mediated anti-tumor activities.
untitled h1
hv vaccine 1.2.2.1

PDS0101 is a novel nanoparticle-based immunotherapy (therapeutic vaccine) developed to treat genital cancers such as anal, cervical, vulvar cancer etc. and their precancers (late-stage dysplasia/neoplasia), as well as head and neck cancers caused by infection with the human papilloma virus (HPV). PDS0101 is a subcutaneously administered therapy consisting of two vialed components mixed by the investigator at the time of administration. The immunotherapy is composed of liposomal nanoparticles of the pure immunologically active R enantiomer of the cationic (positively charged) lipid DOTAP (dioleoyl-trimethylammonium propane) mixed with 6 lipidated antigenic peptides derived from the HPV-16 E6 and E7 proteins.  
In-vivo and in-vitro studies performed in normal and transgenic mouse models as well as in-vitro  
  
studies using human lymphocytes in culture demonstrated potent immune modulating effects by R-DOTAP. The pure R stereoisomer of DOTAP acts as a potent activator of the MAP kinase signaling pathway leading to activation and maturation of dendritic cells and subsequent induction of the production and release of cytokines and chemokines critical for promotion of an effective immune response. The positive charge on the surface of R-DOTAP liposomes facilitates their uptake by the dendritic cell. This in turn facilitates uptake of the HPV peptide antigen micelles and the antigenic processing and presentation by dendritic cells to T-cells. The ability of cationic liposomes to destabilize endosomes facilitates antigen delivery into the cytoplasm of the dendritic cells and other antigen presenting cells [13]. Effective induction of cytotoxic T-lymphocyte (CTL) responses and tumor regression has been demonstrated in the pre-clinical studies. Toxicology studies of R-DOTAP and a prototype R-DOTAP/HPV16-E7 single peptide formulation were evaluated in rats and in non-human primates (monkeys). No deaths or clinical signs related to treatment were observed in any of the studies. No treatment effects on body weight were noted. No treatment-related and/or toxicologically significant effects for any clinical chemistry parameters evaluated were noted during any of the studies. All hematology parameters, red blood cell counts, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration remained normal. Toxicology of the PDS0101 clinical formulation (6-peptide HPVmix and ImmunoMAPKRDOTAP) was evaluated in humanized transgenic mice. The transgenic mice expressed the human HLA-A2 antigen recognition and presentation molecule, and their antigen presenting cells were therefore capable of recognizing and responding to the HPV A2 epitope human peptide antigens in the drug formulation. Similar to toxicology studies in rats and monkeys with the prototype drug formulation, few clinically relevant toxicological effects were observed at any dose level in the transgenic humanized mice. However, for the clinical PDS0101 formulation mean hematocrit was decreased in the low R-DOTAP + HPVmix and high R-DOTAP + HPV mix groups by approximately 3% and 5%, respectively; and although minimal, may be related to treatment. No treatment-related toxicological findings were observed during gross necropsy. Microscopic histopathologic findings (minimal fibrosis) considered related to treatment with the test article were limited to the skin at the injection site in 3 out of 8 in the high R-DOTAP + HPV mix group only. In 2014-2016, a Phase I/IIA human clinical trial was performed at 3 sites in the US in 12 subjects with high-risk HPV infection and biopsy-proven cervical intraepithelial neoplasia (CIN1) to study the safety and immunogenicity of PDS0101.The study initiated with Cohort 1 and progressed through Cohort 3, with each subsequent cohort receiving a higher dose of PDS0101 (i.e., 1.0, 3.0, and 10.0 mg R-DOTAP); 3 in Cohort 1 (1-mg R-DOTAP dose), 3 in Cohort 2 (3 mg R-DOTAP dose), and 6 in Cohort 3 (10-mg RDOTAP dose). Successive cohorts received a constant dose of 2.4 mg HPV-16 E6 and E7 peptides (400 μg/peptide) with 1 of 3 escalating doses of R-DOTAP. All subjects received 3 doses of vaccine given SC, approximately 21 days apart. 10/12 subjects had a positive vaccine-induced response (a ≥3-fold increase over baseline at ≥1 of the 4 postvaccination visits by either the IFN-γ or granzyme B ELISpot assays, with background counts subtracted). Individual subject results from both the IFN-γ and the granzyme B assays showed that all doses of RDOTAP were effective in inducing HPV-specific T-cell responses (**Figure 2A**). IFN-γ (all T-cells) and the granzyme B (CD8+ T-cells) responses were elicited in both HPV-16+ and HPV-16- subjects, and in subjects with various HLA types. Administration of PDS0101 was also well tolerated. The majority of AEs were due to administration site reactions. Most administration site reactions were mild or moderate in severity. Some more severe administration site reactions including swelling and redness were reported in the 3- and 10-mg R-DOTAP cohorts. Most administration site reactions resolved the same day or within a few days. Per the subject symptoms diary, administration site reactions were more severe and of longer duration in subjects in the 10-mg R-DOTAP cohort. No clinically significant differences in the types and pattern of TEAEs were observed between Vaccinations 1, 2, or 3. No DLTs were observed; thus, the MTD was the 10-mg R-DOTAP dose. No serious adverse events (SAEs), study discontinuations due to adverse events, or deaths occurred. No clinically relevant abnormal hematology, blood chemistry, urinalysis, or physical findings were observed. Regression of CIN was noted in a number of patients (**Figure 2B**). Regression was seen in CIN related to a variety of HPV types including HPV-16, HPV-18 as well as other high-risk HPV types. The 3-mg R-DOTAP PDS0101 dose (containing 2.7 mg HPV-16 E6 & E7 antigens) has been chosen for further evaluation in larger Phase II trials given it seems to afford the best combination of safety and potency. The original dose of HPV-16 E6 & E7 antigens has been minimally increased from 2.4 mg to 2.7 mg for planned phase II studies. This was done due to some concern with possible oxidation of one of the peptides at the original lower dose. For simplicity the dose of all peptides has been minimally increased.  
  
Versamune®-HPV (PDS0101) Phase 1 Clinical Trial: Confirmation of unique potential to induce rapid and strong CD4 and CD8 T-cell responses against a viral target (HPV16) 14 days post-vaccination  
| Evaluable   | Clearance of CIN         |     |                |
|-------------|--------------------------|-----|----------------|
| Patients    | 12 Months Post Treatment |     |                |
| Dose Cohort | N =                      | N   | % of Evaluable |
| 1 mg        | 3 of 3                   | 67% |                |
| 3 mg        | 2 of 3                   | 1   | 50%            |
| 10 mg       | 5 of 6                   | 3   | 60%            |
| Total       | 10                       | 60% |                |
| 6           |                          |     |                |  
Two of twelve patients were not evaluable; one patient, who demonstrated a strong immune response (high dose cohort), was lost to follow up and another standard of care LEEP excision therapy at 4 months (mid dose cohort)
Two of ten evaluable patients who had clearance of CIN by cytology were not considered as clinical responders: one patient regressed from CIN to atypical squarno cells of undetermined significance (ASCUS) with detectable virus, and the other showed consistent disease elimination by cyclogy, but showed residual disease by clposcopy Figure 2 A (top). ELISpot responses in patients treated with PDS0101. B (bottom). CIN
Regression in patients treated with PDS0101 [ 14 ].
untitled h1
avelumab (anti-pd-l1) and m7824 (anti-pd-l1/tgf-

Studies in the LITB were the first to describe [ 15 , 16 ] avelumab, a fully human anti-PD-L1 IgG1 MAb, and its ability to mediate ADCC for a range of human carcinoma cells employing NK cells as effectors. Avelumab (Bavencio) has since been approved by the FDA for the therapy of Merkel cell carcinoma (the first drug approved for this indication) and second line urothelial carcinomas.  
We have also shown that endogenous IL-12 will enhance avelumab-mediated NK lysis, thus forming the basis for the combined use of avelumab or M7824 with NHS-IL12. The first-in-human clinical study of avelumab with over 1,700 patients was led by Gulley with the dose escalation and initial PK and PD done entirely within the CCR [16]. Because of the activity this study was amended to include multiple expansion cohorts internationally. Because PD-L1 is also expressed on some immune cell subsets, our finding [17] that there were no adverse events in the patients treated with avelumab above that seen with other anti-PD-L/PD-L1 MAbs was important for planning further studies with avelumab or M7824. TGF-β is a well-known inhibitor of immune cell function, especially in the TME [18-23]. Prior  
 studies have indicated the advantage in the use of a TGF-β inhibitor in combination with checkpoint therapy [24, 25] In collaboration with our CRADA partner EMD Serono, we have supported the development of a bifunctional fusion protein aimed at bringing a TGF-β TRAP to tumor cells via binding to PD-L1. M7824 is a novel fusion protein consisting of anti PD-L1 antibody linked to the extracellular domain of two TGF-βR2 (serving as a TGF-β TRAP) (Figure 3).  
We were the first to report [26, 27] that M7824 (a) mediates ADCC, (b) increases tumor cell gene expression of molecules involved in T-cell trafficking to the tumor, (c) enhances TRAIL- and antigen-specific CD8+ T-cell lysis of tumor cells, and (d) reduces TGF-β‒induced immunosuppressive activity. We have also recently demonstrated [28] in in vivo preclinical murine carcinoma models **(Figure 4A and B)** that M7824 has the ability to decrease TGF-β‒
induced signaling in the TME. Specifically, phosphorylation of SMAD2 was significantly decreased in tumors following M7824 treatment, and not with an M7824(mut) molecule, devoid of the PD-L1 binding site **(Figure 4C)**. In both breast and colon carcinoma murine models, M7824 decreased tumor burden and increased survival, which required both CD8+ and NK cell activity
(Figure 4D).  
  
Two recent studies [29, 30] demonstrated the advantage in anti-tumor activity in the use of the bifunctional agent vs. the use of a PD- 1/PD-L1 checkpoint in combination with a systemic TGFβ inhibitor. TGF-β is also known to alter human carcinoma cells from a more epithelial to a more mesenchymal phenotype and such tumor cells are more resistant to a range of therapies (Figure 5). In LTIB studies, TGF-β‒induced mesenchymalization of carcinoma cells was shown [31] to be reversed by M7824, rendering tumor cells more susceptible to chemotherapy and immunemediated killing [26, 28, 31]
The first-in-human trial of M7824 (anti-PD-L1/TGF-βR2) was conducted at the CCR [32]. Nineteen heavily pretreated patients with solid tumors (non-melanoma) were treated; the MTD was not reached. M7824 was shown to saturate peripheral PD-L1 and sequester all released (acid released) plasma TGFβ1, -β2, and -β3 throughout the dosing period at >1 mg/kg (recommended phase 2 dose is 1200 mg or about 10 mg/kg). There were signs of clinical efficacy across all dose levels **(Figure 6** and **Figure 7)**, including one ongoing CR (cervical cancer), two durable PRs
(pancreatic and anal cancers), one near PR (cervical) and two cases of prolonged stable disease.  
  
  
Figure 6. Phase I trial of M7824, spider plot of tumor responses and reduction of serum levels of TGFP1.  
  
