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Abbreviated Title: Phase II M7824 in UC NIH Protocol \#: 20C0142 Version Date: 06/23/2021 NCT Number: NCT04501094 Title: A Phase II Study of Bintrafusp alfa (M7824) in Checkpoint Inhibitor Naïve and Refractory Subjects with Urothelial Carcinoma NCI Principal Investigator: Andrea B. Apolo, M.D.  
Genitourinary Malignancies Branch (GMB) Center for Cancer Research National Cancer Institute Building 10, Room 13N240 Bethesda, MD 20892 Ph: 301-480-0536 Email: andrea.apolo@nih.gov  
| Drug Name:    | Bintrafusp alfa (M7824)                     |
|---------------|---------------------------------------------|
| IND Number:   | 149968                                      |
| Sponsor:      | Center for Cancer Research  (CCR), NCI, NIH |
| Manufacturer: | EMD Serono                                  |
| Supplier      | EMD Serono                                  |  
Commercial Agents: None 1
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precis background

Metastatic urothelial carcinoma is lethal and incurable with a median overall survival of 14 months from diagnosis.  
Immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway have greatly changed clinical management of metastatic urothelial carcinoma (mUC) improving survival by 3 months in the second-line setting.  
Five PD-1/PD-L1 inhibitors are FDA-approved for for second-line mUC, two agents for first-line cisplatin-ineligible mUC. However, response rates are modest, ranging from15– 20% in the second-line and 24% in the first-line cisplatin-ineligible.
Therefore, novel strategies are needed to extend benefit of immunotherapy to the remaining
~75% of non-responders.  
Higher levels of transforming growth factor-beta (TGF-β) are associated with immune escape, therapy resistance and poor outcomes in advanced malignancies. Non-responders to anti-PD-1/PD-L1 antibodies have also been found to have increased TGF-β in the tumor microenvironment.  
Bintrafusp alfa (M7824) is a novel first-in-class bifunctional fusion protein composed of a monoclonal antibody against PD-L1 fused to the extracellular domain of human TGF-β receptor II (TGFβRII), which effectively functions to sequester or "trap" all three TGF-β isoforms. A phase I study of M7824 (NCT02517398) demonstrated a manageable safety profile and clinical efficacy among patients with heavily pre-treated advanced solid tumors.  
We hypothesize that M7824 is safe and improves outcomes in patients with checkpoint naïve or refractory urothelial carcinoma.  
Objectives:
To evaluate the activity of M7824 as determined by objective response rate (ORR) in two metastatic urothelial carcinoma cohorts:
o Cohort 1: Checkpoint inhibitor naïve Cohort 1A: cisplatin ineligible Cohort 1B: refractory post-platinum therapy o Cohort 2: Checkpoint inhibitor previously treated patients Cohort 2A: previously achieved a CR/PR Cohort 2B: previously had SD/PD
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eligibility

Patients must have a histologically confirmed diagnosis of metastatic urothelial cancer.  
Patients may have been previously treated with prior cytotoxic chemotherapy regimen or targeted agent. Patients may have received any number of prior cytotoxic agents.  
18 years of age or older
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design

This is an open label, non-randomized, single arm phase II trial of M7824 in checkpoint inhibitor naïve and previously treated patients with urothelial carcinoma of the bladder.  
M7824 (intravenous 1200 mg fixed dose) will be delivered every 2 weeks
Patients will receive treatment in cycles consisting of 4 weeks. A maximum of 75 subjects will be enrolled in this trial.  
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| TABLE OF CONTENTS                                                                                                                                  |                                                                                                                                                                     |
|----------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS                                                                                                                                             |                                                                                                                                                                     |
| TABLE OF CONTENTS ...............................................................................................................................4 |                                                                                                                                                                     |
| STATEMENT OF COMPLIANCE                                                                                                                            |                                                                                                                                                                     |
| 1                                                                                                                                                  | INTRODUCTION .                                                                                                                                                      |
| 1.1                                                                                                                                                | Study Objectives .                                                                                                                                                  |
| 1.2                                                                                                                                                | Background .......................................................................................................................................................8 |
| 2                                                                                                                                                  | ELIGIBILITY ASSESSMENT AND ENROLLMENT ...............................................................14                                                             |
| 2.1                                                                                                                                                | Eligibility Criteria ................................................................................................................................14             |
| 2.2                                                                                                                                                | Screening Evaluation .....................................................................................................................18                        |
| 2.3                                                                                                                                                | Participant Registration and Status Update Procedures                                                                                                               |
| 2.4                                                                                                                                                | Baseline Evaluation                                                                                                                                                 |
| 3                                                                                                                                                  | STUDY IMPLEMENTATION ..........................................................................................................................20                   |
| Study Design .                                                                                                                                     |                                                                                                                                                                     |
| 3.1                                                                                                                                                |                                                                                                                                                                     |
| 3.2                                                                                                                                                | D Drug Administration                                                                                                                                               |
| 3.3                                                                                                                                                | Dose Modifications .                                                                                                                                                |
| 3.4                                                                                                                                                | Study Calendar .                                                                                                                                                    |
| 3.5                                                                                                                                                | Cost and Compensation ............................................................................................................................33                |
| 3.6                                                                                                                                                | Criteria for Removal from Protocol Therapy and Off Study Criteria                                                                                                   |
| 4                                                                                                                                                  | CONCOMITANT MEDICATIONS/MEASURES.....................................................................35                                                             |
| Permitted Medicines .....................................................................................................................35        |                                                                                                                                                                     |
| 4.1                                                                                                                                                |                                                                                                                                                                     |
| 4.2                                                                                                                                                | Prohibited Medicines ......................................................................................................................35                       |
| S                                                                                                                                                  | Correlative Studies for Research .......................................................................................................35                          |
| 5.1                                                                                                                                                | Biospecimen Collection ..................................................................................................................35                         |
| 5.2                                                                                                                                                | Sample collection schedule ......................................................................................................46                                 |
| 5.3                                                                                                                                                | Sample Storage, Tracking and Disposition ...........................................................................48                                              |
| 5.4                                                                                                                                                | Samples for Genetic/Genomic Analysis ............................................................................................50                                 |
| 6                                                                                                                                                  | DATA COLLECTION AND EVALUATION .                                                                                                                                    |
| DATA COLLECTION                                                                                                                                    |                                                                                                                                                                     |
| 6.1                                                                                                                                                |                                                                                                                                                                     |
| 6.2                                                                                                                                                | DATA SHARING Plans                                                                                                                                                  |
| 6.3                                                                                                                                                | Response Criteria                                                                                                                                                   |
| 6.4                                                                                                                                                | Toxicity Criteria                                                                                                                                                   |  
Version Date: 06/23/2021  
| 7    | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN 59         |
|------|-----------------------------------------------------------------------|
| 7.1  | Definitions 59                                                        |
| 7.2  | OHSRP Office of Compliance and Training / IRB Reporting 59            |
| 7.3  | NCI Clinical Director Reporting 59                                    |
| 7.4  | NIH Required Data and Safety Monitoring Plan 60                       |
| 8    | SPONSOR PROTOCOL/SAFETY REPORTING 60                                  |
| 8.1  | Definitions 60                                                        |
| 8.2  | Assessment of Safety Events 61                                        |
| 8.3  | Reporting of Serious Adverse Events 62                                |
| 8.4  | Waiver of expedited reporting to CCR 62                               |
| 8.5  | Safety Reporting Criteria to the Pharmaceutical Collaborators 62      |
| 8.6  | Reporting Pregnancy 63                                                |
| 8.7  | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND 63 |
| 8.8  | Sponsor Protocol Non-Adherence Reporting 63                           |
| 9    | Clinical Monitoring 64                                                |
| 10   | STATISTICAL CONSIDERATIONS 64                                         |
| 10.1 | Statistical Hypothesis 64                                             |
| 10.2 | Sample Size Determination 65                                          |
| 10.3 | Populations for Analyses 66                                           |
| 10.4 | Statistical Analyses 67                                               |
| 11   | COLLABORATIVE AGREEMENTS 68                                           |
| 11.1 | CRADA 68                                                              |
| 12   | HUMAN SUBJECTS PROTECTIONS 68                                         |
| 12.1 | Rationale For Subject Selection 68                                    |
| 12.2 | Participation of Children 68                                          |
| 12.3 | Participation of Subjects Unable to Give Consent 68                   |
| 12.4 | Evaluation of Benefits and Risks/Discomforts 69                       |
| 12.5 | Risks/Benefits Assessment 69                                          |
| 12.6 | Consent Process and Documentation 71                                  |
| 13   | REGULATORY AND OPERATIONAL CONSIDERATIONS 72                          |
| 13.1 | Study Discontinuation and Closure 72                                  |
| 13.2 | Quality Assurance and Quality Control 73                              |  
5 Version Date: 06/23/2021 13.3 Conflict of Interest Policy ...............................................................................................73  
| 13.4   | Confidentiality and Privacy 73                           |
|--------|----------------------------------------------------------|
| 14     | PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION 74 |
| 14.1   | M7824 (Bintrafusp alfa, MSB0011359C) (IND # 149968) 74   |
| 15     | REFERENCES 79                                            |
| 16     | Appendices 84                                            |
| 16.1   | Appendix A: Study Calendar 84                            |
| 16.2   | Appendix B: Performance Status Criteria 86               |  
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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.
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1 introduction 1.1 study objectives

To evaluate the activity of Bintrafusp alfa (M7824) as determined by objective response rate (ORR) in two metastatic urothelial carcinoma cohorts:
o Cohort 1: Checkpoint inhibitor naïve Cohort 1A: cisplatin ineligible Cohort 1B: refractory post-platinum therapy o Cohort 2: Checkpoint inhibitor previously treated patients Cohort 2A: previously achieved a CR/PR Cohort 2B: previously had SD/PD
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1.1.2 secondary objective(s)

To evaluate the activity of Bintrafusp alfa (M7824) in metastatic urothelial carcinoma patients as determined by progression free survival (PFS) and overall survival (OS).  
To evaluate the safety of bintrafusp alfa (M7824) in metastatic urothelial carcinoma patients.  
1.1.3 Exploratory Objective(s)
(1) Determine peripheral immune modulation using:
Multi-parametric flow cytometry - immune subset analysis Cytokine/chemokine analysis (i.e TNF, IFN- IL-6, IL-8, etc.)
Blood-based immune parameters (neutrophil-to-lymphocyte ratio)
C-Met analysis Circulating Tumor Cells (CTCs), and circulating cell-free tumor DNA (cfDNA)
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(2) Determine immune status of the tumor microenvironment using:
Multiplex IHC for PD-L1 expression, CD8, CD4, FoxP3 T-cell infiltration RNA-Seq transcriptome sequencing for tumor/immune gene expression profile
(and peripheral blood mononuclear cells)
T-cell receptor (TCR) sequencing for T-cell clonality and MET protein levels Next generation sequencing (TSO-500) for tumor biomarkers in tumor samples
(3) Determine the diagnostic accuracy of diffusion weighted whole body MRI in this patient population, compared to standard imaging
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1.2 background 1.2.1 immunotherapy and

For decades, the standard-of-care for advanced urothelial carcinoma (UC) has almost exclusively revolved around cisplatin-based chemotherapy [1]. However, approximately 50% of patients are deemed cisplatin-ineligible and patients with adverse prognostic features derive marginal benefit from cisplatin-based chemotherapy [2-4]. Cisplatin-ineligible and platinum-refractory patients have limited options with an unclear impact on prolonging survival [5]. As such, less toxic and more efficacious therapies are needed. Immunotherapy in the form of Bacillus Calmette-Guérin (BCG), an attenuated strain of *Mycobacterium bovis* has been a long-standing pillar in the management of non-muscle invasive bladder cancer (NMIBC) and has provided some insights that immune-based approaches may be effective in more advanced and lethal forms of urothelial carcinoma [6]. The advent of immune checkpoint blockade has revolutionized cancer immunotherapy leading to an explosion of FDA approvals in UC. This class of agents disrupts immune-inhibitory molecular pathways, termed immune checkpoints, via blockade of ligand-receptor interactions thereby promoting T-cell activation and effector function [7]. At present, monoclonal antibodies targeting immune checkpoints, cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death protein-1 (PD-1) or cognate ligand, programmed cell death-ligand 1 (PD-L1), are the only FDAapproved immune checkpoint blocking agents. There are currently five PD-1/PD-L1 inhibitors approved for second-line advanced UC [8-13], two additional indications in the first-line setting [14, 15]. Despite altering the prognostic landscape for a subset of patients with limited and toxic treatment options, many patients fail to respond to upfront therapy with immune checkpoint blockade and additional subsets of patients ultimately develop acquired resistance to this approach. To underscore this point, objective response rates in the second-line UC setting range from 15-21%
with modestly higher response rates in the upfront UC setting (23.5-24%). As shown by Keynote45 PD-1/PD-L1 check point blockade can have a durable response in metastatic urothelial patients.  
When compared to chemotherapy in the second line setting patients receiving pembrolizumab had an overall survival of 10.3 months (95% confidence interval [CI], 8.0 to 11.8) compared with 7.4 months (95% CI, 6.1 to 8.3). Novel combination strategies are needed to extend the benefit of immunotherapy to the remaining 75-85% of non-responders with metastatic non-prostate genitourinary cancer [8]. This population represents a clear unmet clinical need as these patients continue to succumb to rapid and lethal progression of disease despite currently approved therapies. 1.2.2 Rationale to Target Dual Immunosuppressive Pathways: PD-L1 and TGF-β blockade Given that only a subset of patients (~20%) derive clinical benefit from anti-PD-1/PD-L1 immune checkpoint blockade, there is growing interest in combination approaches to improve outcomes.  
The concurrent targeting of additional immune suppressive pathways to enhance responses to antiPD-1/PD-L1 blockade is emerging as an attractive therapeutic strategy.  
TGF-β is pleotropic cytokine with potent immunosuppressive activity within the tumor microenvironment (TME) in advanced cancer. Several recent preclinical reports have demonstrated that TGF-β signaling can directly and  
 indirectly restrain effector T-cell function which promotes tumor immune evasion and therapeutic resistance to immunotherapy [16-18]. Poor response to anti-PDL-1 agent was associated with TGF- β signaling in fibroblasts, particularly in patients with CD8+ T cells that were excluded from the tumor parenchyma and instead found in the fibroblasts and collagen-rich peritumoral stroma, which is common in patients with metastatic UC [19]. Blockade of surface-bound TGF-β on regulatory T-cells (Tregs) has been shown to abrogate suppression of effector T-cell function within the TME [20]. Recent clinical investigation in metastatic urothelial cancer patients has demonstrated that TGF-β attenuates response to PD-L1 blockade (atezolizumab) via T-cell exclusion and reduced T-cell tumor infiltration. *TGFBR2* gene expression was significantly associated with clinical non-response and with reduced overall survival (see Figure 1).  
overall survival when split by quartiles (P=0.022) [top panel]. EMT6 and MC38 murine models demonstrate tumor regression and TME changes following therapeutic anti-TGF-β and anti PD-L1 treatment [middle and lower panel] [21].  
Preclinical murine models demonstrated that stromal TGF-β signaling recapitulates an immuneexcluded phenotype common to anti-PD-L1 non-responders. Further, the combination of PD-L1 blockade and TGF-β blockade facilitated T cell penetration into the center of the tumor, causing immune-mediated tumor regression and led to superior survival to either monotherapy in EMT6 and MC38 tumor models (see **Figure 1**). Additional, preclinical studies modeling human microsatellite-stable colorectal cancers which traditionally are characterized by low mutational load and poor response rates to anti-PD-1/PD-L1 checkpoint blockade have demonstrated that TGF-β inhibition promotes effector T-cell driven anti-tumor immune response and renders tumors susceptible to anti-PD-1/PD-L1 immunotherapy [22]. Other groups have independently confirmed the correlation of high mRNA expression of TGF-β1-3 with adverse prognosis and with reduced disease-free survival. Taken together, TGF-β (stromal and non-stromal) signaling within the TME appears to be a dominant mechanism of immune evasion and therapeutic targeting of this immunosuppressive axis may enrich the proportion of patients responding to anti-PD-1/PD-L1 immunotherapy and/or re-invigorate responses to PD-1/PD-L1 blockade. 1.2.3 Preclinical Activity with M7284, a bifunctional molecule targeting PD-L1 and TGF-β  
 M7824 (MSB0011359C) is a novel first-in-class bifunctional fusion protein composed of a monoclonal antibody against PD-L1 fused to the extracellular domain of human TGF-β receptor II (TGFβRII), which effectively functions to sequester or "trap" all three TGF-β isoforms [23]. Preclinical studies have shown that M7824 can simultaneously bind PD-L1 and TGF-β *in vitro*  
  