In the Phase I study [32] 15/43 (34.9%) of patients with checkpoint naïve HPV associated malignancies had radiographic tumor responses according to RECIST 1.1 or iRECIST with M7824 (anti-PD-L1/TGF-βR2) therapy. Prior studies by others have shown approximately a 15% objective response in this population employing checkpoint inhibition therapy. In an ongoing phase II study (NCT03427411) 3/22 (13.6%) patients with checkpoint refractory advanced HPV associated malignancies have had responses to M7824 which compares well to the less than 5% response rate seen in the literature with further checkpoint therapy. Sufficient peripheral blood mononuclear cells were available from 12 patients on the phase I trial to test for HPV-specific T-cell responses before and after M7824 treatment. Considering all posttherapy timepoints examined, 7 of 12 patients (58%) had an increase in HPV-specific T cells postvs pre-M7824 therapy. Immune responses after 1 and 3 cycles of M7824 were compared between patients who developed a clinical response of stable disease or better vs those with progressive disease (**Figure 8**). In clinical responders, HPV-specific T cells developed in 3 of 4 patients (75%)
after 1 cycle of M7824 and in 4 of 4 patients (100%) after 3 cycles. On the other hand, in patients with progressive disease, HPV-specific T cells developed in only 1 of 5 patients (20%) after 1 cycle of M7824 and in 1 of 4 patients (25%) after 3 cycles (Supplementary **Table 3**). There was also an increased magnitude of HPV-specific T cells generated in clinical responders vs nonresponders: clinical responders had on average 12- to 27-fold more HPV-specific T cells producing cytokines or positive for CD107a (LAMP1, a functional marker of T and NK cell activity) after 1 and 3 cycles of M7824 compared with nonresponders (**Figure 3A**).  
Another observation worth noting is that 2 of the 3 patients with HPV+ cancer who received prior  
 lymphodepleting chemotherapy (e.g., cyclophosphamide, fludarabine) were assessed for immune responses post- vs pre-M7824 therapy, and neither had evidence of an HPV T-cell‒specific immune response. In contrast, in patients with HPV+ cancer who did not receive prior lymphodepleting chemotherapy, 6 of 9 (67%) had a positive T-cell‒specific immune response.  
Figure 8. Immune responses in HPV+ patients with disease control (BOR of SD, PR, or CR) vs nonresponders (BOR of PD) to M7824 therapy by intracellular cytokine staining following in vitro stimulation with a mixture of overlapping 15-mer peptide pools encoding HPV-16 E6 and E7, as previously described.21
untitled h1
nhs-il12 immunocytokine (m9241)

NHS-IL12 is a tumor targeting immunocytokine that binds to DNA-histone in necrotic areas of  
  
tumor. Studies by others have shown the ability of radiolabeled NHS-IL12 to target murine and human tumors **(Figure 9)**. Prior studies in the LTIB have shown the ability of this agent to elicit anti-tumor activity in multiple murine models, and in combination with anti-PD-L1 and with radiation and chemotherapy [33]. Gulley (GMB, CCR) and the LTIB conducted the Phase I dose escalation trial of the NHS-IL12 immunocytokine [34]. Due to known prior toxicities using a rec. IL-12 protein, multiple ascending dose levels were employed and the agent was administered every 4 weeks. Time-dependent increase in serum IFN-γ and subsequent rise in IL-10 increased with dose and varied greatly among patients **(Figure 10A)**. Increases in TCR diversity and TIL density in the TME following NHS-IL12 dosing were observed in those patients with elevation IFN-γ serum levels **(Figure 10B)**. Toxicity was acceptable with an MTD of 16.8 mcg/kg and this total dose was also found to be safe when given split in half on an every 2 week schedule. Although no objective tumor responses were seen, of 30 patients with measurable disease, 15 had stable disease, with some durable (from 6‒30+ months).  
  
Our recent preclinical studies have also shown the enhanced anti-tumor effect of avelumab when  
 used in combination with NHS-IL12 in syngeneic mice bearing an intravesical bladder tumor
(Figure 11, left panel) and that this effect is mediated via an IFN-γ‒dependent mechanism.  
Studies in NSG-β2m‒/
‒ mice bearing HTB1, a human bladder carcinoma, and reconstituted with human PBMC, also showed the enhanced anti-tumor effect of the combination that coincided with increased NK and CD8+ in the TME (**Figure 11, right panel**).  
This further provided the rationale for an ongoing CCR led clinical study of avelumab in combination with NHS-IL12 (NCT02994953), currently evaluating safety of the combination. In addition, to the combination being found to be safe and well-tolerated, evidence of radiographic tumor responses has been observed. As an example, a patient with checkpoint refractory metastatic urothelial cancer with a large sacral mass and retroperitoneal adenopathy at enrollment has had an ongoing complete response for 18+ months.
untitled h1
combination immunotherapies

In addition to harnessing the potential multi-functionality of M7824, we have conducted several combination studies employing this agent with other immune modulating agents. We have previously shown [27] enhanced anti-tumor activity when M7824 was combined with vaccine in EMT6, a metastatic breast carcinoma murine model **(Figure 12)**.  
  
Furthermore, combining M7824 with NHS-IL12 also greatly enhanced anti-tumor activity in the EMT6 breast carcinoma model ( Figure 13 ). Tumor challenge studies showed the presence of immune memory in mice that received the combination of M7824 and NHS-IL12, which conferred protection against re-challenge with tumor. Similar results were also seen in the MB38-CEA
murine colorectal carcinoma model.  
  
Multimodal therapies were also carried out in mice bearing HPV + tumors. The combination of PDS0101 vaccine and M7824 showed significant anti-tumor efficacy in the NSG β2m ′′ humanized mouse model reconstituted with human PBMC and bearing human cervical HPV+ cancer (SiHa)
subcutaneously (Figure 14).  
  
In contrast to the previous clinical trials, where we saw anti-tumor efficacy of M7824 or PDS0101 alone in HPV-associated tumors, in this model there was no single therapy effect of M7824 or PDS0101, but the combination significantly decreased tumor burden. Furthermore, in C57BL/6J mice bearing TC-1 (a murine lung carcinoma line transfected with HPV16 E6 and E7 oncoproteins), which is a very aggressive model of HPV+ carcinoma, the combination of PDS0101 vaccine, M7824 and NHS-IL12 resulted in the greatest level of anti-tumor activity compared to the use of individual agents or dual combinations **(Figure 15)**. The use of the combination of PDS0101 vaccine, M7824 and NHS-IL12 also resulted in an intense influx of CD4+ and CD8+ T
cells in tumors **(Figure 16, upper panels** and **Figure 17)**, increased activation and proliferation of T-cells (**Figure 16, lower panels**), as well as an enhanced HPV specific T cell response measured by ELISpot not seen with lesser combinations **(Figure 18)**.  
  
  
  
  
  
Figure 17. Immunohistochemistry shows increased T-cell infiltration in C57BL/6J mice bearing TC-1 HPV+ tumors, and treated with the combination of PDS0101, M7824, and NHSIL12.  
  
| Treatment         | Tumor Volume  <300mm3   | E7 ELISpot   |
|-------------------|-------------------------|--------------|
| PBS Control       | 0/16                    | -            |
| DOTAP             | 0/8                     | -            |
| M7824             | 0/16                    | -            |
| PDS0101           | 3/16                    | +            |
| NHS               | 6/16                    | -            |
| M7824+NHS         | 8/16                    | -            |
| PDS0101+NHS       | 10/16                   | +            |
| PDS0101+M7824     | 5/16                    | +            |
| PDS0101+M7824+NHS | 13/17                   | -            |  
Figure 18. PDS0101 generated HPV-specific T-cells, as measured by ELISpot. Table shows a summary of tumor volume and E7-specific IFNγ ELISpot for C57BL/6J mice bearing TC-1 HPV+ tumors, and treated with combinations of PDS0101, M7824, and NHSIL12 1.2.3 Safety and Efficacy of I/O Combinations in Humans Table 3: Previously evaluated combinations of I/O agents demonstrating an acceptable safety profile.  
| Avelumab (anti-PD-L1) + NHS-IL12   | NCT02994953   |
|------------------------------------|---------------|
| CEA/ MUC1 vaccine + M7824          | NCT03315871   |  
Dose Rationale: In the phase I dose escalation trial of combination Avelumab (anti-PD-L1) + NHS-IL12, escalating doses of NHS-IL12 were evaluated at 8,12, and 16.8 mcg/kg given every 4 weeks in combination with standard dosing of Avelumab. The combination including NHS-IL12 at the highest dose evaluated (16.8 mcg/kg given every 4 weeks) was found to be well tolerated. The only DLT observed on the trial was a transient grade 3 AST/ALT elevation at NHS-IL12 at 12 mcg/kg but otherwise no other DLT was observed and the MTD was not reached. Therefore, for this protocol, NHS-IL12 was originally used at a dose of 16.8 mcg/kg every 4 weeks in combination with the standard phase II dose of M7824 of 1200 mg given every two weeks. While we observed no DLTs with the combination of NHSIL12 at 16.8 mcg/kg along with M7824 and PDS0101 in the six participant lead in safety cohort, after treating 19 participants on protocol we observed that the majority of participants (11/19; 57.9%) needed dose reductions of NHSIL12 when given in combination with PDS0101 and M7824 due mostly to grade 2 fever and flu like symptoms. At this point (with Protocol Amendment Version 12.11.2020) we lowered the starting dose of NHS-IL12 in this protocol from 16.8 mcg/kg to 8 mcg/kg to try and minimize toxicity. After evaluating a total of 39 patients on this protocol including 21 with starting dose of 16.8 mcg/kg and 18 patients with starting dose of 8 mcg/kg we observed what looks to be reduced efficacy in patients receiving 8 mcg/kg as a starting dose with only 3/18 patients responding with the 8 mcg/kg starting dose as compared to 11/21 with the 16.8 mcg/kg starting dose. Therefore, based on this observation that the possibility that a lowering starting dose of NHS-IL12 may reduce efficacy we are amending to allow treatment (effective with Protocol Amendment Version 09.20.2021) with a limited number of doses of NHS-IL12 at 16.8 mcg/kg (four doses total), followed by dosing at 8 mcg/kg every 4 weeks thereafter. This is based upon responding patients in the first 21 patients which received anywhere between 1-4 doses of NHS-IL12 at 16.8 mcg/kg before dose reductions.
untitled h1
1.2.4 immune assays

Multiple immune assays have been developed in the LTIB to better define the mechanism(s) involved in the use of specific novel agents, as monotherapy or in combination therapies, both for preclinical and clinical studies. In addition to analyses of biopsies, analyses of the peripheral immunome can provide valuable information in that multiple samples can be analyzed over the course of a given therapy vs. pre-therapy. The LTIB has now developed and employed [15, 16, 31, 35] a flow cytometry‒based assay that can analyze 123 immune cell subsets in human PBMC from one vial of processed PBMC (approximately 107 cells). This assay will be used in this clinical trial to detect multiple (n=32) subsets of CD4+ T cells, CD8+ T cells (n=29), Tregs (n=7), B cells (n=5), NK (n=20), NKT (n=4), DC (n=10), and MDSC (n=16) to better understand the role of each agent. We also plan to evaluate changes in TCR clonal diversity both in biopsies and the periphery.  
One example involves an ongoing first-in-human trial of the NHS-IL12 tumor-targeting immunocytokine. TCR diversity increased 6-14‒fold in biopsies of patients with a high or intermediate IFN-γ response but were unchanged or decreased with a low IFN-γ response. These findings also correlated with TIL in biopsies. We are also currently employing NanoString analyses to identify a gene signature in biopsies and PBMC, pre- and post-treatment. The GMB will conduct multiplexed, multispectral imaging of FFPE tissue to evaluate multiple immune parameters within the TME before and after treatment in patients with available tissue. 1.2.5 Summary
- We are proposing a novel I/O combination to help overcome multiple barriers in an effective anti-tumor immune response.  
o (1) HPV therapeutic vaccine targeting tumor associated antigens to prime an antitumor immune response o (2) NHS-IL12 and M7824 to enable activated immune cells to work better in the TME
- Preclinical studies have shown that treatment with the combination of an HPV
therapeutic vaccine, M7824 and NHS-IL12 leads to greater anti-tumor activity than any single or double combination of these agents
untitled h1
2 eligibility assessment and enrollment