(**Figure 2**) and this bifunctional immunomodulatory capacity has translated to enhanced antitumor efficacy *in vivo* relative to blockade of either immune-inhibitory protein alone. Specifically, treatment with M7284 (IV 482µg) suppressed tumor growth, reduced lung metastases and prolonged survival in MC38 or EMT-6 tumor-bearing mice in comparison to treatment with isotype control, anti-PD-L1, Trap control and anti-PD-L1/Trap control. Furthermore, treatment with M7824 led to the development of long-term anti-tumor immunity in mice initially cured by M7284 that were subsequently re-challenged with EMT-6 or MC38 tumors - 100% and 80% of mice rejected tumor and survived re-challenge, respectively [23]. Quantitative and immunophenotypic analysis has shown that M7824 treatment is associated with robust CD8+ TME infiltration and a pro-inflammatory immune signature consistent with active innate and adaptive immunity (**Figure 3**). M7824 has also demonstrated encouraging efficacy across an array of human urothelial carcinoma cell lines (UMUC-3, UMUC-5, HTB-4, HTB-1, HTB-2, HTB-9 and HTB-5) [24]. Mechanistically, M7284 enhanced immune recognition of human urothelial cells via immunogenic modulation, increases in chemokine expression associated with T-cell trafficking, upregulation of TRAIL and antigen-specific CD8+ T-cell mediated lysis. The NCI led the first-in-human phase I trial of M7824 (NCT02517398) which demonstrated a manageable safety profile and nascent yet encouraging evidence of clinical activity in cohort of patients with heavily pre-treated advanced solid tumors [25]. In this standard 3+3 design phase I dose-escalation study, patients with tissue-confirmed metastatic or locally advanced solid tumors that were refractory to or ineligible for standard therapy (ineligible if previously treated with prior immune checkpoint blockade) were treated with infusional M7824 doses of 1mg/kg, 3mg/kg, 10mg/kg and 20mg/kg once every two weeks (Q2W). A separate cohort of patients was initially treated with 0.3mg/kg to establish a pharmacokinetic/pharmacodynamic relationship followed by 10mg/kg dosing thereafter. A total of 19 patients were enrolled with maximum tolerated dose (MTD) not reached at the highest dose level of 20mg/kg Q2W. A total of 9 patients (47.4%) experienced any grade adverse event (AE) and four patients (21.1%) experienced > grade 3 AE. Ultimately, three patients discontinued therapy due to treatment-related AEs (bullous pemphigoid, colitis, gastroparesis) leading the authors to conclude that M7824 had a manageable and acceptable toxicity/side effect profile across all evaluated dose levels. The side effect profile of M7824 is similar to other anti-PD-1/PD-L1 agents with the exception of increased incidence of keratocanthomas presumed to be related to TGF-β axis modulation which resolved following discontinuation of M7824 [26]. PK/PD studies demonstrated saturation of PD-L1 target occupancy and potent sequestration of all TGF-β isoforms at doses >1mg/kg. Clinical activity was observed across all M7824 dose levels (**Figure 4**). One durable complete response (CR), two durable/confirmed partial responses (PR), one near PR, and two patients with stabilization of disease were observed. A single patient with progression of disease ultimately achieved a 45% decline in tumor burden on day 280 restaging suggestive of a treatment response with delayed kinetics [25]. As MTD was not reached, ongoing dose-expansion cohorts are evaluating several dose schedules including 1200mg Q2W and 10mg/kg Q2W.  
  
In line with early clinical experience of similar anti-TGFβ agents, the toxicity profile of M7824 is predominantly manageable and highly comparable to that of checkpoint inhibitors. In the recently released results of the phase I study NCT02517398 (M7824) 'Phase 1 Trial of M7824, a Bifunctional Fusion Protein Targeting PD-L1 and TGFβ, in Advanced Solid Tumors' with dose escalation cohorts of 1, 3, 10, and 20mg/kg (exceeding the 1,200mg flat dose in this study), M7824 was given every two weeks and the MTD was not reached. Data suggested overall good tolerance. One out of 16 patients developed a keratoacanthoma which could be related to the TGFβ inhibition mechanism of M7824 [25]. There was no grade 4-5 TEAEs. The only dose-limiting toxicity (DLT)
observed was colitis. The highest dose for M7824 tested in EMR200647-001 was 30 mg/kg once every 2 weeks and the maximum tolerated dose was not reached. Three participants in EMR200647-001 dose escalation cohorts received the actual dose of at least 2400 mg q2w: 2 in 30 mg/kg cohort and 1 in 20 mg/kg. All 3 participants who received ≥ 2400 mg q2w had no DLTs. The overall safety profile of M7824 in Phase I was considered well tolerated, can be adequately managed and consistent across various tumor types. Refer to Investigator's Brochure for more details.
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1.2.5 hypothesis and summary

Although PD-1/PD-L1 immune check-point inhibitors can lead to a durable response in advanced urothelial cancer patients, only a subset ranging from 15-20% have a clinical "response." Therefor enhancing PD-1/PD-L1 blockage to increase the responder rates is a logical strategy. Higher levels of transforming growth factor (TGF-β) are associated with immune escape, therapy resistance, and poor outcomes in advanced malignancies[27, 28]. Non-responders to anti-PD-L1 antibodies have been found to have increased TGF-β in the tumor microenvironment as well. [23, 29] Thus, TGF-β plays an important role in immune resistance to checkpoint inhibitors and other therapies. M7824 is a bifunctional fusion protein composed of the extracellular domain of two TGF-β sequestering or trap molecule fused to a fully humanized monoclonal antibody against PD-L1 [30]. M7824 simultaneous blocks the PD-1 and TGF-β pathways of immune evasion which effects both the innate and adaptive immune systems. Preclinical studies show that M7824 enables immunecell infiltration and can overcome resistance seen with other antibodies targeting the PD-1/PD-L1 interaction through phenotypic modifications[23, 31]. Early data from phase 1 trials demonstrated that M7824 has an acceptable safety profile and higher responses than expected in heavily pretreated patients with immunologically "cold" tumors [25, 31]. M7824-induced combined PDL1 inhibition and TGF-β sequestration has the potential to overcome immune escape thereby increasing anti-tumor activity. This could potentially increase clinical response rates compared to standard PD-1/PD-L1 checkpoint blockage. There is currently no standard therapy for advanced urothelial cancer patients who have received prior PD-1/PD-L1 therapy that allows for challenging with a subsequent immunotherapy therapy. These patients are left with 2nd/3rd line chemotherapy such as taxanes and pemetrexed which have low response rates. Thus, M7824 could represent a potential treatment option for these patients. We hypothesize that M7824 is safe and improves outcomes in urothelial carcinoma of the bladder patients who are checkpoint naïve or refractory.
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2 eligibility assessment and enrollment

2.1.1.1 Ability to understand the purpose of the study, provide signed and dated informed consent, and able to comply with all procedures.  
2.1.1.2 Male or female patients aged ≥ 18 years of age at time of consent.  
2.1.1.3 Patients with histologically confirmed diagnosis of urothelial carcinoma of the urinary tract, including the renal pelvis, ureter, bladder, or urethra. Differentiation with variant
histologies (e.g. squamous cell differentiated) will be permitted. Mixed histologies are required to have a dominant urothelial/transitional cell pattern.  
2.1.1.4 Patients must have metastatic disease defined as new or progressive lesions on crosssectional imaging. Radiological evaluation should occur within 21 days prior to enrollment.  
2.1.1.5 Patient must have evaluable and measurable disease, per RECIST 1.1. See Section **6.3**
for the evaluation of measurable disease.  
2.1.1.6 Patients may have been previously treated with prior cytotoxic chemotherapy regimen or targeted agent. Patients may have received any number of prior cytotoxic agents.  
2.1.1.7 Patients may have had prior immunomodulating therapy including therapy targeting the PD-1/PDL-1 axis (cohort 2A and B) but excluding prior treatment with M7824.  
2.1.1.8 Pre-treatment tissue biopsy and/or archival tissue availability for PD-L1 expression testing is mandatory for enrollment.  
2.1.1.9 Eastern Cooperative Oncology Group (ECOG) performance status ≤2(see Section **16.2)** 2.1.1.10 Required laboratory values reflective of organ function are listed below:
Absolute neutrophil count ≥ 1500/µL
Platelets ≥ 100,000/µL
Hemoglobin ≥ 9 g/dL (erythrocyte tranfusions are allowed to achieve acceptable Hgb)
AST(SGOT)/ALT(SGPT) ≤ 1.5 × institutional upper limit of normal (ULN) with the following exception:
o Patients with liver involvement who have AST and ALT ≤ 5 × ULN may be enrolled.  
Total bilirubin within normal limits with the following exceptions:
o Patients with known Gilbert disease who have serum bilirubin level ≤ 3 ULN may be enrolled.  
o Patients with tumor liver involvement bilirubin with ≤ 3.0 × ULN.  
INR and aPTT ≤ 1.5 × ULN
o This applies only to patients who are not receiving therapeutic anticoagulation; patients receiving therapeutic anticoagulation (such as low-molecular-weight heparin or warfarin) should be on a stable dose.  
Creatinine clearance (CrCl) ≥ 30 mL/min/1.73 m2 (GFR may be used in place of CrCl.  
Creatinine clearance or eGFR should be calculated per institutional standard)
2.1.1.11 The effects of M7824 on the developing human fetus are unknown. For this reason, women of child-bearing potential and men must agree to use strict and effective contraception (hormonal or barrier method of birth control; abstinence) during treatment and for at least 65 days for women and 125 days for men, after the last dose of M7824 administration. 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.  
2.1.1.12 Human immunodeficiency virus (HIV) positive patients are eligible if on stable dose of highly active antiretroviral therapy (HAART), CD4 counts are greater than 350 cells/mm3 and viral load is undetectable.  
2.1.1.13 Patients with previously treated brain or central nervous system (CNS) metastases are eligible provided that the subjects have recovered from any acute effects of radiotherapy and is not requiring steroids, and any whole brain radiation therapy or any stereotactic radiosurgery was completed at least 2 weeks prior to M7824 administration.  
2.1.1.14 Hepatitis B virus (HBV) positive patients are eligible-they must have been treated and on a stable dose of antivirals (eg, entecavir, tenofovir, or lamivudine; adefovir or interferon are not allowed) at study entry and with planned monitoring and management according to appropriate labeling guidance.  
2.1.1.15 Hepatitis C virus (HCV) positive patients are eligible if participants are on active HCV
therapy at study entry and on a stable dose without documented clinically significant impaired liver function test or hematologic abnormalities and with planned monitoring and management according to appropriate labeling guidance.  
2.1.2 Cohort 1A Cisplatin Ineligible Specific Inclusion Criteria (first-line for metastatic cisplatin-ineligible)
No prior chemotherapy for inoperable locally advanced or metastatic or recurrent UC
o For patients who received prior adjuvant/neoadjuvant chemotherapy or chemoradiation for UC, a treatment-free interval > 12 months between the last treatment administration and the date of recurrence is required in order to be considered treatment naive in the metastatic setting. Prior local intravesical chemotherapy or immunotherapy is allowed if completed at least 4 weeks prior to the initiation of study treatment.  
Ineligible ("unfit") for chemotherapy or cisplatin-based chemotherapy as defined by any one of the following criteria [32]:
o Impaired renal function (CrCl > 30 but < 60 mL/min); GFR should be calculated per institutional standard.  
o A hearing loss (measured by audiometry) of 25 dB at two contiguous frequencies o Grade ≥ 2 peripheral neuropathy (i.e., sensory alteration or paresthesias including tingling)
o ECOG performance score of 2 o Patient declines chemotherapy after informed discussion with the study doctor 2.1.3 Cohort 1B Refractory Post-platinum Therapy Specific Inclusion Criteria (second-line for metastatic disease)
Disease progression during or following treatment with a platinum-containing regimen for inoperable locally advanced or metastatic urothelial carcinoma or disease recurrence. Examples of regimens include cisplatin + gemcitabine (GC), methotrexate + vinblastine sulfate + doxorubicin + cisplatin (MVAC), and carboplatin + gemcitabine (CarboGem).  
o Patients who received prior adjuvant/neoadjuvant chemotherapy and progressed within 12 months of treatment with a platinum-containing adjuvant/neoadjuvant regimen will be considered as second-line patients.  
2.1.4 Cohort 2A Checkpoint Inhibitor Previously Treated Patients that Previously Achieved a Complete Response (CR) or Partial Respsonse (PR) Specific Inclusion Criteria Patients must have been treated with at least one treatment of a PD-1/PD-L1 checkpoint inhibitor for advance or metastatic UC and achieved a complete response or partial respsonse by RECIST 1.1 criteria.  
2.1.5 Cohort 2B Checkpoint inhibitor previously treated patients that previously had stable disease (SD) or progressive disease (PD)
2.1.6 Specific Inclusion Criteria Patients must have been treated with at least one treatment of a PD-1/PD-L1 checkpoint inhibitor for advance or metastatic UC and had stable disease or a progressive disease by RECIST 1.1 criteria.  
2.1.7 Exclusion Criteria 2.1.7.1 History of allergic reactions attributed to compounds of similar chemical or biologic composition to M7824 investigational agents used in the study.  
2.1.7.2 Uncontrolled intercurrent illness including, but not limited to, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements.  
2.1.7.3 Symptomatic central nervous system metastasis. 2.1.7.4 Subjects unwilling to accept blood products as medically indicated 2.1.7.5 Pregnant women are excluded from this study because M7824 is an agent with the 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 M7824, breastfeeding should be discontinued if the mother is treated with these agents.  
2.1.7.6 Patients with any active or recent history of a known or suspected autoimmune disease
(with the exception of diabetes type I, vitiligo, psoriasis, or hypo- or hyperthyroid diseases not requiring immunosuppressive treatment) or recent history of a syndrome that required treatment with either systemic corticosteroids (>10 mg daily prednisone equivalent) or immunosuppressive medications. Inhaled steroids and adrenal replacement steroid doses up to 10 mg daily prednisone equivalents are permitted in the absence of active autoimmune disease.  
2.1.7.7 Patients with a "currently active" second malignancy other than non-melanoma skin cancers or cervical carcinoma in situ or incidental organ-confined prostate cancer found on cystoprostatectomy (provided that the following criteria are met: Stage T2N0M0 or lower; Gleason score ≤ 3+4, PSA undetectable). Patients are not considered to have a "currently active" malignancy if they have completed therapy and are free of disease for ≥ 2 years and currently do not require systemic therapy.  
2.1.7.8 Patients who have received or will receive a live vaccine within 30 days prior to the first administration of study intervention. Seasonal flu vaccines that do not contain a live virus are permitted. Locally approved COVID vaccines are permitted.
2.1.7.9 Patients having tumor lesion(s) in the liver or chest which are 10 cm or larger. 2.1.7.10 Patients previously treated with M7824. 2.1.7.11 Patients previously treated with PD-1/PD-L1 checkpoint inhibitors (for Cohorts 1A and 1B only)
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2.1.8 recruitment strategies