2.1.1 Inclusion Criteria 2.1.1.1 Subjects with cytologically or histologically confirmed locally advanced or metastatic HPV associated malignancies:
- Cervical cancers;
- P16+ Oropharyngeal cancers;
- Anal cancers;
- Vulvar, vaginal, penile, and squamous cell rectal cancers;
- Other locally advanced or metastatic solid tumors (e.g., lung, esophagus) that are known HPV+.  
2.1.1.2 Subjects must have measurable disease, per RECIST 1.1. See Section 6.3 for the evaluation of measurable disease.  
2.1.1.3 Subjects must have received one prior line of systemic chemotherapy as well as checkpoint therapy if checkpoint therapy is FDA approved for that specific tumor type (e.g., HNSCC and PDL1+ cervical cancer). Prior checkpoint therapy is not needed where checkpoint therapy has not been FDA approved for that specific tumor type (e.g. anal, vaginal, vulvar, penile, PDL1 negative cervical). Exceptions to the above include participant who are not eligible to receive the above therapies or who decline these standard treatment options after appropriate counseling has been provided.  
2.1.1.4 Age >18 years. 2.1.1.5 ECOG performance status < 2. 2.1.1.6 Adequate hematologic function at screening, as follows:
- Absolute neutrophil count (ANC) ≥1 x 109/L;
- Hemoglobin ≥ 9 g/dL;
- Platelets ≥ 75,000/microliter.  
2.1.1.7 Adequate renal and hepatic function at screening, as follows:
- Serum creatinine ≤ 1.5 x upper limit of normal (ULN) OR
Measured or calculated creatinine clearance ≥ 40 mL/min for participant with creatinine levels > 1.5 X institutional ULN (GFR can also be used in place of creatinine or CrCl);
- Bilirubin ≤ 1.5 x ULN OR in subjects with Gilbert's syndrome, a total bilirubin ≤ 3.0 x ULN;
- Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 x ULN,
unless liver metastases are present, then values must be ≤ 3 x ULN).  
2.1.1.8 The effects of the immunotherapies on the developing human fetus are unknown. For this reason and because immunotherapeutic agents as well as other therapeutic agents used in this trial are known to be teratogenic, women of child-bearing potential and men must agree to use highly effective contraception (hormonal or barrier method of birth control; abstinence) prior to study entry and for two months after study treatment. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.1.9 Participants serologically positive for HIV, Hep B, Hep C are eligible as long as the viral loads are undetectable by quantitative PCR. HIV positive participants must have CD4 count ≥ 200 cells per cubic millimeter at enrollment, be on stable antiretroviral therapy for at least 4 weeks and have no reported opportunistic infections or Castleman's disease within 12 months prior to enrollment.  
2.1.1.10 Ability of subject to understand and the willingness to sign a written informed consent document.  
2.1.2 Exclusion Criteria 2.1.2.1 Participants with prior investigational drug, chemotherapy, immunotherapy or any prior radiotherapy (except for palliative bone directed therapy) within the past 28 days prior to the first drug administration except if the investigator has assessed that all residual treatment-related toxicities have resolved or are minimal and feel the participant is otherwise suitable for enrollment. Participants may continue adjuvant hormonal therapy in the setting of a definitively treated cancer (e.g., breast cancer).  
2.1.2.2 Known intolerance to or life threatening side effects resulting from prior checkpoint inhibitor therapy.  
2.1.2.3 Major surgery within 28 days prior to the first drug administration (minimally invasive procedures such as diagnostic biopsies are permitted).  
2.1.2.4 Known active brain or central nervous system metastasis (less than a month out from definitive radiotherapy or surgery), seizures requiring anticonvulsant treatment (<3 months) or clinically significant cerebrovascular accident (<3 months). In order to be eligible participants must have repeat CNS imaging at least a month after definitive treatment showing stable CNS disease. Participants with evidence of intratumoral or peritumoral hemorrhage on baseline imaging are also excluded unless the hemorrhage is grade ≤ 1 and has been shown to be stable on two consecutive imaging scans.  
2.1.2.5 Pregnant women are excluded from this study because these drugs have not been tested in pregnant women and there is potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with these immunotherapies, breastfeeding should be discontinued if the mother is treated on this protocol.  
2.1.2.6 Active autoimmune disease that might deteriorate when receiving an immunostimulatory agent with exception of:
- Diabetes type I, eczema, vitiligo, alopecia, psoriasis, hypo- or hyperthyroid disease or other mild autoimmune disorders not requiring immunosuppressive treatment;
- Subjects requiring hormone replacement with corticosteroids are eligible if the steroids are administered only for the purpose of hormonal replacement and at doses ≤ 10 mg of prednisone or equivalent per day;  
- Administration of steroids for other conditions through a route known to result in a minimal systemic exposure (topical, intranasal, intro-ocular, or inhalation) is acceptable;
- Subjects on systemic intravenous or oral corticosteroid therapy with the exception of physiologic doses of corticosteroids (≤ the equivalent of prednisone 10 mg/day) or other immunosuppressives such as azathioprine or cyclosporin A are excluded on the basis of potential immune suppression. For these subjects these excluded treatments must be discontinued at least 1 weeks prior to enrollment for recent short course use (≤ 14 days) or discontinued at least 4 weeks prior to enrollment for long term use (> 14 days). In addition, the use of corticosteroids as premedication for contrast-enhanced studies is allowed prior to enrollment and on study.  
2.1.2.7 Subjects with a history of serious intercurrent chronic or acute illness, such as cardiac or pulmonary disease, hepatic disease, bleeding diathesis or recent (within 3 months) clinically significant bleeding events, or other illness considered by the Investigator as high risk for investigational drug treatment.  
2.1.2.8 Subjects unwilling to accept blood products as medically indicated. 2.1.2.9 History of non-HPV associated second malignancy within 3 years of enrollment except localized malignancy which has been adequately treated or malignancy which does not require active systemic treatment (e.g., low risk CCL). Patients taking adjuvant hormonal therapy for definitively treated cancers (e.g. breast cancer) are eligible.  
2.1.2.10 Subjects with a known severe hypersensitivity reaction to a monoclonal antibodies (grade
>/= 3 NCI-CTCAE v5) will be evaluated by the allergy/immunology team prior to enrollment.  
2.1.2.11 Receipt of prior lymphodepleting chemotherapy (e.g. cyclophosphamide, fludarabine) or any organ transplantation requiring ongoing immunosuppression.
untitled h1
2.1.3 recruitment strategies

This study will be posted on www.clinicaltrials.gov, NIH websites and on NIH social media forums. After IRB approval of the recruitment material, this study may be advertised at national conferences including AACR and ASCO and may also be shared with the HPV and Anal Cancer Foundation who may advertise it further. Participants will be recruited from the current participant population at NIH. Participants with HPV related cancers are currently being treated on another trial through LTIB, CCR, NIH, where 2-3 participants per month are being screening and enrolled. Some of these participants come from established community networks, some of these participants are self-referrals, and some of these participants are referred by another group (Dr. Hinrichs's group, ETIB, CCR, NIH) who also have a large referral base for HPV related cancers.
untitled h1
2.2 screening evaluation 2.2.1 screening

Minimal risk activities that may be performed before the subject has signed a consent include the following:
- Email, written, in person or telephone communications with prospective subjects  
- Review of existing medical records to include H&P, laboratory studies, etc.  
- Review of existing MRI, x-ray, or CT images
- Review of existing photographs or videos
- Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes 2.2.2 Screening activities performed after a consent for screening has been signed
The following activities will be performed only after the subject has signed the study consent or the consent for study \# 01C0129 (provided the procedure(s) are permitted on that study) on which screening activities will be performed. Assessments performed at outside facilities or on another NIH protocol within these timeframes below may also be used to determine eligibility once a participant has signed the consent. Within 28 days prior to enrollment, unless otherwise specified:
- Complete medical history and physical examination (including height, weight, vital signs, and ECOG performance status).  
- CT of chest, abdomen and pelvis.  
- A brain CT scan in participants with known CNS disease as described in Section **2.1.2.4**.  
- For skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion.  
- EKG
- Clinical laboratory tests (within 16 days prior to enrollment).  
− Chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN,
creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and direct bilirubin.
− Hematology: complete blood count (CBC) with differential.  
− CD4 (if clinically indicated).
− HLA typing (where feasible)
- Urine or serum pregnancy test (β-HCG) for females of childbearing-potential and women
< 12 months since the onset of menopause (within 16 days prior to enrollment).  
- Urinalysis.
- HBV, HCV, HIV testing including viral load if clinically indicated (within 3 months prior to enrollment).  
- Histologic confirmation (at any time point prior to enrollment). If there is no available tumor sample or pathology report, a biopsy will be performed to confirm the diagnosis.
untitled h1
2.3 participant registration and status

Registration and status updates (e.g., when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found at: https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=73203825. 2.3.1 Screen Failure Screen failures are defined as participants who consent to participate in the clinical trial but are not subsequently assigned to the study intervention or entered in the study. A minimal set of screen failure information is required to ensure transparent reporting of screen failure participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities. Minimal information includes demography, screen failure details, eligibility criteria, and any serious adverse event (SAE).  
Individuals who do not meet the criteria for participation in this trial (screen failure) because of a transient lab abnormality may be rescreened. 2.3.2 Treatment Assignment Procedures  
| Number   | Name     | Description                                                                        |
|----------|----------|------------------------------------------------------------------------------------|
| 1        | Cohort 1 | Subjects with HPV associated malignancies                                          |
| 2        | Cohort 2 | Subjects with cervical cancer with prior pelvic radiation  and boost brachytherapy |  
2
untitled h1
arms arm assignment

| Number   | Name   | Description                                     |
|----------|--------|-------------------------------------------------|
| 1        | Arm 1  | HPV vaccine + NHS-IL12 + M7824                  |
| 2        | Arm 2  | HPV vaccine + NHS-IL12 + M7824 at reduced doses |  
Participants in Cohort 1 will be assigned to Arm 1, and patients in Cohort 2 will be assigned to Arm 2. If more than three of eight patients have an objective response then accrual will be expanded to enroll 20 evaluable participants for cohort 1, while cohort 2 may enroll up to 12 patients for a safety evaluation and up to 12 additional patients for preliminary evaluation of efficacy as well as further evaluation of safety
untitled h1
2.4 baseline evaluation

All subjects are required to complete baseline evaluations within one week prior to the first planned dosing of the study drug (any screening evaluation done within this time period can also serve for the baseline evaluation):
- Physical exam including weight, ECOG performance status and vital signs.  
- Concomitant Medications and Baseline Signs and Symptoms evaluation.
- Urine or serum pregnancy test (β-HCG) for females of childbearing-potential and women < 12 months since the onset of menopause.  
- Chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN,
creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and direct bilirubin.
- ACTH, TSH, reflex free T4, lipase, amylase, CRP
- Hematology: CBC with differential
- Coagulation panel: PT, INR, and PTT
untitled h1
3 study implementation 3.1 study