Both men and women and members of all races and ethnic groups are eligible for this trial. This protocol may be abstracted into a plain language announcement posted on NIH websites and on NIH social media. This study will be listed on www.clinicaltrials.gov and participants will be recruited from the current patient population as well as referrals at NIH.
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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 Screening evaluation testing/procedures are conducted under the separate screening protocol, 01- C-0129 (Eligibility Screening and Tissue Procurement for the NIH Intramural Research Program Clinical Protocols). Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent. All screening tests and procedures must be performed within 21 days prior to initiation of study therapy except physical exam with skin assessment, ECOG, vital signs, blood tests and preganacy test (repeated if >7 days):
Complete medical history and physical examination (including height, weight, vital signs, and ECOG performance status) and skin assessment.  
Electrocardiogram (ECG)  
CT scan of chest, abdomen and pelvis with IV and PO (as indicated) contrast, or CT
chest and MRI abdomen/pelvis may be performed, and [18F]-Sodium Fluoride (NaF) PET/CT bone scan (if indicated).  
Laboratory assessments: CBC with differential, acute care panel (sodium, potassium, chloride, total CO2 (Bicarbonate), creatinine, glucose, urea nitrogen), mineral panel
(albumin, calcium total, magnesium total, phosphorus), hepatic panel (alkaline phosphatase, ALT/GPT, AST/GOT, total bilirubin, direct bilirubin), ionized calcium, amylase, lipase, LDH, total protein, GGT, Hepatitis B and C, PT/INR, PTT, urine analysis.
Hepatitis B and C antibody panel Thyroid function tests - TSH, reflex T3 and T4 Pre-treatment tissue biopsy and/or archival tissue availability for PD-L1 expression retrospective testing is mandatory for enrollment. PD-L1 will be performed retrospectively by EMD Serono using a test determined by EMD Serono.  
Pregnancy test for females of childbearing-potential (within 3 days prior to first dose of study drug).  
Lymphocyte Phenotyping - CD4/CD8 will be performed on known HIV-positive patients.
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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 here. 2.3.1 Treatment Assignment Procedures
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cohorts

Patients will be assigned to the appropriate cohort depending on prior checkpoint inhibitor therapy and documented or reviewed radiologic responses to prior checkpoint inhibitor therapy. Whenever possible prior scans will be loaded to our PACS and reviewed for chort 2A and 2B. Subjects from all cohorts will be treated with a flat dose of M7824 (1200 mg) every 2 weeks (+/- 3 days for scheduling issues).  
| Number   | Name      | Description                                                                                                                              |
|----------|-----------|------------------------------------------------------------------------------------------------------------------------------------------|
| 1        | Cohort 1A | Checkpoint inhibitor naïve patients who are cisplatinineligible                                                                                                                                          |
| 2        | Cohort 1B | Checkpoint inhibitor naïve patients who are refractory  post-platinum therapy                                                            |
| 3        | Cohort 2A | Checkpoint inhibitor refractory patients with prior  Complete Response (CR) or Partial Response (PR) as  per RECIST 1.1 as best response |
| 4        | Cohort 2B | Checkpoint inhibitor refractory patients with Stable  Disease (SD) or Progressive Disease (PD) as per  RECIST 1.1 as best response       |  
Arms  
| Number   | Name   | Description                                                 |
|----------|--------|-------------------------------------------------------------|
| 1        | Arm 1  | Treatment with fixed dose of M7824 (1200  mg) every 2 weeks |  
Arm assignment Subjects from all cohorts (1A, 1B, 2A and 2B) will be directly assigned to Arm 1.
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2.4 baseline evaluation

Tests done at screening do not need to be repeated on baseline (C1D1) if performed within 21 days prior to initiation of study therapy except physical exam, ECOG, vital signs, blood tests (CBC with differential, acute care, mineral and hepatic panels, amylase, lipase, LDH, GGT, thyroid function tests) and pregnancy test (repeated if >7 days). Please refer to Section 2.2 for details. Correlative Studies: Please refer to Section 5.2 for information regarding collection of blood, and tissue for correlative research studies.
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3 study implementation 3.1 study

This is an open label, non-randomized, single arm phase II trial of M7824 in checkpoint inhibitor naïve and refractory patients with urothelial carcinoma.  
We will plan to use a fixed dose of 1200 mg delivered IV every 2 weeks (Q2W) based on the recent NCI-led phase I dose-escalation trial of M7824 in advanced solid tumors (NCT02517398) [33]. M7284 appeared to have a manageable safety profile and early evidence of clinically efficacy. The MTD was not reached in this study at 20mg/kg and PD-L1 receptor occupancy and sequestration of TGF-β isoforms 1-3 was achieved at M7284 dose levels >1mg/kg.  
A maximum of 68 evaluable subjects will be enrolled in checkpoint naïve [Cohort 1] and checkpoint inhibitor refractory [Cohort 2] patients with urothelial carcinoma of the bladder.  
o Cohort 1A: Checkpoint inhibitor naïve patients who are cisplatin-ineligible o Cohort 1B: Checkpoint inhibitor naïve patients who are refractory postplatinum therapy o Cohort 2A: Checkpoint inhibitor refractory patients with Complete Response
(CR) or Partial Response (PR) as per RECIST 1.1 o Cohort 2B: Checkpoint inhibitor refractory patients with Stable Disease (SD)
or Progressive Disease (PD) as per RECIST 1.1 Enrollment of 2-3 patients per month is expected on this trial; thus, 2 to 3 years is anticipated as the accrual period.  
Restaging will be performed every 2 cycles (8 weeks). Safety/tolerability analysis and secondary clinical efficacy analysis will be ongoing through cycle 4. Patients can continue study drug in all arms provided there is no confirmed evidence of progression, unacceptable toxicity or withdrawal from the study.
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treatment schema of m7824 28-day

If appropriate screening imaging is done within 21 days of C1D1 repeat screening imaging is not required.  
| Cycles and Day         | C1D1   | C1D15   | C2D1   | C2D15   | C3D1   | C3D15   | C4D1   | C4D15   |
|------------------------|--------|---------|--------|---------|--------|---------|--------|---------|
| M7824 (1200mg IV  Q2W) | X      | X       | X      | X       | X      | X       | X      | X       |  
3.2 DRUG A**DMINISTRATION** 3.2.1 M7824 See Section **14.1** for drug preparation, administration and infusion times.
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3.3 dose modifications 3.3.1 m7824

M7824 is planned to be administered at 1200 mg (flat dose) every two weeks. Dosing may be delayed up to 12 weeks for toxicity or logistical reasons, including the inability of the patient to get to the clinic. Dose reductions are not allowed.
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3.3.1.1 premedications

Current experience revealed that infusion-related reactions (IRRs) to bintrafusp alfa occur seldomly and are generally mild to moderate in severity. Therefore, administration of a premedication is generally not required. If an Investigator deems necessary to administer a premedication to a particular participant, an antihistamine (e.g. 25 to 50 mg diphenhydramine) and paracetamol (acetaminophen, 500 to 650 mg intravenously or equivalent oral dose) 30 to 60 minutes prior to bintrafusp alfa infusion is recommended. Premedication should be administered for subsequent bintrafusp alfa doses based upon clinical judgment and presence/severity of prior infusion reactions. This regimen may be modified based on local treatment standards and guidelines as appropriate, provided it does not include systemic corticosteroids. If Grade ≥ 2 infusion reactions are seen during the first two infusions, premedication should not be stopped. Steroids as premedication are not permitted. 3.3.1.2 Adverse Drug Reactions (ADRs) Requiring Treatment Discontinuation Any Grade 4 ADRs require permanent treatment discontinuation except for single laboratory values out of normal range that do not have any clinical correlate, and resolve to Grade ≤ 1 or baseline grade within 7 days with adequate medical management. Grade 3 or 4 symptomatic endocrinopathies (e.g., thyroiditis or hypophysitis), treatment should be delayed and treatment started according to NCCN guidelines. If condition improves to Grade 1, treatment may be resumed. Subjects with grade 4 amylase or lipase abnormalities must permanently discontinue treatment. If the participant develops any evidence of pancreatitis, it should be managed accordingly.
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any grade 3 adrs require

Transient (≤ 6 hours) Grade 3 flu-like symptoms or fever, which is controlled with medical management.  
Transient (≤ 24 hours) Grade 3 fatigue, local reactions, headache, nausea, emesis that resolves to ≤ Grade 1 or Baseline grade.  
Tumor flare phenomenon defined as local pain, irritation, or rash localized at sites of known or suspected tumor that resolves within 7 days with optimal medical management.  
Any single Grade ≥ 3 drug-related transaminase, alkaline phosphatase, or bilirubin abnormality with no other associated laboratory (or other) abnormality that is not associated with symptoms or clinical manifestations of hepatitis. If the liver function abnormality not associated with symptoms or clinical manifestations of hepatitis has not resolved to Grade ≤ 1 within the subsequent 2 cycles (28 days), the subject should permanently discontinue treatment with M7824.
Subjects with asymptomatic Grade 3 drug-related amylase or lipase abnormalities may continue treatment.  
.  
Grade 3 Hgb decrease (< 8.0 g/dL) that is clinically manageable with blood transfusions or erythroid growth factor use.  
Increases in Eastern Cooperative Oncology Group performance status (ECOG PS) ≥ 3 that resolves to  2 by Day 1 of the next cycle (infusions should not be given if the ECOG PS is ≥ 3 on the day of M7824 administration and should be delayed until ECOG PS ≤ 2).  
Keratoacanthoma and squamous cell carcinoma of the skin. Any suspicious skin lesion should be biopsied for confirmation of diagnosis and be surgically removed as indicated by dermatologist and a dermatological consult obtained.
Other immune-related ADRs, see NCCN guidelines for the management of immune related adverse events 3.3.1.3 Adverse Drug Reactions Requiring Management
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any grade 2 adr should

If a Grade 2 ADR resolves to Grade ≤ 1 by the last day of the current cycle, treatment may continue.  
If a Grade 2 ADR does not resolve to Grade ≤ 1 by the last day of the current cycle but it is manageable and / or not clinically relevant, it is possible the infusion will be given on the following cycle.
3.3.1.4 Infusion-related Reactions (IRR) Signs and symptoms usually develop during or shortly after drug infusion and generally resolve completely within 24 hours of completion of infusion. These possible IRR are identified based on a list of MedDRA PTs and divided into reactions versus signs and symptoms.  
An IRR should be considered when onset is on the day of infusion (during or after the infusion)
or the day after the infusion (irrespective of resolution date) for any infusion related reaction, drug hypersensitivity, anaphylactic reaction, hypersensitivity and/or Type 1 hypersensitivity.  
Signs and symptoms of infusion related reactions and hypersensitivity/allergic reactions should be considered when onset is on the day of infusion (during or after the infusion) and resolved completely with the end date within 2 days after onset. Signs and symptoms may include but not limited to: fever, chills or shaking, rigors, flushing, hypotension, wheezing, pruritus, rash and hypoxemia, shortness of breath, back or neck pain, facial swelling, dizziness, feeling of passing out.  
Infusion-related reactions and hypersensitivity reactions (Grades 1 to 4) should be handled according to the Guidelines provided in **Table 1**.  
Management of immune-mediated reactions is described in **Table 2**.  
| Hypersensitivity NCI-CTCAE Grade                                                                                                                                                                                                                           | Treatment Modification                                                                                                                                                                                                                            |                                                                                                                                                                                   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1 - mild Mild transient reaction; in general, infusion  interruption not indicated; intervention not  indicated                                                                                                                                      |                                                                                                                                                                                                                                                  | Increase monitoring of vital signs as medically  indicated as participants are deemed medically  stable by the attending Investigator.                                            |
|                                                                                                                                                                                                                                                           | Hold infusion if deemed necessary by the  investigator.                                                                                                                                                                                           |                                                                                                                                                                                   |
| Grade 2 - moderate Therapy or infusion interruption indicated but  responds promptly to symptomatic treatment  (for example, antihistamines, nonsteroidal  anti-inflammatory drugs, narcotics, IV fluids);  prophylactic medications indicated for  24 h. |                                                                                                                                                                                                                                                  | Stop the infusion of the study intervention.                                                                                                                                      |
|                                                                                                                                                                                                                                                           | Increase monitoring of vital signs as medically  indicated as participants are deemed medically  stable by the attending Investigator.                                                                                                            |                                                                                                                                                                                   |
|                                                                                                                                                                                                                                                           | If symptoms resolve quickly, resume infusion at  50% of original rate with close monitoring of  any worsening signs and symptoms, otherwise  dosing held until resolution of symptoms with  mandated premedication for the next scheduled  visit. |                                                                                                                                                                                   |
|                                                                                                                                                                                                                                                           | If not improving, consider administration of  glucocorticoids and stop the infusion for that  day.                                                                                                                                                |                                                                                                                                                                                   |
|                                                                                                                                                                                                                                                           | If the participant has a second IRR Grade ≥ 2 on  the slower infusion rate despite premedication,  the infusion should be stopped, and the  investigator may consider withdrawal of this  participant from the study.                             |                                                                                                                                                                                   |
| Grade 3 or Grade 4 - severe or  life-threatening o Grade 3: Prolonged (for example, not  rapidly responsive to symptomatic                                                                                                                                 |                                                                                                                                                                                                                                                  | Stop the infusion of study intervention  immediately and disconnect infusion tubing  from the participant with additional appropriate  medical measures and closely monitor until |  
| medication and/or brief interruption of  infusion); recurrence of symptoms  following initial improvement;  hospitalization indicated for clinical  sequelae.   |                                                                         |                                                                                                                 |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------|
| o                                                                                                                                                               | Grade 4: Life-threatening consequences;  urgent intervention indicated. | deemed medically stable by the attending  Investigator. Hospitalization and/or close  monitoring is recommended |
| Administration of glucocorticoids may be required  For Grade 3 or 4 IRRs, permanent  discontinuation of study intervention is  mandated.                       |                                                                         |                                                                                                                 |
| Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must remain                                                               |                                                                         |                                                                                                                 |  
deemed medically stable by the attending Investigator. Hospitalization and/or close monitoring is recommended  
Administration of glucocorticoids may be required  
For Grade 3 or 4 IRRs, permanent  
discontinuation of study intervention is mandated.  
Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must remain decreased for all subsequent infusions. For all types and grades of infusion reactions, details about drug physical constitution, method of preparation, and infusion must be recorded. Participants should be instructed to report any delayed reaction immediately. 1 IRR=infusion-related reactions, IV=intravenous, NCI-CTCAE=National Cancer Institute-Common Terminology Criteria for Adverse Event, NSAIDs=nonsteroidal anti-inflammatory drugs.
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table 2 management of immune-