- This is a phase I/II trial of combination immunotherapy.  
- The trial will be conducted using a Simon optimal two-stage design.  
- Participants will receive HPV vaccine + NHS-IL12 + M7824 **(Figure 19).**
o The first six participants will be evaluable for dose limiting toxicities (DLTs) and accrual will continue to 8 participants if less than 2 out of the first 6 participants experience a DLT.  
If three or more out of eight patients have objective responses then accrual will be expanded to enroll 20 evaluable participants **(Figure 20).** Additional patients will be enrolled in the cohort with prior pelvic radiation and boost brachytherapy: up to 12 for safety and dose finding and up to 12 for an exploratory evaluation of efficacy.  
- There will be a minimum of six days between the first and second participant. The third through sixth participant may enroll no sooner than 48 hours after the most recent enrollee. Once six participants have enrolled and been found to be safe further participants may enroll at any time.  
- Although positive HPV testing will not be required prior to enrolling, HPV testing will be offered as an exploratory endpoint and participants testing negative for HPV after enrolling or whose HPV status cannot be confirmed may be replaced with other participants for the primary efficacy analysis. Participants testing negative for HPV after enrolling or whose HPV status cannot be confirmed may continue to receive treatment on study.  
- The combination of PDS0101+ NHS-IL12+ M7824 will be evaluated in all HPV serotypes given the clinical remissions PDS Biotechnology has observed in CIN studies with PDS0101 in non-HPV 16 serotypes. However, if it is found that the ORR with non-HPV- 16 serotypes is noticeably less than the ORR observed with HPV 16 serotypes than participants having disease related to non-HPV-16 serotypes may be replaced with participants having disease related to the HPV-16 serotype for the first and second stage of the primary efficacy analyses.  
- In addition, while the primary focus of this protocol is to evaluate the response rate of this I/O combination in participants with a wide array of HPV associated malignancies, if the I/O combination in not deemed to be promising in this larger population (less than three out of eight evaluable participants have a response) than the study will be amended to evaluate this I/O combination specifically in participants with cervical cancer (and participants who have already been enrolled with non-cervical cancers may be replaced with cervical cancer participants for the first stage of the primary efficacy analyses).  
- While checkpoint refractory participants will be eligible to enroll, the primary objective of response rate will be determined among the checkpoint naïve participants and the response rate among checkpoint refractory participants will be an exploratory objective.
untitled h1
3.1.1 dose limiting toxicity dlt

Dose-limiting toxicity (DLT) will be defined as any one of the following adverse events, possibly attributable to study drugs, that occur within 28 days of the start of therapy:
- Any Grade 4 or higher adverse drug reactions (ADRs) as defined by CTCAE v5.0 and assessed as possibly related to that agent by the Investigator, except for laboratory values that are asymptomatic or resolve to Grade ≤ 1 or baseline grade within 7 days without medical intervention.  
- Any Grade 3 ADRs possibly attributed to an agent except for any of the following:
o Grade 3 flu-like symptoms or fever, as well as associated symptoms of fatigue, headaches, nausea, emesis which can be controlled with conservative medical management.  
o Tumor flare phenomenon defined as local pain, irritation, or rash localized at sites of known or suspected tumor.  
o Grade 3 Hgb decrease (< 8.0 g/dL) that is clinically manageable with blood transfusions or erythroid growth factor use does not require treatment discontinuation.  
o Grade 3 laboratory values that are asymptomatic or resolve to Grade ≤ 1 or baseline grade within 14 days.  
o Keratoacanthoma and squamous cell carcinoma of the skin.  
o Any endocrinopathy that can be medically managed with hormone replacement o Any grade 3 adverse drug reaction which can be medically managed with minimal risk to the participant (e.g., placement of a pleural catheter for recurrent inflammatory pleural effusions) and resolves to at least grade one or baseline grade within 14 days.  
Criteria are based on the NCI Common Terminology Criteria for Adverse Events, Version 5. Where clinically appropriate subjects will receive one year of treatment during which subjects will be followed with surveillance scans every 6-12 weeks. On a case by case basis treatment beyond a year is allowed per investigator discretion. Subjects with evidence of disease progression after completing a year of treatment will be allowed retreatment. Patents might be taken off treatment for disease progression prior to completing one year of treatment, but where clinically appropriate, treatment beyond radiographic progression is allowed if in the opinion of the investigator the subject is benefiting from treatment. Subjects will be taken off treatment if unacceptable toxicity occurs and is attributed to all therapeutic agents. If a single agent is tolerated that agent may be continued. This trial will be conducted using a Simon two-stage phase II trial design in which 8 evaluable participants will enroll and if 3 or more of the 8 have an objective response, then accrual would continue until a total of 20 evaluable participants have been treated. If there are 9 or more of 20 (45.0%) who experience a response, this would be sufficiently interesting to warrant further study. In the event that a participant develops a response (>30% decrease in target lesions as compared with baseline scan) following initial progression or "pseudo-progression", this response will be considered as an objective response in the determination to expand accrual.
untitled h1
3.2 drug administration general rule

| Dosage Form:                   | Sterile injectable liquid                                                                                          |
|--------------------------------|--------------------------------------------------------------------------------------------------------------------|
| Number of vials:               | 2-vial formulation                                                                                                 |
| Preparation:                   | 1:1 v/v mixture of the 2 vial formulation. 0.6mL of vial #1,  ImmunoMAPK-RDOTAP added to vial# 2, HPVmix (0.6  mL) |
| Total Administered             | 2.7 mg of total peptide and 3 mg of R-DOTAP                                                                        |
| dose:  Total Injection Volume: | 1.0mL (administered as two individual 0.5mL injections)                                                            |
| Injection route:               | Subcutaneous                                                                                                       |  
A window of +/- 14 days for a scheduled treatment is allowed in the event of scheduling issues
(i.e., holiday, bad weather or other scheduling issues). The minimum time between administrations of a similar agent is 11 days. Where feasible PDS0101 should be administered first, followed by NHS-IL12 (when given), followed by M7824. PDS0101 will be administered every 4 weeks for 6 doses followed by every three months (i.e. every 12 weeks) for an additional two doses.  
- 0.6 ml will be drawn up from each vial (Vial \#1 Immuno MAPK-RDOTAP, Vial \#2 HPV
mix) and mixed (total = 1.2ml for the PDS0101 admixture). Two separate 0.5 ml doses will be injected into the participant. These should preferably be given ~6cm apart in the same limb. Each subsequent vaccination should preferably be administered alternating between limbs (e.g., L arm  R thigh).  
Subjects will receive M7824 via IV infusion over 1 hour (-10 minutes / +20 minutes, that is, over 50 to 80 minutes) once every 2 weeks. M7824 will be administered as a "flat" dose of 1,200 mg independent of body weight. However lower doses may also be used as per Section **3.4.3**. M7824 is administered as an intravenous infusion with a mandatory 0.2 micron in-line filter. In order to mitigate potential infusion-related reactions, premedication with an antihistamine and with acetaminophen (for example, 25-50 mg diphenhydramine and 500-650 mg acetaminophen) within approximately 30 to 60 minutes prior to dosing of M7824 is optional and at the discretion of the Investigator. NHS-IL12 will be administered at as dose of 16.8mcg/kg by SC injection every 4 weeks for up to 4 consecutive doses then 8mcg/kg every 4 weeks thereafter. However lower doses may also be used as per Section **3.4.3**. The dose of NHS-IL12 will be calculated based on the weight of the subject determined within 2 weeks prior to the day of drug administration. The dose of NHS-IL12 used for the previous administration can be repeated if the change in the subject's weight is 10% or less than the weight used for the last dose calculation.
untitled h1
3.3 study intervention compliance

All study interventions will be given at NIH and documented in the electronic medical record.
untitled h1
3.4 dose modifications 3.4.1 discontinuation

Treatment with individual agents will be discontinued in the case of:
- Any Grade 4 or higher adverse drug reactions (ADRs) as defined by CTCAE v5.0 and assessed as possibly related to that agent by the Investigator, except for laboratory values that are asymptomatic or resolve to Grade ≤ 1 or baseline grade within 7 days
- Any Grade 3 ADRs possibly attributed to an agent except for any of the following:
o Grade 3 flu-like symptoms or fever, as well as associated symptoms of fatigue, headaches, nausea, emesis which can be controlled with conservative medical management.  
o Tumor flare phenomenon defined as local pain, irritation, or rash localized at sites of known or suspected tumor.  
o Grade 3 Hgb decrease (< 8.0 g/dL) that is clinically manageable with blood transfusions or erythroid growth factor use does not require treatment discontinuation.  
o Grade 3 laboratory values that are asymptomatic or resolve to Grade ≤ 1 or baseline grade within 14 days.  
o Keratoacanthoma and squamous cell carcinoma of the skin. o Any endocrinopathy that can be medically managed with hormone replacement o Any grade 3 adverse drug reaction which can be medically managed with minimal risk to the participant (e.g., placement of a pleural catheter for recurrent inflammatory pleural effusions) and resolves to at least grade one or baseline grade within 14 days.
untitled h1
3.4.2 dose delay

Individual agents should be withheld for any Grade 2 or 3 ADR possibly attributed to that agent until resolution to Grade ≤ 1 unless the ADR in the opinion of the investigator is not clinically relevant or can be medically managed with minimal risk to the participant. NHS-IL12 may be restarted at any scheduled visit as long as there is at least 11 days between doses of this agent. Should a clinically relevant grade 2 or 3 ADR persist for more than 4 weeks, consideration should be given to discontinuing treatment with that individual agent at the discretion of the investigator. For non-medical logistical reasons, unrelated acute illnesses, or palliative radiation, scheduled assessments and dosing can be delayed up to 2 months. Where at all possible, dosing should be restarted to keep in line with the original treatment schedule.
untitled h1
3.4.3 dose modification