Immune-related AEs are specific to immunotherapies and vary by organ system. The following immune-related AEs are important identified risks for M7824: - Immune-related pneumonitis - Immune-related hepatitis - Immune-related colitis - Other immune-related events (myositis, myocarditis, encephalitis) The following immune-related AEs are important potential risks for M7824: - Guillain-Barré syndrome - Uveitis - Pancreatitis - Myasthenia gravis/myasthenic syndrome Recommended guidance and management for specific irAEs are provided in the current National Comprehensive Cancer Network (NCCN) guideline available at http://www.nccn.org. Requirements in addition to NCCN guidelines:  
Permanent treatment discontinuation is required in case of immune-related Grade 4 rash/inflammatory dermatitis, nephritis, autoimmune hemolytic anemia, hemolytic uremic
- Immune-related nephritis and renal dysfunction  
| -   | Immune-related endocrinopathies                                                           |
|-----|-------------------------------------------------------------------------------------------|
| -   | (thyroid disorders, adrenal insufficiency, type 1 diabetes mellitus, pituitary disorders) |
| -   | Immune related rash                                                                       |  
syndrome, aplastic anemia, immune thrombocytopenia, acquired thrombotic thrombocytopenic purpura inflammatory arthritis, myositis and polymyalgia-like syndrome.  
For Grade 4 immune-related lymphopenia, permanent treatment discontinuation will be required, if lymphopenia is considered immune-related in nature, no clear alternative explanation exists for the event, and it does not resolve within 14 days. Permanent treatment discontinuation is not required when the AE is manifested by a single laboratory value out of normal range without any clinical correlates. In this case, treatment should be held until the etiology is determined. If the event is not considered immune-related and resolves to Grade ≤ 1, restarting treatment may be considered.  
For Grade 1 immune-related pneumonitis: continue treatment. If clinically indicated, monitor participants weekly or more frequently as needed with history, physical examination and pulse oximetry. If symptoms appear and/or changes in the physical exam are noted, treat as Grade 2.  
For myositis: in case of management with rituximab, treatment should be discontinued. For Grade 3 or 4 endocrinopathies: withhold until clinically stable or permanently discontinue depending on severity.  
For hepatitis with no tumor involvement of the liver: withhold if total bilirubin increases to more than 1.5 and up to 3 times ULN, permanently discontinue if more than 3 times ULN
Hepatitis with tumor involvement of the liver: permanently discontinue if total bilirubin increases to more than 3 times ULN.
3.3.1.5 Additional Modifications for Subjects with Grade 2 Infusion-related Reactions If, in the event of a Grade 2 infusion-related reaction that does not improve or worsens after implementation of the modifications indicated in Table 1.  
Management of immune-mediated reactions is described in **Table 2.**  
| Hypersensitivity NCI-CTCAE Grade                                                                                                                          | Treatment Modification                                                                                                                 |                                                                                                                                        |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1 - mild Mild transient reaction; in general, infusion  interruption not indicated; intervention not  indicated                                     |                                                                                                                                       | Increase monitoring of vital signs as medically  indicated as participants are deemed medically  stable by the attending Investigator. |
|                                                                                                                                                          | Hold infusion if deemed necessary by the  investigator.                                                                                |                                                                                                                                        |
| Grade 2 - moderate Therapy or infusion interruption indicated but  responds promptly to symptomatic treatment  (for example, antihistamines, nonsteroidal |                                                                                                                                       | Stop the infusion of the study intervention.                                                                                           |
|                                                                                                                                                          | Increase monitoring of vital signs as medically  indicated as participants are deemed medically  stable by the attending Investigator. |                                                                                                                                        | Version Date: 06/23/2021  
| Version Date: 06/23/2021 anti-inflammatory drugs, narcotics, IV fluids);                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                      | If symptoms resolve quickly, resume infusion at                                                                                                                                                                                                                                                    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| prophylactic medications indicated for  24 h.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | 50% of original rate with close monitoring of  any worsening signs and symptoms, otherwise  dosing held until resolution of symptoms with  mandated premedication for the next scheduled  visit.                      |                                                                                                                                                                                                                                                                                                    |
|                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | If not improving, consider administration of  glucocorticoids and stop the infusion for that  day.                                                                                                                    |                                                                                                                                                                                                                                                                                                    |
|                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | If the participant has a second IRR Grade ≥ 2 on  the slower infusion rate despite premedication,  the infusion should be stopped, and the  investigator may consider withdrawal of this  participant from the study. |                                                                                                                                                                                                                                                                                                    |
| Grade 3 or Grade 4 - severe or  life-threatening o Grade 3: Prolonged (for example, not  rapidly responsive to symptomatic  medication and/or brief interruption of  infusion); recurrence of symptoms  following initial improvement;  hospitalization indicated for clinical  sequelae. o Grade 4: Life-threatening consequences;  urgent intervention indicated.                                                                                                                                                                                           |                                                                                                                                                                                                                      | Stop the infusion of study intervention  immediately and disconnect infusion tubing  from the participant with additional appropriate  medical measures and closely monitor until  deemed medically stable by the attending  Investigator. Hospitalization and/or close  monitoring is recommended |
| Administration of glucocorticoids may be required  For Grade 3 or 4 IRRs, permanent  discontinuation of study intervention is  mandated.                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                    |
| Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must remain  decreased for all subsequent infusions.  For all types and grades of infusion reactions, details about drug physical constitution, method of  preparation, and infusion must be recorded. Participants should be instructed to report any delayed reaction immediately. 2 IRR=infusion-related reactions, IV=intravenous, NCI-CTCAE=National Cancer Institute-Common  Terminology Criteria for Adverse Event, NSAIDs=nonsteroidal anti-inflammatory drugs. |                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                    |  
(including reducing the infusion rate by 50%), the investigator may consider treatment with corticosteroids and the infusion of M7824 should be stopped for that day. At the next infusion, the investigator may consider the addition of H2-blocker antihistamines (for example, famotidine), in addition to premedication, for select subjects. However, prophylactic steroids are NOT permitted. If the subject has a second infusion-related reaction Grade ≥ 2 on the slower infusion rate, with or without the addition of further medication to premedication, the infusion should be stopped and the subject may be removed from M7824 treatment at the discretion of the investigator. 3.3.1.6 Immediate Hypersensitivity Reaction Hypersensitivity reactions may require immediate intensive care. Bintrafusp alfa should be administered in a setting that allows immediate access to an intensive care unit or equivalent environment and administration of therapy for anaphylaxis, such as the ability to implement immediate resuscitation measures. Potent steroids (e.g. dexamethasone), catecholamines (e.g. epinephrine), allergy medications (IV antihistamines), bronchodilators, or equivalents and oxygen should be available for immediate access. A complete guideline for the emergency treatment of anaphylactic reactions according to the Working Group of the Resuscitation Council United Kingdom and can be found at https://www.resus.org.uk/pages/reaction.pdf. 3.3.1.7 Severe Hypersensitivity Reactions and Flu-like Symptoms If a hypersensitivity reaction occurs, the subject must be treated according to the best available medical practice including ACLS guidelines. Subjects should be instructed to report any delayed reactions to the investigator immediately. A. Symptoms  
Impaired airway Decreased oxygen saturation (< 92%)
Confusion Lethargy
Hypotension  
Pale / clammy skin Cyanosis B. Management Epinephrine injection and IV dexamethasone Patient should be placed on cardiac, blood pressure, heart rate, and oxygen saturation monitor immediately Alert intensive care unit for possible transfer if required
Treatment is based on clinical assessment and at the discretion of the Investigator. For prophylaxis of flu-like symptoms, a NSAID, for example, ibuprofen 400 mg or comparable NSAID dose, may be administered 2 hours before and 8 hours after the start of each dose of M7824 IV infusion. 3.3.1.8 Immune-Related Adverse Events Immune-related adverse events (irAEs) are specific to immunotherapy. IrAEs are important identified risks for M7824. Immune-related adverse events should be documented as an 'Adverse Event of Special Interest (AESI),' and it is recommended to involve the principal investigator at first incidence and subsequently as needed for follow-up. Details of the diagnostic work-up will be requested by the study team. An immune related adverse event (irAE) is defined as off target side effects associated with exposure of immunogenic drug and is consistent with immune mechanism. In the process of identification of irAEs, any possible etiology of neoplastic, infectious, metabolic, toxin, or any other factor should be ruled out. Serologic, histologic (biopsy), and/or immunologic results should be obtained to evaluate the differential diagnosis and/or support an immune-mediated cause. The recommendations for irAE management, in accordance with the joint American Society of Clinical Oncology (ASCO) Clinical Practice Guidelines (Brahmer, 2018) and National Comprehensive Cancer Network (NCCN Guidelines®). General management by CTCAE v5.0 grading, as per ASCO, is listed below (ASCO organ/system specific toxicity management takes precident over below general guidelines): Grade 1: study treatment should be continued with close monitoring, with the exception of some neurologic, hematologic, and cardiac toxicities. Grade 2: study treatment may be suspended for most Grade 2 toxicities, with consideration of resuming when symptoms revert to Grade 1 or less. Corticosteroids may be administered (initial dose of 0.5 to 1 mg/kg/d of prednisone or equivalent). Grade 3: study treatment is generally suspended and the high-dose corticosteroids (prednisone 1 to 2 mg/kg/d or methylprednisolone 1 to 2 mg/kg/d) treatment should initiated. Corticosteroids should be tapered over the course of at least 4 to 6 weeks. Some refractory cases may require infliximab or other immunosuppressive therapy. Grade 4: in general, permanent discontinuation of study treatment is recommended, with the exception of endocrinopathies that have been controlled by hormone replacement.
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3.3.1.9 anemia

For anemia events assessed as treatment-related, items queried may include but are not limited to detailed relevant past medical and treatment history, bruising tendency, history of blood transfusions and/or dependency, details such as concomitant medications, all laboratory data, updated dosing information and recent tumor evaluation scans. M7824 treatment-related anemia is an important risk (see Investigators' Brochure). Notably, there are many reasons for anemia in patients with advanced cancer, and Hb level of at least 9 g/dl is required for this study. A thorough investigation of new anemia cases of unspecified etiology is requested. Safety laboratory testing of relevant blood parameters is conducted per schedule of assessment. General Guidance for anemia management and evaluation:
Participants must enter the study with Hgb values at least 9 g/dL and baseline anemia evaluation is conducted per **Table 3.**
All relevant hematologic testing for treatment related anemias should be done prior to blood transfusion, if clinically feasible.  
If a participant experiences significant anemia (<8g/dl), then the amount of blood to be drawn may be reduced by not taking blood at selected time points for -exploratory biomarkers. The decision to reduce the time points for these biomarkers will be taken by the investigator in consultation with the medical monitor. This will be documented.  
Transfusion should be performed at the discretion of the investigator, based on clinical assessment and considered when participant experiences significant anemia. Attempt should be made to initiate work-up (as specified below) for cause of anemia prior to transfusion if clinically feasible to not confound this workup. Version Date: 06/23/2021 Guidance for evaluation of suspected treatment-related anemias is provided in **Table 3.**
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management of treatment-related anemias is

Table 3: Evaluation Guidance of Suspected Treatment-related Anemia AEs Basic anemia evaluation (prior to transfusion, if feasible) CBC with differential (e.g. MCV, RDW, ANC, hemoglobin, hematocrit) Reticulocytes counts Peripheral blood smear for cell morphological assessment Complete metabolic panel including liver panel-LFTs, bilirubin, LDH, renal function, and serum folate, B12 values and other chemistries Coagulation factors (PT, PTT, INR) Urinalysis including culture Iron panel (TIBC, ferritin, Fe) TSH/thyroid panel Fecal-occult blood testing Erythropoietin Haptoglobin Further recommendation based on suspected etiology (in addition to baseline anemia testing)  
| Unknown etiology, suspect   | Coombs test, fibrinogen, d-dimer                                                                                                                                                 |         |                         |
|-----------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------|-------------------------|
| possible hemolysis          | Consider hematology consultation. Consider blood transfusion at clinical discretion.                                                                                             |         |                         |
| Unknown etiology, suspect   | Consider blood transfusion at clinical discretion.                                                                                                                               |         |                         |
| possible bleeding           | Consider surgical/interventional radiology consultation. Consider imaging, as clinically indicated (e.g. FAST scan, CT scan, MRI, angiography). Consider endoscopy (upper/lower) |         |                         |
| Unknown                     | etiology                                                                                                                                                                         | despite | Hematology consultation |
| above work-up               | Consider bone marrow aspiration/morphologic evaluation                                                                                                                           |         |                         |  
Table 4: Management of Treatment-Related Anemia Hematology assessment must be performed at baseline, prior to each bintrafusp alfa dose, at the end of treatment visit and at 28 (±5 days) days post-treatment safety follow-up.  
Participants must enter the study with Hgb values at least 9g/dl All relevant hematological testing for treatment-related anemias should be done prior to a blood transfusion, if clinically feasible
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diagnostic work-up

|    | Baseline Anemia Evaluation  CBC with emphasis on red cell indices                                                                                                                                                                                  |
|-----|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
|    | If indicated and at clinical discretion, the following should be considered:  Iron studies  Serum Folate and Vit B12 values  Coagulation factors  Fecal occult blood  Urinalysis  Hormone panel: TSH, Erythropoietin  Peripheral blood smear |  
Further recommendation based on suspected etiology (in addition to basic anemia testing)
Suspected Hemolysis bilirubin, LDH, Coombs test, haptoglobin Suspected bleeding:
Consider imaging/interventional radiology consultation as indicated Consider imaging and/or endoscopy as clinically indicated Suspected aplastic anemia:
Hematology consultation Consider bone marrow aspiration/morphologic evaluation Additional consideration In general, blood transfusions and erythroid growth factors are permitted as clinically indicated.  
3.3.1.10 Rash with Hyperkeratosis / Keratoacanthoma / Squamous Cell Carcinoma of the Skin Monitoring will include skin assessments every 4 weeks with biopsy of suspicious lesions. Dermatological consults should be requested as needed. Treatment modifications for reactions caused by M7824 are addressed in Table 5.  
Table 5: Management of M7824 mediated Skin Reactions Hyperkeratosis Keratoacanthoma Cutaneous squamous cell carcinoma (cSCC)
Basal cell carcinoma Actinic keratosis Management Discontinuation or termination not required in most cases. Continuation of treatment should be evaluated by the Investigator.  
Emollients may be used Develop diagnostic and treatment plan in collaboration with Investigator and dermatologist Treatment follow-up will depend on number and localization of lesions.  
o Single lesion: full excision may be recommended o Multiple lesion or location not suitable for full excision: Mohrs surgery, cryotherapy or other standard treatment options depending on pathology. Retinoids may be used per Investigator decision.  
Close clinical follow-up for re-evaluation, resolution and potential recurrence should be implemented In general, treatment of M7824 mediated skin lesions should be based on local guidelines/standard of care.  
Additional consideration: Keratoacanthoma lesions may resolve spontaneously without surgical intervention within weeks after discontinuing bintrafusp alfa. Consult with Medical Monitor as needed for management of M7824 mediated skin lesions.
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3.3.1.11 impaired wound healing o