Participants with dose modifications or skipped doses within DLT period for reasons other than a DLT (e.g., logistical reasons) who do not experience a DLT within the DLT window will not be evaluable for DLTs and will be replaced for the six-participant lead in DLT assessment. Participants who have dose modifications or skipped doses within the DLT period for logistical reasons and nevertheless experience a DLT within the DLT period will be considered DLT evaluable. PDS0101 HPV vaccine: Vaccine doses will not be modified but doses may be skipped per investigator discretion. For example, PDS0101 (which targets HPV 16 specifically) may be held for non-HPV 16 tumors per investigator discretion. M7824: Doses (including starting doses) may be reduced to a 300 or 600 mg flat dose or doses may be skipped. NHS-IL12: All patients, except patients with cervical cancer and prior pelvic radiation and brachytherapy (described in the next paragraph), will receive NHS-IL12 at a starting dose of 16.8 mcg/kg for up to a total of 4 consecutive doses at which time the dose will be reduced to 8 mcg/kg thereafter. Doses may be reduced to 8 mcg/kg at any time after the first dose for grade ≥ 2 flu-like symptoms, fever, or other NHS-IL12 related toxicities. Additionally, doses may be skipped or further dose-reduced to 4 or 6 mcg/kg. For patients who have already received initial starting doses of NHS-IL12 at 8 mcg/kg they may receive NHS-IL12 at 16.8 mcg/kg for up to a total of 4 consecutive doses. In select participants receiving doses of 8 mcg/kg or lower, consideration may be given to every 2 week dosing of this agent. In patients with cervical cancer with prior pelvic radiation and boost brachytherapy where a potential higher risk of grade 3 hematuria may exist with the full starting doses of M7824 at 1200 mg and NHS-IL12 at 16.8 mcg/kg, a staring dose of M7824 at 600 mg and NHS-IL12 at 4 mcg/kg will be used instead. If grade 3 or greater hematuria occurs in ≥ 2 out of four of these patients then the starting dose for these patients will be lowered to M7824 at 300 mg and NHS-IL12 at 4 mcg/kg. If grade 3 or greater hematuria occurs in ≥ 2 out of four of these patients when given M7824 at 300 mg and NHS-IL12 at 4 mcg/kg then these patients will no longer to be eligible to enroll on this protocol. On the other hand, if < 2 patients out of four have grade 3 or greater bleeding events when given M7824 at 600 mg and NHS-IL12 at 4 mcg/kg but objective responses are also observed in < 2 out of these four patients then a dose of M7824 at 1200 mg in combination with NHS-IL12 at 4 mcg/kg will be evaluated in these patients. If ≥ 2 patients out of four have grade 3 or greater bleeding events or < 2 out of four patients have objective responses when given M7824 at 1200 mg and NHS-IL12 at 4 mcg/kg then the combination of M7824 at 600 mg and NHS-IL12 at 6 mcg/kg may be evaluated. Stopping Rule: If more than one third of enrolled patients have permanently discontinued one or more study agents due to grade 3 or 4 toxicity per guidelines in Section **3.4.1** or any death has occurred which is at least possibly related to study regimen this will prompt a suspension of accrual and reassessment of available safety information prior to reopening study to accrual. Version Date: *05.12.2023* 3.4.4 Guidelines for Management of Infusion Reactions  
| NCI-CTCAE Grade                                                                                                                                                                                          | Treatment Modification for Infused Agent  (M7824)                                                                                                                                                                     |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1 - mild                                                                                                                                                                                           | Consider decreasing the infusion rate of the  particular agent by 50% and monitoring  closely for any worsening.                                                                                                      |
| Mild transient reaction; infusion interruption  not indicated; intervention not indicated.  Grade 2 - moderate                                                                                           | Consider temporarily discontinuing infusion  of the particular agent.                                                                                                                                                 |
| Therapy or infusion interruption indicated but  responds promptly to symptomatic treatment  (e.g., antihistamines, NSAIDs, narcotics, i.v.  fluids); prophylactic medications indicated for  ≤ 24 hours. | Consider resuming infusion of the particular  agent at 50% of previous rate once infusion  related reaction has resolved or decreased to  at least Grade 1 in severity, and monitor  closely for any worsening.       |
| Grade 3 or Grade 4 - severe or lifethreatening                                                                                                                                                                                                          | Stop the infusion immediately and disconnect  infusion tubing from the subject.                                                                                                                                       |
| Grade 3: Prolonged (e.g., not rapidly  responsive to symptomatic medication and/or  brief interruption of infusion); hospitalization  indicated for clinical sequelae.                                   | For grade 3 events: Consider withdrawing  immediately from treatment with that  particular agent and not offering any further  treatment with that agent based upon if the  clinical condition can be safely managed. |
| Grade 4: Life-threatening consequences;  urgent intervention indicated.                                                                                                                                  | For grade 4 events: Withdraw immediately  from treatment and do not offer further  treatment with that agent.                                                                                                         |
| If the infusion rate of M7824 has been decreased by 50% or interrupted due to an infusion reaction,  keep it decreased for the next scheduled infusion. If no infusion reaction is observed in the next  |                                                                                                                                                                                                                       |  
If the infusion rate of M7824 has been decreased by 50% or interrupted due to an infusion reaction, keep it decreased for the next scheduled infusion. If no infusion reaction is observed in the next scheduled infusion, the infusion rate may be returned to baseline at the subsequent infusions based on investigator's medical judgment.  
If hypersensitivity reaction occurs, the subject should be treated according to the best available medical practice. 3.4.5 Guidelines for Management of Immune-Mediated Adverse Reactions (In addition to the below guidelines, investigators may also use NCCN, ASCO, SITC or FDA guidelines for Immune Related Adverse Events)  
| Version Date: 05.12.2023                                                                                                                                                                                                                                      | Gastrointestinal irAEs                                                                                                                                                                              |                                                                                                                                                                                 |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Severity of Diarrhea/Colitis (NCI-CTCAE v5)                                                                                                                                                                                                                   | Initial Management                                                                                                                                                                                  | Follow-up Management                                                                                                                                                            |
| Grade 1 Diarrhea: < 4 stools/day over Baseline Colitis: asymptomatic                                                                                                                                                                                          | Consider continuing M7824  Symptomatic treatment (e.g.  loperamide)                                                                                                                                 | Consider close monitoring for  worsening symptoms Consider educating subject to  report worsening immediately If worsens: Treat as Grade 2, 3 or 4.                             |
| Grade 2  Diarrhea: 4 to 6 stools per day over  Baseline; IV fluids indicated < 24  hours; not interfering with ADL Colitis: abdominal pain; blood in stool                                                                                                    | Consider withholding M7824                                                                                                                                                                          | If improves to Grade ≤ 1:                                                                                                                                                       |
| Symptomatic treatment                                                                                                                                                                                                                                         | Consider resuming therapy If persists > 7 days or recurs: Consider treating as Grade 3 or 4.                                                                                                        |                                                                                                                                                                                 |
| Grade 3 to 4 Diarrhea (Grade 3): ≥ 7 stools per day  over Baseline; incontinence; IV fluids  ≥ 24 h; interfering with ADL Colitis (Grade 3): severe abdominal  pain, medical intervention indicated,  peritoneal signs Grade 4: life-threatening, perforation | Consider withholding or permanently  discontinuing M7824 for Grade 3  events based upon if the clinical  condition can be safely managed. For  grade 4 events permanently  discontinue treatment.   | If improves:  Consider continuing steroids  until Grade ≤ 1, then tapering  over at least 1 month; consider  resuming therapy following  steroids taper (for initial Grade  3). |
| Consider 1.0 to 2.0 mg/kg/day  prednisone IV or equivalent                                                                                                                                                                                                    | If worsens, persists > 7 days, or  recurs after improvement: Consider adding infliximab  5mg/kg (if no contraindication).  Note: infliximab should not be  used in cases of perforation or  sepsis. |                                                                                                                                                                                 |
| Consider adding prophylactic  antibiotics for opportunistic  infections  Consider lower endoscopy                                                                                                                                                             |                                                                                                                                                                                                     |                                                                                                                                                                                 |  
Version Date: *05.12.2023*  
| Version Date: 05.12.2023                                                                          | Dermatological irAEs                                                                                                                                                                                                                                                                                          |                                                                                                                                                           |
|---------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade of Rash                                                                                     |                                                                                                                                                                                                                                                                                                               |                                                                                                                                                           |
| (NCI-CTCAE v5)                                                                                    | Initial Management                                                                                                                                                                                                                                                                                            | Follow-up Management                                                                                                                                      |
| Grade 1 to 2                                                                                      | Consider continuing M7824                                                                                                                                                                                                                                                                                     |                                                                                                                                                           |
| Covering ≤ 30% body surface area                                                                  | If persists > 1 to 2 weeks or  recurs:  Consider withholding M7824  therapy  Consider skin biopsy                                                                                                                                                                                                             |                                                                                                                                                           |
| Consider symptomatic therapy (for  example, antihistamines, topical  steroids)                    | Consider 0.5-1.0 mg/kg/day  prednisone or equivalent. Once  improving, consider tapering  steroids over at least 1 month,  consider prophylactic antibiotics  for opportunistic infections, and  consider resuming M7824  therapy following steroids taper. If worsens:  Consider treating as Grade 3 to 4.   |                                                                                                                                                           |
| Grade 3 to 4 Grade 3: Covering > 30% body  surface area;  Grade 4: Life threatening  consequences | Consider withholding or permanently  discontinuing M7824 for Grade 3  events based upon if the clinical  condition can be safely managed. For  grade 4 events permanently  discontinue treatment. Consider skin biopsy  Consider dermatology consult  Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent | If improves to Grade ≤ 1:  Consider tapering steroids over at  least 1 month; consider resuming  therapy following steroids taper  (for initial Grade 3). |
| Consider adding prophylactic  antibiotics for opportunistic  infections  Pulmonary irAEs          |                                                                                                                                                                                                                                                                                                               |                                                                                                                                                           |
| Grade of Pneumonitis (NCI-CTCAE v5)                                                               | Initial Management                                                                                                                                                                                                                                                                                            | Follow-up Management                                                                                                                                      |
| Grade 1                                                                                           | Consider withholding M7824                                                                                                                                                                                                                                                                                    |                                                                                                                                                           |
| Radiographic changes only                                                                         | Consider Pulmonary and Infectious  Disease consults                                                                                                                                                                                                                                                           | Consider re-assessing at least  every 3 weeks If worsens:  Consider treating as Grade 2 or  Grade 3 to 4.                                                 |
| Grade 2                                                                                           | Consider withholding M7824                                                                                                                                                                                                                                                                                    | If improves:                                                                                                                                              |
| Mild to moderate new symptoms                                                                     |                                                                                                                                                                                                                                                                                                               |                                                                                                                                                           | Version Date: *05.12.2023*  
| Version Date: 05.12.2023                                                                     | Consider pulmonary and Infectious  Disease consults Consider monitoring symptoms  daily; consider hospitalization  Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent Consider adding prophylactic  antibiotics for opportunistic  infections  Consider bronchoscopy, lung biopsy                                                                                                                                                                | When symptoms return to Grade  ≤ 1, consider tapering steroids  over at least 1 month, and then  consider resuming therapy  following steroids taper  If not improving after 2 weeks or  worsening:  Consider treating as Grade 3 to 4.                                  |
|----------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 3 to 4 Grade 3: Severe new symptoms;  New/worsening hypoxia; Grade 4: Life-threatening | Consider withholding or permanently  discontinuing M7824 for Grade 3  events based upon if the clinical  condition can be safely managed. For  grade 4 events permanently  discontinue treatment. Consider hospitalization. Consider pulmonary and Infectious  Disease consults. Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent  Consider adding prophylactic  antibiotics for opportunistic  infections  Consider bronchoscopy, lung biopsy | If improves to Grade ≤ 1:  Consider tapering steroids over at  least 1 month  If not improving after 48 hours or  worsening:  Consider adding additional  immunosuppression (for  example, infliximab,  cyclophosphamide, IV  immunoglobulin, or  mycophenolate mofetil) |
| Hepatic irAEs                                                                                |                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                          |
| Grade of Liver Test Elevation (NCI-CTCAE v5)                                                 | Initial Management                                                                                                                                                                                                                                                                                                                                                                                                                                    | Follow-up Management                                                                                                                                                                                                                                                     |
| Grade 1 Grade 1 AST or ALT > ULN to 3.0 x  ULN and/or Total bilirubin > ULN to  1.5 x ULN    | Consider continuing M7824                                                                                                                                                                                                                                                                                                                                                                                                                             | Consider continued liver function  monitoring  If worsens:  Consider treating as Grade 2 or 3  to 4.                                                                                                                                                                     |
| Grade 2 AST or ALT > 3.0 to ≤ 5 x ULN  and/or total bilirubin > 1.5 to ≤ 3 x  ULN            | Consider withholding M7824                                                                                                                                                                                                                                                                                                                                                                                                                            | If returns to Grade ≤ 1: Consider resuming therapy. If elevation persists > 7 days or  worsens:  Consider treating as Grade 3 to 4.                                                                                                                                      |
| Grade 3 to 4 AST or ALT > 5 x ULN and/or total  bilirubin > 3 x ULN                          | Consider withholding or permanently  discontinuing M7824 for Grade 3  events based upon if the clinical  condition can be safely managed. For  grade 4 events permanently  discontinue treatment.                                                                                                                                                                                                                                                     | If returns to Grade ≤ 1:  Consider tapering steroids over at  least 1 month  If does not improve in > 7 days,  worsens or rebounds:                                                                                                                                      | Version Date: *05.12.2023*  
| Version Date: 05.12.2023                                                                | Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent Consider adding prophylactic  antibiotics for opportunistic  infections  Consider consulting  gastroenterologist/ hepatologist Consider obtaining MRI/CT scan of  liver and liver biopsy if clinically  warranted                                                                                                                                                                                                                                                                                             | Consider adding mycophenolate  mofetil 1 gram (g) twice daily  If no response within an  additional 7 days, consider other  immunosuppressants per local  guidelines.   |
|-----------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Renal irAEs                                                                             |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                         |
| Grade of Creatinine Increased  (NCI-CTCAE v5)                                           | Initial Management                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Follow-up Management                                                                                                                                                    |
| Continue renal function monitoring If worsens: Consider treating as Grade 2 to 3 or  4. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                         |
| Grade 1 Creatinine increased > ULN to 1.5 x                                             | Consider continuing M7824                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                         |
| ULN                                                                                     | If returns to Grade ≤1: Consider tapering steroids over at  least 1 month, and consider  resuming therapy following  steroids taper. If worsens:  Treat as Grade 4.                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                         |
| Grade 2  Creatinine increased > 1.5 and ≤ 6 x  ULN                                      | Consider withholding M7824  Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent. Consider adding prophylactic  antibiotics for opportunistic infections Consider renal biopsy Consider withholding or permanently  discontinuing M7824 for Grade 3  events based upon if the clinical  condition can be safely managed. For  grade 4 events permanently  discontinue treatment. Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent. Consider adding prophylactic  antibiotics for opportunistic infections Consider renal biopsy Consider Nephrology consult | If returns to Grade ≤1: Consider tapering steroids over at  least 1 month.                                                                                              |
| Grade 3-4  Creatinine increased > 6 x ULN                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                         |  
| Cardiac irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                 |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Myocarditis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Initial Management                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Follow-up Management                                                                                                                                                                                                                                                                                            |
| New onset of cardiac signs or  symptoms and / or new laboratory  cardiac biomarker elevations (e.g.  troponin, CK-MB, BNP) or cardiac  imaging abnormalities suggestive of  myocarditis.                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Consider withholding or permanently  discontinuing M7824 based upon if  the clinical condition can be safely  managed. Consider hospitalization In the presence of life threatening  cardiac decompensation, consider  transfer to a facility experienced in  advanced heart failure and  arrhythmia management. Consider cardiology consult to  establish etiology and rule-out  immune-mediated myocarditis.  Consider myocardial biopsy if  recommended per cardiology consult. | If symptoms improve and  immune-mediated etiology is  ruled out, consider re-starting  therapy. If symptoms do not  improve/worsen, viral  myocarditis is excluded, and  immune-mediated etiology is  suspected or confirmed following  cardiology consult, consider  managing as immune-mediated  myocarditis. |
| Immune-mediated myocarditis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Consider withholding or permanently  discontinuing M7824 based upon if  the clinical condition can be safely  managed. Consider guideline based supportive  treatment as appropriate as per  cardiology consult. Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent Consider adding prophylactic  antibiotics for opportunistic  infections.                                                                                                                                  | Once improving, consider  tapering steroids over at least 1  month. If no improvement or worsening,  consider additional  immunosuppressants (e.g.  azathioprine, cyclosporine A).                                                                                                                              |
| *Local guidelines, or eg. ESC or AHA guidelines  ESC guidelines website: https://www.escardio.org/Guidelines/Clinical-Practice-Guidelines AHA guidelines website:  http://professional.heart.org/professional/GuidelinesStatements/searchresults.jsp?q=&y=&t=1001 Endocrine irAEs Endocrine Disorder Initial Management Follow-up Management Grade 1 or Grade 2  endocrinopathies (hypothyroidism,  hyperthyroidism, adrenal  insufficiency, type I diabetes  mellitus) Consider continuing M7824  Consider endocrinology consult if  needed Consider continuing hormone  replacement/suppression and  monitoring of endocrine function  as appropriate. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                 | Version Date: *05.12.2023*  
| Version Date: 05.12.2023                                                                                                                                                                                                                                                                                                      | Consider starting thyroid hormone  replacement therapy (for  hypothyroidism), anti-thyroid  treatment (for hyperthyroidism),  corticosteroids (for adrenal  insufficiency) or insulin (for Type I  diabetes mellitus) as appropriate.  Consider ruling-out secondary  endocrinopathies (i.e.  hypopituitarism / hypophysitis)                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                               |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 3 or Grade 4  endocrinopathies (hypothyroidism,  hyperthyroidism, adrenal  insufficiency, type I diabetes  mellitus)                                                                                                                                                                                                    | Consider withholding or  permanently discontinuing M7824 for Grade 3-4 events based upon if  the clinical condition can be safely  managed.  Consider hospitalization Consider endocrinology consult                                                                                                                                                                                                                                                                                    | Consider resuming therapy once  symptoms and/or laboratory tests  improve to Grade ≤ 1 (with or  without hormone  replacement/suppression). Consider continuing hormone  replacement/suppression and  monitoring of endocrine function  as appropriate.                                                                                                       |
| Consider starting thyroid hormone  replacement therapy (for  hypothyroidism), anti-thyroid  treatment (for hyperthyroidism),  corticosteroids (for adrenal  insufficiency) or insulin (for type I  diabetes mellitus) as appropriate.  Consider ruling-out secondary  endocrinopathies (i.e.  hypopituitarism / hypophysitis) |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                                                               |
| Hypopituitarism/Hypophysitis                                                                                                                                                                                                                                                                                                  | If secondary thyroid and/or adrenal                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                               |
| (secondary endocrinopathies)                                                                                                                                                                                                                                                                                                  | insufficiency is confirmed (i.e. subnormal serum FT4 with  inappropriately low TSH and/or low  serum cortisol with inappropriately  low ACTH): - Consider referring to  endocrinologist for dynamic  testing as indicated and  measurement of other hormones  (FSH, LH, GH/IGF-1, PRL,  testosterone in men, estrogens  in women)  - Consider hormone  replacement/suppressive therapy  as appropriate  - Consider performing pituitary  MRI and visual field  examination as indicated | Consider resuming therapy once  symptoms and hormone tests  improve to Grade ≤ 1 (with or  without hormone replacement). In addition, for hypophysitis  with abnormal MRI, consider  resuming M7824 only once  shrinkage of the pituitary gland  on MRI/CT scan is documented.  Consider continuing hormone  replacement/suppression therapy  as appropriate. |
| If hypophysitis confirmed:                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                                                               | Version Date: *05.12.2023*  
| Version Date: 05.12.2023                                                       | -                                                                                                                                                                                                                                                                        | Continue M7824 if mild  symptoms                                                                                            |
|--------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------|
| -                                                                              | Consider withholding M7824 and if moderate, severe or lifethreatening symptoms of  hypophysitis. Consider  hospitalization. Consider  initiating corticosteroids (1 to 2  mg/kg/day prednisone or  equivalent) followed by  corticosteroids taper during at  least 1 month.                                                                                                                                                                                                                                                                          |                                                                                                                             |
| -                                                                              | Consider adding prophylactic  antibiotics for opportunistic  infections.                                                                                                                                                                                                 |                                                                                                                             |
| Other irAEs (not described above)                                              |                                                                                                                                                                                                                                                                          |                                                                                                                             |
| Grade of other irAEs                                                           | Initial Management                                                                                                                                                                                                                                                       | Follow-up Management                                                                                                        |
| (NCI-CTCAE v5)                                                                 |                                                                                                                                                                                                                                                                          |                                                                                                                             |
| Grade 2 or Grade 3 clinical signs or  symptoms suggestive of a potential  irAE | Consider withholding M7824                                                                                                                                                                                                                                               | If irAE is ruled out, consider                                                                                              |
| pending clinical investigation                                                 | managing as appropriate  according to the diagnosis and  consider re-starting therapy If irAE is confirmed, consider  treating as Grade 2 or 3 irAE.                                                                                                                     |                                                                                                                             |
| Consider withholding M7824 based                                               |                                                                                                                                                                                                                                                                          |                                                                                                                             |
| Grade 2 irAE or first occurrence of  Grade 3 irAE                              | upon if the clinical condition can be  safely managed. Consider 1.0 to 2.0 mg/kg/day  prednisone or equivalent  Consider adding prophylactic  antibiotics for opportunistic  infections  Specialty consult as appropriate                                                | If improves to Grade ≤ 1:  Consider tapering steroids over  at least 1 month and resuming therapy following steroids taper. |
| Recurrence of same Grade 3 irAEs                                               | Consider permanently discontinuing  M7824 based upon if the clinical  condition can be safely managed;  Consider 1.0 to 2.0 mg/kg/day prednisone or equivalent Consider adding prophylactic  antibiotics for opportunistic  infections  Specialty consult as appropriate | If improves to Grade ≤ 1:  Consider tapering steroids over  at least 1 month.                                               |  
| Grade 4                                                                                                                                                            | Permanently discontinue M7824  Consider to 2.0 mg/kg/day prednisone or equivalent and/or  other immunosuppressant as needed Consider adding prophylactic  antibiotics for opportunistic  infections  Consider specialty consult as  appropriate   | If improves to Grade ≤ 1:  Consider tapering steroids over  at least 1 month   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------|
| Requirement for 10 mg per day or  greater prednisone or equivalent  for more than 12 weeks for reasons  other than hormonal replacement  for adrenal insufficiency | Consider permanently discontinuing M7824  Consider specialty consult as  appropriate                                                                                                                                                              |                                                                                |
| Persistent Grade 2 or 3 irAE  lasting 12 weeks or longer                                                                                                           |                                                                                                                                                                                                                                                   |                                                                                |  
Abbreviations: ACTH=adrenocorticotropic hormone; ADL=activities of daily living; ALT=alanine aminotransferase; AST=aspartate aminotransferase; BNP=B-type natriuretic peptide; CK-MB=creatine kinase MB; CT= computed tomography; FSH=follicle-stimulating hormone; GH=growth hormone; IGF-1=insulin-like growth factor 1; irAE=immune related adverse event; IV=intravenous; LH=luteinizing hormone; MRI=magnetic resonance imaging; NCI CTCAE=National Cancer Institute Common Terminology Criteria for Adverse Events; PRL=prolactin;T4=thyroxine; TSH=thyroid stimulating hormone; ULN=upper limit of normal  
| 3.5                                              | STUDY CALENDAR   | Safety     |               |      |        |
|--------------------------------------------------|------------------|------------|---------------|------|--------|
| Procedure                                        | Screening1       | Baseline / | Week  (2N+1)3 | EOT4 | follow |
| Week 12                                          | up5              | Long  Term |               |      |        |
| follow up6                                       |                  |            |               |      |        |
| Treatment3                                       | X                | X          |               |      |        |
| NIH Advance Directives Form7                     | X                |            |               |      |        |
| Medical History                                  | X                |            |               |      |        |
| Height                                           | X                |            |               |      |        |
| Physical exam, weight, vital  signs, ECOG        | X                | X          | X             | X    | X      |
| HIV, HCV, HepB                                   | X                |            |               |      |        |
| EKG                                              | X                | X          | X             |      |        |
| CBC with differential                            | X                | X          | X             | X    | X      |
| Biochemical profile8                             | X                | X          | X             | X    | X      |
| Tumor Markers9                                   | X                | X          | X             |      |        |
| CD4 10                                           | X                |            |               |      |        |
| HLA typing                                       | X                |            |               |      |        |
| ACTH, TSH, reflex Free T4,  lipase, amylase, CRP | X                | X11        |               |      |        |
| Urinalysis                                       | X                |            |               |      |        |
| Urine or serum pregnancy  testing in women of    | X                | X          | X             |      |        |
| childbearing potential  PT, INR, aPTT            | X                |            |               |      |        |
| Tumor evaluation (CT Scan)12                     | X                | X          | X             |      |        |
| Brain CT 13                                      | X                |            |               |      |        |
| Concomitant Medications                          | X                | X          | X             | X    |        |
| Adverse events                                   | X                | X          | X             | X    |        |
| Optional biopsy for immune  analysis 14          | X                | X          |               |      |        |
| Research Blood 15                                | X                | X          | X             |      |        |
| Telephone Follow Up                              | X                | X          |               |      |        |  
1. Screening evaluations performed within 28 days prior to the first drug administration, unless specified in Section 2.2.  
2. Baseline evaluations performed within 1 week prior to first drug administration.  
3. Schedule of therapeutic agents administered will be as outlined in Section 3.1, **Figure 19.**. NHS-IL12 will be given every 4 weeks, M7824 infusion will be every two weeks and HPV vaccine will be given every 4 weeks for the first 6 doses followed by every 12 weeks thereafter. (The first administration of HPV vaccine should preferably be given 12 weeks before the first administration of NHS-IL12 and M7824.) Administration of study agents and indicated evaluations can be done up to 14 days earlier or delayed up to 14 days due to holidays, inclement weather, conflicts, or similar reasons. The timing of subsequent administrations is adjusted to maintain a minimum of 11 days between treatments of similar agents. 4. EOT - End of treatment visit: Where feasible, on the day of or within 7 days of the decision to discontinue treatment prematurely before completion of one year of treatment. Does not need to be completed if drug is withheld after one year of treatment.  
5. 28 days (+/- 7 days) after last treatment. If subjects are not willing to come to NIH to FU visit, they will be contacted by phone to assess adverse events. 6. Participants who have come off treatment for disease progression will be followed by phone annually for survival. Participants who have not progressed on treatment will continue to be followed and scanned per investigator discretion until progression. Those that completed one year of treatment will be invited for an additional year of treatment at the time of progression. Initial and follow up courses of treatment may extend beyond a year per investigator discretion.  
7. As indicated in Section **12.3**, all subjects will be offered the opportunity to complete an NIH advanced directives form. This should be done preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly recommended, but is not required. 8. Biochemical profile: mineral panel, sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN, creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and direct bilirubin. Note: the mineral panel will start from baseline onward.  
9. Evaluate CEA, CA19-9, CA125 and CA15-3 at baseline and follow elevated values thereafter. 10. In HIV positive participants.
untitled h1
11. every 8 weeks.