Management should be discussed on a case-by-case basis. Dermatological consults should be requested as needed.  
Impaired wound healing is considered an important potential risk for bintrafusp alfa Management should be discussed with Medical Monitor for participants requiring surgery on study.  
It is recommended to hold study intervention for approximately 4 weeks post major surgery for observation.  
Post-operative wound healing should be closely monitored 3.3.1.12 Dose Interruptions for Adverse Events not Related to Study Drug In case of Grade 3 and Grade 4 AEs not study drug related, the study treatment may be interrupted based on the investigator assessment and the subject will be medically treated for the event. If the AE reduces to a lower tolerable grade the study treatment might be resumed in the subsequent cycle. If the AE remains the same despite the medical treatment until the next treatment (second cycle after the AE occurred) a consideration of a possible extension of the dose interruption for up to 1 additional cycle or a permanent withdrawal from the study treatment should be considered. If upon the resumed study treatment, the subject experiences the same AE, permanent withdrawal from the study treatment should be considered. Grade 3 and 4 laboratory abnormalities that do not have clinical significance and are not related to study drugs do not require dose interruption.
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3.3.1.13 embryo-fetal toxicities

Embryo-fetal toxicities are a known risk of the PD-1/PD-L1 targeting class and are considered important potential risk for M7824. Animal models link the PD-1/PD-L1 signaling pathway with maintenance of pregnancy through induction of maternal immune tolerance to fetal tissue [34-37].  
Embryofetal toxicity is an important potential risk of bintrafusp alfa. Embryo-fetal and reproductive toxicities have also been investigated in animal models for a humanized mAb targeting TGFβ1. At doses as high as 30 mg/kg, no maternal reproductive toxicity or embryo-fetal lethality were observed in rabbits [38]. To mitigate these potential risks, pregnant participants are excluded from the study, and all participants of childbearing/conceiving potential must use highly effective contraception (See Section **2.1.1.11**).
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3.3.1.14 mild to moderate bleeding

Bleeding events of mild to moderate severity were observed in participants treated with M7824 in ongoing studies and are a potential risk for M7824. Events may include epistaxis, hemoptysis, gingival bleeding, or hematuria among others. In general, majority of these events were observed to be self-limiting, did not require intervention, and resolved without discontinuation of study treatment. Management of bleeding is provided in **Table 6**.  
| Table 6: Management of Bleeding Adverse Events Bleeding Adverse Events  Bleeding adverse events are considered  important identified risk for bintrafusp  alfa.  In general, mild and moderate mucosal  bleedings resolve without  discontinuation of treatment.  These events may include, but are not  limited to the following: o Epistaxis o Hemoptysis  o Gingival bleeding o Hematuria Non-tumor Bleeding Grading   | Management                                                                                                                                                                                                              |                                                                                                                                                                    |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 2                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                        | If resolves to Grade ≤ 1 by the day before the next infusion, study  intervention may be continued                                                                 |
|                                                                                                                                                                                                                                                                                                                                                                                                                             | If not resolved to Grade ≤ 1 by the day before the next infusion, but is  manageable and /or not clinically relevant, consult Medical Monitor to  assess if clinically reasonable to administer the following infusion. |                                                                                                                                                                    |
| Grade 3                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                        | Permanently discontinue treatment unless an alternative explanation  can be identified (such as concomitant use of antithrombotic agents,  traumatic events, etc.) |
|                                                                                                                                                                                                                                                                                                                                                                                                                             | In case of alternative explanations, hold study treatment until the  event recovers to Grade ≤ 1                                                                                                                        |                                                                                                                                                                    |
| Grade 4                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                        | Treatment must be permanently discontinued if no alternative  explanation is identified.                                                                           |
| Tumor Bleeding Grade ≥ 2                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                        | Study treatment must be held till the event recovers to Grade ≤ 1                                                                                                  |
|                                                                                                                                                                                                                                                                                                                                                                                                                             | Permanently discontinue treatment if the Investigator considers the  participant to be at risk for additional severe bleeding.                                                                                          |                                                                                                                                                                    |
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3.5 cost and compensation 3.5.1

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 performed outside the NIH Clinical Center, participants may have to pay for these costs if they are not covered by insurance company. Medicines that are not part of the study treatment will not be provided or paid for by the NIH Clinical Center. 3.5.2 Compensation No compensation will be provided for study participation.  
3.5.3 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..
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3.6 criteria for removal from

Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 28-30 days after the last dose of study therapy.  
3.6.1 Criteria for removal from protocol therapy  
Completion of study therapy
Progressive disease  
DLT if clincially indicated.
Intercurrent illness or medical circumstances. Voluntary withdrawal from the treatment regimen  
PI determines further treatment on this study is not in patient's best interest Patients who are off-treatment but still on-study will be followed as per the schedule in Appendix A: Study Calendar, until they begin alternative therapy or voluntarily withdrawal from follow-up testing. Thereafter, they may be followed on-study for survival and other secondary endpoints.
Positive pregnancy test 3.6.2 Off-Study Criteria Death Patient request to be withdrawn from study  
Non-compliance with study treatment and/or testing, or lost to follow-up, at discretion of PI
3.6.3 Lost to Follow-up A participant will be considered lost to follow-up if he or she fails to return for 6 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 3 months 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, a 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.
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4 concomitant medications/measures 4.1 permitted

Any medications (other than those excluded by the clinical trial protocol) that are considered necessary to protect subject welfare and will not interfere with the trial medication may be given at the Investigator's discretion.
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4.2 prohibited medicines the following

Immunotherapy including interferon, immunosuppressive drugs (for example, systemic corticosteroids except for short term treatment of allergic reactions, endocrine replacement therapy at low dose prednisone [>10 mg daily] or equivalent, or for the treatment of irAEs or other appropriate short-term steroid use), or other experimental pharmaceutical products. Short term administration of systemic steroid or other immunosuppressant such as infliximab or mycophenolate (that is, for allergic reactions or the management of irAEs) is allowed. Steroids with no or minimal systemic effect (topical, inhalation) are allowed.  
Prophylactic use of corticosteroids for infusion related reactions is prohibited.  
Herbal remedies with immunostimulating properties (for example, mistletoe extract) or known to potentially interfere with major organ function (for example, hypericin).  
Any live vaccine therapies for the prevention of infectious disease. Administration of inactivated vaccines is allowed (for example, inactivated influenza or SARS-CoV-2 vaccines, and locally approved COVID vaccines).  
If the administration of a non-permitted concomitant drug becomes necessary during the trial, the subject will be withdrawn from trial treatment.
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5 correlative studies for research

Please refer to Section 5.2 for sample collection and timepoints. 5.1.1 Immune Monitoring and Peripheral Immune Subset Analysis Peripheral Blood Mononuclear Cells (PBMCs) will be analyzed for changes in standard immune cell types (T-cell receptor clonality, 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, as described elsewhere [39, 40].  
PBMC analysis may include phenotypic and functional analysis of immune-cell subsets (such as CD4 and CD8 T cells, NK cells, Tregs, and MDSCs) including proliferation, cytokine production, lysis, and suppression using multicolor flow cytometry based assays.  
For PBMCs collection, six 10mL sodium heparin (green top) tubes will be used.
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sample transport/storage

Samples will be stored by Clinical Services Program - Leidos Biomedical Research, Inc. (CSP).. Attn: Theresa Burks/Dr. Inglefield 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. Samples can be picked up Monday through Friday, three times per day (~9, 11, 2). Samples will be analyzed by the Jeffery Schlom, Laboratory of Tumor Immunology and Biology (LTIB)NCI. 5.1.2 Comprehensive Cytokine/Chemokine analysis To evaluate for circulating systemic levels of cytokines, chemokines of patients on treatment and correlate levels and changes with clinical activity of the study drug, plasma will be collected per PI discretion:  
1. Plasma may be analyzed pre- and post-therapy for the following soluble factors: sCD27, sCD40 ligand using assays adapted to ECL platform.  
2. Plasma may be analyzed for changes in cytokines (IFN-γ, IL-6, IL-8, TNF-α , etc.),
chemokines, antibodies, tumor-associated antigens, and/or other markers using ELISA or multiplexed assays (e.g. Mesoscale, Luminex, cytokine bead array).
For plasma collection, one 4 ml lavender top EDTA tube will be used..  
Blood samples will be stored by Dr. Figg's laboratory and processed and analyzed by Dr. Liang Cao's laboratory. For Figg lab, please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov by e-mail or at 240-858-3191.  
5.1.3 Circulating cell-free DNA (cfDNA) isolation The primary purpose of these studies is to evaluate for circulating cell-free DNA (cfDNA) of patients on treatment and correlate levels and changes with clinical activity of the study drug combination.  
Blood draw and storage o Draw 2 X 10 mL blood samples to EDTA tubes (lavender tops) follow the manufacture's instruction.  
o Remove tube from adaptor and immediately mix by gentle inversion 8 to 10 times.  
CRITICAL STEP: Do not shake or vortex tubes as cellular lysis could occur.  
o Tubes should be stored and transport at room temperature for up to four (4) hrs.  
Do not freeze.  
Blood/plasma samples will be stored by Dr. Figg's laboratory and processed and analyzed by Dr. Liang Cao's laboratory. For Figg lab, please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov by e-mail or at 240-858-3191.
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5.1.4 hgf and met

Plasma will be collected to determine whether plasma HGF and Met levels are biomarkers for metastatic non-prostate genitourinary tumors and/or response to treatment.  
10mL of whole blood collected in one 10mL EDTA tube at room temperature will be collected at each time point. Plasma samples will be stored by Dr. Figg's laboratory and processed and analyzed by Dr. Cao's laboratory. 5.1.5 Circulating Tumor Cells (CTCs) To evaluate and characterize changes in expression of immune-related markers on CTCs. All patients enrolled on study will have CTC's measured using Epic Science's "no cell left behind" platform [41].  
CTC Samples should be collected in the 10 mL Streck Cell-Free DNA BCT. These tubes are commercially available through Streck (Omaha, NE). IMPORTANT: The first 5 mL of blood collected from the fresh venipuncture cannot be used for the collection into the Streck tubes due to possibility of contaminating epithelial cells during venipuncture. Please ensure that at least one blood tube of 5 mL or more is collected prior to collection of the CTC sample to avoid adversely affecting the test results. Prevention of Backflow: Since Streck Cell-Free DNA BCT tubes contain chemical additives, it is important to avoid possible backflow from the tube. To guard against backflow, observe the following precautions:
Keep patient's arm in the downward position during the collection procedure.  
Hold the tube with the stopper uppermost.  
Release tourniquet once the blood starts to flow into the tube, or within 2 minutes of application.  
Tube contents should not touch stopper or the end of the needle during the collection procedure.
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blood collection instructions

**Arrange for the same day shipment!  
Confirm blood tube is not expired. Expired tubes should not be used for blood collection.  
Draw whole blood sample into 10 mL Streck Cell-Free DNA BCT tube (*see note regarding prevention of backflow). Fill tube until blood flow stops. NOTE: Epic requires a minimum of 4 mL blood per sample, but a full 10 mL tube of blood should be provided when possible.  
Remove tube from adapter and immediately mix by gentle inversion 8 to 10 times. Tube inversion prevents clotting. Inadequate or delayed mixing may result in inaccurate test results.  
Label the tube with subject's identification and date and time of blood draw. Unlabeled blood tubes may not be processed.  
Keep sample at room temperature and ship on day of collection in shipper with ambient gel packs.
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specimen shipment logistics

All shipments must include requisition forms that contain Patient ID, Collection Date and time, Collection Site (including address), Time Point (if applicable), and the appropriate trial code (20C0012/ Epic Internal ID: NE-003). Clinical sites should provide email notification of sample shipment to Epic Sciences on the day of collection. The email should contain:
Trial codes (20C0012/ Epic Internal ID: NE-003)
Patient ID  
Collection date and time* Time point/Visit
Tracking information If possible, include a scanned copy of the completed sample requisition form. Partner will be responsible for all blood collection supplies, shipping materials, and shipping expenses.  
* When collection time is not provided, Epic Sciences will assume the sample was collected at 8:00AM (local time) on the date of collection.  
Send notification to: partners@epicsciences.com Ship to: Epic Science Attn: (20C0012/Epic internal ID: NE-003) 9381 Judicial Dr. Suite 200 Sand Diego, CA 92121 Contact phone number is: 1-858-356-6610 Epic Science will promptly notify the project stakeholders via email of any holiday closures. 5.1.6 c-Met analysis by RT-PCR Peripheral blood for analysis of c-Met mRNA will be collected in a PAXgene tube (PreAnalytix; 2.5 cc peripheral blood per tube) per the manufacturer's instructions. After the blood is drawn, the tube should be inverted several times, placed at 4°C. RNA will be isolated using the PAXgene Blood RNA Kit according to the manufacturer's instructions. The first strand cDNA will be synthesized from total RNA by using a HC reverse transcription kit (Applied Biosystems). The reaction mixture is diluted to 100 μL with TE. PCR will be performed using the c-met primers).
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sample transport/storage

Blood/plasma samples will be stored by Dr. Figg's laboratory and processed and analyzed by the Dr. Liang Cao's laboratory. For Figg lab, please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred).  
For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov by e-mail or at 240-858-3191. 5.1.7 Tumor Studies 5.1.7.1 RNA-Seq, TCR sequencing, and MET Protein
**Single Cell RNA-Seq**: from RNA extracted from fresh frozen tumor tissue for transcriptome sequencing (RNA-Seq) to evaluate if RNA gene expression profiling that measure responsive genes relative to antigen presentation, chemokine expression, cytotoxic activity and adaptive immune resistance are correlated with clinical response.  
**TCR Sequencing**: from RNA extracted from fresh frozen tumor tissue: To evaluate and characterize the clonality of the T-cell population as a way to infer the peripheral expansion of tumor-reactive T-cell clones in response to study drug combination.  
**MET protein**: MET protein evaluated from tumor biopsies will be correlated with clinical responses Tumor tissue will be prioritized (highest to lowest): 1) Formalin fixed for diagnostic and DNA/RNA extraction from FFPE (20 slides total)  
- 10 unstained FFPE slides (room temperature) will be sent to Laboratory of Pathology.  
Please contact Dr. Jun Wei (weij@mail.nih.gov), prior to transport. Telephone: 240760-7428.  
- 10 unstained FFPE slides (room temperature) will be stored in the Laboratory of Pathology for additional tumor testing
2) Three cores will be collected when possible and flash frozen for RNA extraction
- For frozen tumor, place 1 core in 1.5mL Eppendorf or 15mL conical tube or 1-2 blocks on dry ice. DNA and RNA extracted by Laboratory of Pathology and sent to Oncogenomics Section for exome sequencing, and TCR sequencing (contact Dr. Young Song, songyo@mail.nih.gov). Single Cell RNA Seq will be performed in the Single Cell Analysis Facility (SCAF) (contact Dr. Michael Kelly, michael.kelly3@nih.gov / 240-760-6301).  
- 1-2 frozen cores will be stored by Dr. Bottaro's lab for additional testing. Please contact Rene Costello (rene.costello@nih.gov / 301-443-6915) to arrange for pick-up of sample)
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ffpe

In the case of exisiting (pre-treatment) FFPE slide/blocks already available for the consenting patients, the sample will be transferred to the Laboratory of Pathology (LP).  
For DNA and RNA to be extracted from FFPE tissues the Laboratory of Pathology in consultation with the surgical pathologist will evaluate specimen for adequacy and diagnosis. For the majority of cases the Laboratory of Pathology will perform both the extraction and quality control (QC) of nucleic acids. This will include DNA and RNA from tumor, and DNA from germline tissue (matched normal, blood,or other tissues). The DNA will be used for exome sequencing, and RNA sequencing will be performed in a research setting. Final approval to proceed to sequencing will be determined by passing all QCs according to the SOPs.
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fresh/frozen tissue

If a pre-treatment biopsy is performed, the surgical sample will be obtained by the interventional radiology or surgical dissection/resection, and the sample collection will be processed as follows: Interventional Radiologist: Recommend 19 gauge needle or larger in caliber after discussion and at the discretion of the intervention radiologist Contact Research Nurse or Rene Costello to be present at time of procedure.  
1. 1-2 cores snap frozen sent to OncoGenomics (contact Dr. Young Song, songyo@mail.nih.gov). Transport to OncoGenomics in 4°C wet ice.  
2. 1-2 cores in container cooled in dry ice for flash freezing. Transport to Don Bottaro's lab in dry ice. (contact Rene Costello, rene.costello@nih.gov).
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sample transport/storage

Laboratory of Pathology (LP) will arrange transportation of the DNA (and RNA sample) to the Genetics Branch. Building 37, Room 6144, Bethesda, MD 20892. Please contact Dr. Jun Wei (weij@mail.nih.gov), prior to transport. Telephone: 240-760-7428. Dr. Wei will ensure that the patient has been consented and that he has been emailed a copy of the consent form before the samples are processed for RNA sequencing The primary purpose of the studies is to utilize Omics high throughput techniques to identify novel biomarkers and targets of response to this study drug.  
**Transcriptome sequencing (RNA-Seq)** from RNA extracted from blood and fresh frozen tumor tissue: To evaluate if RNA gene expression profiling that measure responsive genes relative to antigen presentation, chemokine expression, cytotoxic activity and adaptive immune resistance are correlated with clinical response.  
**T-cell receptor (TCR) sequencing** from RNA extracted from fresh frozen tumor tissue:
To evaluate and characterize the clonality of the T-cell population as a way to infer the peripheral expansion of tumor-reactive T-cell clones in response to study drug.  
5.1.7.2 Genomics studies– TrueSight Oncology Pre-treatment tissue biopsy and/or tissue availability is mandatory. On-treatment biopsy is required unless obtaining biopsy is not technically or clinically feasible. Post-treatment biopsy is optional.  
10 unstained FFPE slides (room temperature) from in-house surgery and outside materials will be brought directly to General Surgical Pathology office - 10/Room 2S262.  
Paraffin-embedded tissue specimens from outside must be formalin fixed. They will be submitted either as a tissue block containing >20% tumor, or a minimum of 10 FFPE unstained slides for molecular pathology testing and surgical pathology review. Alterations to the above requirements on sample submission will be at the discretion of the Laboratory of Pathology. The TruSight Oncology 500 (TSO-500) is a next-generation sequencing (NGS) assay that analyzes cancer-relevant genes from both DNA and RNA in one integrated workflow. During library preparation, enrichment chemistry is optimized to capture nucleic acid targets from formalin-fixed, paraffin-embedded (FFPE) tissues. With simultaneous analysis of both DNA and RNA, various types of biomarkers relevant to a given tumor type (single nucleotide variants (SNVs), indels, fusions, splice variants, tumor mutation burden (TMB), and microsatellite instability (MSI)) can be assessed from the same sample in a single assay. The RNA panel uses a probe design that enables capture of both known fusions and novel fusion partners. The TSO500 panel includes 523 genes for DNA mutation detection and 55 genes for fusion and splice variant detection. The new molecular pathology report is also incorporated with reporting software for clinical actionability as Tier levels of FDA-approved drug and clinical trials, and pathogenicity based on AMP/ASCO/CAP/ACMG guidelines. NIH Clinical Record Interactive System (CRIS) orders will be placed to notify the laboratory of pathology.  
Please contact Mark Raffeld, mraff@mail.nih.gov at 301-480-8927. 5.1.8 Multiplex Immunohistochemistry To correlate baseline expression of PD-L1 (on tumor cells, tumor-infiltrating immune cells or both) with activity of study drug. To evaluate modulation of intratumoral immune contexture / immunophenotype (i.e. CD3, CD8, CD4, FoxP3) in response to study drug compared with baseline.  
- In the case of exisiting (pre-treatment) FFPE slide/blocks already available for the consenting patients, the sample will be transferred to the Laboratory of Pathology (LP).  
- For patients undergoing dedicated research biopsies (baseline or on-treatment C3D1),
tissue will be sent to the Laboratory of Pathology (LP) for accessioning, processing overnight in formalin and embedding in paraffin (FFPE).
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plan for analysis and quantification

Multiplex Immunohistochemistry: 4-5 micron thick sections of tissue (from FFPE blocks mentioned above) will be mounted on 25 x 75mm, charged glass slides (routinely used in Laboratory of Pathology). These unstained slides (US) will undergo several cycles of immunostaining using the Opal Method after initial deparaffinization and hydration. In brief, the tissue will be subject to heat induced epitope retrieval (HIER), followed by blocking agent, then primary and secondary (HRP) antibody incubation. This is followed by signal amplification using tyramide based fluorophores which will covalently bind to tissue at the site of primary antibody attachement (antigen of interest). This is followed by stripping of antibodies using HIER, to allow for 2nd round of new antibodies of intetest. These steps are repeated for total of 6-7 times to allow detection of 6-7 markers. This is followed by DAPI (4',6-diamidino-2-phenylindole) counterstain to allow for nuclear detection. Once all antigens of interest are stained, a multiplex scanning protocol will be created on Vectra Polaris scanner (Perkinelmer), to allow for whole slide imaging at 10-20X. This is followed by higher resolution of regions of interest (ROIs) picked by trained research pathologist (Dr. H. Sater). The scans genetrated at high resolution are called multispectral images (MSIs) and have the im3 format. These can only be analyzed using inform software (available in Dr. Sater's Lab). The software allows for spectral unmixing based on a spectral library built from single stains with each fluorophore used and scanned with multiplex protocol as MSI. The initial step will be to create monoplex IHC using CD3 antibody on all available cases. These will be scanned and MSIs will be generated to achieve the primary endpoint analysis (CD3+ cell density). Quantification of cells expressing CD3 will be perfomed on inform using training sets of images to create an algorithm. The algorithm, will be used for quantifying the cell number per area. Representative ROIs (planned to be 5 in number and randomly selected) might vary based on tissue size. Once CD3+ staining is verified for accuracy and integrity of images, we will move to next step which is multiplexing. The antibodies of interest are based on preclinical data or hypothesis driven. We plan to investigate a panel of multiple immune cell susbets such as T-helper cells (CD4+), T regulatory cells (CD4+FOXP3+), Cytotoxic T cells (CD3+CD8+), NK cells (CD56+), macrophages (CD68), B cells (CD19+) and other immune activation/immunsuppresive markers (IDO, PD-1, PD-L1, Ki67, ICOS, CD69, CD27, CD16 and other T cell activation markers) as well as tumor cell markers (Cytokeratin, Vimentin, etc) in a multiplex staining method. This method ensures simultaneous staining of different markers in the same tissue plane for colocaliztion and near neighbor analysis. The staining will be done in a panel like fashion where 6-7 immunohistochemical (IHC) markers plus DAPI are chosen in any single slide. All images are analyzed and saved in a secure server, accessible and monitored by Dr. H. Sater.  
The Sater Lab will request 4-5 micron thick recuts of 5-10 FFPE slides by submitting a Tissue Resource Committee (TRC) request for Human Biological Materials for Research form. These biopsies will be examined by a research pathologist (Dr. H Sater) for the presence of tumor and tissue integrity and at least 5 consecutive recuts of each biopsy is deemed optimal for analysis. Hematoxyln and Eosin staining will be first done on the deepest level cut to reassess integrity of tissue for further analysis. Please contact: Dr. Houssein A. Sater MD / Beatriz Walters (GMC, CCR/NCI) 10 Center Drive, Building 10, Room 6B12 Bethesda, MD 20892 E-mail: houssein.abdulsater@nih.gov 5.1.9 Imaging
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background

Magnetic resonance imaging (MRI) offers a high tissue contrast which allows direct imaging of the bones and soft tissues. Application of whole-body MRI approach has been shown to be clinically useful in certain diseases such as myeloma, lung cancer, lymphoma at both initial diagnosis/staging and treatment follow up [42-44]. In addition to anatomic pulse sequences, inclusion of diffusion weighted MRI (DW MRI) has also been documented to improve disease detection and monitoring steps. In this protocol the whole-body MR imaging protocol will include two different anatomic pulse sequences in coronal planes (T1W MRI and Short TI Inversion Recovery [STIR]) and a functional imaging pulse sequence (b800 diffusion weighted MRI). The DW MRI enables detection of lesions/foci with increased cellularity whereas the two anatomic pulse sequences will enable characterization of these lesions/foci detected on DW MRI. There will be no intravenous gadolinium injection-based imaging sequences, therefore it is a relatively less invasive novel imaging technique for patients with urothelial carcinoma especially considering the need for follow up imaging.
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methods

Whole body MRIs will be performed in B3 Molecular Imaging Clinic using a clinical 3 Tesla scanner. The patients will be imaged in supine position using surface coils and no intravenous contrast injection will be utilized. The imaging will be done in 5 stacks (head/neck, chest, abdomen, pelvis, thighs) for T1W MRI and STIR and in 4 stacks (head/neck, chest, abdomen, pelvis/thighs) for DW MRI. For chest, abdomen and pelvis stacks the patient will be asked to hold Version Date: 06/23/2021 breath for a few times. The active scan time is planned to be 18-25 minutes. The entire imaging session is planned to be 40 minutes. Timepoints: Please refer to Section **5.2** and **Appendix A: Study Calendar** for timepoints.  
| Sequence   | Plane    | FOV            | Slice   |             |              |         |     |            |
|------------|----------|----------------|---------|-------------|--------------|---------|-----|------------|
| Thickness  | TR/TE/TI | Voxel size     | Matrix  | Flip  Angle | Scan Time    |         |     |            |
| T1W        | Coronal  | 450 L-R        | 6 mm    | 694/8/N/A   | Scan 1.6x1.6 | 188x247 | 90  | 4 minutes  |
| 5 stacks   | 300 H-F  | Recon1.17x1.17 |         |             |              |         |     |            |
| STIR       | Coronal  | 450 L-R        | 6 mm    | 8399/70/230 | Scan 1.6x1.6 | 188x248 | 125 | 4 minutes  |
| 5 stacks   | 300 H-F  | Recon 1.0x1.0  |         |             |              |         |     |            |
| DWIBS  (DWI bvalue 800)            | Axial    | 450 L-R        | 6 mm    | 7730/66/220 | Scan 3.5x3.5 | 128x81  | N/A | 10 minutes |
| 4 stacks   | 300 H-F  | Recon 1.7x1.7  |         |             |              |         |     |            |
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mpi mri wb protocol parameters