12. Every 8 weeks. In the event of a PR or CR tumor imaging assessments may be performed every 3 months (+/- 2 weeks) at the discretion of the investigator. Tumor assessment should be continued beyond end of treatment in participants who have not experienced PD until they experience PD in order to assess PFS. MRI will be used when CT scan is not an option to follow the disease clinically. Gadolinium will be used only for clinically indicated MRIs. Bone scans and other imaging assessments may also be performed as clinically indicated. 13. In participants with known CNS disease as described in Section **2.1.2.4**. MRIs may be performed as clinically indicated in this population. 14. Optional biopsies at baseline and as close as possible to first imaging restaging.  
15. Where feasible, research blood for study assessments (see Section 5) will be collected at baseline, 3 days and 1 week after the first M7824 + NHS-IL12 dose) as well as at weeks 3 and 7 and at restaging visits in selected participants, per PI discretion.
untitled h1
3.6 cost and compensation 3.6.1

NIH does not bill health insurance companies or participants for any research or related clinical care that participants receive at the NIH Clinical Center. If some tests and procedures are performed outside the NIH Clinical Center, participants may have to pay for these costs if they are not covered by an insurance company. Medicines that are not part of the study treatment will not be provided or paid for by the NIH Clinical Center.
untitled h1
3.6.3 reimbursement

The NCI will cover the costs of some expenses associated with protocol participation. Some of these costs may be paid directly by the NIH and some may be reimbursed to the participant/guardian as appropriate. The amount and form of these payments are determined by the NCI Travel and Lodging Reimbursement Policy.
untitled h1
3.7 criteria for removal from

Regardless of reason for removal from study therapy, participants will be asked to have a 28 day follow up safety visit. Participants who refuse to return for this visit will be asked to review any safety concerns by phone within this time period. 3.7.1 Criteria for Removal from Protocol Therapy  
- Clinical or radiographic progression of disease except when the investigator feels the subject is still benefiting from treatment. (It is generally preferable for participants to remain on treatment past initial radiographic progression in case there is pseudo - progression, except when the investigator feels that the clinical picture warrants changing therapy at initial progression).
- Unacceptable Toxicity possibly attributed to all active therapies. - Participant requests to be withdrawn from active therapy.  
- Start of another systemic anticancer treatment or participation in another investigational therapeutic trial. Focal palliative radiotherapy, ablation, or surgery to a site of disease will not necessitate removal from protocol therapy.  
- Investigator discretion.  
- Positive pregnancy test.
3.7.2 Off-Study Criteria
- Screen failure.  
- PI decision to end the study.  
- Participant requests to be withdrawn from study. - Participant lost to follow up.
- Investigator discretion.  
- Death.  
3.7.3 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 for 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.
untitled h1
4 concomitant medications/measures

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-spearing technique may be administered during the trial. The assessment of PD will not be based on the necessity for palliative radiotherapy.
untitled h1
4.1 the following treatments should

- 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 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.
untitled h1
5 correlative studies for research/pharmacokinetic

| 5.1                                                             | BIOSPECIMEN COLLECTION                          |                                   |                  |             |
|-----------------------------------------------------------------|-------------------------------------------------|-----------------------------------|------------------|-------------|
| Test/assay                                                      | Volume  (approx.) per  Timepoint                | Type of                           | Collection point | Location of |
| tubea                                                           | specimen  analysis*                             |                                   |                  |             |
| Standard and 123  immune cell  subsets by FACS                  | See study calendar                              | LTIB                              |                  |             |
| 3.5                                                             |                                                 |                                   |                  |             |
| Functional  Analysis of  immune cell  subsets by FACS           | LTIB                                            |                                   |                  |             |
| 60-80 mL  blood for  PBMCs                                      | Sodium  heparin  (green top)  tubes             |                                   |                  |             |
| Antigen Specific  Immune Response  by cytokine  staining assay  | LTIB                                            |                                   |                  |             |
| T cell clonality by  immunseq  platform                         | LTIB and NCI  Frederick  Genomic Core  Facility |                                   |                  |             |
| RNA expression                                                  | LTIB and NCI                                    |                                   |                  |             |
| level of 770 genes                                              | Frederick  Genomic Core  Facility               |                                   |                  |             |
| Soluble Factors  (to include sCD27  and sCD40  ligand) by ELISA | 16 mL blood                                     | SST                               | LTIB             |             |
| for serum                                                       |                                                 |                                   |                  |             |
| Immune Markers                                                  | Tumor                                           | N/A                               | GMB TIME Lab     |             |
| by IHC                                                          | samples                                         |                                   |                  |             |
| RNA expression                                                  | Tumor                                           | N/A                               | LTIB and NCI     |             |
| level of 770 genes                                              | samples                                         | Frederick  Genomic Core  Facility |                  |             |  
| Test/assay                                    | Volume  (approx.) per  Timepoint   | Type of               | Collection point            | Location of   |
|-----------------------------------------------|------------------------------------|-----------------------|-----------------------------|---------------|
| tubea                                         | specimen  analysis*                |                       |                             |               |
| HPV status by                                 | Tumor                              | N/A                   | Dr. Hinrich's Lab           |               |
| PCR of DNA                                    | samples                            |                       |                             |               |
| Circulating tumor  DNA (ctDNA) by  PCR system | Plasma                             | EDTA  (lavender  top) | LTIB and NIH  Core Facility |               |
| T cell clonality by  immunoseq  platform      | Tumor                              | N/A                   | LTIB and NCI                |               |
| samples                                       | Frederick  Genomic Core  Facility  |                       |                             |               |  
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 to the Clinical Services Program - Leidos Biomedical Research, Inc. (CSP) (Section **5.3.1**) for barcoding, initial processing and storage. Tissue will be sent to the Laboratory of Pathology as described in Section **5.3.2**. From these facilities, coded, linked samples will be sent to the designated labs for analysis upon request.
untitled h1
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 Study Calendar 3.5. 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 [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 such as CEA, MUC-1 and Brachyury; 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.
untitled h1
5.1.2 soluble factors

Samples for soluble factor analysis will be collected as per Study Calendar 3.5.  
-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).
untitled h1
5.1.3 analyses of tumor tissue

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 Lab. Where available, archival tumor samples will be requested (preferably tissue samples from the last 6 months). For participants with lesions amenable to biopsy, two optional biopsies may be performed at baseline and at first restaging. 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 **5.3.1**.
untitled h1
5.1.4 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. Hinrich's Lab.
untitled h1
5.1.5 circulating tumor dna

If sufficient plasma is available, selected participant samples may be analyzed for circulating tumor DNA (LTIB and NIH Core Facility). Plasma DNA will be isolated with an automated purification system by Promega Corp using a system-attached bar-code reader to track samples and DNA products. 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.
untitled h1
5.2 samples for genetic/genomic analysis

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: Dr. Xiaolin Wu ATRF, Rm C3016 8560 Progress Drive Frederick, MD 21701 Ph. 301-846-7677 5.2.2 Description of how privacy and confidentiality of medical information/biological specimens will be maximized Confidentiality for genetic samples will be maintained as described (Section **5.3.1** and **5.3.2**).  
5.2.3 Management of Results Subjects will be contacted if a clinically actionable gene variant is discovered. Clinically actionable findings for the purpose of this study are defined as disorders appearing in the American College of Medical Genetics and Genomics recommendations for the return of incidental findings that is current at the time of primary analysis. (A list of current guidelines is maintained on the CCR intranet: https://ccrod.cancer.gov/confluence/display/CCRCRO/Incidental+Findings+Lists). Subjects will be contacted at this time with a request to provide a blood sample to be sent to a CLIA certified laboratory. If the research findings are verified in the CLIA certified lab, the subject will be offered the opportunity to come to NIH (at our expense) to have genetic education and counseling to explain this result. If the subject does not want to come to NIH, a referral to a local genetic healthcare provider will be provided (at their expense). This is the only time during the course of the study that incidental findings will be returned. No interrogations regarding clinically actionable findings will be made after the primary analysis.
untitled h1
5.3 sample storage tracking and