Coronal 5 stacks: Thighs, Pelvis, Abdomen, Chest, Head/neck Axial DWIBS 4 stacks: Head/Neck, Chest, Abdomen, Pelvis/thighs.  
| 5.2                                                                      | SAMPLE COLLECTION SCHEDULE  Test/Location   | All cycles (28 days)               |                                    |                                    |                                    |                                    |                                    |                                    |
|--------------------------------------------------------------------------|---------------------------------------------|------------------------------------|------------------------------------|------------------------------------|------------------------------------|------------------------------------|------------------------------------|------------------------------------|
| Baseline (C1D1)                                                          | C1D15                                       | C2D1                               | C3D1                               | C4D1                               | C5D1                               | C6D1 and  beyond                   | EOT visit                          |                                    |
| Correlative Research Studies (Blood-based) Immune subsets X X            | X                                           | X                                  | X                                  | X                                  | X                                  | X                                  |                                    |                                    |
| 5.1.1 Peripheral Blood  Mononuclear Cells  (PBMCs) Frederick/Schlom  Lab | 6x10 mL Sodium  Heparin  green top          | 6x10 mL Sodium  Heparin  green top | 6x10 mL Sodium  Heparin  green top | 6x10 mL Sodium  Heparin  green top | 6x10 mL Sodium  Heparin  green top | 6x10 mL Sodium  Heparin  green top | 6x10 mL Sodium  Heparin  green top | 6x10 mL Sodium  Heparin  green top |
| X                                                                        | X                                           | X                                  | X                                  | X                                  | X                                  | X                                  | X                                  |                                    |
| Cytokine / Chemokine  Plasma 5.1.2 Figg/Cao Lab                          | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                             | 1x4 mL  EDTAVacutain er tube  (lavender  top)                                    | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                    | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                    | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                    | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                    | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                    | 1x4 mL  EDTAVacutaine r tube  (lavender  top)                                    |
| X                                                                        | X                                           | X                                  | X                                  | X                                  |                                    |                                    |                                    |                                    |
| cfDNA Plasma 5.1.3 Figg/Cao Lab                                          | 1x10 mL EDTAVacutaine r tube  (lavender  top)                                             | 1x10 mL EDTAVacutainer  tube  (lavender  top)                                    | 1x10 mL EDTAVacutainer  tube  (lavender  top)                                    | 1x10 mL EDTAVacutainer  tube  (lavender  top)                                    | 1x10 mL EDTAVacutainer  tube  (lavender  top)                                    |                                    |                                    |                                    |
| X                                                                        | X                                           | X                                  | X                                  | X                                  | X                                  | X                                  | X                                  |                                    |
| HGF and MET Plasma 5.1.4 Figg/Cao                                        | 1x10mL                                      | 1x10mL                             | 1x10mL                             | 1x10mL                             | 1x10mL                             | 1x10mL                             | 1x10mL                             | 1x10mL                             |
| EDTAVacutaine r tube  (lavender  top)                                                                          | EDTAVacutaine r tube  (lavender  top)                                             | EDTAVacutainer  tube  (lavender  top)                                    | EDTAVacutainer  tube  (lavender  top)                                    | EDTAVacutainer  tube  (lavender  top)                                    | EDTAVacutainer  tube  (lavender  top)                                    | EDTAVacutainer  tube  (lavender  top)                                    | EDTAVacutainer  tube  (lavender  top                                    |                                    |
| X                                                                        | X                                           | X                                  | X                                  |                                    |                                    |                                    |                                    |                                    |
| c-Met analysis Blood                                                     | 2x 5ml                                      |                                    |                                    |                                    |                                    |                                    |                                    |                                    |
| 5.1.6 Figg/Cao Lab                                                       | PAXGene  RNA                                | 2x 5ml                             | 2x 5ml                             | 2x 5ml                             |                                    |                                    |                                    |                                    |
| PAXGene  RNA                                                             | PAXGene  RNA                                | PAXGene  RNA                       |                                    |                                    |                                    |                                    |                                    |                                    |
| X                                                                        | X                                           | X                                  | X                                  |                                    |                                    |                                    |                                    |                                    |
| Circulating Tumor  Cells (CTCs)  5.1.5 Peripheral Blood EPIC             | 1x10mL Streck  BrownBlack  tube                                             |                                    |                                    |                                    |                                    |                                    |                                    |                                    |
| 1x10mL Streck  BrownBlack tube                                                                          | 1x10mL Streck  BrownBlack tube                                             | 1x10mL Streck  BrownBlack tube                                    |                                    |                                    |                                    |                                    |                                    |                                    |  
| Test/Location                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | All cycles (28 days)   |      |      |      |      |                  |           |     |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------|------|------|------|------|------------------|-----------|-----|
| Baseline (C1D1)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | C1D15                  | C2D1 | C3D1 | C4D1 | C5D1 | C6D1 and  beyond | EOT visit |     |
| Total Research Blood  Volume per                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | 118                    | 88   | 98   | 118  | 78   | 108              | 78        | 118 |
| Timepoint (mL) Correlative Research Studies (Tumor-based) Single cell RNA-Seq 5.1.7.2 X X* 3 cores snap frozen  tissue Kelly Lab TCR-Seq 5.1.4 Fresh frozen 1 core  biopsy tissue LP X X* TSO 5.1.7.2 FFPE/Core biopsy  fresh tissue Raffeld lab ** X MET protein 5.1.7.1 10 Slides and/or 1-2  cores Bottaro lab X X* Multiplex IHC 5.1.8 FFPE tissue, at least 5  consecutive slides Sater lab X X* Correlative Research Studies (Imaging-based) Whole body  MRI*** X X 5.1.9 MIP, Turkbey *Between cycles 2 and 4 ** Optional collection based on history of prior tumor sequencing or clinical indication at the PI's discretion. |                        |      |      |      |      |                  |           |     |  
*Between cycles 2 and 4 ** Optional collection based on history of prior tumor sequencing or clinical indication at the PI's discretion. *** Mandatory unless the participant is unable to lay flat or be in a confined space for the duration of the procedure.
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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) without appropriate approvals and/or agreements, if required.
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5.3.1 laboratory of pathology

All consenting patients with available tissue will undergo comprehensive Omics analysis on the tumor and normal DNA, RNA. Samples collected from patients for Omics studies will be labeled with the medical record number, patient name, accessioning with SoftPath, barcode generated by Labmatrix and an assigned unique identifier. This information will not be released to patients or families as these results are considered exploratory and of no established clinical utility. Research samples will be labeled with the Study ID number, barcode from Labmatrix and samples ID which is sequentially determined (e.g. CL0001, CL0002, etc., which is automatically coded by the LIMS system utilized ). Laboratory personnel will store the key to the code in a secure, password protected database accessible only to them. The entire DNA and RNA sequence data will be deposited in secure OncoGenomics server which exists within the NIH firewall. All data is coded and stripped of identifiable information. The code is kept by the principal investigator and the companion protocol PI and/or enrolling investigator. The data is stored in the form of coded BAM files, and processed vcf files contacting all calls in the germline and tumor DNA. The investigator will have access only to the subdirectory that contains the data from his/her patients. The folder can only be accessed using the investigators NIH username and password. All data will be coded and stripped of identifiable information. 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 tissue that is not placed in paraffin blocks is stored in formalin 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 patient was enrolled.
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5.3.2 bottaro laboratory

Biospecimens will be collected and processed using validated SOPs that will ensure both specimen quality and patient confidentiality. Using a computerized inventory system and a backup hardcopy process, all specimen collection and processing steps will be documented and the specific location of each specimen will be tracked. Please contact Dr. Bottaro to notify of biopsy date and time at 240-858-3967 or Rene Costello at 240-858-3889 and to arrange for pick-up of sample.
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5.3.3 figg lab

All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and stored in the Labmatrix utilized by the BPC. This is a secure program, with access to Labmatrix limited to defined Figg lab personnel, who are issued individual user accounts. Installation of Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without Labmatrix access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer locations. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (e.g. delay in sample processing, storage conditions on the ward, etc.). Barcoded samples are stored in barcoded boxes in locked freezers at either -20 C or -80 C according to stability requirements. These freezers are located onsite in the CPP and offsite at NCI Frederick Central Repository Services in Frederick, MD. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in Labmatrix. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per IRB approved protocol) and that any unused samples must be returned to the BPC. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. 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 below. 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. If, at any time, a patient withdraws consent, the participants 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. The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of Section 7.2. Sample barcodes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of the Labmatrix. It is critical that the sample remains linked to patient information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate pharmacokinetic data with these variables.
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5.3.4 clinical services program

All data associated with the patient 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, limitedaccess 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 patient 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 patient-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.
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5.4 samples for genetic/genomic analysis

5.4.1 Description of the scope of genetic/genomic analysis Using the tissue, and blood samples we will perform next research generation sequencing (NGS) of DNA (Exome or Panel). 5.4.2 Privacy and Confidentiality of medical information/biological specimens Initially the samples of each patient will be barcoded. At no time will patient's names be used on the blood and tissue samples. Sometimes, because a group collaboration or journal policy requires it, a subject's genetic data will be deposited in a database such as dbGaP. Although there is controlled access to such a database, such a submission carries theoretical risks of revealing the identity of the subject. This is discussed in the consent.
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5.4.3 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 with the NCI Genetics Branch 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. 5.4.4 Genetic counseling The costs of CLIA testing will be paid for by the Center for Cancer Research, the Branch, or the Principal Investigator. If the health history, family history, or tumor diagnosis from the Laboratory of Pathology at the NIH Clinical Center suggests that the participant might benefit from genetic testing, we will discuss this with him/her.
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6 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) 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 30 days after the end of treatment. Beyond 30 days after the last dose of study drug, 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 patient'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**. 6.1.1 Tumor data The tumor disease information that will be documented and verified at the screening visit for each subject includes the following. All these details will be documented in patient's history and physical exam and also recorded in the C3D database.  
Detailed history of the tumor, including histopathological diagnosis, grading and staging in accordance with the Union Internationale Contre le Cancer Tumor Node Metastasis Classification at diagnosis (UICC TNM).
o The T and M category (T1-3 vs T4 and M0 or M1) of the tumor at the time of study entry, based on screening assessments All therapy used for prior treatment of the tumor (including surgery, radiotherapy and chemotherapy, immunotherapy).  
Any other conditions that were treated with chemotherapy, radiation therapy, or immunotherapy.  
6.1.2 Concomitant Medications and Therapies All concomitant medications taken by the subject during the trial, from the date of signature of informed consent are to be recorded in the appropriate section of the eCRF, noting the name, dose, duration and indication of each drug. Nondrug interventions (other than vitamins) and any changes to a concomitant medication or other intervention should also be recorded in the eCRF.
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6.1.3 data elements

Documentation of dosage and timing of drug administration Outside laboratory, radiologic, and pathology results will be sent to the PI and entered into NCI C3D at NIH
NIH labs and tests will be downloaded into C3D
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6.2 data sharing plans

6.2.1 Human Data Sharing Plan Human data generated in this research for future research will be shared as follows:
Coded, linked data in an NIH-funded or approved public repository.  
Coded, linked data in another public repository.
Identified or coded, linked data in BTRIS (automatic for activities in the Clinical Center)
Data will be shared through:
An NIH-funded or approved public repository: clinicaltrials.gov, dbGaP
BTRIS (automatic for activities in the Clinical Center)  
Approved outside collaborators under appropriate individual agreements. Publication and/or public presentations.
Data will be shared:
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.
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6.3 response criteria

For the purposes of this study, patients should be re-evaluated for response every 8 weeks. After one year response evaluation will be done every 12 weeks. In addition to a baseline scan, confirmatory scans should also be obtained 4-8 weeks (not less than 4 weeks) following initial documentation of objective response. Response and progression will be evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) [45]. Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria. 6.3.1 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:
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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 patient, 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. 6.3.2 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. 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. 6.3.3 Response Criteria 6.3.3.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study. 6.3.3.2 Evaluation of Non-Target Lesions Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal progression* of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
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6.3.3.3 evaluation of best overall

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| For Patients with Measurable Disease (i.e., Target Disease) Target  Non-Target  New  Overall  Best Overall Response when  Lesions Lesions Lesions Response Confirmation is Required* CR CR No CR >4 wks. Confirmation** CR NonCR/Non-PD No PR CR Not  No PR evaluated >4 wks. Confirmation** PR NonCR/NonPD/not  evaluated No PR SD NonCR/NonPD/not  evaluated No SD Documented at least once >4  wks. from baseline** PD Any Yes or  PD No Any PD*** Yes or  PD No no prior SD, PR or CR Any Any Yes PD * See RECIST 1.1 manuscript for further details on what is evidence of a  new lesion. ** Only for non-randomized trials with response as primary endpoint. *** In exceptional circumstances, unequivocal progression in non-target  lesions may be accepted as disease progression. Note: Patients with a global deterioration of health status requiring  discontinuation of treatment without objective evidence of disease  progression at that time should be reported as "symptomatic  deterioration." Every effort should be made to document the objective  progression even after discontinuation of treatment.   |
|---|  
| Non-Target Lesions                                                                                                                                                                                                                             | New Lesions   | Overall Response   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------------|
| CR                                                                                                                                                                                                                                             | No            | CR                 |
| Non-CR/non-PD                                                                                                                                                                                                                                  | No            | Non-CR/non-PD*     |
| Not all evaluated                                                                                                                                                                                                                              | No            | not evaluated      |
| Unequivocal PD                                                                                                                                                                                                                                 | Yes or No     | PD                 |
| Any                                                                                                                                                                                                                                            | Yes           | PD                 |
| * 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease  since SD is increasingly used as an endpoint for assessment of efficacy in some  trials so to assign this category when no lesions can be measured is not advised |               |                    |
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6.3.4 duration of response

Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. 6.3.5 Progression-Free Survival PFS will be defined as the duration of time from start of treatment to time of progression or death, whichever occurs first. 6.3.6 Response Review 6.3.6.1 Best overall response (BOR) The duration of best overall response is measured from the time measurement criteria are met for CR, PR or SD (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. 6.3.6.2 Duration of stable disease Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. Stable Disease (SD) when sum of all target lesions does not qualify for CR/PR/PD for Target Lesion Response and Persistence of non-target lesions on Non-Target Lesion Response.
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6.4 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm).
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7 nih reporting requirements/data and

Please refer to definitions provided in Policy 801: Reporting Research Events found here.
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7.2 ohsrp office of compliance

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found here. Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies. 7.2.2 IRB Requirements for PI Reporting at Continuing Review Please refer to the reporting requirements in Policy 801: Reporting Research Events found here.
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7.3 nci clinical director reporting

Problems expeditiously reported to the OHSRP in iRIS will also be reported to the NCI Clinical Director. A separate submission is not necessary as reports in iRIS will be available to the Clinical Director. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email to the Clinical Director unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to Dr. Dahut at NCICCRQA@mail.nih.gov within one business day of learning of the death.
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7.4 nih required data and

The clinical research team will meet on a weekly basis when patients are being actively treated on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. Events meeting requirements for expedited reporting as described in Section 7.2.1 will be submitted within the appropriate timelines. The principal investigator will review adverse event and response data on each patient to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff.
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8 sponsor protocol/safety reporting 8.1

Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including a clinically significant abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product [(ICH E6 (R2)].
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8.1.2 serious adverse events (sae)

An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
Death, A life-threatening adverse event (see Section **8.1.3**) Inpatient hospitalization or prolongation of existing hospitalization  
o A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event.  
o A hospitalization/admission that is solely driven by non-medical reasons (e.g.,
hospitalization for patient convenience) is not considered a serious adverse event.  
o Emergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria.
Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions A congenital anomaly/birth defect.  
Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.
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8.1.3 life-threatening

An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death. (21CFR312.32). 8.1.4 Severity The severity of each Adverse Event will be assessed utilizing the CTCAE version 5. 8.1.5 Relationship to Study Product All AEs will have their relationship to study product assessed using the terms: related or not related.  
Related - There is a reasonable possibility that the study product caused the adverse event.  
Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event.  
Not Related - There is not a reasonable possibility that the administration of the study product caused the event.
8.1.6 Adverse Events of Special Interest (AESI) Adverse events of special interest (AESIs) are serious or nonserious AEs that are of clinical interest and should be closely followed. AESIs include following:  
Infusion-related reactions including immediate hypersensitivity.  
Immune-related adverse events. M7824 mediated skin reactions.  
Anemia.  
Bleeding AEs
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8.2 assessment of safety events