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) appropriate approvals and/or agreements, if required.  
5.3.1 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. All data associated with the participant samples is protected by using a secure database. All Clinical Support Laboratory Staff receive annual training in maintaining records' confidentiality. All samples drawn at the NIH Clinical Center will be transported to the Clinical Support Laboratory at the Frederick National Laboratory for Cancer Research by couriers.  
Samples will be tracked and managed by Central Repository database, where there is no link to personal identifiable information. All samples will be stored in either a -80ºC freezer or vapor phase liquid nitrogen. These freezers are located at NCI Frederick Central Repository in Frederick, Maryland. NCI Frederick Central Repositories (managed under a subcontract) store, among other things, biological specimens in support of NIH clinical studies. All specimens are stored in secure, limited-access facilities with sufficient security, backup, and emergency support capability and monitoring to ensure long-term integrity of the specimens for research. Specimens are stored in accordance with applicable HHS and FDA Protection of Human Subjects Regulations in accordance with the subcontractor's Federal-wide Assurance. The subcontractor's role limited to clinical research databases and repositories containing participant specimens. The subcontractor does not conduct or have any vested interest in research on human subjects, but does provide services and support the efforts of its customers, many of which are involved in research on human subjects. It is the intent and purpose of the subcontractor to accept only coded, linked samples and sample information. To the limit of our ability, every effort will be made to ensure that protected information is not sent electronically or by hard copy or on vial labels. Sample data is stored in the BioSpecimen Inventory System II (BSI). This inventory tracking system is used to manage the storage and retrieval of specimens as well as to maintain specimen data. BSI is designed for controlled, concurrent access. It provides a real-time, multi-user environment for tracking millions of specimens. The system controls how and in what order database updates and searches are performed. This control prevents deadlocks and race conditions. For security, BSI has user password access, 3 types of user access levels, and 36 user permissions (levels of access) that can be set to control access to the system functions. BSI provides audit tracking for processes that are done to specimens including shipping, returning to inventory, aliquoting, thawing, additives, and other processes. BSI tracks the ancestry of specimens as they are aliquoted, as well as discrepancies and discrepancy resolution for specimens received by the repository. If a specimen goes out of the inventory, the system maintains data associated with the withdrawal request. Vials are labeled with a unique BSI ID which is printed in both eye-readable and bar-coded format. No participant -specific information is encoded in this ID. Investigators are granted view, input, and withdrawal authority only for their specimens. They may not view specimen data or access specimens for which they have not been authorized. Access to specimen storage is confined to repository staff. Visitors to the repositories are escorted by repository staff at all times. 5.3.2 Procedures for Storage of Tissue Specimens in the Laboratory of Pathology Tissues designated for clinical diagnostics are transported to the Laboratory of Pathology (LP) where they are examined grossly and relevant portions are fixed, embedded in paraffin and sectioned and stained for diagnostic interpretation. Unutilized excess tissues are stored for up to three months, in accordance with College of American Pathologists/Joint Commission on Accreditation of Healthcare Organizations (CAP/JCAHO) guidelines, and then discarded. Following completion of the diagnostic workup, the slides and tissue blocks are stored indefinitely in the LP's clinical archives. All specimens are catalogued and retrieved utilizing the clinical laboratory information systems, in accordance with CAP/JCAHO regulations. The use of any stored specimens for research purposes is only allowed when the appropriate IRB approval has been obtained. In some cases, this approval has been obtained via the original protocol on which the participant was enrolled.
untitled h1
5.3.3 protocol completion/sample destruction

All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described above. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. The PI will report any loss or unanticipated destruction of samples per Section **7.2.1**. If the patient withdraws consent the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. Samples will also be reported as lost if they are lost in transit between facilities or misplaced by a researcher. Freezer problems, lost samples, or other problems associated with samples that meet expedited reporting requirements (see Section **7.2.1** ) will also be reported.
untitled h1
6 data collection and evaluation

The PI will be responsible for overseeing entry of data into a 21 CFR Part 11-compliant data capture system provided by the NCI CCR and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Document AEs from the first study drug administration, Study Day 1 through 28 days after removal from study treatment or until off-study, whichever comes first. Adverse events that are serious need to be recorded through 28 days after the last intervention. Beyond 28 days after the last intervention and through long term follow up (survival of subject), only adverse events which are serious and related to the study intervention need to be recorded.  
An abnormal laboratory value will be recorded in the database as an AE **only** if the laboratory abnormality is characterized by any of the following:
- Results in discontinuation from the study  
- Is associated with clinical signs or symptoms - Requires treatment or any other therapeutic intervention - Is associated with death or another serious adverse event, including hospitalization. - Is judged by the Investigator to be of significant clinical impact
- If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the participant's outcome.  
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per requirements in Section **7.2.1**.
untitled h1
6.2 data sharing plans 6.2.1

I will share human data generated in this research for future research as follows:  
- Coded, linked data in an NIH-funded or approved public repository.  
- Coded, linked data in BTRIS (automatic for activities in the Clinical Center)
- Identified or coded, linked data with approved outside collaborators under appropriate agreements.
untitled h1
how and where will the

Data will be shared through:
- An NIH-funded or approved public repository: clinicaltrials.gov - BTRIS (automatic for activities in the Clinical Center). - Approved outside collaborators under appropriate individual agreements. - Publication and/or public presentations.
untitled h1
when will the data be

- Before publication. - At the time of publication or shortly thereafter.  
6.2.2 Genomic Data Sharing Plan Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.
untitled h1
6.3 response criteria 6.3.1 antitumor

Tumor assessments may include the following evaluations: physical examination (with photograph and measurement of skin lesions, as applicable); cross-sectional imaging using computed tomography (CT) or magnetic resonance imaging (MRI) scan of the chest, abdomen, and pelvis (pelvis scan is optional unless known pelvic disease is present at baseline); nuclear bone scan for subjects with known/suspected bone lesions; and CT or MRI scan of the brain (only as clinically warranted based on symptoms/findings). The preferred method of disease assessment is CT with contrast. If CT with contrast is contraindicated, CT of the chest without contrast and MRI scan of the abdomen/pelvis is preferred.  
At baseline, tumor lesions will be selected and categorized as target or non-target lesions. Target lesions include those lesions that can be accurately measured in at least 1 dimension as ≥ 20 mm with conventional techniques or ≥ 10 mm with CT scan. Malignant lymph nodes with a short axis diameter ≥ 15 mm can be considered target lesions. Up to a maximum of 2 target lesions per organ and 5 target lesions in total will be identified at baseline. These lesions should be representative of all involved organs and selected based on their size (those with the longest diameter) and their suitability for accurate repeated measurements. A sum of the longest lesion diameter (LLD) for all target lesions will be calculated and reported as the baseline sum LLD. For malignant lymph nodes identified as target lesions, the short axis diameter will be used in the sum of LLD calculation. All other lesions (or sites of disease) should be identified as non-target lesions (including bone lesions). All post-baseline response assessments should follow the same lesions identified at baseline. The same mode of assessment (e.g., CT) used to identify/evaluate lesions at baseline should be used throughout the course of the study unless subject safety necessitates a change (e.g., allergic reaction to contrast media). For the primary endpoint antitumor activity will be evaluated with target and/or non-target lesions according to RECIST Version 1.1.
untitled h1
6.3.2 disease parameters

Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as:
- By chest x-ray: > 20 mm;
- By CT scan:
o Scan slice thickness 5 mm or under: as > 10 mm o Scan slice thickness > 5 mm: double the slice thickness
- With calipers on clinical exam: > 10 mm.  
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Malignant lymph nodes. To be considered pathologically enlarged and measurable, a lymph node must be > 15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. Non-measurable disease. All other lesions (or sites of disease), including small lesions (longest diameter < 10 mm or pathological lymph nodes with ≥ 10 to < 15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same participant, these are preferred for selection as target lesions. Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up. 6.3.3 Methods for Evaluation of Measurable Disease All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam. Clinical lesions: Clinical lesions will only be considered measurable when they are superficial
(e.g., skin nodules and palpable lymph nodes) and ≥10 mm diameter as assessed using calipers
(e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended. Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable. Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g., for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible.
untitled h1
6.3.4 response criteria

All the scans performed at Baseline and other imaging performed as clinically required (other supportive imaging) need to be repeated at subsequent visits. In general, lesions detected at Baseline need to be followed using the same imaging methodology and preferably the same imaging equipment at subsequent tumor evaluation visits. Brain CT / MRI scan should be performed, if clinically indicated by development of new specific symptoms or on the discretion of the Principal Investigator. For each subject, the Investigator will designate 1 or more of the following measures of tumor status to follow for determining response: CT or MRI images of primary and / or metastatic tumor masses, physical examination findings, and the results of other assessments. All available images collected during the trial period will be considered. The most appropriate measures to evaluate the tumor status of a subject should be used. The measure(s) to be chosen for sequential evaluation during the trial have to correspond to the measures used to document the progressive tumor status that qualifies the subject for enrollment. The tumor response assessment will be assessed and listed according to the Study Calendar 3.5.  
The foreseen treatment duration is until disease progression verified by a scan subsequent to the initial documentation of PD, unacceptable toxicity, or any criterion for withdrawal from the trial occurs (see Section 6.3). Before stopping the treatment, progressive disease should be confirmed by imaging 4 to 6 weeks (preferably 6 weeks, but not later) after progression has been diagnosed according to RECIST 1.1 [36]. If progression is based on the occurrence of a new lesion in an area not scanned at Baseline, a further on-study scan 6 weeks later should be considered before performing the 28-Day Safety Follow-up visit. Treatment may be continued despite progression according to RECIST 1.1 at any time if: - There are no new or concerning symptoms. - There is no decrease in ECOG PS. - The Investigator does not consider it necessary to administer a salvage therapy. The treatment should be stopped immediately, if the subject does not tolerate M7824 or if therapeutic failure occurs, which requires urgent treatment with an additional drug or results in clinically significant progression / deterioration. Tumor responses to treatment will be assigned based on the evaluation of the response of target, non-target, and new lesions according to RECIST 1.1 (all measurements should be recorded in metric notation). •To assess objective response, the tumor burden at baseline will be estimated and used for comparison with subsequent measurements. At baseline, tumor lesions will be categorized in target and non-target lesions according to RECIST 1.1. Results for these evaluations will be recorded with as much specificity as possible so that pre-and post-treatment results will provide the best opportunity for evaluating tumor response. Any CR or PR should be confirmed according to RECIST 1.1. In the case of a PR or CR, a confirmatory CT or MRI scan should be done no sooner than 4 weeks (preferably at the scheduled 6-week interval). The Investigator may perform scans in addition to a scheduled trial scan for medical reasons or if the Investigator suspects PD.  
As an exploratory endpoint antitumor activity will also be evaluated according to iRECIST [37].  
Using iRECIST criteria the following will be incorporated into assessment:
1. An increase in the sum of target lesions of more than 20%, unequivocal increase in nontarget lesions or new lesions result in iUPD (unconfirmed progressive disease); iUPD can be assigned multiple times as long as iCPD (confirmed progressive disease) is not confirmed at the next assessment. 2. Progression is confirmed in the target lesion category if the next imaging assessment after iUPD (4–8 weeks later) confirms a further increase in sum of measures of target disease from iUPD, with an increase of at least 5 mm. Progression is confirmed in the non-target lesion category if subsequent imaging, done 4–8 weeks after iUPD, shows a further unequivocal increase in non-target lesions. Progression is confirmed in the new lesions category if at next assessment additional new lesions appear or an increase in size of previously seen new lesions is seen (≥5 mm for sum of new lesion target). 3. However, the criteria for iCPD (after iUPD) are not considered to have been met if complete response, partial response, or stable disease criteria (compared with baseline and as defined by RECIST 1.1) are met at the next assessment after iUPD. The status is reset (unlike RECIST 1.1, in which any progression precludes later complete response, partial response, or stable disease). iCR, iPR, or iSD should then be assigned; and if no change is detected, then the timepoint response is iUPD.  
6.3.5 Response Criteria by RECIST 1.1