AE information collected will include event description, date of onset, assessment of severity and relationship to study product and alternate etiology (if not related to study product), date of resolution of the event, seriousness and outcome. The assessment of severity and relationship to the study product will be done only by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal investigator or sub-investigator. AEs occurring during the collection and reporting period will be documented appropriately regardless of relationship. AEs will be followed through resolution.  
SAEs will be:
Assessed for severity and relationship to study product and alternate etiology (if not related to study product) by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  
Recorded on the appropriate SAE report form, the medical record and captured in the clinical database.  
Followed through resolution by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to Section 6.1. All serious adverse events recorded from the time of first investigational product administration must be reported to the sponsor with the exception of any listed in Section 8.4.
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8.3 reporting of serious adverse

Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of Special Interest that require expedited reporting must be submitted immediately (within 24 hours of awareness) to OSRO Safety using the CCR SAE report form. Any exceptions to the expedited reporting requirements are found in Section 8.4. All SAE reporting must include the elements described in Section **8.1.6**. SAE reports will be submitted to the Center for Cancer Research (CCR) at: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. CCR SAE report form and instructions can be found at: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions. Following the assessment of the SAE by OSRO, other supporting documentation of the event may be requested by the OSRO Safety and should be provided as soon as possible.
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8.4 waiver of expedited reporting

As overall survival which includes death due to disease progression is part of the study objectives, and captured as an endpoint in this study, death due to disease progression will not be reported in expedited manner to the sponsor. However, if there is evidence suggesting a casual relationship between the study drug and the event, report the event in an expedited manner according to Section 8.3.
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8.6 reporting pregnancy 8.7 regulatory

All required pregnancy reports/follow-up to OSRO will be submitted to:
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. Forms and instructions can be found here: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions. 8.6.1 Maternal Exposure If a patient becomes pregnant during the course of the study, the study treatment should be discontinued immediately, and the pregnancy reported to the Sponsor no later than 24 hours of when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later than 24 hours of when the outcome of the Pregnancy become known, Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects and spontaneous miscarriages that meet serious criteria (Section **8.1.2**) should be reported as SAEs. The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up and documented. 8.6.2 Paternal Exposure Male patients should refrain from fathering a child or donating sperm during the study and for 125 days after treatment. Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 125 days after the last dose should, if possible, be followed up and documented. Following notification from the investigator, CCR, the IND sponsor, will report any suspected adverse reaction that is both serious and unexpected in expedited manner to the FDA in accordance to 21 CFR 31.2.32. CCR will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the study product and the adverse event. CCR will notify FDA and all participating investigators (i.e., all investigators to whom the sponsor is providing drug under its INDs or under any investigator's IND) in an IND safety report of potential serious risks from clinical trials or any other source, as soon as possible, in accordance to 21 CFR Part 312.32. All serious events will be reported to the FDA at least annually in a summary format. Protocol non-adherence is defined as any noncompliance with the clinical trial protocol, GCP, or protocol-specific procedural requirements on the part of the participant, the Investigator, or the study site staff inclusive of site personnel performing procedures or providing services in support of the clinical trial. It is the responsibility of the study Staff to document any protocol non-adherence identified by the Staff or the site Monitor on the OSRO Site Protocol Non-Adherence Log. The protocol-specific, cumulative non-adherence log should be maintained in the site essential documents file and submitted to OSRO via OSROMonitoring@mail.NIH.gov on the first business day of each month over the duration of the study. In addition, any non-adherence to the protocol should be documented in the participant's source records and reported to the local IRB per their guidelines. OSRO required protocol non-adherence reporting is consistent with E6(R2) GCP: Integrated Addendum to ICH E6(R1): 4.5 Compliance with Protocol; 5.18.3 (a), and 5.20 Noncompliance; and ICH E3 16.2.2 Protocol deviations
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9 clinical monitoring

Clinical site monitoring is conducted to ensure that the rights of the participants are protected, that the study is implemented per the approved protocol, Good Clinical Practice and standard operating procedures, and that the quality and integrity of study data and data collection methods are maintained. Monitoring for this study will be performed by NCI CCR Office of Sponsor and Regulatory Oversight (OSRO) Monitoring based on OSRO standards, FDA Guidance E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable regulatory requirements. Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human subject protections and integrity of the study data. The intensity and frequency of monitoring will be based on several factors, including study type, phase, risk, complexity, expected enrollment rate, and any unique attributes of the study and the site. OSRO Monitoring visits and related activities will be conducted throughout the life cycle of each protocol, with the first activity being before study start to conduct a Site Assessment Visit (SAV) (as warranted), followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s) (IMVs), and a study Close-Out Visit (COV). Some monitoring activities may be performed remotely, while others will take place at the study site(s). Monitoring visit reports will describe visit activities, observations, findings of protocol non-adherence and associated action items or follow-up required for resolution of findings. Monitoring reports will be distributed to the study PI, NCI CCR QA, coordinating center (if applicable) and the OSRO regulatory file. If protocol non-adherence is identified by the Monitor (i.e., any noncompliance with the clinical trial protocol, GCP, or protocol-specific procedural requirements on the part of the participant, the Investigator, or the site Staff) the Monitor will note the observation, review with site Staff and if unresolved, request that the Staff document the non-adherence on the protocol-specific OSRO Site Protocol Non-Adherence Log (see Section 8.8).
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10 statistical considerations 10.1 statistical

10.1.1 Primary Endpoint(s) The primary endpoint of this trial is to evaluate the activity of bintrafusp alfa (M7824) as determined by objective response rate (ORR) in two metastatic urothelial carcinoma cohorts:
o Cohort 1: Checkpoint inhibitor naïve Cohort 1A: cisplatin ineligible Cohort 1B: refractory post-platinum therapy o Cohort 2: Checkpoint inhibitor previously treated patients Cohort 2A: previously achieved a CR/PR Cohort 2B: previously had SD/PD
10.1.2 Secondary Endpoint(s)
To evaluate the activity of bintrafusp alfa (M7824) in urothelial carcinoma patients as determined by progression free survival (PFS) and overall survival (OS).  
To obtain additional data to evaluate the safety of bintrafusp alfa (M7824) in urothelial carcinoma
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10.2 sample size determination

10.2.1 Cohort 1A: Checkpoint inhibitor naïve patients who are cisplatin-ineligible In this cohort, it would be desirable to demonstrate that the treatment exceeds a 30% response rate. As such, in this cohort, this trial will be conducted using a Simon minimax two-stage phase II trial design (Simon R, Controlled Clinical Trials 10:1-10, 1989) to rule out an unacceptably low PR+CR rate of 15% (p0=0.15) in favor of an improved response rate of 40% (p1=0.40). With alpha=0.10 (probability of accepting a poor treatment=0.10) and beta = 0.20 (probability of rejecting a good treatment=0.20), the first stage will enroll 9 evaluable patients, and if 0 to 1 of 9 have a clinical response, then no further patients will be accrued in this trial. If 2 or more of the first 9 patients have a response, then accrual would continue until a total of 16 evaluable patients have been treated. As it may take up to several months to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 2 to 4 patients with a response out of 16 patients, this would be an uninterestingly low response rate. If there were 5 or more of 16 (31.3%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (15% response rate), the probability of early termination is 60.0%. 10.2.2 Cohort 1B: Checkpoint inhibitor naïve patients who are refractory post-platinum therapy In this cohort, it would be desirable to demonstrate that the treatment exceeds a 20% response rate. As such, in this cohort, this trial will be conducted using a Simon minimax two-stage phase II trial design (Simon R, Controlled Clinical Trials 10:1-10, 1989) to rule out an unacceptably low PR+CR rate of 10% (p0=0.10) in favor of an improved response rate of 40% (p1=0.40). With alpha=0.10 (probability of accepting a poor treatment=0.10) and beta = 0.10 (probability of rejecting a good treatment=0.10), the first stage will enroll 8 evaluable patients, and if 0 of 8 have a clinical response, then no further patients will be accrued in this cohort. If 1 or more of the first 8 patients have a response, then accrual would continue until a total of 15 evaluable patients have been treated. As it may take up to several months to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 1 to 3 patients with a response out of 15 patients, this would be an uninterestingly low response rate. If there were 4 or more of 15 (26.7%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (10% response rate), the probability of early termination is 43.1%. 10.2.3 Cohort 2A: Checkpoint inhibitor refractory patients with Complete Response (CR) or Partial Response (PR) as per RECIST 1.1 In this cohort, it would be desirable to demonstrate that the treatment exceeds a 15% response rate.  
As such, in this cohort, this trial will be conducted using a Simon minimax two-stage phase II trial design (Simon R, Controlled Clinical Trials 10:1-10, 1989) to rule out an unacceptably low PR+CR rate of 5% (p0=0.05) in favor of an improved response rate of 25% (p1=0.25). With alpha=0.10 (probability of accepting a poor treatment=0.10) and beta = 0.20 (probability of rejecting a good treatment=0.20), the first stage will enroll 12 evaluable patients, and if 0 of the 12 have a clinical response, then no further patients will be accrued in this trial. If 1 or more of the first 12 patients have a response, then accrual would continue until a total of 16 evaluable patients have been treated. As it may take up to several months to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 1 to 2 patients with a response out of 16 patients, this would be an uninterestingly low response rate. If there were 3 or more of 16 (18.8%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (5% response rate), the probability of early termination is 54.0%. 10.2.4 Cohort 2B. Checkpoint inhibitor refractory patients with Stable Disease (SD) or Progressive Disease (PD) as per RECIST 1.1 In this cohort, it would be desirable to demonstrate that the treatment exceeds a 5% response rate. As such, in this cohort, this trial will be conducted using a Simon minimax two-stage phase II trial design (Simon R, Controlled Clinical Trials 10:1-10, 1989) to rule out an unacceptably low PR+CR rate of 5% (p0=0.05) in favor of an improved response rate of 20% (p1=0.20). With alpha=0.10 (probability of accepting a poor treatment=0.10) and beta = 0.20 (probability of rejecting a good treatment=0.20), the first stage will enroll 12 evaluable patients, and if 0 of the 12 have a clinical response, then no further patients will be accrued in this trial. If 1 or more of the first 12 patients have a response, then accrual would continue until a total of 21 evaluable patients have been treated. As it may take up to several months to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 1 to 2 patients with a response out of 21 patients, this would be an uninterestingly low response rate. If there were 3 or more of 21 (14.3%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (5% response rate), the probability of early termination is 54.0%. These phase II cohorts treated with TGFB trap alone may require up to 15+16+16+21=68 evaluable patients. To allow for a small number of inevaluable patients, this trial of up to 4 different cohorts will have an accrual ceiling of 75 patients. It is expected that 2-3 patients per month may enroll onto the trial; thus, it is expected that this trial may complete accrual in 2 to 3 years.
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10.3 populations for analyses

Modified intention to treat: all patients who receive at least one dose of each agent will be included in the statistical analyses performed.
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10.4 statistical analyses 10.4.1 general

Response fractions, and time to event endpoints will be reported along with appropriate confidence intervals. 10.4.2 Analysis of the Primary Endpoints Separately by cohort, the fraction of patients in phase II cohorts who experience a PR or CR using the study treatment will be determined by dividing the number responders by the total evaluable patients. The fraction will be reported along with 80% and 95% two-sided confidence intervals. 10.4.3 Analysis of the secondary efficacy endpoints Progression-free survival (PFS) will be determined using the Kaplan-Meier method, considering those who progress or die without progression as failures, and censoring those who do not. Overall survival (OS) will be determined using the Kaplan-Meier method. Each of these will be calculated starting from the date the patient enrolled onto the trial. Appropriate confidence intervals will be reported for each of these measures. 10.4.4 Baseline Descriptive Statistics Demographic and baseline clinical characteristics of all patients will be reported. 10.4.5 Planned Interim Analyses As indicated above, for each phase II cohort, using a Simon two-stage design, after the required number of evaluable patients have been enrolled in the first stage, an analysis of the response rate will be undertaken to determine if there are sufficient responses to proceed to the second stage. 10.4.6 Sub-Group Analyses Analyses will be performed by arm without further sub-analyses. 10.4.7 Tabulation of Individual Participant Data None will be provided. 10.4.8 Exploratory Analyses 1. Determine peripheral immune modulation using:  
Multi-parametric flow cytometry - immune subset analysis Cytokine/chemokine analysis (i.e TGF-, IFN-, IL-6, IL-8, etc.)
Blood-based immune parameters (neutrophil-to-lymphocyte ratio)
Circulating Tumor Cells (CTCs) and circulating cell-free tumor DNA (cfDNA)
2. Determine immune status of the tumor microenvironment using:  
Multiplex IHC for PD-L1 expression, CD8, CD4, FoxP3 T-cell infiltration RNA-Seq transcriptome sequencing for tumor/immune gene expression profile
(and peripheral blood mononuclear cells T-cell receptor (TCR) sequencing for T-cell clonality and MET protein levels Next generation sequencing (TSO-500) for tumor biomarkers in tumor samples
Any of these exploratory evaluations which generate quantitative measures will be done using descriptive statistics including confidence intervals when appropriate. Any statistical tests performed for evaluation of exploratory objectives will be done without formal adjustment for multiple comparisons, but in the context of the number of tests performed.
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11 collaborative agreements 11.1 crada

This study will receive the investigational study agent under a Cooperative Research and Development Agreement (CRADA) with EMD Serono. CRADA Number: 02666.
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12 human subjects protections 12.1

Cisplatin ineligible or platinum refractory metastatic urothelial cancer patients may have received pior checkpoint inhibitor therapy or be checkpoint inhibitor naïve to enroll. Patients with metastatic urothelial cancer who received prior checkpoint inhibitors are left with limited options and the effect of subsequent immunotherapy treatment is unknown. While current PD-1/PD-L1 checkpoint inhibtors have shown efficacy in checkpoint naïve patients who are either cisplatin ineligible or refractory response rates response rates are about 20% [46]. Therefore, it is imperative to improve on this response rate with drugs like M7824 that have potential of increasing response rates through immunomodulation. These reasons express the need to determine M7824 activity in our patient population. Participants who do not accept blood transfusions will be excluded. As there is a risk of severe bleeding with this study drug, participants must be willing to receive blood transfusions if medically necessary for their own safety. Participants must be able to receive blood transfusions in order to minimize the risks of receiving M7824. Another reason for this is that including these patients could compromise the scientific validity of the study. For example, death from blood loss could make it difficult to assess other aspects of the investigational immunotherapy's safety—a primary scientific goal in this early-phase immunotherapy trial, which are carried out in small numbers of participants.
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12.2 participation of children

Because no dosing or adverse event data are currently available on the use of M7824 in patients less than 18 years of age, children are excluded from this study.